reportes - elfosscientiae.cigb.edu.cu · @corresponding author 1st international and 2 nd national...

@ Corresponding author

1st International and 2nd National Workshopon Vaccine Adjuvants. 5-8 December 2001, Havana, Cuba

@ Tamara Rodríguez , Oliver Pérez

Finlay Institute, AP 16017, Ciudad de La Habana, Cuba.E-mail: [email protected]; [email protected]

ABSTRACTThis workshop was organized by the Cuban Society for Immunology (CSI) with the cooperation of the Latin Ameri-can Association for Immunology (ALAI), the Finlay Institute, the Center for Genetic Engineering and Biotechnology(CIGB), the American Society for Microbiology (ASM), and Alfa Universal Invest., Corp.Adjuvants help antigens to elicit an early, high and long-lasting immune response with less antigens. In recentyears, adjuvants received much attention because of the development of purified recombinant and syntheticpeptide vaccine candidates, many of which are poor immunogens and require adjuvants to evoke the desiredimmune response. Besides, for many years, the only adjuvants available for general use in human vaccines werealuminium salts; but new adjuvants like proteoliposomes are already extensively used.With the use of adjuvants the immune response can be selectively modulated to a Th1 or Th2 response which isvery important for protection against diseases caused by intracellular and extracellular pathogens, respectively. Todiscuss updated aspects concerning the development of adjuvants of importance in human and veterinary vaccines,the Cuban Society for Immunology (CSI) organized from December 5-8 of 2001 The 1st International and 2nd

National Workshop on Vaccine Adjuvants with the cooperation of the Finlay Institute, the Center for GeneticEngineering and Biotechnology (CIGB), The Latin American Association for Immunology (ALAI) and the AmericanSociety for Microbiology (ASM) at the Neptune-Triton Hotel in Havana, Cuba.Dr. Gustavo Sierra (President of the CSI and ALAI, Vicepresident of the Finlay Institute and President of the ExpertVaccines Committee) chaired the meeting that he organized together with Dr. Oliver Pérez (General Secretary ofCSI and ALAI). The meeting was opened by stating that the discussion and proceedings should involve the screen-ing and application of new adjuvants for many different kinds of vaccines. The program emphasized vaccination forprophylaxis or therapy in infectious diseases and in therapy of cancer. It also focused on adjuvants as carrierproteins, immunomodulators, appropriate delivery systems, whole organism vectors, liposomes and proteolipo-somes. Future meetings may also consider procedures for safetly testing of new adjuvants and regulatory aspectsof their application in humans as well as emphasize in vaccine adjuvants already in, or very close to, enteringclinical trials. Another interesting point suggested for future meetings was the use of cytolysins as a new approachfor immune deviation and the induction of CTL response in vaccine development.Topics of special interest were the role of dendritic cells as natural adjuvants, the ability of adjuvants to selectivelymodulate the immune response to Th1 or Th2, the advances in adjuvants for cancer vaccines, the rational design ofnew vectors as antigen-delivery systems, adjuvants for mucosal vaccine delivery, adjuvants that can be used invaccines against viral or parasitic vaccines and the adjuvant capacity of liposomes and proteoliposomes.Researchers representing twenty-three different academic institutions and three pharmaceutical companies in-volved in adjuvant research and development attended to the meeting. The most import aim of this workshop wasto discuss updated results in the field of adjuvants and to strengthen relationships between researchers and com-panies dedicated to research and production of new vaccines.The present report is structured in topics which include oral presentations and posters after a summarized explana-tion of each topic by the chairpersons.

Keywords: adjuvants, immunology, vaccines

Biotecnología Aplicada 2002;19:49-96

RESUMEN1er Taller Internacional y 2do Taller Nacional Sobre Adyuvantes Vacunales. Diciembre 5-8 del 2001,La Habana, Cuba. Este taller fue organizado por la Sociedad Cubana de Inmunología (SCI) con la cooperación dela Asociación Latino Americana de Inmunología (ALAI), el Instituto Finlay, el Centro de Ingeniería Genética yBiotecnología (CIGB), la Sociedad Americana de Microbiología (ASM) y la compañía Alfa Universal Invest.Los adyuvantes ayudan a inducir una respuesta inmune temprana, elevada y de larga duración contra el antígeno,siendo necesario incluso menores concentraciones del mismo. En los últimos años los adyuvantes han recibidomucha atención debido al desarrollo de péptidos sintéticos y recombinantes purificados como candidatos vacunales,muchos de los cuales son pobres inmunógenos y requieren adyuvantes para evocar la respuesta inmune deseada.Por muchos años, el único adyuvante disponible para el uso general en vacunas humanas fue las sales de aluminio,sin embargo, nuevos adyuvantes como los proteoliposomas son ahora extensivamente usados.Con el uso de los adyuvantes la respuesta inmune puede ser selectivamente modulada a Th1 o Th2 lo cual es muyimportante para la protección contra enfermedades causadas por patógenos intracelulares y extracelulares

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respectivamente. Con el objetivo de discutir los más actuales aspectos concernientes al desarrollo de adyuvantesde importancia para vacunas humanas y veterinarias la SCI organizó del 5-8 de Diciembre de 2001 el 1er TallerInternacional y 2do Taller Nacional Sobre Adyuvantes Vacunales que tuvo lugar en el Hotel Neptuno-Tritón enCiudad de La Habana, Cuba.El Dr. Gustavo Sierra (Presidente de la SCI y de la ALAI, Vicepresidente del Instituto Finlay y Presidente del Comitéde Expertos en Vacunas) presidió el taller junto al Dr. Oliver Pérez (Secretario General de la SCI y de la ALAI). Lareunión fue inaugurada sobre la base de propiciar que las discusiones y procedimientos involucraran principalmentela selección y aplicación de nuevos adyuvantes para diferentes tipos de vacunas. Tópicos de especial interés fueronel papel de las células dendríticas como adyuvantes naturales, la habilidad de los adyuvantes para modularselectivamente una respuesta inmune Th1 o Th2, los avances en adyuvantes para vacunas contra cáncer, el diseñoracional de nuevos vectores como sistemas liberadores de antígenos, adyuvantes para vacunas mucosales, adyuvantesque pueden ser usados en vacunas contra enfermedades virales o parasíticas y la capacidad adyuvante de liposomasy proteoliposomas.Se propusieron como temas a discutir en futuras reuniones los procedimientos para evaluar de forma seguranuevos adyuvantes y los aspectos regulatorios para su aplicación en humanos, así como enfatizar en los adyuvantesvacunales que ya están en estudios clínicos. Otro punto interesante sugerido para reuniones futuras fue discutir eluso de citolisinas en el desarrollo de nuevas vacunas como una novedosa aproximación para desviar al sistemainmune hacia la inducción de respuesta CTLInvestigadores de veintitrés instituciones académicas y tres compañías farmacéuticas asistieron a la reunión, siendoel objetivo más importante de este taller discutir los resultados más actuales en el campo de los adyuvantes yestrechar las relaciones entre los investigadores y compañías dedicadas a la investigación y producción de vacunas.El presente reporte está estructurado por temas los cuales incluyen una explicación resumida de cada uno elaboradapor los presidentes de cada sesión seguida de los resúmenes de las presentaciones orales y en carteles.

Palabras claves: adyuvantes, inmunología, vacunas

IntroductionFive talks within the programmed workshop plusan additional talk on the subject delivered in thesecond day of the meeting constituted the core ofthe presentations dealing with DC. However, theissue of the function of DC was brought up in mostof the papers presented at the meeting, alluding totheir potential as the initiators of the immune re-sponse elicited by adjuvants/vaccines. Althoughthere was no a poster session explicitly devoted tothe topic, discussions that took place around theposter presentation in other sections often includedDC as a subject. The speakers presenting in theworkshop were:

Workshop on Dendritic Cells as Natural Adjuvants

Chairpersons: José Alejandro López,1 Circe Mesa2

1Mater Medical Research Institute, Brisbane, Australia. E-mail: [email protected] of Molecular Immunology, Havana, Cuba. E-mail: [email protected]

ABSTRACTThe fast growing field of research on dendritic cell (DC) biology is the focus of attention by vaccinologists andscientists developing optimal adjuvants for currently utilized/developed vaccines. This workshop was aimed todiscuss basic aspects of the biology of DC for its application in the practice of current adjuvant technology in thedevelopment of more efficient vaccines. The overall take-home message transmitted during the meeting can besummarized in that DC are the main targets of a vaccination program and that it can be achieved in two mainforms. First, the delivery of �danger signals� to DC generates their activation and, therefore, their more efficientperformance in the initiation of the immune response. Products derived from bacteria, like proteoliposomes, fromvirus-like particles (VLP), or from DNA contain elements that prompt DC to be activated and therefore to presentantigens to both naïve and cognate lymphocytes. The second element that is crucial in the optimal utilization of DCis the choice of DC aimed vehicles that may directly target one (or more) of the functions of DC. Specific receptorshave been now identified and may play a major role in the re-direction of the immune response. Specifically, theperformance of G protein-coupled receptors and their integrative role as neurotransmitter and cytokine is anexciting field of research that may be conducive to the best dissection of the Th1/Th2 dichotomy in the generationof an immune response to a vaccine.

Johannes Hoebeke from the C.N.R.S. “Immunologieet Chimie Thérapeutiques”, I.B.M.C., 15, rue Descartes,F-67084 Strasbourg (France) with the title: “G proteincoupled receptors: from dendritic cells to lymphocytes”.

José Alejandro López from the Mater Medical Re-search Institute, South Brisbane, 4101, (Australia) withthe titles: “A human blood dendritic cell purificationplatform for clinical applications” and “Dendritic celltherapy of melanoma” (Presented on behalf of Dr. ChrisSchmidt from the Queensland Institute of Medical Re-search, Brisbane, Australia).

Claude Leclerc from the Unité de Biologie desRégulations Immunitaires, Institut Pasteur, Paris,

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(France) with the title: “Cross-priming of CTL re-sponses by exogenous virus-like particles”.

Ann Hill from the Department of Microbiologyand Immunology, L220 Oregon Health Sciences Uni-versity, Portland, OR 97201 (USA) with the title:“Coupling apoptosis to antigen expression in a DNAvaccine: augmenting immunity or tolerance?”.

Circe Mesa from the Vaccine Department, Centreof Molecular Immunology, PO Box 16040, Havana,Cuba, with the title: “VSSP: an ideal adjuvant for DCactivation”.

Main BodyThe field of research on DC biology is currently in afruitful period that resembles that of research on AIDS10 years ago. Furthermore, since the discovery of asystem to produce large quantities of DC-like cells de-rived from monocytes in 1994 [Romani et al., 1994, J.Exp. Medicine, 180:83; Sallusto et al., 1994, J. Exp. Medi-cine, 179:1109], the use of DC in immunotherapy bothin animal models and, more recently, in human clinicaltrials has increased exponentially. In the Figure, the num-ber of publications containing the words “DC” and “im-munotherapy” in the last years has augmentedsubstantially, in parallel to a significant decrease in thepublications related to AIDS. This indirect indication ofthe success in this area of research reflects the impor-tance of DC in various areas of the immune response.

The evaluation of G-protein receptors, the type ofRhodopsin has been extended now to DC. Chemok-ine receptors belonging to the family of moleculeswith similar structure, have been identified in DC andtheir function in maturation and migration is beingcurrently studied. Immature DC express α1b adren-ergic receptors that may participate in the cross talkbetween the immune and the nervous systems. A num-ber of neurotransmitters such as histamine, SubstanceP, or various chemokines, may be some of the smallmolecules interacting with these receptors on DC. Thedimerization of the molecules upon activation triggersthe NF-κβ activation pathway leading to lympho-cyte specific responses that may alter the Th1/Th2balance of the response (Hoebeke, France).

A new approach for the generation of DC derivedfrom blood (BDC) was presented and it utilizes mag-netic beads and biotinylated monoclonal antibodies.Data presented during the workshop suggest that thephysiology of DC derived by this mechanism differsfrom those obtained via cytokine meditated matura-tion of monocyte (or CD34+) precursors. The induc-tion of primary responses in the context of both class Iand II MHC molecules appears to be more efficientby BDC; furthermore, the expression of costimulatorymolecules [Hart et al., 2000, 7th leukocyte differen-tiation antigen workshop DC section summary, inpress] and antigen uptake [Ho et al., 2002, Blood 99:in press] differ between the two DC types. Evidenceshown here demonstrates that it is possible to gener-ate sufficient number of BDC for immunotherapyusing a combination of apheresis and a single stepmagnetic bead isolation (CliniMACS) in a closed, ster-ile, clinical grade protocol (López, Australia).

In studies in a mouse system, the presentation ofantigen to CD8 lymphocytes (CTL) mediated by viruslike particles (VLP) was shown to be mediated by DC;

specifically, CD8+ in particular those from the CD11csubtype are the cells responsible for the powerful adju-vant capacity of VLP. Using the ovalbumin as an antigenmodel, the recognition of the famous Kb restrictedSIINFEKL CTL epitope by CTL was achieved by in-jecting recombinant porcine parvovirus (PPV)-VLP-OVA; the construct, derived from the VP2 capsid proteinof the virus formed 24 nm particles after their expressionin insect cells that were used in the absence of any addi-tional adjuvant. These findings highlight the importanceof directing antigens to DC in order to optimize theadjuvant capacity of these cells (Leclerc, France).

DC can also induce immuno-regulatory responses thatmodulate the recognition of cognate antigens. A follow-up study of a DNA vaccination protocol includingapoptosis-inducing genes and a model antigen showedinduction of regulatory T-lymphocytes upon a secondinjection of the cognate antigen in the absence of specificCTL activity. In fact, the expected splenomegaly in-duced by the vaccinia infection was absent in animalspreviously immunized s.c. with a DNA construct con-taining the apoptosis-inducing gene in the presence ofthe specific cognate antigen. These results could be in-terpreted as the function of tissue DC that, lacking theexpression of a second “danger” signal, induced regula-tory responses to cognate antigens (Hill, USA).

The use of less toxic novel adjuvant formulations iscurrently a subject of intense research. Bacterial prod-ucts such as CpG and LPS are known to activate DCand, therefore, explain an adjuvantory capacity of thesecompounds. The outer membrane protein complex ofNeisseria meningitidis was coupled to form very smallsize proteoliposomes (VSSP) with gangliosides, a self-antigen the immune system is normally tolerant to. Thisnew formulation proved to be safe and effective in hu-mans. It induced both the maturation of mouse (bonemarrow) and human (monocyte)-derived DC in vitroand the activated DC stimulated IFN-γ producing CD4+

T-lymphocytes. The fact that LPS hyporesponsiveC3H/HeJ also exhibits similar sensitivity suggests thatthe activation observed in this strain was LPS indepen-dent (Mesa, Cuba).

SummaryWe are begining to have a fuller glimpse of the vast fieldof DC biology and in the process we realize the im-mense potential of these cells to modulate the immuneresponse. The main features of the vaccination proto-cols in the coming future will, necessarily, include DC asa target. Moreover, as we better understand their per-formance, we can also exploit their immunoregulatorypotential in the treatment of autoimmune diseases.Clearly, the use of DC as natural adjuvants is not apractical alternative for many immunization protocols;however, our understanding of the ways in which adju-vants (and candidates) affect DC will be crucial in theirevaluation and judicious use for vaccine development.

Oral PresentationsG protein-coupled receptors: from dendriticcells to lymphocytesJ Hoebeke. UPR9021 du C.N.R.S. Immunologie etChimie Thérapeutiques, I.B.M.C., 15, rue Descartes,F-67084 Strasbourg (France).E-mail: [email protected]

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Increasing evidence suggests the importance ofneurotransmitters in the regulation of the immuneresponse. Such neurotransmitters mostly act on Gprotein-coupled membrane receptors. Since chemok-ine receptors are also included in this family, I shallgive an introduction over what we know about thestructure of G protein-coupled receptors at the handof the 3D structure of rhodopsin, about their activa-tion mechanisms with emphasis on dimerisation,about their signal transduction mechanisms and theirregulation by desensitization mechanisms. I shallfurther explain how the transduced signals can posi-tively or negatively interfere with the activation ofimmune cells by the NF-kB pathway. I shall furthersummarize our knowledge about the secretion of dif-ferent neurotransmitters by immune cells such asdendritic cells and lymphocytes and about the pres-ence of functional G protein-coupled receptors inthe same cells. I shall then present recent findingsabout the regulatory mechanisms on Th1/Th2 re-sponses by catecholamines and histamine. These re-sults will be discussed in the general context ofimmune status and in the particular context of vacci-nation protocols.

A human blood dendritic cell purificationplatform for clinical applicationsLópez JA, Bioley G, Ho CSK, Turtle CJ, Vuckovic S,Crosbie GV, Munster D, Wright S, Taylor K, Rodwell R,Hart DNJ. Mater Medical Research Institute, SouthBrisbane, 4101, Australia. Phone: +61-7-38402562;Fax: +61-7-38402562. [email protected]

Objective: To establish a blood dendritic cells (DC)isolation strategy for cancer immunotherapy. DCgenerated in vitro by transforming monocytes withexogenous cytokines (Mo-DC), are functionally dif-ferent from blood DC. We describe a platform forisolation of blood DC that are: 1) in a defined stateof differentiation, 2) able to respond to physiologi-cal stimuli with the capacity to take up and processantigen, 3) more homogeneous and, 4) not exposedto exogenously added cytokines. Methods: Afterovernight culture of PBMC, biotinylated CMRF-44/56 were used to purify DC in a single step mag-netic beads (Miltenyi Biotec, Germany) procedure.DC composition was evaluated by FACS in 10L and15L COBE Spectra PBMC apheresis product ob-tained from 16 healthy individuals. Results: Usingbuffy coats as a source of PBMC, magnetic beadseparations yielded up to 99% CMRF-44+ cells in-cluding up to 67% CMRF-44+ CD14- CD19- DC,i.e. a 100 fold enrichment from the 0.5% starting DCpopulation. The yield of CMRF-44+ cells variedwith individuals (n=53) with a mean of 52% (range19%-99%). Blood DC (CD14- CD19- CMRF-44+)averaged 17% (range 3% - 67%). Similar results wereobtained using CMRF-56 mAb (n = 10); an averageof 46% CMRF-56 cells were obtained, of which 17%(range 4% – 31%) were blood DC. Overall, the pro-cedure isolated an average of 2.2% (CMRF-44) and1.3% (CMRF-56) of the starting PBMC, respec-tively. The purified CMRF-44+ cell fraction wasmore potent in eliciting an allogeneic MLR comparedto whole cultured PBMC and no allo-stimulatoryactivity was observed in the CMRF-44- fraction.

Moreover, MLR stimulatory capacity was identicalto that found with FACS sorted CMRF-44+ CD14-CD19- cells. CMRF-56+ isolated cells efficientlygenerated primary immune responses restricted byboth class II (KLH) and class I (HLA-A*2001) MHCmolecules. We explored the yield of blood DC ob-tained from apheresis product in 16 healthy volun-teers and established that the automated softwarecontrol (6.0 Auto PBPC) yielded the better results.DC subset and activation status were not altered bythe procedure, and DC numbers obtained weregreater than 20 million in all cases, representing atleast 20 DC immunization doses. CMRF-44+ cellswere also successfully isolated from apheresis prod-ucts and showed intact allo-stimulatory capacitybefore and after freezing. Conclusions: We describe aclinically applicable procedure for obtaining bloodDC in sufficient quantity and quality for cancer im-munotherapy.

Cross-priming of CTL responsesby exogenous virus-like particlesG Morón,1 GP Rueda,2 I Casal,2 C. Leclerc.1 1Unitéde Biologie des Régulations Immunitaires, InstitutPasteur, Paris, France, and 2INGENASA, Madrid,Spain. [email protected]

Virus-like particles (VLPs) have revealed an excep-tional capacity to trigger CTL responses. However,the mechanisms of uptake, processing and presenta-tion of these exogenous particles remain unclear. Inparticular, the antigen-presenting cells (APC) involvedin the induction of CTL response by VLPs is stillunknown. We have developed an antigen-deliverysystem based on non-replicative, recombinant par-vovirus-virus-like particles (PPV-VLPs) formed bythe self-assembly of the VP2 capsid protein of por-cine parvovirus (PPV). The VP2 protein, the mostabundant structural viral protein of the porcine par-vovirus capsid, carrying foreign CD8+ T cell epitopesself-assembles into 25 nm pseudo viral particles afterexpression in insect cells. Mice immunized with thesePPV-VLPs carrying a CD8+ T cell epitope from theLCMV nucleoprotein, in the absence of adjuvant, de-veloped a CTL response against LCMV, which pro-tected mice against a lethal intracerebral injection ofLCMV, based on the induction of high-frequency ofCTLs of high-avidity. In the present study, we deter-mined the APC involved in the presentation of PPV-VLPs by MHC class I molecules, using PPV-VLPscarrying the Kb-restricted CD8+ OVA epitopeSIINFEKL (PPV-VLPs-OVA). Spleen dendritic cellsfrom mice injected with PPV-VLPs-OVA and trans-ferred onto naïve mice elicited CTL response againstSIINFEKL-coated target cells. Moreover, after i.v.injection of C57BL/6 mice with PPV-VLPs-OVA, ex-vivo antigen presentation assays evidenced thatCD11c+ cells dendritic cells (DC) were very efficientAPC and that CD8a- DCs rather than CD8a+ DCswere able to present the CTL-OVA epitope to a spe-cific CD8+ hybridoma. Flow cytometric assays dem-onstrated that in vivo PPV-VLPs are mainly uptakenby DCs, with no differences between CD8a+ andCD8a- DCs. Therefore, PPV-VLPs are selectivelypresented by CD8a- DCs, and this differential anti-gen presenting capacity is not mediated by differ-

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ences in the uptake capacity, or by on maturationlevel between both DCs sub-populations.

Apoptosis coupled to antigen expressionin a DNA vaccine induces an antigen-dependent bystander anti-inflammatoryeffectD Mourich, A Hill. Dept of Microbiology and Immu-nology, L220 Oregon Health Sciences University, Port-land, OR 97201. [email protected]

Dendritic cells can engulf dying cells, take up an-tigen and present it on their own MHC class I mol-ecules, a process known as cross-presentation. Wesought to exploit this phenomenon to improve theefficacy of DNA vaccination. To this end, we coupledmodel antigens to an apoptosis inducing gene(E4ORF4) in a DNA vaccine in an attempt to aug-ment cross priming of CTL. However, we found noinfluence on the numbers of CTL generated after in-tramuscular injection. When the vaccine was admin-istered subcutaneously (sc), no CTL response couldbe detected. Surprisingly, when these sc-immunizedanimals were subsequently challenged with a vac-cinia virus expressing the cognate antigen, the nor-mal splenomegalic response to vaccinia failed todevelop. Control animals (primed or challenged withirrelevant antigen, or with vaccine expressing anti-sense E4ORF4) developed large spleens. The differ-ence was not due to better control of vaccinia virus,as all animals developed similar titers. We believethat we have induced antigen specific regulatory Tcells that respond to antigen by secreting anti-in-flammatory factors. We believe that this responsewas induced because dendritic cells perceive cellsdying of apoptosis as “normal”, and in the absenceof other “danger” signals induce a protective, regula-tory response which acts to suppress inflammation.These results highlight the importance of the contextin which antigen is presented in the ability to primean immune response. In addition, these results indi-cate that this methodology could be exploited to

modulate undesirable inflammatory processes suchas autoimmune disease.

VSSP: an ideal adjuvant for DC activationC Mesa,1 K Rigley,2 LE Fernández.1 1Vaccine Depart-ment, Centre of Molecular Immunology. PO Box 16040,Havana, Cuba; [email protected] 2Dendritic CellGroup, Edward Jenner Institute for Vaccine Research,Compton, Newbury, RG20 7NN, UK.

Adjuvants are an important component of many vac-cine formulations. Recently a new generation of adju-vants which provide ‘danger’ signals that switch theimmune system on has arisen. However, many of themhave adverse side effects and are therefore not acceptedin humans for routine vaccines. We have described anew approach in the cancer vaccines field, in whichgangliosides are incorporated into the outer membraneprotein complex of Neisseria meningitidis (Nm) to formVery Small Size Proteoliposomes (VSSP). This novelvaccine has shown the ability to overcome the tolerancenormally associated with gangliosides and its use is safein humans. This results drove our attention to theimmunopotentiatory properties of VSSP. Given thatDC are required for the initiation of adaptive immuneresponses we investigated the effects of VSSP on den-dritic cell (DC) maturation. Because VSSP are essen-tially bacterial membranes we also designed experimentsin which the effects of LPS in the VSSP could be dis-sected from molecules distinct from LPS. Immature DC,derived from human monocytes or mouse bone marrowwere exposed to either LPS from Nm, or VSSP. BothVSSP and LPS induce similar DC maturation and stimu-late the production of similar patterns of cytokines. DCmatured by VSSP and LPS induce CD4+ T cell activa-tion “in vitro” and “in vivo” and INFγ production. How-ever, the adjuvanticity of VSSP can not be explained bythe LPS component alone since VSSP induces IL12p40,IL1b, MIF and IL6 in C3H/HeJ mice which are hypore-sponsive to LPS. This result also suggest that, perhapsanother molecule in addition to Toll4 is required forsome aspects of VSSP signaling.

Workshop on Cellular Adjuvants

Chairpersons: Agnes Le Bon1, Oliver Pérez2

1The Edward Jenner Institute for Vaccine Research, Compton, Newbury, Berkshire, England.E-mail: [email protected] 2Finlay Institute, Havana, Cuba. E-mail: [email protected]

ABSTRACTThe aim of the workshop �Cellular Adjuvants� was to highlight new findings concerning different aspects of thecellular immune response. In addition, results regarding the use of novel adjuvants with the capacity to stimulatedendritic cells, or to direct antigens to the cytosol and the use of components of an effective vaccine as an adjuvantwere presented.

IntroductionThree talks and six posters covered the scope of thisworkshop. The oral presentations were presented by:• Dr. Anselmo Otero from Havana University, Cuba,

with the title: “Cytolysins: a new approach forimmune deviation in vaccine development”.

• Dr. Agnes Le Bon from The Edward Jenner Insti-tute for Vaccine Research, England, with the title:

“The adjuvant activity of type I interferons: a linkbetween natural and adaptive immunity”.

• Dr. Oliver Pérez from the Finlay Institute, Cuba,with the title “Th1 response induced by the B com-ponent of VA-MENGOC-BC overcomes the thy-mus independence of polysaccharide C and primesfor memory in toddler”.

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Oral Sessions

A new antigen delivery system?Dr. Otero addressed a very interesting question re-garding how to direct the presentation of exogenousprotein to the class I pathway in order to generate agood CD8 T cell response. The approach of his workwas to use cytolysins. These proteins commonly ex-pressed by bacteria are porines and allow pathogensto escape to the cytoplasm. Part of this family, Lyste-riolysin from Lysteria monocytogene offer a good strat-egy to direct purified antigens or DNA in a solubleform or encapsulated into liposomes towards the class Ipathway. However, the bacterial origin of this porinemight raise some question about its safety for use inhumans. Therefore, Dr. Otero’s group looked for othersources of porines derived from non-bacterial organ-isms. They managed to isolate and characterize twonew cytolysins derived from the sea anemone:Stichodactyla helianthus. The biochemical character-ization of these new cytolysins revealed that their ac-tivity was pH dependent, which is compatible withtheir utilization to delivery antigens encapsulated inlow pH sensitive liposomes. Therefore, these resultsare quite promising for the future utilization of thesenew proteins as a novel system for vaccine delivery.

A new adjuvant for humoral and cellularresponse?I myself, Dr. Le Bon, presented a talk concerning theadjuvant activity of type I IFN. Based on the obser-vation that natural infections generally induce strongimmune responses and that innate cytokines such astype I interferons are normally induced to very highlevel shortly after infection, we have investigated thepotential role of type I interferons as an adjuvant. Weshowed that the co-injection of type I IFN with anantigen (CGG) was greatly enhancing the antibodyresponse specific for CGG leading to a far broaderresponse in terms of isotype switching with the emer-gence of IgG2a and IgG3 isotypes. The potency oftype I IFN as an adjuvant was comparable to CFA.Using mice deficient for type I IFN receptor we showedthat the adjuvant activity of CFA was indeed mainlytype I IFN dependent. We also showed that the pres-ence of type I IFN during the primary response wasleading to a long lasting production of antibodies aswell as a good memory response. Finally, we demon-strated that the adjuvant activity of type I IFN wasmediated by dendritic cells. Then, we investigated theeffect of type I IFN on T cell responses. We showedthat type I IFN was clearly enhancing the priming ofCD4 T cells as well as their ability to produce IFN-γ .More interestingly, we showed that type I IFN hadthe ability to induce CD8 T cell responses to exog-enous protein (OVA). Indeed, the addition of type IIFN to OVA during the priming was sufficient to in-duce OVA specific T cells to produce IFN-γ or to dif-ferentiate into CTL. Taken together, these data couldsuggest type I IFN as a very potent adjuvant.

Is VA-MENGOC-BCTM vaccine doing morethan antibodies?So far, the protective activity of vaccines against Neis-seria meningitidis has always been related to the pres-

ence of bactericidal antibodies. Dr. Pérez presentedstrong evidence that the Cuban vaccine VA-MENGOC-BCTM was not simply generating antibody responsesbut also cellular responses. It clearly showed thatyoung vaccinated babies were clearly able to mount aDTH response. Moreover, PBMC from young vacci-nated adult were able to produce IL-2 and IFN-γ afterrestimulation in vitro but no IL-4 nor IL-5 indicatingthe development of a Th1 type T cell response. Asecond aspect of his talk concerned the response topolysaccharide C. This polysaccharide derived fromserogroup C has been non covalently incorporated inthe OMV based vaccine specific from N. meningitidisB. This polysaccharide is a T-independent antigenonly able to induce the IgG2 isotype when given onits own. When incorporated to OMV from Neisseriaserogroup B, the response to polysaccharide C ismodified towards IgG4 and IgG3. Moreover, the mainproblem with the use of polysaccharide as antigen isthat the response that it generates is inadequate interms of isotype switching and not long lasting.Dr. Pérez showed that the immunization with VA-MENGOC-BCTM was sufficient to induce a long last-ing immune response to EMV and C components.Overall, Dr. Pérez presented new data highliting newunderstanding of the protective activity of VA-MENGOC-BCTM.

Poster SessionThe poster session covered several aspects of thecellular immune response and will be summarizedbriefly:

Possible role of neutrophils in the inductionof immune response against Neisseriameningitidis BM Lastre. Finlay Institute, Cuba.

The main results of this poster reveals that neutro-phils might play a role in the defense against N. men-ingitidis B by producing inflammatory cytokines andchemokines (TNF-γ, IL-1b, IL-8, and MIP-1α/β). In-terestingly, OMVs were also able to trigger neutro-phils to produce these pro-inflammatory cytokines.The possible role of neutrophils in the immune re-sponse induction is also addressed.

Bactericidal activity against N. meningitidis Bin relation to the Th1 response elicited byVA-MENGOC-BCTM

T Rodríguez. Finlay Institute, Cuba.This poster showed a clear correlation between the

bactericidal activity of antibodies directed against N.Meningitidis B in vaccinated subjects and their isotypesubclasses. The sera with the highest bactericidal ac-tivity were containing the highest titres of IgG1 andIgG3, which are good-complement fixing isotypes.

T-cell epitope mapping of P64Kmeningococcal protein in Balb/c miceS González. Centro de Ingenieria Genética y Biotec-nología, Cuba.

The aim of this work was to map dominant epitopesof p64K. Using overlapping peptides, it was shownthat p64k contained a dominant T-cell epitope in a15-amino acids sequence namely 470-485.

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Enhanced IFN-γ secreting CTL activity againstthe HIV-1 multi-epitope TAB9 by a priming-recom-binant Fowlpox virus boosting regimen after plamid-backbone manipulation (EG. Rodríguez, Centro deIngeniería Genética y Biotecnología, Cuba.). Thisposter showed results concerning the improvementof the plasmid coding for HIV-1 multi-epitope TAB9used for DNA vaccination. Indeed, insertion of mul-tiple copies of immunostimulatory sequences intothe backbone of the plamid was clearly enhancingthe CD8 T cell response as measured by IFN-γElispot Assays.

A truncated HCV core protein formulatedin different adjuvants is highly immunogenicinvolving a strong participation of cellularimmunity components in miceJC Álvarez-Obregón. Centro de Ingeniería Genéticay Biotecnología, Cuba.

This poster presented a study of the immune re-sponse induced in mice against a recombinant HCV-core protein. When administrated in Alum or in CFA,this vaccine was leading to a high production of anti-bodies, an increased expression of IFN-γ gene as wellas a good secondary proliferative response.

TNF-α and nitric oxide as targets in adjuvantscreeningD Gil. Finlay Institute, Cuba.

This poster showed the possibility to use TNFαand nitric oxide quantification in the screening of adju-vants. The induction of TNF? and NO by differentLPS was studied in vitro. The production of TNFaand nitric oxide by spleen cells after immunisationwith different adjuvants was also examined.

As a summary the Cellular Adjuvants session wasclearly covering a lot of different aspects of the cellu-lar response from innate response (eg: neutrophils,macrophages) to the generation of good Th1 responsenaturally induced (using Type I IFN as an adjuvant orby VA-MENGOC-BCTM), engineered (recombinantprotein, DNA plasmid) or by using cytolysins to di-rect antigen to the cytosol and allow presentation onClass I molecules. As an overall, the results presentedduring this session were high quality and raised someinteresting discussions.

Oral Presentations

Cytolysins: A new approach for immunedeviation in vaccine developmentAJ Otero. Lab of Immunology, Faculty of Biology,Havana University. [email protected]

Current strategies for vaccine development mainlyfocus the immune response in terms of protective anti-bodies by antigen processing and presentation viaphago-lysosome in the context of MHC class II mol-ecules. The assessment of long-lasting cytotoxic T lym-phocyte responses require the antigen to arrive into thecytosol for an adequate processing and presentation viaMHC Class I. Bacterial porines like Lysteriolysin fromListeria monocytogenes, as a clear evasive mechanism,facilitate the escape of this pathogen to cytoplasm. Thesemolecules have been used under novel vaccine approachesfor re-directing purified antigens, attenuated bacteria,

DNA sequences in a soluble form or encapsulated intoliposomes. Other cytolysins from non-bacterial sourcescould be used for the same purposes.

Th1 response induced by the B componentof VA-MENGOC-BC overcomesthe thymus independence of polysaccharide Cand primes for memory in toddlerO Pérez, M lastre, G Bracho, JM del Campo, TRodríguez, M, Díaz, C Zayas, G Sierra. Finlay Insti-tute, PO Box 16017, Havana, Cuba. Fax: (53-7)2086075. [email protected]

VA-MENGOC-BC is an outer membrane protein-based vaccine of serogroup B of Neisseria meningitidis.This also contain the serogroup C polysaccharide (PsC)which is non-covalently incorporated. We recently dem-onstrated the participation of other mechanisms pos-sible involved in their protection (1). Here we showfirst, the characteristic Th1 response induce by thisvaccine in young adults and toddler demonstrated atdifferent levels: functional (presence of delayed-typehypersensitivity, opsonophagocytic and bactericidalactivities and not immediate anaphylaxis nor passivecutaneous anaphylaxis); class and subclasses (IgG andIgG1 in human); molecular (induction of IL-2 and IFNγRNAm and not IL-4 nor IL-5 RNAm); prost-transcrip-tional (production of IL-2 and IFNγ and not IL-4 norIL-5 in supernatant of re-stimulated peripheral cells);and biological activity of γ-IFN produced. Second, atypical anti-PsC IgG secondary response was inducedin toddler which demonstrated that this vaccine over-come the absence of response of the PsC, a thymusindependent antigens. The main IgG subclass inducedby plain PsC vaccines in human is IgG2 which is verifyin our work. This response is changed by VA-MEN-GOC-BC to IgG4 and IgG3. Third, the priming in-duce by PsC not induced hyporesponsiveness for anatural challenge or a booster dose. Four, the long-last-ing immune memory against the B and C componentswas demonstrated before and after a booster dose.

1. Infect and Immun. 2001;69(7):4502-8.

The adjuvant activity of type I interferons:a link between natural and adaptive immunityA Le Bon,1 N Etchart,1 G Schiavoni,2 GD’Agostino,2 IGresser,3 F Belardelli,2 S Hou,1 DF Tough1. 1The Ed-ward Jenner Institute for Vaccine Research, Compton,Newbury, Berkshire, England. 2Istituto Superiore diSanità, Laboratorio di Virologia, Rome, Italy. 3InstitutCurie, Paris, France.

Type I interferons (IFN-I) are rapidly induced fol-lowing infection and play a key role in non-specificinhibition of virus replication. Here we have investi-gated the effects of IFN-I on the generation of antigen-specific responses. We show that IFN-I potentlyenhance the primary antibody response to a solubleprotein, stimulating the production of all subclasses ofIgG, and induce long-lived antibody production andimmunological memory. As well as enhancing antibodyproduction, IFN-I also augmented the priming of IFN-γ-producing CD4+ T cells. In addition, endogenous pro-duction of IFN-I was shown to be essential for theadjuvant activity of CFA. Finally, IFN-I enhanced theantibody response and induced isotype switching whendendritic cells were the only cell type responding to

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IFN-I. We are currently investigating the effect of IFN-Ion CD8+ T cell responses and our preliminary datasuggest that IFN-I strongly enhance the generation of aCTL response to co-injected protein. The data revealthe potent adjuvant activity of IFN-I and their impor-tant role in linking innate and adaptive immunity.

Le Bon A, Schiavoni G, D’Agostino G, Gresser I,Bellardelli P, David F. Tough. Immunity 2001;April:461-70.

Can the protective effect of a M. tuberculosisproteins cocktail be predicted or improved byimmunomodulation?AH Hovav, Y Fishman, H Bercovier. Department ofClinical Microbiology, the Faculty of Medicine, TheHebrew University, Jerusalem, Israel. [email protected]

We have cloned and purified four proteins of Myco-bacterium tuberculosis, which are immunogenic andpotential protective antigens, the 27 kDa, the L7/L12,the 85B and the ESAT-6 proteins. The cloned M.tuberculosis 27 kDa, L7/L12, 85B and the ESAT-6genes were subcloned in pcDNA3.1, a DNA vectorfor DNA vaccination in addition to the cloning in theplasmids pQE for antigen preparation. We obtainedwith the pQE expression vectors large amount (1-6 mg/mL) of the four recombinant proteins. Groupsof mice (female Balb/c 6-8 weeks, 18-20 g) were in-jected subcutaneously twice or three times, 3 weeksapart. Recombinant M. tuberculosis proteins, riboso-mal L7/L12; ESAT-6 (6 kDa); 85B (30 kDa); and thelipoprotein 27 kDa, were used as such, adjuvanted byRibi or entrapped in liposomes. IFN-γ was added atdifferent doses whether free or entrapped in liposomes.Identical amount of empty liposomes was injected inthe control group. The challenge was performed 4weeks after the last immunization by injecting i.v. adose of 106 CFU of the strain BCG Pasteur. The im-mune response resulting from the different formula-tions was evaluated by determining the ratios of IgG1/IgG2a and by profiles of lymphokines secreted byspleen lymphocytes during in vitro stimulation. Ani-mals were sacrificed 5 weeks after the challenge. Ingeneral the immune response was a mixed Th1/Th2response. Protection against BCG varied from noth-ing to a 2.5 log decrease in spleen CFU. The evalua-tion of the efficiency of protection according to thedifferent ways used to present the mycobacterial an-tigens will be discussed.

Poster Presentations

A truncated HCV core protein formulatedin different adjuvants is highly immunogenicinvolving a strong participation of cellularimmunity components in miceJC Álvarez-Obregón,1 S Dueñas-Carrera,1 CValenzuela,2 J Morales1. 1HCV Department, VaccineDivision; 2Clinical Trials Division. Centro deIngeniería Genética y Biotecnología. PO Box 6162,10600 Havana City, Cuba. Fax: (53-7) 2714764;E-mail: [email protected]

HCV infection leads to viral persistence and chronicdisease in at least 70% of cases, among which a sig-nificant proportion eventually develops cirrhosis andhepatocellular carcinoma. Current therapies are only

minimally effective and no vaccines have been devel-oped. The knowledge of anti-HCV-core responsescould be of prophylactic or therapeutical concern fortreatment of HCV infection. In the present study, theimmunogenicity of a truncated recombinant derivedHCV core protein (Co-120) in Balb/c and C57BL/6mice was examined. Three doses of protein wereadjuvanted onto either aluminum hydroxide or Freund´sadjuvant, and injected intramuscularly into Balb/c andC57BL/6 mice. The antibody response rose rapidlyafter the first dose, and at the end of the program bothstrains of mice exhibited comparable levels of anti-core IgG (titers above 1: 100 000), and a mixed IgG1/IgG2a subclass pattern of response was found. Spleencells from Co.120 immunized mice gave a significantspecific proliferative response with respect to con-trol (P < 0.01). An appreciable induction of IFN-γgene expression was also detected after an ex vivospecific stimulation of spleen cells from all immu-nized mice. The response showed to be independentof dose, H-2 genetic background or type of adjuvant.The results suggested that immunization with theCo.120 protein elicits a potent immune response witha strong participation of cellular immunity compo-nents, which could be taken into account to inducehumoral and cellular anti-HCV responses.

Enhanced IFN-γ -secreting CTL activity againstthe HIV-1 multi-epitope TAB9 by a DNApriming-recombinant Fowlpox virus boostingregimen after plasmid-backbone manipulationEG Rodríguez, DM Vázquez, AM Herrera, DQuintana, CA Duarte. Centro de Ingeniería Genéticay Biotecnología. Ave 31 e/ 158 y 190. Cubanacán.Playa. Ciudad de La Habana. AP 6162, CP [email protected]

Combined DNA priming-recombinant poxvirusboosting strategies have proven to be effective forgenerating antiviral immunity. In this work, we stud-ied the effect of two different expression cassettesand the number of plasmid-backbone immunostimu-latory sequences on the immune responses elicitedby this vaccination regimen. The pAEC’s transcrip-tional unit was replaced by another, containing the hu-man cytomegalovirus promoter, a synthetic intron anda synthetic RBG-based termination/polyadenilationsequences. Further constructs were generated by theinsertion of 5, 10, and 20 copies of the 5’-AACGTT-3’ immunostimulatory motif. Balb/c micewere immunized by intramuscular injection of 200mg of each plasmid, coding for the HIV-1 multi-epitope TAB9. After three doses of DNA, a fourthboost with plasmid DNA or a TAB9-expressing re-combinant Fowlpox virus (FPTAB9LZ) was admin-istered. The immune responses were evaluated byELISA and ELISPOT assays. Serum IgG antibodiesagainst TAB9 could not be detected. Only the ani-mals receiving the new set of DNA constructs in-duced a detectable effector CTL response, whichwas further increased by the prime-boost regimen.The response was dependent on the number of im-munostimulatory sequences. After in vitro re-stimu-lation with the IIIB peptide, significant CTL levelswere evidenced for all the groups immunized withthe new set of vectors.

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T-cell epitope mapping of the p64kmeningococcal protein in BALB/c miceS González,1 C Nazábal,1 KVS Rao,2 O Reyes,1 HEGaray,1 G Sardiñas,1 E Caballero,1 R Silva1. 1Centerfor Genetic Engineering and Biotechnology. PO Box6162, Havana 10600, Cuba. Tel: (53-7) 2716221, Fax:(53-7) 2714764; E-mail: [email protected] Center for Genetic Engineering andBiotechnology, Aruna Asaf Ali Marg, New Delhi110067, India.

Our group has previously characterized the P64kprotein of Neisseria meningitidis. Cloning and expres-sion in Escherichia coli of the lpdA gene, which en-codes for this protein, yielded a soluble antigenaccounting for more than 20% of the total host pro-tein [1]. Due to its relatively high molecular weight,demonstrated immunogenicity and availability, P64kwas employed as a carrier protein for poorly immu-nogenic peptides and N. meningitidis serogroup Cpolysaccharide with good results [2]. In this study,59 overlapping synthetic peptides that encompassedthe full-length 596 amino acids of the protein weretested for proliferation in P64k-sensitized mice. Thehighest proliferative responses were induced againstpeptides P1 (amino acids 1-20) and P48 (amino acids470-490). However, only lymph node cells obtainedfrom either P48- or P64k-sensitized mice, produced astatistically significant proliferative response whenchallenged with the homologous peptide or the re-combinant protein, respectively. In addition, threeoverlapping peptides spanning the P48 sequence weretested for proliferation in homologous peptide- andP48-sensitized mice. The highest proliferative re-sponse was obtained against a peptide that includesamino acids 470-485. We can conclude that this 15-amino-acid peptide (IPGVAYTSPEVAWVG) of P64kcontains an immunodominant T-cell epitope forBALB/c mice.1. Guillén G et al. Biotechnol Appl Biochem 1998;

27:189-96.2. González S et al. Scand J Immunol 2000;52:113-6.3. González S et al. Biotechnol Appl Biochem 2000;

32:1-8.

TNFα and nitric oxide as targets in adjuvantscreeningD Gil, M Lastre, R Delgado, J del Campo, O Pérez.Finlay Institute. PO Box 16017, Havana, Cuba. Fax:(53-7) 2086075; E-mail: [email protected]

The tumour necrosis factor α and the nitric oxide(NO) are molecules related with the proinflammatoryresponses and probably with Th1 responses. For thatreason we introduce the L-929 bioassay (TNFα) andGriess (NO) test for adjuvant screening. Different li-popolysaccharides (LPS, N. meningitidis B, V.cholerae, and S. Typhimurium) were included. VibrioLPS produced less TNFα than the other two and theproduction of NO was similar with all LPS. The EMVinduce the early production of TNFa and NO in peri-toneal macrophages from immunised (with anti-Neis-seria vaccine) and non immunised mice. TNFα wasinduced with lower doses of EMV than NO. TNFalevels from cells of immunised animals were higherthan from cells of not immunised animals, but theproduction of NO was similar. In addition, these as-

says were used in combination with antigenicity forthe evaluation of adjuvant stability.

Possible role of neutrophils in the inductionof immune response against Neisseriameningitidis BM Lastre,1 J Lapinet,1 G Bracho,1 C Zayas,1 M Díaz,1

G Sierra,1 MA Cassatella,2 O Pérez1. Depart. of Basicand Clinical Immunology, Finlay Institute. PO Box16017, Havana, Cuba; E-mail: [email protected]. of Pathology, Section of General Pathology,Verona, Italy.

The role of neutrophils (PMN) as effector cellsagainst bacterial infection and the production ofcytokines in response to polysaccharide (LPS) is welldocumented. However, no conclusive data of the im-portance of PMN as effector cell in Neisseria infec-tion and no data about the possible role of PMN inthe afferent arm of the immune response were re-ported. Therefore, we used a neutropenic rat modeland infected theme with N. meningitidis B. The treatedanimals showed a 100% of mortality in less than 12 h.The bacteriolytical activity of co-cultures of humanPMN and N. meningitidis B was demonstrated, whichwas increased with LPS and γIFN. In the afferent arm,the presence of high concentration of PMN aroundthe site of immunization with anti-N. meningitidis Bvaccine, VA-MENGOC-BC™, in hystopathologicstudies was observed. In addition, the outer mem-brane vesicles (OMV) from N. meningitidis B stimu-lated PMN and produced pro-inflammatory cytokinesand chemokines including TNFa, IL-1b, IL-8, MIP-1a/b which are increased by γIFN. IP-10 was alsoproduced by OMV+γIFN. IL-10, a regulatorycytokine, potently inhibited TNFα, IL-1b, IL-8, andMIP-1a/b production triggered by OMV. Finally, aneutralizing anti-TNFα mAb did not influence therelease of IL-8 and MIP-1b induced by OMV, there-fore excluding a role for endogenous TNFa in mediat-ing the induction of chemokine release by OMV. Incontrast, the ability of LPS from N. meningitidis B toinduce the production of IL-8 and MIP-1b was sig-nificantly inhibited by anti-TNFα mAb. In conclu-sion, our results establish the role of PMN as essentialeffector cells and that in response to OMV, PMNproduce a pro-inflammatory profile of cytokines andchemokines which may not only play a role in thepathogenesis of meningitis, but may also contributeto the development of protective immunity to sero-group B meningococci.

Bactericidal activity against N. meningitidis Bin relation to the Th1 response elicitedby VA-MENGOC-BCRodríguez T, del Campo J, Lastre M, Bracho G, ZayasC, Díaz M, Sierra G, Pérez O. Finlay Institute, POBox 16017, Havana, Cuba. Fax: (53-7) 2086075;Phone: (53-7) 2718221; [email protected]

The Cuban anti-Neisseria meningitidis B and C vac-cine, VA-MENGOC-BC induces opsonophagociticantibodies, lymph proliferation, positive DTH reac-tions and production of INFg in humans suggesting apreferential cellular (Th1) stimulation. However, thecorrelate of protection for serogroup B meningococciis not currently known but for serogroup C is belived

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to be the serum bactericidal assay (SBA).That is why,we evaluated the bactericidal activity against sero-group B in relation with the IgG subclasses elicited bythis vaccine in healthy adults. A new colorimetric se-rum bactericidal assay (cSBA) was standardized andused for the determination of the bactericidal activity(1) and the IgG subclasses were measured by ELISA.73% of evaluated sera showed high bactericidal re-sponse, corresponding to the cases with higher tittersof complement fixing IgG1 subclass that was the mainsubclass induced fallowed by IgG3. In summary, thehigh levels of IgG1 and IgG3, both complement-fixingsubclasses and it’s functionally efficiency in the acti-vation of the bactericidal response were in agreementwith our theory that the Th1 pattern induced by VA-

MENGOC-BC and the group of mechanisms elic-ited in this environment are the responsible of theefficiency showed by this vaccine [2].1. Rodríguez T, Lastre M, Cedré B, del Campo J,

Bracho G, Zayas C, Taboada C, Díaz M, Sierra G,Pérez O. Standardization of Neisseria meningitidisserogroup B colorimetric serum bactericidal assay.Clinical and Diagnostic Laboratory Immunology2002. In press.

2. Pérez O, Lastre M, Lapinet J, Bracho G, Padrón J,Díaz M, Zayas C, Taboada C, Sierra G. Immuneresponse induction and new effector mechanismspossibly involved in protection of Cuban anti-men-ingococcal BC vaccine. Infectious and Immunity.2001;4502-8.

Workshop on Carriers and Immunomodulators

Chairpersons: César Pérez-Maldonado,1 Gustavo Bracho2

1Cátedra de Inmunología. Facultad de Farmacia, Universidad de Los Andes. Mérida, Venezuela.E-mail: [email protected]. 2Finlay Institute, Havana, Cuba. E-mail: [email protected]

ABSTRACTAn important step in the development of effective vaccines is to assure the activation of cellular populations bymeans of specific and potent antigens. Recent advances on vaccination approaches show that the nature of adju-vants is of great importance for the immune system�s ability to fight against a broad array of infectious andpathological agents. The protective effect of certain antigenic proteins is only obtained when an appropriate pre-sentation/recognition is carried out by antigen presenting cells. Adjuvants must accomplish specific features, spe-cially those related to a lack of toxicity as well as the non-induction of tolerance. The composition and molecularstructure of the carrier have been established as �of extreme importance� for these achievements.

IntroductionMost of the oral and poster presentations in thissession (3 and 11, respectively) were focused onthe growing importance recently obtained by re-search on vaccination strategies. In our session, theindustrial sector was most highly represented (9posters and 1 oral presentation) followed by theuniversity (3 and 1, respectively). A significant num-ber of topics discussed were in accordance to presentday worldwide research publications which stressthe role of new vaccine adjuvants in the pursuit ofreliable and more effective ways to improve immu-nological functions. The following paragraphs high-light the most important features contained in thepresentations.

Impact of cytokines (IL-2, IL-4, IL-7 and γ-IFN)on the replication of primary HIV isolatesfrom infants in the thymusL Pedroza-Martins.University of California, Los An-geles, 10833 LeConte Avenue, Los Angeles CA, 90095,USA.

HIV replication was inhibited by IFN-γ , IL-2,and IL-4, together, enhanced primary isolates pro-duction in thymocytes. A significantly increased rep-lication of the X4 isolates as compared to R5 isolateswas observed when stimulated with IL-4 and IL-7.These cytokines effects were related to their induc-tion of T cell differentiation by increased chemokinereceptor expression in specific subsets. IFN-γ inhib-ited HIV replication in thymocytes through differ-ent mechanisms.

Characterization of P64k as a carrier proteinS González. Centro de Ingeniería Genética y Biotec-nología. Apdo 6162, La Habana 10600.

P64k increased the immune response against sixout of seven peptides and against the serogroup Ccapsular polysaccharide of Neisseria meningitidischemically coupled to this antigen. It was also foundthat P64k induced a specific humoral immune responsein 15 out of 18 individuals inoculated with no induc-tion of anti-mitochondrial antibodies.

Chemokines and their receptors in HIV-1infected childrenC Pérez-Maldonado. Cátedra de Inmunología. Escuelade Bioanálisis. Facultad de Farmacia. Universidadde Los Andes, Mérida. Venezuela.5101-A. Laboratoriode Inmunología. Hospital General. Hospitals Valld’hebron. Barcelona, España.

RANTES is used by HIV-1 and increased plasmalevels of it have been associated with HIV-1 infectionresistance and immune recovery. An importantRANTES augment in the infected children groups wasaccompanied by an improvement of the CD4+ / CD8+index during the post HAART period which indicatesthat an assesment o it levels could a reliable indicatorof immune recovery in the HIV-1 infection.

Cytokines production by M. tuberculosisH37Rv strainAY Arce Mendoza. Facultad de Medicina. U.A.N.L.NL, México.

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The way by which cytokine participates in thepathogenesis and disease, is not well understood. Thisstudy offers new information for the therapeutic man-agement.

The biological activity of IFNα 2b in liquidand freeze-dried formulationsLl Ruíz. Center for Genetic Engineering and Biotech-nology. Havana. Cuba.

The results obtained proved the physical, chemicaland biological equivalence between all of the examinedformulations

IL-2 biological activity in liquid lyophilizedformulationsN Reyes. Center for Genetic Engineering and Biotech-nology. Havana. Cuba.

As judged by SDS/PAGE, RP-HPLC and the bio-activity assay, formulations resulting from this workare equivalent to others previously described by re-searchers.

Immune response induced in mice by twomeningoccocal polysacharide-proteinconjugatesM Cuello. Finlay Institute. Havana, Cuba.

In conclusion, at the level of IgG subclass antibod-ies, the conjugate PsC-OMP was able to generate aTh1 immune response, whereas the conjugate PsC-TTT generated a Th2 immune response.

Comparative immunogenecity of conjugatescomposed of Neisseria meningitidis serogroupC polysaccharide to tetanus toxoid bydifferent spacer armsO Cabrera. Finlay Institute. Havana. Cuba.

Higher levels of anti-TT antibodies were elicitedby conjugates than TT itself.

P64k as a carrier protein of Neisseriameningitidis PorA peptidesG Sardiñas. Center for Genetic Engineering and Bio-technology. Havana. Cuba.

In two out of three conjugates, the anti-peptidesera reacted with native meningoccocal outer mem-brane vesicles in ELISA, suggesting that chemical con-jugation to the carrier granted, in addition to T-cellhelp, a proper folding to these synthetic peptides.

Comparison of the immune response inmice against meningoccocal group Cpolysaccharides conjugated to three carrierproteinsA Álvarez. Center for Genetic Engineering and Bio-technology. Havana. Cuba.

The carrier protein present in the C-Ps conjugatesinfluenced the levels of the IgG antibodies elicited tothe Ps. In this regard, P64k was superior to TT andBSA.

Immunization with a peptide sequencederived from Neisseria meningitidis usingdifferent vaccine formulationsT Menéndez. Center for Genetic Engineering and Bio-technology. Havana. Cuba.

The best results were achieved in the group immu-nized with the three doses assayed of the MAP con-jugated to P64k using the carbodiimide method.

Peptide synthesis containing a B-celland a T-cell epitopes on dextran beadsand evaluation of the humoral responseagainst bead-peptide constuctLJ Cruz. Center for Genetic Engineering and Biotech-nology. Havana. Cuba.

This experiment suggest that peptide synthesis ondextran beads can be used to raise their immunogeni-cuty in order to use them on vaccines or therapeuticsapproaches.

Effect of conjugation methodologyof the immunogenicity and protectiveefficacy of meningoccocal group Cpolysaccharide P64k protein conjugatesT Carmenate. Center for Genetic Engenieering andBiotechnology. Havana. Cuba.

This experiment proves that is possible to gener-ate a protective T-cell dependent response againsmeningoccocal C polysaccharide using P64k proteinas carrier.

SummaryOral and poster sessions pointed towards the P64kability as a suitable carrier. Dr. S González (CIGB.Cuba) exposed its ability to increase the immuneresponse whether attached to peptides or to bacte-rial antigens, and Dr. G Sardiñas, (CIGB, Cuba)showed its capacity to elicit a potent Th1 and Th2response and high reactivity against native menin-goccocal antigens. Dr. C Pérez-Maldonado (ULA,Venezuela) presented chemokines as immunomodu-lators that have a “protective effect” on certain patho-logical entities. Although the value of cytokineproduction during different stages of M. tuberculo-sis infection needs further research work, Dr. AY,Arce Mendoza (UANL, Mexico) also showed theirpotential impact for future therapeutical approaches.Dr. Ll, Ruiz and Dr. N, Reyes, (CIGB, Cuba) provedthat new storage strategies provided important find-ings about the long-standing biological activity offrozen cytokine formulations. The possible peptidesynthesis on dextran beads exposed by Dr. LJ Cruz(CIGB, Cuba) offers novel and very suitable methodto improve a more potent and specific array of syn-thetic peptides.

Oral Presentations

Impact of cytokines (IL-2, IL-4, IL-7 and γ-IFN)on the replication of primary HIV isolatesfrom infants in the thymusL Pedroza-Martins,1 WJ Boscardin,1 D Schols,2 YJBryson,1 C Uittenbogaart1. 1University of CaliforniaLos Angeles, 10833 LeConte Avenue, Los Angeles CA,90095, USA. 2Rega Institute for Medical Research,Katolieke Universiteit, Leuven, [email protected]

HIV infection of the thymus disrupts T cell devel-opment and may impact the outcome of disease inchildren. Cytokines such as IL-2, IL-4 and IL-7 play

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a role in thymopoiesis and could be manipulated toincrease T cell reconstitution or as adjuvants to aug-ment vaccine efficacy. Some of these cytokines andIFN-γ are indeed induced, intentionally or not, byvaccination and in reaction to boosters and/or chal-lenge with the infectious agents. Notwithstanding theirbeneficial effects in immunity, we and others havefound that IL-2, IL-4 and IL-7, alone or in combina-tions, can enhance the replication of HIV in thy-mocytes, cord and peripheral blood lymphocytes. Incontrast we found that IFN-γ inhibits HIV replicationand CD4 depletion in thymocytes, while others foundno negative effects of IFN-γ on HIV replication inperipheral blood mononuclear cells. Consequently, itis important to determine whether immunotherapyand vaccine strategies for HIV-infected children thatinvolve cytokines could impact HIV replication in thethymus. We addressed this issue by analyzing theimpact of cytokines on the replication of HIV in thepost-natal thymus using well-defined in vitro sys-tems (thymus organ cultures and suspension cultures).Ten HIV primary isolates obtained at /or close to birthwere characterized according to their replication ki-netics, response to cytokines and patterns of viralexpression in thymocyte subsets at different stagesof maturation. The use of CCR5 and CXCR4 ascoreceptors was determined directly in the thymusby blocking studies with monoclonal antibodies andchemokine receptor antagonists.Viral tropism, thy-mocyte differentiation, and chemokine receptor ex-pression were analyzed by flow cytometry.ELISPOT was used to assess cytokine production.Viral production was measured by ELISA (p24). Wefound that all 10 HIV primary isolates were able toreplicate in thymocytes. Loss of CD4 expressionwas observed after infection with primary isolatesusing CXCR4 (X4) as well as CCR5 (R5) ascoreceptors. Replication of most HIV isolates fromneonates was inhibited by IFN-γ , but large differ-ences in sensitivity were observed. IL-2 and IL-4,together, enhanced the production of all primary iso-lates in thymocytes. In contrast IL-4 and IL-7, sig-nificantly increased the replication of the X4 isolatesas compared to R5 isolates. These effects of IL-2,IL-4 and IL-7 were related to their ability to induceT cell differentiation and increase chemokine recep-tor expression in specific thymocyte subsets, there-fore expanding the range of thymocytes able to fullysupport HIV replication in the thymus. In contrast,IFN-γ inhibited HIV replication in thymocytesthrough mechanisms that did not involve T cell matu-ration or major changes in chemokine-receptor ex-pression.

Characterization of P64k as a carrier proteinS González,1 R Silva,1 A Álvarez,1 G Guillén,1 E Ca-ballero,1 A Pérez,2 Z Cinza,1 A Ruíz,2 F Dickinson,2 CNazábal,1 KVS Rao,3 G Sardiñas,1 T Serrano,2 J Sosa,2

D Guzmán,2 O Gutiérrez2. 1Centro de IngenieríaGenética y Biotecnología. Apdo 6162, La Habana10600; E-mail: [email protected] 2Institutode Medicina Tropical “Pedro Kourí”, Autopista Noviadel Mediodía, Km 6½, La Habana, Cuba. 3Interna-tional Center for Genetic Engineering and Biotechnol-ogy, Aruna Asaf Ali Marg, New Delhi 110067, India.

The P64k meningococcal protein, an antigen of64 kDa expressed in Escherichia coli, has been exten-sively characterized [1]. Its lipoyl-binding site wasgenetically modified to allow the use of P64k in pro-phylactic vaccines [2]. Here, we show some of theP64k properties that support its use as a carrier pro-tein for conjugate vaccines. First, we investigated theimmunogenicity in mice of several hapten-P64k con-jugates. P64k increased the immune response againstsix out of seven peptides and against the serogroup Ccapsular polysaccharide of Neisseria meningitidischemically coupled to this antigen. Moreover, it wasdemonstrated that P64k-induced epitope-specific sup-pression might occur, depending on the hapten andthe pre-existing levels of immunity. Studying the cel-lular immune response, it was shown that lymph nodecells from sensitized mice proliferated in a dose-de-pendent manner. Besides, a peptide containing a Tcell epitope was localized, by using 59 overlappingsynthetic peptides. In a Phase I clinical study, con-ducted in 26 healthy volunteers, the safety of twoformulations of P64k, adsorbed onto aluminum hy-droxide was demonstrated. Furthermore, it was foundthat P64k induced a specific humoral immune responsein 15 out of 18 individuals inoculated with it and didnot elicit anti-mitochondrial antibodies.1. Guillén G, Álvarez A, Silva R, et al. Biotechnol

Appl Biochem 1998;27:189-96.2. Guillén G. Expresión en Escherichia coli y

caracterización del antígeno P64k de Neisseria men-ingitidis. Ph.D. Thesis

Role of the macrophages and cytokinesinhibiting or stimulating their activationin experimental infection by the helminthEchinococcus granulosusG Ferragut. Laboratorio de Inmunología, RegionalNorte-Sede Salto. Universidad de la República, Salto,Uruguay. [email protected]

The infection by E. granulosus is a worlwide zoono-sis that affects not only humans but also domesticanimals and has a high degree of incidence in Uruguay[1]. The parasites biological cycle includes the trans-mission between dogs (definitive host) and cattle orsheep (intermediate hosts). Although humans are notnatural intermediate hosts, they can infect byoncospheres intake due to the close contact with thedefinitive host, which occurs more frequently in ruralareas [2]. The protoscoleces can differentiate to cystsin humans (secondary infection) which happens whenthe containing cyst breaks due to traumatisms or acci-dentally during surgeries. The experimental secondaryinfection model used for the host-parasite relationshipstudy is the Balb/c mice infection by intraperitonealinoculation of protoscoleces [3]. In E. granulosus in-fected mice the parasite stimulate a strong local cellu-lar response which is not able to eliminate the parasite.However, the induction of protection is possible usingproteic extracts of protoscoleces [4] or irradiated pro-toscoleces [5]. What is the role in the host-parasiteinteraction of the cytokines? It is a question that hasbeen answered in some infection by helminths. Thepublished results have not always agreed in such away that there are different hypotheses on the rolethat these cytokines would be playing on the host

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resistance or susceptibility to a determined infectionby helminths. The Balb/c animals susceptible to the E.granulosus infection developed an early Th2 type re-sponse which is kept until the third week of infection.This response was characterised through splenocytesstimulation of infected mice with protoscoleces anti-gens in vitro. The results showed that the splonocytesproduced high levels of IL-4, IL-5 and IL-10 and didnot produced IFN-γ [6]. On the other hand, we haveobserved that: a) the protoscoleces are susceptible tonormal macrophages in vitro activated with INFγ andLPS and b) molecules such as the hydrogen peroxideand nitric oxide donors such as SNAP are toxic for thein vitro protoscoleces. This suggests that the pro-toscoleces might be eliminated in vivo through a cellu-lar defence mechanism that involves the macrophagesas effect cells, with the condition, that these showedbe activated during the first stages of the infection.The macrophages activation gets better in the contextof a Th1 type response. Therefore, the next hypoth-esis is suggested: in the experimental hydatidosis aTh1 response might be associated to minor levels ofinfection contrary to a Th2 type response. During thefirst stages of the experimental infection by E.granulosus a cytokine type 2 profile is induced whoserelationship with resistance or susceptibility has notbeen established6. The main aim of our study is toobtain results that help to clarify this point. The pos-sible strategies to reach this aim basically consist of:1) to in vivo selectively block each one of the type 2cytokines and to evaluate the result of this treatmenton the infection and on the host immune response;2) to evaluate the susceptibility to the infection by E.granulosus and the cytokines response on knock outmice on type 2 cytokines; 3) to induce a response oftype 1 cytokines that negatively regulates the produc-tion of type 2 cytokines and to evaluate on the immu-nomodulation mice the results of the infection.1. Araj G, Matossian R, Frayha G. Z Parasitenk 1977;

57:23.2. Hernández A, Nieto A. P Immunol 1994;16:537.3. Thompson RCA. In: Echinococcus and hydatid

disease. UK: RCA Thompson and A.3. LymberyCab. International; 1995.

4. Molan A, Saeed 1. J Parasitol 1988;37:203.5. Dematteis S, et al. P Immunol 1999;21:19.6. Velupillai P, Sypek J, Harn D. Inf Immunity 1996;

64:4557.

Chemokines and their receptors in HIV-1infected childrenC Pérez-Maldonado,1 M Hernandez, 2 I Caragol,2 TEspañol.2 1Cátedra de Inmunología. Escuela deBioanálisis. Facultad de Farmacia. Universidad de LosAndes, Mérida. Venezuela.5101-A. 2Laboratorio deInmunología. Hospital General. Hospitals Valld’hebron. Barcelona, España. [email protected]

Chemokines are proinflammatory cytokines thatattract and activate specific leukocyte subsets whichreceptors are used as coreceptors by some AIDS-re-lated viral strains. CCR5 the especific ligand for β-chemokine RANTES is used by HIV-1. Increasedlevels of plasma RANTES have been associated withHIV-1 infection resistance and immune recovery. In agroup of 28 non-infected children (age-control) and

35 vertically infected children under HAART (mean:9 years), we evaluated CCR5 expression and RANTESsynthesis. Also CD4+, CD8+, CD8/RA-, CD8/38+,CD8/28-, CD8/45HLA lymphocytes subsets, CCR5expression and intracellular production of IL-2 andIFN-γ were determined by flow cytometry (FCM).Plasma and cell culture supernatant´s levels ofRANTES were assesed by ELISA. Mean values inGroup I (1 to 6 years) and Group II (> of 6 years) ofinfected children were: CD4+: 901 and 538 cel./mL.CD8+: 1443 and 1043 cel./mL., respectively. Basalexpression of CCR5 was 18% and RANTES plasmalevel was 1025 pg/mL. Control groups mean valueswere: CD4+: 1172 and 849 cel./mL. CD8+: 839 and703 cel./mL., respectively and their CCR5 basal ex-pression was 38% with plasma level of RANTES was687 pg/mL. An important RANTES augment in theinfected children groups was accompained by an im-provement of the CD4+ / CD8+ index during the postHAART period. RANTES levels during HAARTcould a reliable indicator of immune recovery in theHIV-1 infection.1. Cocchi F, et al. Science 1995;270:1811-5.2. Auskurt P, Muller F, Froland SS. J Infect Dis

1998;177(4):1091-6.


Citokines production by Mycobacteriumtuberculosis H37Rv StrainAY Arce Mendoza, A Mendiola Jiménez, and MC Sali-nas Carmona. Facultad de Medicina U.A.N.L.Gonzalitos #235 Nte. Col. Mitras Centro Mty N.L.México. [email protected]

The immune response against Mycobaterium tu-berculosis is mediated by different cells of the im-mune system with several functions. The cytokinesproduced by the macrophage are mainly IL-1, IL-6and TNF-α. The molecules TNF-β, IFN-γ and IL-2release by TH1 lymphocytes are very importantduring the cellular immune response, on the otherhand, the cytokines derived from TH2 lymphocytesare: IL-4, IL-5, IL-10, IL-13. The aim of this studywas to determinate the kinetics of the productionof TH1, TH2 and proinflammatory cytokines stimu-lated with Mycobaterium tuberculosis. This studywas performed using a bacillar load of 8 X 106 bact/mL equivalent to a ratio of 50 bacterial per mac-rophage, and the cytokines was determined byELISA immunosay. The results demonstrated thatthe production of different cytokines was dependedof time of stimulation; first appearing proinflam-matory, and thereafter TH1 and TH2 cytokines.We supouse that during the infection with Myco-bacterium tuberculosis occurs the same phenomenonthat we demonstrated in this in vitro experimentalanalysis. First are released proinflamatory citokinesand later TH1 and TH2 citokines involved in thecellular and humoral immune response. It is becom-ing strong evidence that cytokines play an importantrole during the different stages of the infection but,the way by which cytokine participates in the patho-genesis and disease, is not well understand. How-ever, this study offer new information for thetherapeutic management.

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The biological activity of IFNα 2b in liquidand freeze-dried formulationsLl Ruiz, N Reyes, J Sotolongo, L Duany, J Ferrero, KAroche, E Hardy. Center for Genetic Engineering andBiotechnology. PO Box 6162, Havana 10600, Cuba.Tel: (53-7) 2716221; Fax: (53-7) 2714764; E-mail:[email protected]

The Center for Genetic Engineering and Biotech-nology (Havana, Cuba) produces interferon alpha 2b(IFN alpha 2b) since 1987 as an active ingredient andalso, as a formulation. This cytokine have been for-mulated for parenteral use. In fact, IFN alpha 2b hasbeen frequently used in Cuba and worldwide, as anadjuvant therapy for several carcinogenic pathologiessuch as respiratory papillomatosis [1].Many investi-gators have contributed to find storage conditions ableto maintain the biological activity of this cytokine forlong periods. This is an essential requirement for theachievement of the desired IFN alpha 2b in vivo ef-fect. As a result, several formulations that keep intactthe IFNα 2b biological activity have been developedsince 1970 [2].In this work we present our IFN alpha 2bformulations, and compare them with previously de-veloped liquid and lyophilized IFN formulations fromother relevant investigations. The biological activityof IFN alpha 2b was determined by the inhibition ofthe cytopathic effect (ECP) produced by the Mengovirus on Hep-2 cells (ATCC No. CCL23). The physi-cal-chemical characteristics of our products were de-termined by RP-HPLC and SDS/PAGE. The resultsobtained proved the physical, chemical and biologicalequivalence between all of the examined formulations.1. Quiney RE, et al. Clin Otolaryngol 1989;14:217-25.2. Gross G, et al. Stabilized interferon alpha solu-

tions. Hoffmann-La Roche Inc., US5762923; 1998.

IL-2 biological activity in liquid and lyophilizedformulationsN Reyes, Ll Ruiz, K Aroche, J Sotolongo, K Soto, HGerónimo, E Hardy. Center for Genetic Engineeringand Biotechnology. PO Box 6162, Havana 10600,Cuba. Tel: (53-7) 2716221; Fax: (53-7) 2714764;E-mail: [email protected]

Antiviral therapies in a given combination of twoor three antiviral agents are able to decrease the chargeof the type I human immunodeficiency virus (HIV-1)to undetectable levels [1]. However, this treatmentdoes not become definitive yet, and the restorationof the immune system has not been completed [1].In order to assist this situation interleukin 2 (IL-2)has been investigated as adjuvant for the therapywith antiviral drugs, for attempting to induce a sig-nificant increment on the immunological activity [1].The biological activity of this cytokine is a essentialcharacteristic for an effective treatment; thus, thisfunction should be carefully preserved during puri-fication, formulation and long-term storage. For IL-2 the major degradation routes are oxidation andaggregation. Aggregation not only decreases thetherapeutic potential of the product but also mayincrease an immune response against this cytokine.In this work we demonstrated that IL-2 produced atCGEB (Havana, Cuba), efficiently retains its bio-logical activity even when stressing factors (e.g.freeze-drying and high temperatures) are applied to

the molecule. To find conditions guaranteeing maxi-mum stability of IL-2, a great number of solutes(aminoacids, sugars, detergents and polymers) wereevaluated. The biological activity of this cytokine wasestimated after freeze-drying or storage at high tem-peratures by the induction of the in vitro proliferationof CTLL-2 cells. As judged by SDS/PAGE, RP-HPLCand the bio-activity assay, formulations resulting fromthis work are equivalent to others previously describedby other researchers.1. Davey R, et al. JAMA 2000;284:183-9.2. Volkin DB, Mach H, Middaugh R. Degradative co-

valent reactions important to protein stability.Chapter 2. Meth Mol Biol 1995;40:35-59.

Immune response induced in mice by twomeningococcal polysaccharide-proteinconjugatesM Cuello, O Cabrera, CR Soto, O Pérez, J del Campo,ME Martínez, M Lastre, JF Infante, G Sierra. FinlayInstitute. PO Box 16017, Havana, Cuba. Fax: (53-7)2086075, 2086754; E-mail: [email protected]

Meningococcal infections are an important causeof morbidity and mortality worldwide. Serogroup Band C strains are responsible for most cases in thedeveloped world. Meningococcal vaccines contain-ing purified serogroup C capsular polysaccharide(PsC) induce protective serum bactericidal antibod-ies in adults but are poorly immunogenic in youngchildren and may induce tolerance. The immunoge-nicity of PsC can be improved by conjugation to acarrier protein. In this study the immunogenicity andtype of immune response: humoral (Th2, IgG1) orcellular (Th1, IgG2a) to two meningococcal PsC-pro-tein conjugates was evaluated in Balb/c mice. Thepurified PsC was linked to a carrier protein (tetanustoxoid (TT) or Outer Membrane Protein from N.meningitidis serogroup B (OMP)), via carbodiímida-mediated reaction. The IgG and IgG subclass anti-bodies (IgG1 and IgG2a) anti PsC and anti OMPwas evaluated in the serum of animals by an indirectELISA. All mice that were inoculated with conju-gates, induced a high titers of IgG anti PsC and antiproteins. The use of TT as carrier induced mainlyIgG1 antiPsC subclass, but the OMP as carrier in-duced also IgG2a anti PsC. The PsC-OMP conju-gate shows high titers of both IgG anti OMPsubclasses, like the mice immunized with nativeOMP. In conclusion, at the level of IgG subclassantibodies, the conjugate PsC-OMP was able to gen-erate a Th1 immune response, whereas the conjugatePsC-TT generated a Th2 immune response.

Comparative immunogenicity of conjugatescomposed of Neisseria meningitidisserogroup C polysaccharide bound to tetanustoxoid by different spacer armsO Cabrera, M Cuello, O Pérez, J del Campo, TRodríguez, CR Soto, ME Martínez, JF Infante, MFariñas, G Sierra. Instituto Finlay. PO Box 16017,Havana, Cuba. Fax: (53-7) 2086075, 2086754.E-mail: [email protected]

The influence in immune response of structurespacer arms used in Neisseria meningitidis serogroup Cpolysaccharide (MGCP) – Tetanus toxoid (TT)

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conjugate was evaluated in Balb/c mouse. 1,6- di-aminohexane (AH), 1,8-diaminooctane (AO), 6-ami-nohexanoic acid (AA) and adipic acid dihydrazide(ADH) were used like spacer arms with differentstructure linked to MGCP and TT using carbodiim-ide –mediated coupling. The IgM antibody antiMGCP, IgG anti MGCP and IgG anti TT was evalu-ated in serums of animals by an indirect ELISA. Wealso evaluated the IgG subclasses (IgG1 and IgG2a)anti MGCP and Bactericidal activity of antibodiespresent in sera. All mice that were inoculated withconjugates, made high titers of IgG anti MGCP, butthe higher titers were found for ADH. In this groupwas found too the higher titers of IgG2a and thehigher Bactericidal titers. Higher levels of anti TTantibodies were elicited by conjugates than TT initself. The possible basis for differences in the im-munogenicities of these conjugates is discussed.

P64k as a carrier protein of Neisseriameningitidis por A peptidesG Sardiñas, S González, HE Garay, C Nazábal, OReyes, R Silva. Centro de Ing. Genética y Biotecnología,PO Box 6162, Havana 10600, Cuba. Tel.: 2716221;Fax: 2714764; E-mail: [email protected]

Class 1 protein from the outer membrane of Neis-seria meningitidis is an attractive target for vaccinedevelopment. Previously, cyclic peptides correspond-ing to VR1 and VR2 regions of the meningococcalclass1 protein have been designed and employed ascandidate antigens with good results [1]. To increasethe humoral immune response against these cyclicsynthetic peptides, we conjugated the peptides toP64k, a novel carrier protein from the same bacte-rium expressed in Escherichia coli [2]. In addition,one of these peptides was restricted to a linear con-formation before it was chemically coupled to thecarrier. The conjugates were administered to mice ina three-dose immunization schedule, resulting in apotent anti-peptide immune response, which sug-gested that chemical conjugation to this carrier pro-vided T-cell help. Antisera directed to the conjugatesreacted with N. meningitidis outer membrane PorAupon immunoblot analysis. Moreover, in two out ofthree conjugates, the anti-peptide sera reacted withnative meningococcal outer membrane vesicles inELISA, suggesting that chemical conjugation to thecarrier granted, in addition to T-cell help, a properfolding to these synthetic peptides.1. Hoogerhout P, et al. Infect Immun 1995;63:3473-8.2. Guillén G, et al. Biotechnol Appl Biochem 1998;27:

189-96.

Comparison of the immune responsein mice against meningococcal group Cpolysaccharides conjugate to three carrierproteinsÁlvarez A, Canaan L, Carmenate T, Menéndez T,Delgado D, Rodés L, Guillén G. Centro de IngenieríaGenética y Biotecnología. PO Box 6162, Habana 10600Cuba. [email protected]

Objective: To study the nature and kinetics of theserum antibody response in mice to meningococcalgroup C polysaccharide (C-Ps) conjugates to threecarriers: Bovine serum albumin (BSA), Tetanus tox-

oids (TT) and recombinant P64k. Design: Meningo-coccal group C polysaccharide was conjugated usingcarbodiimide as coupling reagent and adipic acid(ADH) as spacer between the C-Ps and the carrier.Carrier proteins, conjugates and free C-Ps were em-ployed to immunize Balb/C mice. Aluminum hydrox-ide was used as adjuvant. The murine humoral immuneresponses were evaluated by ELISA after the thirddose. Result: The polysaccharide-protein ratios of thethree conjugates were: TT-1.26, BSA-1.13 and P64k-0.4 mg/mL. The immune response against the three C-Ps conjugates was higher than against free C-Ps. Thelevels of antibodies detected in the sera of mice immu-nized with C-Ps-P64k conjugate were higher than thosedetected against C-Ps-TT and C-Ps-BSA conjugates.Conclusions: The carrier protein present in the C-Psconjugates influenced the levels of the IgG antibodieselicited against to the Ps. In this regard P64k wassuperior to TT and BSA.

Immunization with a peptide sequencederived from Neisseria meningitidis usingdifferent vaccine formulationsT Menéndez,1 Y Cruz,1 E Coizeau,1 HE Garay,2 TCarmenate,1 A Álvarez,1 G Guillén.1 1Vaccines, 2Chem-istry-Physics Division. Center for Genetic Engineer-ing and Biotechnology. PO Box 6162, Havana 10600,Cuba. Tel: (53-7) 2716221; Fax: (53-7) 2714764;E-mail: [email protected]

In a previous work we have selected a peptide se-quence after the screening of a phage library with amonoclonal antibody directed against the capsularpolysaccharide from Neisseria meningitidis. In thepresent work, a multi antigen peptide (MAP), contain-ing four copies of the selected peptide, was synthe-sized. The MAP was coupled to the carrier proteinP64K using different conjugation methods. The MAP,conjugated and unconjugated, was employed to immu-nize BALB/c mice, using doses of 5, 25 and 50 ug ofeach immunogen. The preinmune and 4th dose sera an-tisera were evaluated by ELISA, using as coating anti-gen the unconjugated MAP. Specific anti-peptideantibodies were elicited after immunization. The bestresults were achieved in the group of mice immunized,with the three doses assayed, with the MAP conju-gated to P64K using the carbodiimide method (1-ethyl-3-(dimethylaminopropyl)carbodiimide)) combinedwith previous conversion of amine residues fromP64K in acid residues using succinic acid, and in thegroup of mice immunized with the MAP coupled toP64K using maleimide propionic acid N-hydroxysuc-cinimide ester as coupling agent.

Peptide synthesis containing a B-celland a T-cell epitopes on dextran beadsand evaluation of humoral response againstbead-peptide constructLJ Cruz, R Padrón, LJ González, JC Aguilar, EIglesias, HE Garay, V Falcón, E Rodríguez, O Reyes.Centro de Ingeniería Genética y Biotecnología.Apartado Postal 6162, CP 10600, La Habana, Cuba.E-mail: [email protected]

Cross-linked dextran beads were chemically modi-fied with Fmoc-bAla to give an amino functionalizedsupport suitable for solid-phase peptide synthesis.

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On this support, a peptide comprising B-cell and T-cell epitopes in tandem was synthesized manuallyby Fmoc procedure monitoring the coupling reac-tion by bromophenol blue procedure and standardKaiser test. The quality of peptide synthesis wasverified by RP-HPLC and mass spectrometry. Thesize and morphologic characteristics of dextran beadswere conserved after peptide synthesis. Furthermore,we analyzed immunogenicity in mice of the T-B pep-tide on dextran beads compared with T-B peptideimmunogenicity when administered in CFA/IFA. Inboth cases, titers were high and there was not a sig-nificant difference in antibody titers between groups(p<0.05). But in contrast CFA they are biocompat-ible and did not induce any adverse reaction at thesite of injection. This experiment suggests that thepeptides synthesis on dextran beads can be used toraise the immunogenicity of synthetic peptide invaccines or therapeutics.

Effect of conjugation methodologyon the immunogenicity and protectiveefficacy of meningococcal group Cpolysaccharide-P64k protein conjugatesT Carmenate, L Canaán, A Álvarez, M Delgado, SGonzález, T Menéndez, L Rodés, G Guillén.E-mail: [email protected]

Neisseria meningitidis serogroup C polysaccharidewas conjugated to the carrier protein P64k using twodifferent conjugation procedures carbodiimide withadipic acid dihydrazide as spacer and reductiveamination method and the condensation mediated bycarbodiimide. BALB/c mice were immunized with theresultant polysaccharide–protein conjugates and theimmune response was evaluated. All conjugates as-sayed generated a higher immune response than thefree polysaccharide. The reductive amination methodrendered the best conjugate named PsC-P64kR. PsC-P64kR was able to elicit an antibody titer statisticallydifferent from the plain polysaccharide (p< 0.001).The bactericidal response obtained against this conju-gate was three folds higher than against the plainpolysaccharide and it was able to protect against thechallenge with the meningococci in the infant rat pro-tection model. The influence of polysaccharide chainlength was also studied. Three different conjugateswere obtained with polysaccharide molecular relativesizes of 20 000 - 40 000 Da, 40 000 - 100 000 Da or100 000 - 500 000 Da but no differences were de-tected in the immune response obtained against thethree conjugates. Our experiment demonstrate that itis possible to generate a protective, T-cell dependentresponse against meningococcal C polysaccharide us-ing the P64k protein as carrier.

Workshop on Adjuvants for Therapeutic Vaccines

Chairpersons: J Koropatnick,1 LE Fernández2

1Cancer Research Laboratories, London Regional Cancer Centre,London, Ontario, Canada. E-mail: [email protected]

2Center of Molecular Immunology, Havana, Cuba. E-mail: [email protected]

Oral Presentations

Hyperthermia and heat shock proteinsas adjuvants for cancer immunotherapyJR Ostberg, SS Evans, JR Subjeck, EA Repasky. De-partments of Immunology and Molecular & Cellu-lar Biophysics, Roswell Park Cancer Institute, Elmand Carlton Streets, Buffalo, NY 14263 USA.Phone: 716-845-3133; Fax: 716-845-8906;E-mail: [email protected]

Increased temperature is a cardinal feature of in-flammatory responses and yet, it is usually overlookedas a potential immunoregulator. We have analyzed theeffects of a mild whole body hyperthermia (WBH)which is similar to that obtained during a normal fe-brile episode (ie: 39.5-40 ºC for 6-8 h) on variousimmunological effector cells and systems testing thehypothesis that this treatment can serve as an adju-vant for cancer therapy. Fever-range WBH results inseveral changes in lymphocytes including alterationsin the organization of the cytoskeleton and in homingand adhesion properties, reorganization of several PKCisoforms, and induction of heat shock proteins. Morerecent studies measuring leukocyte population distri-butions in Balb/c mice after a fever-range WBH treat-ment revealed the loss of lymphocytes from bloodand spleen, suggesting their movement into the pe-riphery. WBH treatment of mice has also been shown

to enhance LPS induced TNF-a and IL-6 levels in theserum. Furthermore, WBH treatment after applica-tion of the elicitation dose of antigen enhanced the earswelling response of a contact hypersensitivity (CHS)model in BALB/c mice. The effects of this treatmentin the CHS response appear to be due to regulation ofboth the dendritic cells in the skin (i.e., Langerhanscells) as well as lymphocytes. In vitro studies usingan ear skin culture system have further revealed thatfever-range hyperthermia (40 ºC for 6 h) enhances thekinetics by which the Langerhans cells are activelyinduced to migrate out of the skin and into the culturesupernatant. Because of this evidence for the capac-ity of WBH to stimulate immune cells and alter theirmotility and homing potential, we have also examinedthe impact of fever-range WBH on tumor growth invivo. Interestingly, treatment with mild WBH aloneresulted in tumor growth delay in SCID mice bearinghuman tumors and BALB/c mice bearing syngeneictumors. This effect appeared to be a result of lym-phocyte and/or NK cell activity. WBH also appearedto induce changes in tumor vasculature that might bepartly responsible for the anti-tumor effects. Overall,these data suggest that this physiological type of “heatstress” can contribute to beneficial anti-tumor re-sponses at the level of the organism’s immunity. A

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phase I clinical trial using this fever-range WBH treat-ment has recently been completed at our institutionand a second is now underway. Supported by the CRI(J.R.O.), NIH grants CA71599 and CA16056 (E.A.R.)and NIH grant CA79765 (S.S.E.)

Antisense targeting of thymidylate synthasein human tumour cells: effects on cell cycle,proliferation, and drug sensitivityJ Koropatnick, R Berg, P Ferguson, M Vincent. Can-cer Research Laboratories, London Regional CancerCentre, London, Ontario. [email protected]

Killing cancer cells by interfering with their in-creased proliferative and metabolic activity has longbeen a goal of cancer chemotherapy. Unfortunately,drug-resistant tumor variants often arise, resulting intreatment failure. To address this problem, we haveused antisense oligodeoxynucleotides (ODNs) thattarget thymidylate synthase (TS), an essential en-zyme for de novo thymidylate production and DNAsynthesis, and a target for a number of chemothera-peutics including 5-fluorouracil and raltitrexed. Anti-sense treatment effectively reduces TS mRNA andprotein, inhibits cell proliferation, and sensitizes hu-man HeLa cervical carcinoma and HT29 colon carci-noma cells to TS-directed chemotherapeutic drugs invitro. Tumor growth in immunocompromised mice isinhibited by intraperitoneal administration of TS an-tisense ODNs, and antisense ODN treatment en-hances the antitumor activity of raltitrexed. Cell cycleanalysis of cultured tumor cell lines using flowcytometry indicates that antisense ODN treatmentresults in accumulation of cells in the G2/M phase, incontrast to G1/S arrest by 5-fluorouracil or raltitrexedtreatment, suggesting a previously unknown connec-tion between TS protein (or mRNA) and the G2/Mcell cycle checkpoint. The current focus is to uncoverthe molecular mechanism mediating G2/M arrest fol-lowing antisense ODN treatment, by investigatingknown cell cycle regulators such as p53 and cyclin/cdk complexes, and by analysing global changes ingene expression using cDNA microarray technology.Our results indicate that TS antisense ODN treat-ment improves the efficacy of anti-TS chemothera-peutic drugs in vitro and in vivo, is effective inovercoming tumor cell resistance to chemotherapeu-tic drugs, and has the potential to become an impor-tant anti-tumor therapy. Supported by a grant fromImperial Oil Canada, Ltd.

VSSP: A novel solution for ganglioside basedcancer vaccinesLE Fernández,1 A Carr,1 C Mesa,1 Z Mazorra,1 OValiente,1 Y Bebelagua,1 C Arango,2 E Noris,2 ERodríguez,2 J Soriano.3 1Center of Molecular Immunol-ogy. Havana. Cuba. 2National Institute of Oncology andRadiobiology. Havana. Cuba. 3"Hermanos Ameijeiras”Hospital. Havana. Cuba [email protected]

Gangliosides are tumor-associated antigens thatconstitute potentials targets for cancer immuno-therapy. A major drawback of ganglioside vaccines,however, is the poor immunogenicity of these gly-colipids. Particularly the presence of substantialamounts of GM3 ganglioside on human melanomasand other tumors, together with its peculiar biologi-cal properties, makes this glycolipid a unique targetfor cancer immunotherapy. Recently, we hydropho-bically incorporated purified GM3 into the outermembrane protein complex from Neisseria menin-gitidis to form Very Small Size Proteoliposomes(GM3/VSSP). The antitumor properties of GM3/VSSP were studied in B16 murine melanoma and arediscussed. The results of a Phase I clinical trial instage III, IV breast cancer patients, testing the GM3/VSSP vaccine, will be also discussed. This clinicaltrial showed that GM3/VSSP/Montanide ISA 51vaccine is safe and remarkably immunogenic, sincemost of the patients were able to generate an spe-cific immune response against the most toleratedganglioside.

Sinusitis autovaccineL Hernández. Cátedra de Microbiología Aplicada.Dpto de Microbiología y Parasitología. Facultad deFarmacia de la Universidad de los Andes. Mérida,Venezuela. Fax 074- 403500. [email protected]

Surgery and the indiscriminate administration ofantibiotics has been the traditional treatment of si-nusitis; sometimes with poor results. Besides therelapsing of the desease has been frequently ob-served; all these unfavorable features go hand inhand with the relatively high cost of the treatment.In this study we prepared a polyvalent autovaccinewith clinical material collected from rinitis and si-nusitis patients. Previous to a clinical and radio-logical diagnosis of the infection a sample was takenfrom each patient taken at the medium meatus andthe laryngopharyngeal zone. This sample was puton agar-blood medium and incubated at 37ºC inaeroplylic conditions. A vaccine was prepared withthe isolated microorganism according to Kolmersmethod. 581 patients were treated with their re-spective vaccine. The dose injected subcutaneouslywas administered during 33 to 44 days, and in-creased every 3 or 4 days; 15 days after treatmentbegan, a reinforcing dose was applied, and 6 monthsafter another and last dose was injected. The medi-cal evaluation revealed an effectivity of 77%. Abacteriological study was conducted on 108 patientsaffected with sinusitis which resulted in the isola-tions and identification of Streptococcus viridansin 23% of these cases. S. viridans has been spo-radically reported in the literature as an etiologicalagent of the sinusitis infection.The autovaccinecould become a potentially more effective and spe-cific alternative for treating sinusitis.

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Workshop on Adjuvants for DNA Vaccines

Chairpersons: Santiago Dueñas-Carrera, Eduardo Pentón

Center for Genetic Engineering and Biotechnology, Havana, Cuba.E-mail: [email protected]

ABSTRACTDNA vaccine technology has provided important hopes and intense investigation for the past decade. DNA vac-cines are typically comprised of plasmid DNA molecules that encode an antigen(s) derived from a pathogen ortumor cell. Following introduction into a vaccine, cells take up the DNA, where expression and immune presenta-tion of the encoded antigen(s) take place. DNA-based immunization of animals with plasmid DNA has been shownto elicit both cellular and humoral immune responses against a wide variety of antigens. However, immune re-sponses generated by using this methodology, remain frequently insufficient. A great deal of effort is now beingapplied to the development of delivery vehicles and adjuvants for DNA-based vaccines. The workshop session�Adjuvants for DNA vaccines� is focused at enhancing DNA vaccine by different approaches including DNA vaccineformulation, vector modification and delivery systems. Different additives and protein combinations were evalu-ated to increase the immune response generated after the administration of DNA based-vaccines. Particularly, theuse of Opc, an outer membrane protein from Neisseria meningitidis complexed to pCMVβ-galactosidase plasmid,expressing β-galactosidase was described. The use of plasmid DNA-protein complexes was associated with theinduction of a humoral response in serum and also with a proliferative response in the spleen. Additionally, a novelpolysaccharide adjuvant acemannan consistently modulated the anti-hepatitis C virus core antibody response elic-ited by DNA immunization. Combinations with other additives like sonicated calf thymus DNA and polyetylenglicolor co-immunization with interleukin15-encoding plasmid also transiently modulated the anti-hepatitis C virus coreimmune response elicited by DNA vaccination. On the other hand, constructs expressing polyprotein variants weresuccessfully evaluated to enhance the immune responses induced after DNA immunization.

IntroductionIt is now well established that the injection of plasmidDNA through a wide range of routes induces bothhumoral and cellular immune responses against theencoded immunogenic proteins in several hosts. More-over, immune responses induced by DNA immuniza-tion have lead to protection against various viral,bacterial and parasitic pathogens. However, while ini-tial human clinical studies demonstrated the primingof immune responses with naked DNA vaccines, po-tency remains a significant limitation. While most ofthe experiments conducted up to date have used phos-phate buffer saline as a diluent for the injected DNA,novel additives are currently investigated. Such re-agents may increase the uptake of DNA, reduce thenumber of doses for immunization, and enhance sub-sequent immune responses. Some systems currentlyunder investigation are dendritic cells, cationic lipo-somes, immunostimulatory oligonucleotide sequences,cytokines, poly(lactide-coglycolide) (PLG) micropar-ticles and monophosphoryl lipid (MPL) A.

The aim of the workshop session “Adjuvants forDNA vaccines” was to discuss different approachesincluding DNA vaccine formulation, vector modifica-tion and delivery systems to enhance the immune re-sponse generated against the encoded antigens. Theworkshop session included two oral presentations andtwo posters.

As a part of the oral presentations Dr. AlexisMusacchio from the Center for Genetic Engineeringand Biotechnology (Havana, Cuba) described the useof plasmid DNA-recombinant Opc protein complexes,for nasal immunization. On the other hand, SantiagoDueñas-Carrera, also from the Center for GeneticEngineering and Biotechnology (Havana, Cuba),showed his work on the enhancement of the immuneresponse generated against the hepatitis C virus enve-

lope proteins after DNA vaccination with plasmidsmanaged to express polyprotein variants of the enve-lope proteins.

At the poster session, Jeny Marante and ArielViña, both from the Center for Genetic Engineeringand Biotechnology (Havana, Cuba) presented in-teresting papers on the adjuvant effect of IL-15 geneand acemannan on the anti-core antibody responseelicited in mice by HCV DNA-based vaccine.

Oral PresentationsImmunization with plasmid DNA-protein complexes.Optimal induction of mucosal immunity in generalrequires targeting antigens to the specialized antigenpresenting cells of mucosal immunity associated tolymphoid tissues. The nasal mucosa may provide asimple, non-invasive route to deliver DNA encodingthe introduced gene to stimulate immunity. In thisway, nucleic acid vaccines represent a new approachto the control of infectious agents. To evaluate pre-liminarily the feasibility of this approach, Dr. AlexisMusacchio investigated a model system, where pro-tein-DNA complexes were used. The outer membraneOpc protein from Neisseria meningitidis, was selecteddue to its role in attachment of meningococci to theendothelial and epithelial cells. Opc-DNA complexesmay facilitate a better interaction between thisinmunogen and mucosal cells. An expression vector,containing the β-galactosidase reporter gene, under thecontrol of the human cytomegalovirus promoter(pCMVβ-galactosidase) was used by Dr. AlexisMusacchio et al. To characterize the complexes, theyperformed gel filtration analysis, transmissionelectronmicroscopy and transfection experiments incos-7 cells. Balb/c mice were intranasally (i.n.) andintramuscularly (i.m.)-immunized to study the im-

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mune response. The humoral immune response againstOpc and β-galactosidase was measured by ELISA andimmunoblot. The proliferative response in the spleenwas also measured. Antibody response was evalu-ated fifteen days after the fourth inoculation. Anti-bodies specific to β-galactosidase were detected ingroups i.m. immunized with pCMV-β galactosidaseDNA, and also in mice where Opc-pCMV-β galactosi-dase DNA complex was i.n.administered. Antibodylevels were significantly higher in animals inoculatedi.m. with pCMV-β galactosidase plasmid. However,after two and a half months this difference in antibodylevels between these groups was not observed. A pro-liferative response specific to β-galactosidase proteinwas also detected, having no appreciable differencesbetween groups. From the resulting data, Dr. AlexisMusacchio concluded that, the use of plasmid DNA-protein complex was associated with the induction ofa humoral response in serum and also with a prolifera-tive response in the spleen.

Immunization with polyprotein-encoding plasmids.The hepatitis C virus (HCV) is the major causativeagent of non-A, non-B viral hepatitis. No prophylac-tic vaccine against HCV is available and therapeutictreatments are effective in less than 40% of the cases.Many studies have been published on the develop-ment of DNA-based vaccines against HCV. However,weak or transient anti-HCV immune responses arefrequently elicited by using this immunization strat-egy. Plasmids expressing variants of the hepatitis Cvirus (HCV) core, E1 and E2 proteins individually oras polyproteins were administered to BALB/c miceby Santiago Dueñas-Carrera et al. All plasmids in-duced a detectable and specific antibody response.Antibody titers against C, E1 and E2, 19 weeks afterthe primary immunization, ranged from 1:50 to 1:4500depending on the inoculated plasmid and the HCVantigen evaluated. Constructs expressing HCV enve-lope proteins as polyprotein variants induced statis-tically stronger antibody response than plasmidsencoding individual E1 and E2. Particularly, thepIDKE2 plasmid, expressing the first 650 aa in theviral polyprotein, induced a potent and multispecificantibody and lymphoproliferative response againstthe HCV core, E1 and E2 proteins. Anti-E2 antibod-ies generated by pIDKE2 immunization were cross-reactive to hypervariable region-1 peptides fromdifferent genotypes. Immunization with the pIDKE2also generated a positive cellular response against thecore antigen, determined by IFN-γ ELISPOT assay,and induced detectable levels of IFN-γ but not IL-4 invaccinated mice. Taking all these data together, SantiagoDueñas-Carrera concludes that plasmids expressingthe HCV structural antigens as polyproteins couldinduce a potent, multispecific and cross-reactive anti-HCV immune response. A transient enhancement ofthe anti-HCV immune response generated by the DNAvaccine was also described by Santiago Duenas-Carrera by the use of additives like sonicated calf thy-mus DNA and polyetylenglicol.

Poster PresentationsIL-15 gene adjuvant. In many animal models for in-fectious diseases, DNA vaccines induced a broad rangeof immune responses, including antibody, CD8+ cy-

totoxic T lymphocyte (CTL) and CD4+ helper T (Th)lymphocyte responses. The magnitude and nature ofthese immune responses to DNA vaccines can be fur-ther manipulated by the use of cytokine genes. Thestudy presented by Jeny Marante (Center for Ge-netic Engineering and Biotechnology, Cuba), was con-ducted to investigate whether the expression of humanIL-15 could enhance the immune response elicited bya DNA vaccine expressing the structural proteins ofHCV (Core, E1 and E2) in mice. Two eukaryotic ex-pression vectors encoding for human IL-15 were con-structed. One of them comprises the coding sequencefor human IL-15 including the own signal peptide; inthe other one, the IL-15 coding sequence is fused tothe erithropoyetin signal peptide. A faster serocon-vertion rate and a stronger antibody response againstthe core protein were observed in mice immunizedwith the IL-15 encoding plasmids compared with ani-mals vaccinated with the HCV antigens-encoding plas-mid alone. These results suggest that the co-expressionof IL-15 influences the specific immune response elic-ited after immunization with HCV antigen-encodingplasmids.

Acemannan as an adjuvant for DNA vaccines.Acemannan has been capable of rising and/or modu-lating the immune response of mice to vaccinal anti-gens at serum and mucus levels, while plasmidexpressing the HCV nucleocapsid alone often elicitedstrong cellular but weak and/or transient humoral im-munity. Ariel Viña (Center for Genetic Engineeringand Biotechnology, Cuba) investigated the influenceof the acemannan on the Ab response elicited by aDNA vaccine. Mice were intramuscularly immunizedwith the pIDKCo plasmid alone or in combinationwith acemannan. The pIDKCo encodes the first 176aa of the hepatitis C virus nucleocapsid protein. Sixweeks after immunization, animals immunized withpIDKCo combined with acemanane 0.1 and 0.3 mg/mLhad statistically higher level of anti-core Ab titers thanthe others (p<0.05). At week 19, 0.3 mg/mLacemannan still increased the anti-core Ab response(p=0.038). However, acemannan did not modify theIgG1/IgG2a ratio of anti-core Ab. Altogether, thesedata indicate that acemannan modulates the anti-coreantibody response elicited by DNA immunization.

SummaryAn important issue for effective vaccines is the devel-opment of potent adjuvants that can facilitate induc-tion or increase of immunity. Adjuvants are known tostrongly enhance immune responses generated by tra-ditional vaccines, but less is known about the effectsof adjuvants on vaccination with DNA. The work-shop summarized here focuses on recent advances inseveral strategies that have been used to improve andmodulate the immune response induced by DNA vac-cines. The combination of DNA vaccines with differ-ent additives transiently influences the immuneresponse elicited in animals. In fact, the use of theacemannan adjuvant or the Opc-DNA complexes evi-denced sustained enhancement of the immune re-sponse against the encoded antigens. While all theseadjuvants are promising, further works are needed tobetter define the mechanisms of adjuvant action. Theaccess of antigens to antigen presenting cells (APCs)

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appears to be a rate-limiting step in the generation ofimmune responses to DNA vaccines. Thus, ultimately,the development of more potent adjuvants, DNA vec-tors or delivery systems targeted to APCs may allowDNA vaccines to be used as both, therapeutic andprophylactic agents.

Oral Presentations

Plasmid DNA�recombinant Opc proteincomplexes, for nasal immunizationA Musacchio, AM Herrera, D Quintana, JC Álvarez,V Falcón, MC la Rosa, B Sandez, O Hayes, DPichardo. Division of Vaccines, Center for GeneticEngineering and Biotechnology. PO Box 6162, Ha-vana 10600, Cuba. Tel: (53-7) 2716221, Fax: (53-7)2714764; E-mail: [email protected]

Optimal induction of mucosal immunity in generalrequires targeting antigens to the specialized antigenpresenting cells of mucosal immunity associated lym-phoid tissues. The nasal mucosa may provide a simple,non-invasive route to deliver DNA encoding the in-troduced gene to stimulate immunity [1]. In this way,nucleic acid vaccines represent a new approach to thecontrol of infectious agents [2]. To evaluate, prelimi-narily, the feasibility of this approach, we have inves-tigated a model system, where protein - DNAcomplexes were used. We selected the outer mem-brane Opc protein from Neisseria meningitidis, dueto its role in attachment of meningococci to the endot-helial and epithelial cells [3]. Opc-DNA complexesmay facilitate a better interaction between thisinmunogen and mucosal cells. An expression vector,containing the β-galactosidase reporter gene, under thecontrol of human citomegalovirus promoter (pCMVβ-galactosidase) was used [4]. Optimal conditions ofinteraction between recombinant Opc protein andpCMVβ-galactosidase DNA were established. Tocharacterize the complexes, gel filtration analysis,transmission electronmicroscopy and transfection ex-periments in cos-7 cells were performed. Balb/c micewere intranasally (i.n.) and intramuscularly (i.m.)-im-munized to study the immune response. The humoralimmune response against Opc and β-galactosidase wasmeasured by ELISA and immunoblot. The prolifera-tive response in the spleen lymph nodes was alsomeasured. Antibody response was evaluated fifteendays after the fourth inoculation. Antibodies specificto β-galactosidase were detected in groups i.m.immunised with pCMV-β galactosidase DNA, andalso in mice where Opc- pCMV-β galactosidase DNAcomplex was i.n. administered. Antibody levels weresignificantly higher in animals inoculated i.m withpCMV-β galactosidase plasmid. However, after twoand half months this difference in antibody levels be-tween these groups was not observed. A proliferativeresponse specific to β-galactosidase protein was alsodetected, having no appreciable differences betweengroups. From the obtained data, we can conclude that,the use of plasmid DNA-protein complexes was as-sociated with the induction of a humoral response inserum and also with a proliferative response in thespleen lymph nodes.1. Klavinskis LS, et al. Vaccine 1997;15(8):818-20.2. Robinson HL. Vaccine 1997;15(8):785-7.

3. Virji M, et al. Mol Microb 1992;10:499-510.4. Herrera AM, et al. Minerva Biotech 1997;9:25-9.

Enhancement of the immune responsegenerated against the hepatitis C virusenvelope proteins after DNA vaccinationwith fusion proteins-encoding plasmidsS Dueñas-Carrera, L Álvarez-Lajonchere, JCÁlvarez-Obregón, A Pérez, N Acosta-Rivero, DMVázquez, G Martínez, A Viña, D Pichardo, J Morales.HCV Department, Vaccine Division, Centro deIngeniería Genética y Biotecnología. PO Box 6162,Havana City, Cuba. Fax: (53-7) 2714764; E-mail:[email protected]

Hepatitis C virus (HCV) is the major causative agentof non-A, non-B viral hepatitis. No prophylactic vac-cine against HCV is available and therapeutic treat-ments are effective in less than 40% of cases. Manystudies have been published on the development ofDNA-based vaccines against HCV [1]. However, weakor transient anti-HCV immune responses are fre-quently elicited by using this immunization strategy[2]. In this work, plasmids expressing variants of thehepatitis C virus (HCV) core, E1 and E2 proteinsindividually or as fusion proteins were administeredto BALB/c mice. All plasmids induced a detectableand specific antibody response. Antibody titersagainst C, E1 and E2, 19 weeks after primary immuni-zation, ranged from 1:50 to 1:4500 depending on theinoculated plasmid and the HCV antigen evaluated.Constructs expressing HCV envelope proteins as fu-sion variants induced statistically stronger antibodyresponse than plasmids encoding individual E1 andE2 proteins. Particularly, the pIDKE2 plasmid, ex-pressing the first 650 aa in the viral polyprotein, in-duced a potent and multispecific antibody andlymphoproliferative response against HCV core, E1and E2 proteins. Anti-E2 antibodies generated bypIDKE2 immunization were cross-reactive to hyper-variable region-1 peptides from different genotypes.Immunization with the pIDKE2 also generated a posi-tive cellular response against the core antigen, deter-mined by IFN-γ ELISPOT assay, and induceddetectable levels of IFN-γ but not IL-4 in vaccinatedmice. These data indicate that plasmids expressingthe HCV structural antigens as fusion proteins couldinduce a potent, multispecific and cross-reactive anti-HCV immune response.1. Lechmann M, Liang TJ. Sem Liver Disease 2000;

20(2):211-26.2. Fournillier A, et al. J Virol 1999;73:7497-504.

Second Generation DNA Vaccine strategiesDA Driver, B Belli, M Singh, M Ugozzoli, J Kazzaz, DO’Hagan, T Dubensky, J Polo. Chiron Corporation,4560 Horton St., Emeryville, CA 94608 [email protected]

DNA vaccine technology has provided great en-thusiasm and intense investigation for the past de-cade. Gene-based vaccination of animals with plasmidDNA has been shown to elicit both cellular and hu-moral immune responses against a wide variety ofantigens. However, while initial human clinical stud-ies demonstrated priming of immune responses withnaked DNA vaccines, potency remains a significant

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limitation. We have sought to address the issue ofDNA vaccine potency using several different ap-proaches, including vector modification, formulationand delivery systems. In one such approach,alphavirus RNA replicon technology was incorpo-rated into plasmid DNA vectors, whereby the RNApolymerase II promoter launches a self-amplifyingRNA transcript encoding the desired antigen. This“layered” expression strategy not only induces anantigen-specific immune response, but also providesa means for stimulating the innate immune responsethrough the presence of cytoplasmic dsRNA inter-mediates. Animal studies with a variety of antigensdemonstrated significantly increased potency, requir-ing as little as 10- to 1000-fold lower dosages ofreplicon plasmid DNA for immune induction, as com-pared to conventional plasmid DNA. In addition,alphavirus replicon and other plasmid DNAs havebeen formulated by adsorption onto the surface ofcationic poly(lactide-coglycolide) (PLG) micropar-ticles to increase the efficiency of delivery. Again,for a variety of antigens, PLG-formulated DNA wasshown to be substantially more potent than corre-sponding naked DNA vaccines, increasing CD8(+)T-cell and antibody responses by approximately 100-to 1,000-fold, respectively. These two strategies rep-resent a powerful means for increasing the overallpotency of DNA vaccines.


Enhancement of the anti-core antibodyresponse elicited in mice by HCV DNA-basedvaccine though the use of IL-15 geneadjuvantJ Marante, A Viña, A Santos, D Urquisa, J Morales, SDueñas-Carrera. Center for Genetic Engineering andBiotechnology, P.O. Box 6162, Havana 10600, Cuba.Tel: (53-7) 2716221; Fax: (53-7) 2714764; E-mail:[email protected]

DNA vaccines containing genes for antigenic por-tions of viruses have been developed as a novel vac-cination technology. An important advantage of thisvaccination method is that in vivo-synthesized viralproteins can enter both major histocompatibilitycomplex (MHC) class I and class II antigen-process-ing pathways to activate specific immunity. In manyanimal models for infectious diseases, DNA vaccinesinduced a broad range of immune responses, includ-ing antibody, CD8+ cytotoxic T lymphocytes (CTL)and CD4+ helper T (Th) lymphocyte responses. Themagnitude and nature of these immune responses toDNA vaccines can be further manipulated by theuse of cytokine genes. We have investigated the util-ity of DNA-based immunization as a strategy forthe development of an effective vaccine against hepa-titis C virus (HCV). The present study was con-ducted to investigate whether expression of humanIL-15 could enhance the immune response elicited

by a DNA vaccine expressing the structural proteinsof HCV (Core, E1 and E2) in mice. Two eukaryoticexpression vectors encoding for human IL-15 wereconstructed. One of them comprises the coding se-quence for human IL-15 including the own signalpeptide; in the other one, the IL-15 coding sequenceis fused to the erithropoyetin signal peptide. A fasterseroconvertion rate and a stronger antibody responseagainst the core protein were observed in mice im-munized with the IL-15 encoding plasmids comparedwith animals vaccinated with the HCV antigens-en-coding plasmid alone. These results suggest that co-expression of IL-15 influence the specific immuneresponse elicited after immunization with HCV an-tigens-encoding plasmids.

Acemannan enhances anti-HCV core Abresponse elicited by DNA immunization inmiceA Viña-Rodríguez, L Crombett, S Dueñas-Carrera1,L Alvarez-Lajonchere1, JC Aguilar2, J Morales1.1HCV and 2Clinical Studies Department, VaccineDivision, Centro de Ingeniería Genética y Biotec-nología. PO Box 6162, Havana, [email protected]

Injection of plasmid DNA induces both humoraland cellular immune responses against the encodedproteins in several hosts (1). However, immune re-sponses generated against several antigens remain in-sufficient (2). A great deal of effort is now beingapplied to the development of delivery vehicles andadjuvants for DNA-based vaccines. Acemanane hasbeen capable of rising and/or modulating the immuneresponse of mice to vaccinal antigens at serum andmucus levels, while plasmid expressing the HCVnucleocapsid alone often elicited strong cellular butweak and/or transient humoral immunity (3). We in-vestigated the influence of the acemanane on the Abresponse elicited by a DNA vaccine. Mice were in-tramuscularly immunized with pIDKCo plasmidalone or in combination with acemanane. ThepIDKCo encodes the first 176 aa of the hepatitis Cvirus nucleocapsid protein. Six weeks after immuni-zation, animals immunized with pIDKCo in combi-nation with acemanane 0.1 and 0.3 mg/mL hadstatistically higher level of anti-core Ab titers thanthe others (p<0.05). At week 19, 0.3 mg/mLacemanane still increased the anti-core Ab response(p=0.038). Additionally, acemanane modified theepitope specificity but not IgG1/IgG2a ratio of anti-core Ab. Altogether; these data indicate acemananemodulates the anti-core antibody response elicitedby DNA immunization.1. Donnelly JJ, Ulmer JB, Shiver JW, Liu MA. Annu

Rev Immunol 1997;15:617-48.2. Hasan UA, Abai AM, Harper DR, Wren BW, Mor-

row WJW. J Immunol Meth 1999;229:1-22.3. Lagging LM, Meyer K, Hoft D, Houghton M,

Belshe RB, Ray R. J Virol 1995;69:5859-63.

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Workshop on Antigen-Delivery Systems

Chairpersons: Claude Leclerc,1 Dania Vázquez Blomquist2

1Pasteur Institute, France. E-mail: [email protected] 2Center for Genetic Engineering and Biotechnol-ogy, Havana, Cuba. E-mail: [email protected]

ABSTRACTConventional vaccines based on inactivated pathogens, live pathogens, subcellular components from bacteria andviruses or recombinant proteins have been successfully used to elicit neutralizing antibodies against surface pro-teins, in order to prevent or detain the infection. In recent years, the vaccine scenario has changed and most of theexperimental vaccines currently tested in humans are based on recombinant viruses, recombinant proteins, DNAvectors, purified subunits, peptides and some others targeting well-defined antigens. In some of the cases, anefficient delivery system should be used to target the antigens to the antigen presenting cells (APC), stabilize theantigen in vivo and/or release the antigen over an extended period of time. The workshop �antigen-deliverysystem� included well-known bacterial, viral or particulated systems for antigen delivery by different routes ofimmunization, including Salmonella typhi, BCG, a toxin from Bordetella pertussis, Fowlpox viruses, Adenoviruses,microparticles and biopolymers. The novel aspects were related to the model antigens and new strategies to controlvery extensively studied diseases such as tetanus, HIV-1, HBV, Cholera and tuberculosis. The study of new naturalpolymers was also shown. The use of bacteria-derived toxins co-administered with antigens is well documented asa potent mucosal adjuvant. The inclusion of CTL epitopes onto a detoxified adenylate cyclase (CyaA) toxin from B.pertussis was shown to be a TAP dependent CD8+ T cell epitope delivery system. The perspective of vaccines basedon antigens expressed in plants was discussed, taking as an example the oral delivery of de HBsAg expressed inpotatoes. One of the most widely discussed topics was Dendritic Cells as professional APC and the ways to improveantigen targeting and delivery to their cytosol, but also the induction of maturation and the expression of MHCclass I molecules to increase CTL epitopes presentation.

monella typhi, for example, is widely used for mu-cosal delivery.

The aim of the “Antigen-Delivery System” work-shop was to discuss different and novel vehicles totarget the antigens to the APC and/or release the anti-gen over an extended period of time. This session ofAdjuvant 2001 included five oral presentations andeight posters.

As a part of the oral presentations Dr. OG Gómez-Duarte from the Department of Pedriatrics in SinaiHospital, Baltimore showed the results after a Phase Iclinical trial with an attenuated strain of S. typhi ex-pressing fragment C from tetanus toxin. Dr. ClaudeLeclerc from Institute Pasteur, France gave an inter-esting presentation on detoxified adenylate cyclase(CyaA) toxin from B. pertussis carrying CTL epitopesand Dania Vázquez from the Center for Genetic Engi-neering, Cuba showed her work using Fowlpox vi-ruses as a carrier vector for HIV-1 multi-CTL epitopepolypeptides. Dr. HM Vordermeier from VLAWeybridge, Addlestone, UK proposed a Mycobacte-rium bovis synthetic peptide vaccine adsorbed ontonano- and microparticles to control tuberculosis incattle. Lastly, Dr. Yasmin Thanavala from RoswellPark Center Institute, Buffalo, USA presented a noveland surprising edible Hepatitis B Virus vaccine inplants, using Agrobacterium plasmid where the HBsAgcoding DNA was cloned.

Dr. W.G Metzger from Max-Planck-Institut fürInfektionsbiology, Berlin, Germany represented theposter session showing a pilot study with the S. typhivaccine expressing Helicobacter pylori urease, fol-lowed by the tetanus vaccination using Adenovirusvectored nasal and epicutaneous vaccines by Dr. B.Bjarnason from University of Alabama, USA. A.Acosta and M.E Sarmiento, both from Finlay Insti-

IntroductionCurrent vaccinology is moving toward the search ofmore defined antigens to be included in vaccines thatsometimes show a lower immunogenicity than theconventional inactivated or attenuated vaccines. Insome infectious diseases the importance of elicitinga cellular response against different antigens or ahumoral/cellular mucosal immunity has become morerelevant. In this sense, the search of new formula-tions, adjuvants and more efficient delivery systemsfor antigens, has been enhanced. The only approvedFDA adjuvant for human trials has been aluminiumsalts, and lately MF59 for the Influenza vaccine.These two adjuvants preferentially promote a Th2-type immune response by a systemic route. Oraladministration is being actively investigated usinglive bacterial vectors and proteins inserted into mu-cosal delivery systems. The use of strong mucosaladjuvants as detoxified toxins such as Cholera Toxin(CT) or E. coli LT is hampered by their very hightoxicity, but point mutation strategies have been car-ried out to dissociate the toxicity and adjuvant prop-erties. Other adjuvants have been tested in somePhase I clinical trials against mortal diseases such asHIV-1, including, MF59 + Muramil Tripeptide, In-complete Freund Adjuvant, Montanide ISA51 orISA720, Ribi and others. Besides the adjuvantswhich stimulate and increase humoral and/or cellu-lar immune responses against antigens through dif-ferent mechanisms, the delivery systems aregenerally, physical structures assuring the presen-tation of the vaccine antigens to the APC or stabi-lizing and releasing the antigen over an extendedperiod of time. Vaccine delivery systems are mostlyparticulated e.g. emulsions, microparticles, ISCOMS,liposomes. Alternatively, we could also consider adelivery system with viral or bacterial vectors; Sal-

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tute, Cuba presented the expression of a B subunit ofcholera toxin in BCG and Salmonella typhi, respec-tively. L. González and E. Muñoz from Finlay Insti-tute, Cuba and LL. Ruiz and A. Pérez from the Centerfor Genetic Engineering and Biotechnology, Cuba pre-sented four posters on particulated systems as ve-hicles to deliver antigens. Those results included naturalbiopolymer A, biodegradable microparticles based onPoly (D, L lactide-co-glycolide), bio-adhesive poly-mers and acemannan plus calcium salts.

Oral PresentationsDr. Oscar Gómez-Duarte et al., used Salmonella as avaccine vector for heterologous antigens and they con-structed an attenuated CVD 908htrA vaccine strain,able to induce strong cellular and humoral immuneresponses in mice and humans following mucosal im-munization. They cloned and expressed a variety offoreign antigens in CVD 908htrA including, tetanusand diphtheria toxoids, and Plasmodium falciparumantigens and mice immunized with these bacterial con-structs by the intranasal route that induced specificimmune responses to these antigens indicating that S.typhi can express and deliver heterologous antigens invivo and successfully present such antigens to themammalian immune system. Phase I clinical studiesshowed that CVD 908htrA expressing fragment C oftetanus toxin also elicited specific antibody responsesnot only to the bacteria itself but also to theFragment C of the tetanus toxin. The titer of anti-tetanus toxin antibody response elicited by the S. typhivaccine construct was equivalent to the titer attainedby the conventional tetanus vaccine.

Dr. Claude Leclerc showed the possibility of therational design of new vectors based on the Adenylatecyclase (CyaA), one of the major toxins produced byBordetella pertussi that is able to enter eukaryotictarget cells. The catalytic domain (AC) is located withinthe first 400 amino acids and the carboxy-terminal1306 residues are responsible for binding the toxin totarget cell membranes and the subsequent delivery ofthe catalytic moiety into the cell cytosol. Exogenouspeptides can be inserted into various permissive siteswithin the catalytic domain of CyaA without ham-pering its ability to enter eukaryotic cells. One par-ticular permissive site located in the center of thecatalytic domain between amino acid 224 and 225 ofCyaA, has been used for the construction of recombi-nant toxins harbouring CD8+ T cell epitopes. Usingvarious APC, they showed that CD8+ T cell epitopesgenetically inserted into the AC domain of detoxifiedCyaA molecules are presented to CD8+ T cells by amechanism requiring 1) proteasome processing;2) TAP and 3) neosynthesis of MHC class I mol-ecules. In vivo, detoxified CyaA toxoids carrying vi-ral or tumoral CD8+ T cell epitopes induce protectiveanti-viral and anti-tumoral CTL responses. Moreover,CyaA hybrid molecules were shown to induce bothCTL and CD4+ T cell responses, characterized byIL-2 and IFN-γ production, indicative of a Th1-likecytokine profile. They also explored the capacity ofthe CyaA vector carrying several different CD8+ T-cell epitopes inserted into sites previously identifiedto stimulate CTL responses. Each of these epitopeswas processed upon delivery by CyaA and in vivo, in

the case of the CyaA toxoid carrying the polyepitopetriggered specific CTL responses for each of the threeepitopes. These results highlighted the potency of theadenylate cyclase vector to induce protective CTLresponses with multiple specificity and/or broadMHC restriction. It was also demonstrated that theCyaA binds to target cells via the aMb2 integrin (CD11b/CD18). Thus, the cellular specificity of CyaA allowsits specific targeting to dendritic cells in vivo.

Another way to deliver antigens into the cytosol ofAPCs is the use of viral vectors such fowlpox virus, amember of Poxvirus family that replicates in the cyto-plasm. Dania Vázquez et al., obtained recombinantfowlpox viruses expressing multi-epitope polypep-tides (MEP) from HIV-1: TAB9 (FPTAB9LZ) andCR3 (FPCR3). Gene tab9 encodes for a protein withsix copies of the V3 loop from HIV-1 isolates: LR150,JY-1, RF, MN, BRVA, IIIB, joined by AGGGA se-quence and fused to the N-terminal of P64K proteinfrom Neisseria meningitidis. Gene cr3 encodes formultiple epitopes from HIV-1 proteins, targeted forhelper or cytotoxic T responses (gp120, gp41, vpr,RT, Nef, p24). Balb/c mice immunized withFPTAB9LZ developed a potent Th1 and cellular re-sponses, demonstrated by Cr51 release assay, IFN-γELISPOT, and ELISA for IFN-γ and IL4 cytokines,mediated by CD8+ lymphocytes and mainly directedagainst a 9 mer immunodominant epitope from IIIBstrain. This response was responsible for the 2.7 to4.7 log decreases in ovary titres, when mice were chal-lenged with a vaccinia virus expressing a similar MEP.Balb/c mice immunization with FPCR3 also inducedan IFN-γ secreting-cells response against CR3, Gagand Nef proteins in ELISPOT. A pilot therapeutictrial in HIV-1+ subjects receiving HAART, with FPCR3will be started soon.

One approach for the efficient delivery of a pep-tide vaccine is the use of structures that ensure thatantigens are slowly released and are protected from arapid clearance. Dr. HMVordermeier talked about theneed for a novel vaccine that protects cattle againstbovine tuberculosis. They chose a peptide vaccinecomposed by promiscuous epitopes adsorbed in nano-and microparticles as a vaccine. They previously iden-tified two immunodominant M. bovis antigens, ESAT-6 and CFP-10 that are frequently recognized by bovineT cells in a MHC promiscuous manner and also dem-onstrated to be ‘xeno-promiscous’. Expanding theirobservation of cross-species promiscuity further, theydemonstrated that human MHC binding predictionprograms were able to predict 8/10 promiscuousepitopes that were empirically identified in M. bovisinfected cattle indicating that such programs can alsobe used to predict bovine promiscuous epitopes.

The new approach of an oral (Edible) vaccineagainst Hepatitis B was presented by Dr. YasminThanavala who exposed the disadvantages of currentneedle stick-based vaccines, mainly in very poor anddistant populations, where the cold chain to preservethe vaccine doses is a limitation. Moreover, the im-possibility to vaccinate at the same time a high num-ber of people, the increased cost of vaccines becauseof the devices or the infections associated with thevaccination procedure in rural and poor areas are addi-tional disadvantages. The possibility to use plants

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expressing antigens as a “vaccine” opens new feasiblepossibilities. She spoke of experience expressing vi-rus-like particles of HBsAg in potatoes. Experimentsin mice showed the appearance of an antibody re-sponse and the establishment of an immunologicalmemory, which supports this strategy as one possi-bility for certain vaccines in the future.

Poster SessionBacteria and viral vectors. Dr. WG Metzger et al.,showed the results of a pilot study of a Salmonellatyphi (Ty21a) vaccine expressing Helicobacter pyloriurease (Ty21a (pDB1)). Ten out of 12 volunteers de-veloped humoral immune responses to the Salmonellacarrier but only two volunteers seroconverted. A to-tal of 5 volunteers showed responses in one or twoout of three assays for cellular responses to the car-rier, and of the volunteers that had received Ty21a(pDB1) three showed a weak but significant T cellresponse to Helicobacter urease, while no volunteerhad detectable humoral responses to urease. Interest-ingly, two of the responders to urease had previouscontact to S. enterica serovar Typhi suggesting thatpre-existing immune responses to the Salmonella car-rier might enhance responses to heterologous anti-gens. Dr. B Bjarnason et al., showed protection againsttetanus by needle-free inoculations of adenovirus-vec-tored nasal and epicutaneous vaccines. They reportedthat the administration of a single intranasal dose ofthis vaccine was 100% protective against the intra-muscular injection of 6 x 103 C. tetani cells, and thatthe administration of a single dose topically was pro-tective in 80% of the mice with the protection risingto 100% when three doses were administered topi-cally. The titres of anti-tetC antibodies of animalselicited by epicutaneous inoculation were relativelylow when compared to those elicited by intranasalinoculations. Intranasal inoculation could completelyovercome the pre-existing immunity to adenovirus,while the epicutaneous route could partially overcomeit. The efficiency of vector absorption into the skinwas very low, with approximately 1 in 4,000 par-ticles being taken within one hour of the topical appli-cation. No adverse effects associated with theinoculation were observed in any of the immunizedmice during the course of these studies. A Acosta etal., expressed the B subunit of cholera toxin in BCGand Balb/c mice immunised with the recombinant BCGshowed a vigorous lymphoproliferative response insplenic lymphocytes in the presence of CTB. A prim-ing effect of the specific humoral response was dem-onstrated in mice immunised with the recombinantBCG followed by a booster with CTB. M.E. Sarmientoet al., also expressed the B subunit of cholera toxinbut in Salmonella typhi Ty21a. The immunisation ofmice with the recombinant vaccine by the oral routeshowed an increased protection capacity after thechallenge with Salmonella typhi Ty2 compared withthe immunisation with Salmonella typhi Ty21a, inac-tivated cells or polysaccharide Vi.

Biopolymers. The biopolymer A is a polysaccha-ride obtained from natural sources to which immuno-stimulating properties have been adduced. L. Gonzálezet al. evaluated its adjuvanticity using the Leptospireparticulate antigen, exploring different concentrations

and routes of immunization. The best results werewith the vaccine formulation containing 2 mg/mL in-tramuscularly and the challenge assay demonstrated aprotecting effect by inducing antibodies in a similarway to that of the preparation adjuvated in aluminiumhydroxide. E. Munoz et al. characterized poly (D, Llactide-co-glycolide)-based microspheres by size,morphology, surface absorbed substances, encapsu-lation efficiency and release kinetics and LL. Ruiz etal. evaluated the influence of some bio-adhesives poly-mers and their viscosity on releasing the IFN alpha2b across synthetic membranes. They showed thatviscosity did not significantly affect the IFN alpha2b biological activity, but the releasing profile nota-bly varied when the viscosity of the polymers wasincreased. The effect of acemannan and calcium saltson the immune response against different antigensexposed by A. Pérez, closed the poster session. Dif-ferent formulations of acemannan (0.3 mg/mL), cal-cium oxalate (0.5 mg/mL) and calcium phosphate(0.5 mg/mL) were administered alone or in combina-tion with the HBsAg and TAB9 antigens and throughdifferent immunization routes in mice. They con-cluded the administration route and the type of anti-gen are important issues in the behaviour of the resultingformulation.

SummaryBesides targeting an antigen or DNA to APCs, deliv-ery systems may enhance vaccine stability in vivoand provide a depot effect, whereby the antigen iskept in the site for continual immune stimulation. Formucosally delivered vaccines, this may enable effi-cient presentation and uptake by M cells, followedby transcytosis into Peyer´s patches and presenta-tion to lymphocytes for the induction of mucosalimmunity. For certain formulations, the vaccine maybe maintained inside a physical structure for a longperiod of time, releasing the antigen slowly in order tofunction as a one-dose vaccine. The discussion of theseissues was fulfilled by this session where differentstrategies were presented. The use of recombinantbacterial or viral vectors for antigen delivery was ac-companied by the use of biopolymers and micropar-ticles as physical vehicles and the oral vaccine forHBV in row potatoes, which open a new path invaccinology. The discovery of and experimentationwith new adjuvants and delivery systems may allowthe development of vaccines against infectious agentsthat do not naturally elicit protective immunity or theimprovement of current vaccines.

Oral Presentations

Attenuated Salmonella vaccine as a vectorfor Heterologous antigensOG Gómez-Duarte,1 M Passeti,2 M Sztein,2 MMLevine.2 1Department of Pediatrics, Sinai HospitalBaltimore, 2410 Belvedere Ave., Baltimore, MD 21215,USA; Fax: 410-601 8766. 2Center for Vaccine Devel-opment, University of Maryland Baltimore, 685 W.Baltimore St., Baltimore, MD 21201, USA. Fax: 410-706 6205. [email protected]

Typhoid fever caused by Salmonella entericaserovar Typhi (S. typhi) is associated with significant

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morbidity and mortality in poor areas around theworld. Typhoid fever vaccines are recognized as animportant public health tool to prevent infections fromhighly antibiotic resistant S. typhi strains. We con-structed the attenuated S. Typhi CVD 908htrA vac-cine strain, an aroA-aroD-htrA triple mutant capableto induce strong cellular as well as humoral immuneresponses in mice and humans following mucosal im-munization. We cloned and expressed a variety of for-eign antigens in CVD 908htrA including, tetanus anddiphtheria toxoids [1], and Plasmodium falciparumantigens [2]. Mice immunized with these bacterialconstructs by the intranasal route induced specificimmune responses to these antigens indicating that S.Typhi can express and deliver heterologous antigensin vivo and successfully present such antigens to themammalian immune system. Phase I clinical studiesshowed that CVD 908htrA expressing fragment C oftetanus toxin also elicited specific antibody responsesnot only to the bacteria itself but also to the fragment Cof tetanus toxin. The titer of anti-tetanus toxin anti-body response elicited by S. typhi vaccine constructwas equivalent to the titer reached by the conven-tional tetanus vaccine [3]. More phase II and phaseIII clinical studies will be necessary to demonstratethat Salmonella vectors may deliver heterologous an-tigens and elicit protective immune responses to mi-crobial pathogens from bacterial and parasitic origin.1. Gómez-Duarte O, et al. Vaccine 1995;13:1596-602.2. Gómez-Duarte O, G. Pasetti M, Santiago A, Sztein

MB, Hoffman SL, Levine MM. Infect Immun 2001;69:1192-8.

3. Tacket CO, Galen J, Sztein MB, Losonsky G,Wyant TL, Nataro J, Wasserman SS, Edelman R,Chatfield S, Dougan G, Levine MM. Clin Immunol2000;Nov97(2):146-53.

Rational design of new vectorsC Leclerc. Biologie des Régulations Immunitaires,Institut Pasteur, 25 rue du Docteur Roux 75724 Pariscedex 15, France, Fax: 33.1.45.68.85.40; Phone:33.1.45.68.86.18; E-mail: [email protected]

Adenylate cyclase (CyaA), one of the major toxinsproduced by Bordetella pertussis, is able to enter eu-karyotic target cells where, upon activation by endog-enous calmodulin (CaM), it synthetizes high levels ofintracellular cAMP, that cause impairment of cellularfunctions. The CaM-dependent adenylate cyclasecatalytic (AC) domain is located within the first 400amino acids. The carboxy-terminal 1306 residues areresponsible for the binding of the toxin to target cellmembranes and the subsequent delivery of the cata-lytic moiety into the cell cytosol. Exogenous pep-tides can be inserted into various permissive siteswithin the catalytic domain of CyaA without ham-pering its ability to enter eukaryotic cells. One par-ticular permissive site located in the middle of thecatalytic domain between amino acid 224 and 225 ofCyaA, has been used for the construction of recombi-nant toxins harboring CD8+ T cell epitopes. Usingvarious APC, we showed that CD8+ T cell epitopesgenetically inserted into the AC domain of detoxifiedCyaA molecules are presented to CD8+ T cells by amechanism requiring 1) proteasome processing; 2)TAP and 3) neosynthesis of MHC class I molecules.

In vivo, detoxified CyaA toxoids carrying viral or tu-moral CD8+ T cell epitopes induce protective anti-viral and anti-tumoral CTL responses. Moreover,CyaA hybrid molecules were shown to induce bothCTL and CD4+ T cell responses, characterized byIL-2 and IFN-γ production, indicative of a Th1-likecytokine profile. We have also explored the capacityof the CyaA vector carrying several different CD8+ T-cell epitopes inserted into sites previously identifiedto stimulate CTL responses. Each of these epitopeswas processed upon delivery by CyaA and in vivo,the CyaA toxoid carrying the polyepitope triggeredspecific CTL responses for each of the three epitope.These results highlighted the potency of the adeny-late cyclase vector to induce protective CTL responseswith multiple specificity and/or broad MHC restric-tion. Very recently, we also demonstrated that theCyaA binds to target cells via the aMb2 integrin (CD11b/CD18). Thus, the cellular specificity of CyaA allowsits specific targeting to dendritic cells in vivo. Thus,altogether, these results demonstrate that CyaA istargeted to professional APC, leading to an excep-tional capacity to induce immune responses.

Induction of specific cellular response afterthe immunization of BALB/c mice withrecombinant fowlpox viruses expressing HIV-1multi-epitope polypeptidesD Vázquez, E Iglesias, A Méndez, C Duarte. Centrode Ingeniería Genética y Biotecnología. PO Box 6162,Ciudad de La Habana, Cuba. Tel: (53-7) 2716022;Fax: (53-7) 2714764; [email protected]

The vaccination with avipoxvirus vectors has beenan effective approach to generate a cellular responseagainst HIV-1, with the additional advantage to besafe in humans where they do not replicate. We ob-tained recombinant fowlpox viruses expressing multi-epitope polypeptides (MEP) from HIV-1: TAB9(FPTAB9LZ) and CR3 (FPCR3). Gene tab9 encodesfor a protein with six copies of the V3 loop from HIV-1 isolates: LR150, JY-1, RF, MN, BRVA, IIIB, joinedby AGGGA sequence and fused to the N-terminal ofP64K protein from Neisseria meningitidis. Gene cr3encodes for multiple epitopes from HIV-1 proteins,targeted for helper or cytotoxic T responses (gp120,gp41, vpr, RT, Nef, p24). Balb/c mice immunized withFPTAB9LZ developed a potent cellular response,demonstrated by Cr51 release assay and IFN-γELISPOT, mediated by CD8+ lymphocytes and mainlydirected against a 9mer immunodominant epitope fromIIIB strain. This response was responsible of the 2.7to 4.7 log decreases in ovaries titres, when mice werechallenged with a vaccinia virus expressing a similarMEP. Balb/c mice immunization with FPCR3 alsoinduced an IFN-γ secreting-cells response against CR3,Gag and Nef proteins in ELISPOT. A pilot therapeu-tic trial in HIV-1+ subjects receiving HAART, withFPCR3 will start very soon.

Oral (Edible) vaccine for hepatitis B: fromlaboratory studies to clinical realityYasmin Thanavala,1 Charles J Arntzen,2,3 Hugh S Ma-son.2 1Department of Immunology, Roswell Park Can-cer Institute, Buffalo, NY, USA. 2The Boyce ThompsonInstitute for Plant Research, Inc., Ithaca, NY, USA. 3De-

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partment of Plant Biology, Arizona State University,Tempe, AZ, USA. [email protected]

The incredible success of vaccination in contribut-ing to public health is attributable, in part, to the sim-plicity of vaccines. For polio, the simple incorporationof attenuated polio virus on sugar cubes resulted in anoral vaccine that was universally acceptable. The im-pressive logistical advantage of orally administeredvaccines were exemplified by two national vaccina-tion days in 1996, when 121 million Indian childrenwere vaccinated against polio at 650,000 immuniza-tion centers. Orally delivered vaccines made this enor-mous endeavor achievable at reasonable costs.Unfortunately, a simple oral formulation is not easilyachieved for the new generation of subunit vaccineswhich hold the greatest promise for disease preven-tion in the twenty-first century. There is an urgentneed to make the technically sophisticated field ofsubunit vaccines (which are almost exclusively bio-technology products) available in the poorer coun-tries of the world where infectious diseases are stillthe primary cause of death.

The Children’s Vaccine Initiative (CVI) called foruniversal immunization against major infectious dis-eases. The CVI focused attention upon the need fornew technologies which would make vaccines costeffective and reliable in both production and delivery,especially for the developing world. However, for sucha program to be effective, it must reduce dependenceon a cold chain, as well as promote the developmentof vaccines that can be administered without needlesand syringes.

In contrast to the large variety of currently avail-able injectable vaccines that provide systemic immu-nity, vaccines administered non systemically are rare.Recently, however, there has been a surge of interestin developing novel strategies for vaccine develop-ment with oral and nasal delivery as the preferredroutes.

In my presentation, I will describe experimentswhere oral immunization with transgenic plants ex-pressing hepatitis B surface antigen has moved suc-cessfully from preclinical efforts through to a phase Iclinical trial.

Micobacterium bovis-derived determinantsare recognized promiscuously by bovine Tcells and can be presented to the immunesystem in the form of synthetic peptidesadsorbed to nano or micro-particlesH M Vordermeier,1 I Saleem,2 AO Whelan,1 A Coombes,2

R Glyn Hewinson.1 1VLA Weybridge, Department ofBacterial Diseases, TB Research Group, Addlestone,New Haw KT 15 3NB, United Kingdom. 2Aston Phar-macy School. Aston University, Birmingham B4 7ET,United Kingdom. [email protected]

Novel vaccines that protect cattle against bovinetuberculosis (BTB) are needed because the only avail-able TB vaccine, Mycobacterium bovis BCG, has vari-able protective efficacy in cattle (as in humans). Inaddition, BCG vaccination compromises the specific-ity of tuberculin-based diagnostic tests, which are cen-tral to current test and slaughter control strategies forBTB in many countries including Great Britain. Sub-unit vaccines, particularly those based on fully syn-

thesized peptides, may have considerable advantagesover other types of vaccines, especially in terms ofquality control and safety to consumers. In order forpeptide vaccines against M. bovis to be a viable op-tion two pre-requisites have to be fulfilled. First, pep-tides that are recognized in the context of multiplecattle MHC alleles (promiscuous epitopes) have tobe identified, and secondly, suitable adjuvants or de-livery systems have to be employed to induce strongand lasting immunity. We have previously identifiedtwo immunodominant M. bovis antigens, ESAT-6 andCFP-10, that are frequently recognized by bovine Tcells in a MHC promiscuous manner (1,2). The iden-tification of these promiscuous peptides satisfies thefirst pre-condition for the development of peptide-based vaccines. Interestingly, T cells from M. bovisinfected guinea pigs, mice and human TB patientsalso recognized the ESAT-6 epitopes that were recog-nized promiscuously in cattle. This suggests that someT cell determinants are recognized not only across theMHC spectrum within one species but also acrosshost species (‘xeno-promiscously’). Expanding ourobservation of cross-species promiscuity further, wehave demonstrated that human MHC binding predic-tion programs were able to predict 8/10 promiscuousepitopes that were empirically identified in M. bovisinfected cattle indicating that such programs can alsobe used to predict bovine promiscuous epitopes. Fi-nally, in the search for adjuvants with which to de-velop peptide vaccines, we have preliminary data toindicate that adsorption of synthetic peptides ontonano- and microparticles improves antigen recogni-tion by bovine T cells and therefore may be useful forthe development of peptide-based vaccines.1.Vordermeier HM, et al. Clinical Infect Dis 2001;

30:S291- 8.2.Vordermeier HM, et al. Clin Diagn Lab Immunol

2001;8:571-8.

Alphavirus replicon vectors for gene-basedantigen delivery to dendritic cellsS Perri, K Thudium, C Greer, J Gardner, Ia Frolov, Jzur Megede, G Otten, T Dubensky, and J Polo. ChironCorporation, 4560 Horton St., Emeryville, CA, 94608USA. [email protected]

Dendritic cells (DCs) are the most potent antigenpresenting cell population and play a pivotal role ineliciting the humoral and cellular immune responsescentral to successful vaccination. We have used twoapproaches to develop alphavirus-based replicon par-ticles for efficient antigen delivery to human DCs.Alphavirus replicon particles provide an exciting plat-form for gene-based vaccines, with high-level tran-sient expression of antigens and availability of stablepackaging cell lines that make these vectors ame-nable to production in sufficient quantities for clini-cal testing. In one approach, variants of theprototype alphavirus, Sindbis virus (SIN) were de-rived, which efficiently transduced immature humanDCs derived from peripheral blood monocytes. Aspecific mutation responsible for DC tropism wasmapped to the E2 envelope glycoprotein gene andthen engineered into SIN replicon particles, resultingin highly efficient vector transduction of immatureDCs. Costimulatory (CD80, CD86) and MHC mol-

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ecules were upregulated on SIN replicon transducedDCs in vitro and in vivo, highlighting the naturaladjuvant activity of these vectors. In a second ap-proach, the natural DC tropism of a pathogenicalphavirus, Venezuelan equine encephalitis virus(VEE), was exploited. Alphavirus replicon particlechimeras were engineered such that SIN repliconRNA molecules were packaged within VEE-derivedenvelope glycoproteins. Although interactions be-tween SIN replicon RNA and VEE structural pro-teins are less efficient than their homologouscounterparts, a series of defined modifications sig-nificantly increased production of such chimeric par-ticles. These novel alphavirus systems represent ahighly efficient modality for gene delivery to DCsand should facilitate the development of potent vac-cines for a variety of human diseases.

Polyphosphazenes: A unique polymerplatform for the rapid synthesis of adjuvantsand delivery vehicles for vaccinesand immunotherapiesBE Roberts, A Andrianov, Parallel Solutions Inc., 763DConcord Ave., Cambridge, MA 02138. USA.Phone: 617 876 2178; Fax: 617 876 0728; E-mail:[email protected]

The development of a number of important vac-cines and immunotherapies has been stymied by theinability to induce appropriate immune responses inthe recipient. Adjuvants are essential componentsof new subunit and killed vaccines that boost theimmune responses to an antigen creating protectiveimmunity in the vaccinated individual. The only ex-isting FDA approved adjuvant, Alum has poor im-munostimulatory properties and some safetyconcerns. Attempts to develop new adjuvants overthe last thirty years in both the academic and com-mercial arenas have not been successful. One of theprincipal problems with adjuvants to date is thatthey induce a specific immune response that cannotbe modulated or enhanced. The increasing use of vac-cines and immunotherapeutics to control a range ofmedical problems including infectious disease, can-cer, neurological and addictive disorders, will requireimproved adjuvants with innovative properties. Thispresentation will outline the development of a flex-ible polymer platform for adjuvants and vaccine de-livery using polyphosphazene polyelectrolytes.Polyphosphazenes are hybrid organic/inorganic poly-mers comprised of a backbone of alternating nitro-gen and phosphorous atoms with two side groupsattached to each phosphorous moiety. Polyphosp-hazenes can be synthesized with properties such ascontrolled architecture, molecular weight and criticalbiological characteristics like biocompatibility, safety,controlled biodegradability and immunostimulation.Moreover polymer polyelectrolytes are potent ad-juvants in the aqueous phase and when conformedinto hydrogel microspheres. The first generation prod-uct, Poly [di(carboxylatophenoxy)phosphazene] re-ferred to as PCPP was licensed to Aventis-Pasteurfor use in the development of injectable vaccines.PCPP in the soluble form was formulated with in-jectable vaccines and shown to be safe and effectivein over 1000 human subjects. Aventis-Pasteur is cur-

rently in Phase II clinical development for RSV uti-lizing PCPP and an existing preclinical package andDMF for PCPP is on file with the FDA. Preliminarydata suggests that PCPP is an effective adjuvant witha variety of viral and bacterial antigens. Parallel chemi-cal synthesis was used to prepare twenty five struc-tural variants of PCPP. Twenty percent of thesepolymers exhibited an enhancement in both the ex-tent and nature of immune responses induced in ani-mals. In fact a polymer was shown to generateimmune responses four hundred times greater thanthose of PCPP. PCPP was also used to develop aproprietary microencapsulation system using aque-ous coacervation and ionic crosslinking which rapidlyproduces uniform and monodisperse microspheres inthe nanometer to micrometer range. The process iscommercially scalable requiring no complex manu-facturing equipment, elevated temperatures, genera-tion of aerosols or the use of organic solvents. Thesemicrospheres were shown to generate mucosal vac-cines combining efficient antigen encapsulation, mu-cosal delivery and adjuvant activity that inducedmucosal and systemic immune responses. These com-bined data imply that a limited group of novel poly-mers will be defined that modulate the TH1/TH2balance and provide an adjuvant and vaccine deliv-ery platform for injectable and mucosal vaccines andimmunotherapies.


Two pilot studies of a Salmonella typhi(Ty21a) vaccine expressing Helicobacter pyloriureaseWG Metzger, D Bumann, E Mansouri, O Palme, MWendland, R Hurwitz, G Haas, T Aebischer, B-U vonSpecht, TF Meyer. Max-Planck-Institut für Infektions-biologie, Abteilung Molekulare Biologie, Schu-mannstraße 21/22, D-10117 Berlin, [email protected]

Helicobacter pylori urease was expressed in thecommon live typhoid vaccine Ty21a yieldingTy21a(pDB1). In a first pilot study, nine volun-teers received Ty21a(pDB1) and three control vol-unteers received Ty21a. No serious adverse effectswere observed in any of the volunteers. Ten out of12 volunteers developed humoral immune responsesto the Salmonella carrier as detected by antigen-specific antibody-secreting cells but only two vol-unteers seroconverted. A total of 5 volunteersshowed responses in one or two out of three assaysfor cellular responses to the carrier (proliferation,IFNγ-secretion, IFNγ-ELISPOT). Three of the vol-unteers that had received Ty21a(pDB1) showed aweak but significant T cell response to Helicobacterurease, while no volunteer had detectable humoralresponses to urease. Ty21a(pDB1) is a suitable pro-totype to optimize Salmonella-based vaccinationfor efficient cellular responses that could mediateprotective immunity against Helicobacter. Interest-ingly, two of the responders to urease had previouscontact to S. enterica serovar Typhi suggesting thatpre-existing immune responses to the Salmonellacarrier might enhance responses to heterologousantigens. In a second pilot study, this hypothesis is

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being tested, and results and conclusions are dis-cussed here.

Protection against tetanus by needle-freeinoculation of adenovirus-vectored nasaland epicutaneous vaccinesZ Shi,1 M Zeng,2 G Yang,1 B Bjarnason,2* F Siegel,1 LJCain,1 KR Van Kampen,1 CA Elmets,2 de-Chu CTang.1,2,3 1Vaxin, Inc., 500 Beacon Parkway West, Bir-mingham, Alabama 35209, USA. 2Department of Der-matology, University of Alabama at Birmingham, 15303rd Ave South, EFH 414, Birmingham, Alabama35294, USA. 3Gene Therapy Center, University of Ala-bama at Birmingham, Birmingham, Alabama 35294,USA. *Hudlaeknastodin ehf, Smaratorg 1, 201Kopavogur, Iceland. Phone: 354 892 3755; Fax 354520 4400; [email protected]

Tetanus continues to be a threat to public healthwith more than half a million fatalities each year beingassociated with infection with Clostridium tetani andvaccination is the most effective medical interventionfor protection of the public against this deadly dis-ease. An alternative new modality is noninvasive vac-cination onto the skin (NIVS) by topical applicationof epicutaneous vaccines. This study was undertakento determine if administration of a vaccine consistingof an adenovirus (Ad) recombinant (AdCMV-tetC)encoding the immunogenic but atoxic tetanus toxin C-fragment (tetC) can protect against a lethal challengewith live C. tetani when administered either intrana-sally or by an epicutaneous patch. We report thatadministration of a single dose of this vaccine intrana-sally was 100% protective against intramuscular in-jection of 6 X 103 C. tetani cells, and that administrationof a single dose topically was protective in 80% of themice with the protection rising to 100% when threedoses were administered topically. The titers of anti-tetC antibodies of animal elicited by epicutaneous in-oculation were relatively low when compared to thoseelicited by intranasal inoculations. Intranasal inocula-tion can overcome the pre-existing immunity to aden-ovirus, while epicutaneous route can partiallyovercome it. The efficiency of vector absorption intothe skin was very low, with approximately 1 in 4,000particles being taken within one hour of the topicalapplication. No adverse effects associated with in-oculation were observed in any of the immunized miceduring the course of these studies. This is the firsttime to demonstrate that topical application ofepicutaneous vaccines can protect recipients againstlive pathogenic bacteria in a disease setting.

Expression of the B subunit of cholera toxinin BCG. Evaluation of the immunogenicityin miceAcosta A, Sarmiento ME, Estévez P, Martínez M, InfanteJF, Benítez J, García L, Pérez JL, Griñán I, Falero G,Pérez O, Lastre M, Sierra G. Finlay Institute. PO Box16017, Havana, Cuba. Phone: 2719542; Fax: (53-7)2086075; E-mail: [email protected] .cu

BCG is one of the most promissory systems ofantigen delivery (1-3). In order to evaluate the feasi-bility to express the B subunit of cholera toxin (CTB)in BCG a shuttle vector E. coli-mycobacteria was con-structed containing the CTB gene with the signal pep-

tide of the labile toxin B of E. coli (LTB) under thecontrol of tac promoter. After the transformation byelectroporation of M. smegmatis and Moreau BCGstrain the expression of CTB was detected in the su-pernatant of culture. Balb/c mice immunised with therecombinant BCG shown a vigorous lymphoprolif-erative response of the splenic lymphocytes in pres-ence of CTB. A priming effect of the specific humoralresponse was demonstrated in mice immunised withthe recombinant followed by a booster with CTB.These results demonstrate the feasibility to expressCTB in BCG as a potential system of delivery ofantigens by different routes.1. Burlein JE, et al. Expression of foreign genes in

mycobactgeria. In: Bloom RB, editor. Tuberculo-sis: pathogenesis, protection and control. Wash-ington: American Society for Microbiology; 1994.p.239-51.

2. Miyaji EN, et al. Induction of neutralizing antibod-ies. 2001.

3. Hayward CM, et al. Vaccine 17:1272-81.

Increase of the protective capabilityof Salmonella typhi Ty21a expresingthe B subunit of cholera toxinSarmiento ME, Acosta A, Díaz J, Vidal T, Riverón LEstévez P, Martínez M, Infante JF, García L, Pérez J,Falero G, Callis A, García H, Sierra G. Finlay Insti-tute. PO Box 16017, Havana, Cuba. Phone: 2719542;Fax: (53-7) 2086075; E-mail: [email protected] .cu

The expression of heterologous antigens in Salmo-nella typhi Ty 21a is one of the options for the devel-opment of antigen delivery systems (1-3). With theobjective to evaluate the impact of the expression ofB cholera toxin (CTB) in the protective capability ofthe attenuated strain, S. typhi Ty 21a was transformedwith a plasmid containing the gene of CTB with thesignal peptide of the labile toxin B of E. coli (LTB)under the control of the inducible tac promoter. Thetransformed strain expressed CTB in the periplasmicspace both constitutively and in an inducible way.After the administration by different routes (O, EV,IP) the persistence in different organs was detected.The administration of the recombinant to Balb/c micehad a priming effect on the specific humoral response.The immunisation of mice with the recombinant bythe oral route shown an increase of the protectionupon challenge with Salmonella typhi Ty2 comparedwith the immunisation with Salmonella typhi Ty21a,inactivated cells or polysaccharide Vi. These resultsdemonstrate the adjuvant effect of the expression ofCTB S. typhi Ty21a.1. Formal SB. Infection and Immunity 1981;34:746-50.2. Tacket CO. Infection and Immunity 1990;58:1620-7.3. Medina E, et al. Eur J Immunol 2000;30:768-77.

Evaluation of immunoadjuvant capacityof biopolymer using Leptospira particulateantigensL González,1 B Tamargo,1 M González,2 L Villachat,1

D Santa Ana,1 JF Infante,2 O Pérez,2 G Sierra.2 1Insti-tute of 1Farmacy and Food (IFAL), University of Ha-vana. 2Instituto Finlay. [email protected]

The biopolymer A is a polysaccharide obtainedfrom natural sources to which have been adduced

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immunostimulating properties. In this paper weevaluated the polymeric adjuvant´s capability bymeans of a Leptospire particulate antigen. We for-mulated vaccine preparations using differentpolysaccharide’s concentrations and they were com-pared with the placebo formulations. We used both,the antigen adjuvanted in gel of aluminium hydrox-ide and without it as controls. The formulations wereadministrated subcutaniusly (sc) and intramuscu-larily (im). When evaluating the responses producedby induced antibodies using microaglutination(MAT), ELISA, and Dot Blotting, we obtained thebest result with the vaccine formulation containing2mg/mL via im. The challenge assay demonstratedthat the protecting effect of induced antibodies wassimilar to that obtained with preparation adjuvantedwith gel of aluminium hydroxide. The toxicity pre-liminary test suggested that polymer A was harm-less and safe in the used dosage.

Influence of formulation variables on thein vitro release from biodegradablemicroparticulate systems in vaccinecandidatesE Muñoz, M Muñoz, C Baldor, M Acosta, R Cabrera, JHernández, JR Aguilar, A Pérez, D Cardoso, A Talavera,V Falcón, O Pérez, G Sierra, C Campa. Finlay Insti-tute. PO Box 16017, Havana, Cuba. Phone: 2718356;Fax: (53-7) 2086075; E-mail: [email protected]

Poly (D,L lactide-co-glycolide) containing BSA(bovine serum albumin) as test, tetanic toxoide,polysaccharide C of Neisseria meningitides and DNAwere prepared by a method of solvent evaporationusing double emulsion. These microspheres werecharacterized for size, morphology, surface absorbedsubstances, encapsulation efficiency and release ki-netics. The influence of two formulation variableslike: (the procedure to obtain the first emulsion andprocedure having dried off once encapsulated theprotein, polysaccharides and DNA) on the physicalcharacteristics and the behavior of release they werealso investigated. The microspheres made with lowerenergy of cavitation were more general spheric al-though there have some changes according to thenature of the substances employee too. The effectsof drying end also had its influences in the physicalcharacteristics from the one encapsulated to otherbeing notably different the process for drying withSpeed Vacuum to Liophilizationn method being sig-nificantly better this last .

Effect of viscosity of bio-adhesive polymerson IFN alpha 2b release across syntheticmembranesLl Ruiz, A Aguilera, N Reyes, L Duany, K Aroche, KSoto, and E Hardy. Center for Genetic Engineeringand Biotechnology. PO Box 6162, Havana 10600,Cuba. Tel: (53-7) 2716221; Fax: (53-7) 2714764;E-mail: [email protected]

Since 1976 scientists began to use interferon (IFN)as an adjuvant therapy after the surgical treatment ofrespiratory papillomatosis, showing effectiveness forthe relapse control of the disease [1]. Other patholo-

gies such as multiple myeloma and colon cancer, havealso been treated with IFN as adjuvant, often using theparenteral via for the delivery of the cytokine. Bio-adhesive polymers have increasingly been thought as agood tool for the development of controlled releasesystems. These polymers can enhance drug releasethrough an optimal contact with adsorption surfaces.In addition, they can extend the drug residence period,reducing the therapeutic doses that are needed in thetreatment [2]. Other administration routes (nasal, sub-lingual, ocular, rectal) are potentially attractive to beevaluated using formulations based on these polymers.In this work, we evaluated the influence of some bio-adhesives polymers as well as their viscosity on releas-ing IFN alpha 2b across synthetic membranes. Releaseof the cytokine was evaluated on a homemade cell de-signed from the Formulation Development Department(CIGB, Havana, Cuba). The concentration of IFN al-pha 2b was determined by a sandwich-type enzyme-linked immunosorbent assay (ELISA). We alsoascertained the effect of these polymers on the physi-cal/chemical (by RP-HPLC and SDS/PAGE) and bio-logical stability of IFN alpha 2b. The later parameterwas estimated by determining the inhibition of the cy-topathic effect (ECP) produced by the Mengo virus onHep-2 cells (ATCC No. CCL23).Viscosity did not sig-nificantly affect the IFN alpha 2b biological activity,but the releasing profile notably varied when the vis-cosity of the assayed polymers increased.1. Quiney RE, et al. Clin Otolaryngol 1989;14:217-

25.2. Van Ooteghem M. In: Préparations Ophtalmiques,

ed. Galenica, Technique and documentation.Lavoisier, Paris; 1995.

Effect of acemannan and calcium salts on theimmune response against different antigensPérez A, Aguilar JC, Herrera AM, Urquiza D, MuzioV, Pentón E, Leal MJ, Guillén GE. Center for GeneticEngineering and Biotechnology. PO Box 6162, Ha-vana 10600, Cuba. Tel: (53-7) 2716221; Fax: (53-7)2714764; E-mail: [email protected]

The development of more effective vaccinesagainst viral diseases is a priority of internationalhealth organizations (WHO, PHO). Efforts are be-ing made to reduce the number of administrationsand to modulate the immune response to vaccineantigens to increase their efficcacy. In this work, westudied the effect of acemannan and calcium salts onthe immune response induced against several pro-teins. Different formulations of acemannan (0.3 mg/mL), calcium oxalate (0.5 mg/mL) and calcium phos-phate (0.5 mg/mL) were administered alone or in com-bination with the antigens and through different routesof immunization, in mice. Total IgG antibody titerswere measured by ELISA, after each dose. A mixedpattern of antibody subclass was evidence for theHBsAg. A prevalence of IgG2a subclass was ob-served for TAB 9, a recombinant polypeptide cod-ing for different V3 peptides. In general we concludethat the route of administration and the type of anti-gens are important issues in the behaviour of theresulting formulation.

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Workshop on Mucosal Adjuvants

Chairpersons: Claude Leclerc,1 Julio Cesar Aguilar2

1Pasteur Institute, France. E-mail: [email protected] for Genetic Engineering and Biotechnology, Havana, Cuba. E-mail: [email protected]

ABSTRACTSeveral adjuvants and adjuvant strategies were presented in the session Mucosal Adjuvants. Two oral presentationswere based mainly on the work with the attenuated strain of Vibrio cholerae 638 (El Tor Ogawa) as a vaccinecandidate for cholera. A new CTX?-negative hemagglutinin/protease defective candidate cholera vaccine strainwas examined for safety and immunogenicity in healthy adult Cuban and Ecuadorian volunteers. The Cuban firstgeneration attenuated strain 81 and JBK70 was inoculated in a group of Cuban volunteers as the reactogeniccontrol. In a double-blind placebo controlled study, no significant adverse reactions were observed in volunteersingesting strain 638. Four volunteers out of 42 which ingested the 638 strain and 1 out of 14 placebo experimentedloose stools. V. cholerae 638 at doses ranging 4 x 107 to 2 x 109, elicited significant serum antibodies shown byvibriocidal and ELISA titers and anti-Ogawa IgA antibody secreting cells in Cuban and Ecuadorian adult volunteers.The mucosal response was evaluated in a second paper on the 638 cholera strain. The IgA secreted in saliva wasanalyzed and detected with maximum values at day 9 after inoculation. These results suggested that the 638 straininduces a good mucosal response.Different vaccine formulations based on HBV antigens were presented. The purpose of one study was the evalua-tion of the use in humoral immunity with a combined formulation of hepatitis B virus antigens in mice administerednasally with different formulations. The systemic and mucosal responses were characterized for the acemannan/HBsAg and HBcAg/HBsAg formulations. A high immunogenicity of recombinant HBcAg after nasal inoculation wasalso evaluated and demonstrated as well as the general high immunogenicity of the formulations under study. Astrong adjuvant effect of HBcAg on HBsAg immunogenicity in serum and vaginal secretions, similar in intensity toCT and acemannan was evidenced. A higher and significant increase was found in the IgG2a/IgG1rate againstHBsAg for the group immunized with the HBcAg/HBsAg combination, following the Th1-like response also observedfor the HBcAg subclass analysis. These results have implications on the design of mucosal therapeutic and preven-tive vaccines against HBV infection.In a last paper, a mucosal administration of the Hepatitis C core was also studied and evidenced the same effectsas the previously described interaction of HbcAg and HbsAg on the resulting immune response.

for detecting anti-Ogawa IgA antibody secreting cells.Vibriocidal test were done against VC12 strain (El TorOgawa) and plates of ELISA and ELISPOT tests werecoated with purified LPS. V. cholerae 638 at doses rang-ing 4 x 107 to 2 x 109, elicited significant serum anti-body showed by vibriocidal and ELISA titers andanti-Ogawa IgA antibody secreting cells in Cuban andEcuadorian adult volunteers.

Mucosal response to 638 attenuated cholerastrainJ del Campo,1 M Lastre,1 G Bracho,1 C Zayas,1 MDíaz,1 C Taboada,1 L García,1 T Serrano,2 A Pérez,2

G Sierra,1 O Pérez.1 1Departments of Basic and Clini-cal Immunology and Enteropathogenic Bacteria, FinlayInstitute. PO Box 16017, Havana, Cuba. 2Clinical Labo-ratory, Pedro Kourí Institute, Havana City, Cuba.3CENIC, Havana City, Cuba. [email protected]

Cholera is a serious health problem in developing coun-tries. Vaccines are not yet available, but they under de-velopment will be administered by mucosal route in orderto induce a protective IgA response. In Cuba, a 638attenuated cholera strain has been developed. This vac-cine candidate induces a good response in animal and inpreliminary human study has proven to be safe andimmunogenic. However, the specific IgA at mucosal sitesand comparison of 638 strain with the known reactogenicJBK70 strain are not demonstrated. Therefore, we evalu-ated the production of anti-LPS (El Tor Ogawa) anti-

Oral Presentations638 attenuated strain cholera candidate vaccine

L García,1* R Fando,2 A Talavera,1 J Benítez,2 HGarcía,1 B Cedré,1 M Díaz,3 BL Rodríguez,2 A Pérez,3

J Campos,2 JL Pérez,1 T Valmaseda,1 A Silva,2 OPérez,1 A Pérez,1 M Ramírez,3 L Bravo,3 T Ledón,2 MLastre,1 Gustavo Sierra.1 1Finlay Institute. PO Box16017, Havana, Cuba. 2National Center of ScientificResearch. 3Tropical Medicine Institute “PedroKourí”Havana, Cuba. E-mail: [email protected]

Vibrio cholerae 638 (El Tor Ogawa) a new CTX?-negative hemagglutinin/protease defective candidatecholera vaccine strain, was examined for safety andimmunogenicity in adult healthy Cuban and Ecuador-ian volunteers. Cuban first generation attenuated strain81 and JBK70 was inoculated in a group of Cubanvolunteers as reactogenic control. In a double-blind pla-cebo controlled study, no significant adverse reactionswere observed in volunteers ingesting strain 638. Fourvolunteers out of 42 which ingested 638 strain and 1out of 14 placebo experimented loose stools. The straincolonized well the human small bowel as evidenced byit isolation from the stools of 37 out of 42 volunteersinoculated. JBK70 and 81 strains orally administeredshowed a significant adverse reaction meanly in thenumber and severity of diarrheic events. No differencewas observed between the isolation from stools of vol-unteers inoculated with 638 or two others strains. Theimmunogenicity was evaluated in inoculated human seraby vibriocidal and ELISA test and in blood by ELISPOT

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body secreting cells (ASC) by ELISPOT in the bloodand the anti-LPS antibodies in the parotid saliva byELISA to explore mucosal responses in human volun-teers inoculated with one or other strains. Three groupsof 638 (109, 108, and 107 CFU), one group of JBK70(109 CFU) and one placebo group were enrolled. TheIgA+ASC response predominated and IgG+ASC wasonly found with the higher doses. The salival secretoryIgA was consistently detected with maximum values atday 9 after inoculation. These results suggest that salivalIgA is locally produced. In conclusion, our results showthat 638 induces a good mucosal response similar tothose induced by JBK70 strain.

Combined formulation of Hepatitis B Virusantigens as a nasal vaccine candidateAguilar JC, Lobaina Y, Pichardo D, Sanchez J, GarcíaD, Iglesias E, Alemán R, Urquiza D, Pentón E, FalcónV, De la Rosa MC, Álvarez F, Muzio V, Guillén G. Vac-cine Division, Biomedical Research, Center for GeneticEngineering and Biotechnology. PO Box 6162, Havana10600, Cuba. Tel: (53-7) 2716221; Fax: (53-7)2714764; E-mail: [email protected]

The purpose of this study is the evaluation of thehumoral immunity raised with a combined formulationof Hepatitis B Virus antigens.We have immunized groupsof 8 to 10 female Balb/c mice with different formulationscontaining HBsAg and HBcAg through the nasal routein a total volume of 50?L per mice. The IgA, IgG, IgG1and 2a titers in sera and vaginal washes were determinedby ELISA. The interaction between HBsAg and HBcAgon HBsAg immunogenicity was carried out using as con-trol adjuvants cholera toxin (CT) and acemannan. Ini-tially we demonstrated a high immunogenicity ofrecombinant HBcAg after nasal inoculation. Then weevaluated the effect of antigen combination on HBsAgimmunogenicity co-administering both antigens nasally.We observed a strong adjuvant effect of HBcAg onHBsAg immunogenicity in serum and vaginal secretions,similar in intensity to CT and acemannan. We alsoachieved a higher and significant increase in the rate IgG2a/IgG1 against HBsAg for the group immunized withHBcAg and HBsAg, following the Th1-like responsealso observed for HBcAg subclass pattern. We concludethat the inoculation of the soluble formulations of HBsAgand HBcAg enhanced the immunogenicity of HBsAg, inserum and mucosal secretions, and modulated the IgGsubclass pattern to the Th1-like pattern.


Intranasal immunisation of Balb/c micewith recombinant Micobacterium tuberculosisantigens using cholera toxin (CT) as adjuvantÁlvarez A,1 Vidal T,1 Fishmann Y,2 Avi-chai Hovav,2

Sarmiento ME,1 González JL,1 López Y,1 Fariñas M,1

Estévez P,1 Infante JF,1 Falero G,1 Sierra G,1 BercovierH,2 Acosta A.1 1Finlay Institute. PO Box 16017, Ha-vana, Cuba. Phone: 219542; Fax: (53-7) 2086075;E-mail: [email protected] .cu 2Hebrew Universityof Jerusalem, Jerusalem, Israel.

Many attempts are being done for the developmentof new generation vaccines against tuberculosis [1, 2]. Inorder to evaluate the immunogenicity and the protectiveeffect of the administration of a mixture of recombinant

M. tuberculosis antigens (85B, L7/L12, 27 kD, esat-6)plus CT, Balb/c mice were immunised by the nasal route.In the immunised animals there were a significative in-crease of the specific IgG antibodies against L7/L12,85B, 27 kD and esat-6 antigens. A protective effectupon challenge with BCG was demonstrated in theimmunised animals although this effect was lower thanthe results obtained in the group immunised with BCG.1. Horwitz MA, et al. Proc Natl Acad Sci USA

97:13853-8.2. Hess J, et al. FEMS Immunol Med Microbiol

27:283-9

Inhibition of anticolonizing effect of positiveserum from humans inoculated with theattenuated strain 638 Vibrio cholerae O1JL Pérez, Y Pino, T Valmaseda, G Año, B Cedré, HGarcía, A Talavera, L García. Finlay Institute. POBox 16017, Havana, Cuba. Fax: (53-7) 2086075;Phone: 2020986; E-mail: [email protected]

As a part of the study to obtain an oral vaccineagainst cholerae, the anti-colonizing capacity of thehuman serum, extracted after an oral dose with the at-tenuated strain 638, was evaluated. Inhibition studieswith somatic antigens of Vibrio cholerae O1 like, Ogawaand Inaba lipopolysaccharide (LPS), outer membraneproteins (OMP) and the 20 kDa antigenic complex aswell as the LPS of the O139 serogroup, were done.Theneonatal mice model was utilized. The positive serumshowed a high anti-colonizing activity. In the antigeninhibition studies, was observed that the Ogawa LPSreduced the anti-colonizing capacity of the positiveserum. The 20 kDa antigenic complex, the Inaba LPSand the OMP showed a low and differential inhibitoryeffect. These results confirm that the most protectiveantigen is the homologous LPS, although there are oth-ers cellular surface antigens involved in the protectiveimmunity induction. The results here described con-tribute to understand the protective immunity mecha-nism against vibrio cholerae and stimulate to use ofconjugated and subunits vaccine.

Nasal monovalent vaccine formulationof HBsAg against Hepatitis B Virus infectionMuzio V, Aguilar JC, Crombet L, Leal MJ, Constante Y,Iglesias E, Lobaina Y, Pichardo D, Pentón E, UrquizaD, Guillén G. Vaccine Division, Biomedical Research,Center for Genetic Engineering and Biotechnology, POBox 6162, Havana 10600, Cuba. Tel: (53-7) 2716221;Fax: (53-7) 2714764; E-mail: [email protected]

The aim of this work was the evaluation of the hu-moral immunity raised with a monovalent formulationof Hepatitis B surface antigen (HBsAg) and the naturalpolysaccharide acemannan to obtain a nasal vaccinecandidate improving the immune response in systemiccompartments and mucosal tissues against the HBV.Groups of 8 Balb/c mice 8 to 12 weeks old, were immu-nized three times, 2 weeks apart by nasal and systemicroutes, with equal doses of HBsAg. The concentrationof acemannan was determined by the Antrona colori-metric method. IgG and IgA titers in sera and vaginalwashes were measured by ELISA. Titers were com-pared using the Student’s t test. Serum IgG and se-creted vaginal IgA response for the acemannan groupwas higher than that obtained with HBsAg in saline-

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phosphate buffer. No difference was observed for theseric IgG titers after i.m. and s.c. inoculations using thesame dose of antigen in alum after three inoculations.Nasal inoculations of HBsAg-acemannan formulationsgave a high length and stronger responses compared tocholera toxin and alum formulations. In conclusion, wehave demonstrated in mice that using acemannan-HBsAgformulation, the nasal route can be as efficient as sys-temic routes in the induction of strong and high lengthanti-HBsAg antibody responses in sera, with the ad-vantage of inducing strong mucosal responses.

Mucosal immunogenicity of HBcAg, a carrierproteinLobaina Y, Aguilar JC, Cruz LJ, Abreu N, Urquiza D,Cabrales A, Muzio V, and Guillén G. Centro deIngeniería Genética y Biotecnología, PO Box 6162. C.Habana, Cuba. [email protected]

In this study we explored the immunogenicity ofthe recombinant hepatitis B virus core antigen(rHBcAg) in Balb/c mice by several mucosal immuni-zation routes. Four doses of 5 µg each were adminis-tered at days 0, 15, 30, and 210. We evaluated the IgGantibody response kinetic and studied the IgG sub-classes response in serum and the IgA antibody re-sponse in mucosal lavages after four doses by ELISA.The immunogenicity of the rHBcAg by the severalmucosal immunization routes was high, resulting theintranasal route the one that generated the highest re-sponse. The intranasal route also generated the higherspecific IgA response at mucosal level. In general, theobtained results strongly suggest that the immunoge-nicity of the rHBcAg by mucosal routes follows thedecreasing order: [intranasal]>> [intrarrectal]> [sub-lingual]> [oral]> [intravaginal], being the intranasalroute able of generating responses of strong intensityas the one generated by systemic route.

HCV core, a mucosal adjuvantN Acosta-Rivero, JC Aguilar, S Dueñas-Carrera, VFalcón, A Mussachio, A Viña, L Álvarez-Lajonchere,J Marante, A Rodríguez, D Pichardo, DUrquiza, MC

de la Rosa, I Menéndez, I Guerra, T Ramos, V Muzio,G Guillén, and J Morales. Center for Genetic Engi-neering and Biotechnology, P.O. Box 6162, Havana10600, Cuba. Tel: 53(7) 2716221, Fax: 53(7) [email protected]

The capacity of both the entire Hepatitis C virus(HCcAg) produced in P. pastoris and a truncated variantcomprising the first 120 aa (HCc.120) produced in E.coli to form particles was studied. Size exclusion chro-matography suggested that HCcAg and HCc.120 as-sembled into high molecular weight structures [1, 2]. Aserum from a chronic HCV carrier patient also specifi-cally recognized them. Both antigens migrated withbuoyant density values similar to those obtained fornative nucleocapsid particles from infected patientswhen analyzed using sucrose density gradient centrifu-gation. The analysis by electron microscopy of purifiedHCcAg and HCc.120 showed aggregates resembling vi-rus-like particles (VLPs) with an average diameter of 30nm. These results indicated that these antigens assembledinto VLPs resembling HCV nucleocapsid particles in amature stage. Afterwards we study the in vitro self-assembly properties of HCcAg. HCcAg was purifiedas a low molecular weight species by electroelusion un-der denaturalizing conditions for confirmation of its self-assembly properties. After renaturalization, the electronmicroscopy showed that HCcAg assembled into spheri-cal particles of 30 nm. HCcAg also showed homogene-ity and was specifically recognized by a serum from achronic HCV carrier patient. The data indicated that invitro assembly of HCcAg into virus-like particles re-sembling HCV nucleocapsid particles in a mature stageis an intrinsic quality of this protein. Finally, HCc.120obtained as a VLP in E. coli was coinoculated nasallywith HBsAg [3]. This formulation evidenced a syner-gistic effect on the immunogenicity of both antigenssuggesting its usefulness as a mucosal adjuvant.1. Acosta-Rivero N, et al. Biochem Biophys Res

Commun. Sep 14;287(1):122-5.2. Lorenzo L, et al. Biochem Biophys Res Commun

281,962-965.3. Aguilar JC, et al. CU 98/ Dec, PCT/CU/99/00006.

Workshop on Adjuvants for Viral Vaccines

Chairpersons: Louise Cosby,1 Enrique Iglesias2

1The Queens University of Belfast, Medical Biology Centre, Belfast, UK. E-mail: [email protected] de Ingeniería Genética y Biotecnología. La Habana, Cuba. E-mail: [email protected]

ABSTRACTThe oral session was a scheduled debate over several strategies to deal with different viral infections in man andanimals. There were presentations on studies in mice to increase cross-reacting antibodies to HIV-1; afterwards,delegates were alerted to a potential drawback of Morbillivirus vaccines; also, DNA vaccination for BHV-1 using alipopolysaccharide from Gram-negative bacteria (RN-205) was subjected to discussion; and finally, also the im-mune regulation of hepatitis B vaccine by MAbs. The poster session was devoted to study the mouse as a model forscreening the inactivated BHV-1 vaccine using different adjuvants (SL-CD/S/W, Acridine, RN-205 and oleous adju-vant); the use of Algammulin as an adjuvant for HBsAg in mice; the use of several additives (CaCl2, PEG-6000,Freund�s Adjuvant, sonicated Calf Thymus DNA and Co.120) for a naked-DNA coding HCcAg; also, the protectioninduced by a recombinant envelope protein from Dengue virus was presented; additionally, the adjuvant effect ofa truncated HCV core protein regarding the E2 protein was illustrated; and finally, preliminary results were shownon the possible mechanisms of virus clearance from the Central Nervous System of mice infected with the Measlesvirus. It was an heterogeneous workshop considering that discussions were on different viruses; but that was in factthe strongest point. This combination provided an exceptional opportunity to exchange experiences.

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IntroductionThe aim of the workshop was to discuss practicalapproaches to viral vaccines stressing the use of clas-sical and novel adjuvants. Chairpersons in this ses-sion were Dr. SL Cosby (The Queens University ofBelfast, United Kingdom) and E Iglesias (Center forGenetic Engineering and Biotechnology, Cuba). Therewere four oral presentations and six posters.

Main bodyThe oral debate started with the lecture “Increasedimmunogenicity and cross-reactivity induced by theconjugation of V3-based multiple antigen peptides(MAP) to carrier proteins” by E Iglesias (Center forGenetic Engineering and Biotechnology, Cuba). Heshowed promising results using synthetic peptidesfor inducing antibodies with a capacity to positivelyrecognize highly variable V3 peptides from HIV-1.From his work it was concluded that a possible ap-proach to increase the generation of cross-reactingantibodies, at least in mice, may be through the conju-gation of MAPs to carrier proteins. However, thecarrier protein exerts its influence on the quality ofantibodies. Because of the flexibility of the syntheticapproach different presentation forms to the immunesystem were explored. It was shown that the mixtureof V3-MAP and CD4bp-MAP (CD4bp: CD4 bind-ing peptide from gp120 of HIV-1) conjugated to dif-ferent carrier molecules enhances the anti-V3 antibodyresponse and the level of cross-reacting antibodies.However, the best response is obtained using a single“heterologous” CD4bp-V3-MAP conjugate.

At the second hour Dr. SL Cosby (The QueensUniversity of Belfast, United Kingdom) gave the talk“Morbillivirus vaccines and virus-induced immuno-suppression”. Morbillivirus infections induce severedisease in their natural host, causing high morbidityand mortality. Virus-induced immune suppression mayinfluence the high mortality. She showed data on theco-expression on the surface of presenter cells of thefusion protein (F) and the hemagglutinin (H) for allmembers of Morvillivirus as the cause of immune sup-pression. Her results showed that vaccine strains ofRPV in cattle and peste des petite ruminants virus(PPRV) in goats, cause immunosuppression by thismechanism. Therefore, H and F protein interactionsmust be considered when designing recombinantmorbillivirus vaccines and the use of adjuvants.

The session continued with Dr. P Zamorano’s(Centro de Investigación en Ciencias Veterinarias,Argentina) talk “DNA Vaccination of mice againstBHV-1: effects of the RN-205 adjuvant in the modu-lation of the immune response”. In her work thespeaker studied the influence of the RN-205 adjuvant(lipopolysaccharide from Gram-negative bacteria) onthe cellular and humoral immune response of threeDNA vaccines for BHV-1. RN-205-treated miceshowed higher levels of proliferation, and IL-4 andγ-IFN secreting cells in comparison to untreated mice.

At the end Dr. A Basalp (Research Institute forGenetic Engineering and Biotechnology, Turkey)talked on “Regulation of the immune response to hepa-titis B virus vaccine by monoclonal antibodies”. Inspite of the success of commercially available HBVvaccines prepared with alum several immunizations

are required to achieve a protective response. Also,they are less effective in the elderly and immune com-promised people. So, there is still a growing need forvaccine systems that effectively enhance the immuneresponse. Dr. Basalp analyzed the antibody responseelicited by both the IgM and IgG-HBV vaccine(GenHevac B Pasteur, France) immunocomplex inmice. An enhanced immune response was observedwhen the HBV vaccine was complexed to IgM in arange from 1 to 5 mg. In the case of IgG an antibodyexcess was necessary; however, at concentrationsequivalents to the vaccine an enhance immune responsewas observed after the second dose. Then, it was con-cluded that monoclonal antibodies coupled to HBVvaccines might be a useful strategy to improve theireffect.

In the poster session Dr. Z Cinza (Center for Ge-netic Engineering and Biotechnology, Cuba) showeddata regarding the use of two Algammulin formulationsas adjuvants for the HBsAg in mice. It was concludedthat the AG-43F (fine formulation; 1 mm) was supe-rior to the AG-38ff (ultra fine formulation; < 1 mm)and to the vaccine in alum. On the other hand, Dr. LÁlvarez from the same institute studied the effect ofdifferent additives on the antibody response to a nakedDNA immunization using the plasmid pIDKCo codingfor a truncated HCV core protein. For the tested addi-tives PEG-6000, Freund´s adjuvant and sonicated calfthymus DNA elicited higher antibody responses re-garding the DNA in PBS and no difference was evidentamong them. A positive IgG2a/IgG1 ratio was found inall cases. In another poster Dr. L Hermida again fromthe same Institute in Havana City assessed the protec-tive effect of a recombinant envelope protein formDengue virus 4 produced in yeast (Pichia pastoris) andadjuvated in alum and Freund´s adjuvant. Mice immu-nized with both formulations were protected after anintra-cranial challenge in more than 60%. Interestingdata were posted by Dr. SL Cosby (The Queens Uni-versity of Belfast, United Kingdom) in her work“Mechanisms of virus clearance from the central ner-vous system of mice persistently infected with measlesvirus”. She unveiled possible mechanisms to clear per-sistent Central Nervous System viral infections in amouse model. Initial results showed that virus clear-ance occurs following treatment with immune serum.The data highlights the importance of an increased neu-tralizing antibody and/or cytokine response. A lastposter by Dr. G Martínez (Center for Genetic Engi-neering and Biotechnology, Cuba) showed the adjuvanteffect of a truncated HCV core protein (Co120) pro-duced in E. coli. Mice immunized with a mixture orconjugate of Co120 and the E2 from the same virusenhance the humoral response to the latter.

Oral Presentations

Immunogenicity and efficacy of subunitvaccines for respiratory syncytial virusinfection in a nonhuman primate modelKK Murthy, MT Salas, KS Rice, MM Leland, JLPatterson. Departments of Virology and Immunology,and Physiology and Medicine, Southwest Foundationfor Biomedical Research, San Antonio, TX, [email protected]

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were set at a similar titer and compared regarding cross-reaction to a panel of several V3 peptides. The analy-sis showed that MAP-based immunogens are moreprompt to elicite cross-reactive Abs than lineal poly-mers or peptides couple to dextran.1. Cruz L, et al. Biotecnol Apl 17:35.2. Cruz L, et al. Letters in Peptide Science 7:229.3. Cruz L, et al. J Pept Sci Accepted for publication.

Non-replicating vaccines for preventionof damaging congenital HCMV infections:Current status and new problemsWJ Britt, S Boppana, M Shimamura. Department ofPediatrics, University of Alabama, Birmingham, Bir-mingham, Ala. USA. [email protected]

Human cytomegalovirus (HCMV) remains an im-portant cause of disease in immunocompromised al-lograft recipients and individuals with AIDS. Inaddition, HCMV is the most common cause of con-genital viral infection in humans and a major cause ofcentral nervous system (CNS) damage in infants andchildren. In the US, congenital HCMV infection oc-curs in 1% of live birth and up to 3000 infants haveCNS damage each year as a result of this infection.Studies have suggested that this infection is poten-tially vaccine modifiable because pre-existing mater-nal immunity has been thought to limit disease in theinfected fetus. Current vaccine approaches include areplicating vaccine and subunit vaccines. A subunitvaccine currently under testing which consists of themajor envelope glycoprotein of HCMV, gB, combinedwith an oil/water emulsion has been shown to inducevirus neutralizing antibodies; however, these antibod-ies wane with time. Virus neutralizing antibodies andT lymphocyte reactivity, including HCMV specificCTL, were induced in mice by immunization with gBcombined with the saponin derivative, QS21. Al-though these conventional approaches are wellfounded, recent data has indicated that immunity fol-lowing natural infection does not prevent fetal infec-tion and disease following congenital infection. Severalpossible reasons could account for these observationsincluding reinfection by antigenically distinct viruses.As an example, gN, which is an abundant viral enve-lope glycoprotein and a target of virus neutralizingantibodies, has been shown to exhibit considerableamino acid divergence in primary isolates. The role ofantigenic variation in this virus and characteristics ofthe natural history of this virus infection in the de-sign of non-replicating vaccines must be reconsideredin the design of protective vaccines for this congeni-tal virus infection.

Morbillivirus vaccines and virus-inducedimmunosuppressionSL Cosby,1 J Heaney,1,2 T Barrett.2 1The Queens Uni-versity of Belfast, Medical Biology Centre, 97 LisburnRoad, Belfast, BT9 7BL, UK, Tel. 44 28 90272127,Fax 44 28 90 236505. 2The Institute of Animal Health,Pirbright, Surrey, GU24 0NF, UK. [email protected]

Morbillivirus infections induce severe disease intheir natural hosts, causing high morbidity and mor-tality. Virus-induced immune suppression may influ-ence the high mortality, allowing secondary bacterialinfections to flourish that are lethal to the host. This

Respiratory syncytial virus (RSV) is the majorcause of upper respiratory system infection in in-fants, elderly, and transplant recipients. Infection ininfants may result in morbidity to mortality depend-ing upon the healthcare support and system. Severalvaccine strategies are being evaluated for prophy-laxis against RSV infection, and include cold adopted,attenuated, and purified subunit F and G proteinvaccines. In the present study we report the immuno-genicity and efficacy of subunit vaccines in the ba-boon model for RSV. Infant baboons (4 week old)exposed to RSV by the intranasal route develop char-acteristic clinical signs and symptoms of infection.Due to delayed development of immunocompetencethey respond poorly to RSV. Therefore, the role ofRSV specific maternal antibodies in prevention or re-duction in severity of infection in infants was evalu-ated by immunizing pregnant baboons with either Fprotein or F + G protein vaccines. Thirty adult femalebaboons seronegative for RSV were intranasally in-oculated with RSV to simulate natural exposure andafter resolution of infection they were placed in abreeding program. Pregnancy was confirmed bysonogram and the animals were immunized with ei-ther the F vaccine (n=10), or F + G vaccine (n=10)and adjuvant alone (n=10). Infants derived from damsin the 3 experimental groups were challenged withRSV by the intranasal route at 4 weeks of age to deter-mine the effect of RSV specific maternal antibodies.Maternal vaccination markedly reduced the numberof infected infants when compared to the rate of in-fection in infants born to mother given adjuvant alone.The results suggest that maternal vaccination strategycan markedly reduce the incidence of RSV infection innewborn infants.

Increased Immunogenicity and cross-reactivityinduced by conjugation of V3 based multipleantigen peptides to carrier proteins.E Iglesias, JC Aguilar, LJ Cruz, O Reyes. Centro deIngeniería Genética y Biotecnología, División deVacunas. PO Box 6162, Cubanacán, Habana 10600,Cuba. Phone: (53-7) 2718008; Fax: (53-7) 2714764;E-mail: [email protected]

Multiple antigenic peptides (MAP) comprisingeight and four synthetic peptides corresponding tothe V3 loop of HIV-1JY1 (subtype D) were com-pared regarding Ab titers and level of cross-reactionto several subtype B V3. The MAP8 induced highertiters and cross-reaction level than MAP4 after fourdoses. The increased recognition of heterologous pep-tides is correlated to a broad interaction with aa resi-dues in the primary sequence. MAP8 did not differ toa quimeric protein comprising several V3 peptides inAb titers to JY1 peptide. But the MAP induced higherlevel of recognition against a panel of several V3 pep-tides in ELISA. To increased the immunogenicity andcross-reaction JY1-MAP8 was coupled to KLH,HBsAg and P64k proteins. The conjugated MAP weremore inmunogenic than the respective peptide-conju-gate and were more or as immunogenic as four timesmore MAP8 immunized alone. Also, conjugates toHBsAg and KLH enhanced the cross-reaction to het-erologous V3 peptides in comparison to JY1-MAP8.Pooled sera from different immunization schedules

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can also give rise to problems with live attenuatedvaccines in susceptible individuals. A hallmark of suchvirus induced immunosuppression is the reduced ca-pability of freshly isolated peripheral leucocytes(PBL) to proliferate in response to a variety of stimuli.We have shown that all members of the morbillivirusexhibit this property and that, as for measles virus,co-expression of both the fusion (F) and thehaemagglutinin (H) proteins of rinderpest virus (RPV),on the surface of the presenter cells is necessary andsufficient to induce immune suppression in vitro [1].Our results show that vaccine strains of RPV in cattleand peste des petits ruminants virus (PPRV) in goats,cause immunosuppression by this mechanism, al-though this is less severe than with wild type strains.Furthermore, challenge with wild type virus, follow-ing vaccination, still gives rise to a degree of immuno-suppression, due to replication of the challenge virus.Therefore, H and F protein-cell surface interactionsmust be considered when designing recombinantmorbillivirus vaccines and use of adjuvants.1. Heaney J, Barrett T, Cosby SL. J Virol (2001, in

press).

DNA Vaccination of mice against BHV-1:effects of the adjuvant RN-205 in themodulation of the immune responseP Zamorano,1 O Taboga,1 M Domínguez,1 A Romera,1

M Puntel,1 C Tami,2 C Mongini,3 C Waldner,3 E Palma,1

A Sadir.1 1Centro de Investigación en CienciasVeterinarias, INTA Castelar, Buenos Aires; Argentina,2Food and Drug Administration, Bethesda, Maryland.3CEFyBO, CONICET. [email protected]

It is well documented that adjuvants improve theimmune response generated by traditional vaccines,but less is known about the effects of adjuvants onthe immune response elicited by DNA vaccines. Inthis study, we have investigated the use of RN-205(lipopolysaccharide from Gram-negative bacteria) asan adjuvant and analyzed the humoral and cellularspecific immune response elicited by DNA vaccinesbased on the glycoprotein D from BHV-1. We havecompared the antibodies induced by a cocktail of threeversions of gD (membrane-anchored, secreted andcytosolic) with and without RN-205. The cellularimmune response of RN-205-treated mice was higher,not only in the indexes of proliferation tests but in thenumber of IL-4 and IFN-γ secreting cells. When totalspleen cells were marked with specific monoclonalantibodies, a significant augment in the macrophagepopulation from all the groups receiving RN-205 havebeen observed, although CD8 and CD4 positive cellswere increased, the differences were not so impor-tant. The antibody titers of RN-205-treated mice de-creased after booster vaccinations compared withthose induced by DNA vaccination without adjuvant(although the differences were not significant). To-gether, our results indicate that the incorporation ofRN-205 into DNA vaccines induces an increase of thecellular specific immune response.

Regulation of immune response to hepatitisB virus vaccine by monoclonal antibodiesA Basalp. Research Institute for Genetic Engineeringand Biotechnology – TUBITAK. PO Box 21, 41470

Gebze – Kocaeli – TURKEY. Tel: 0 (262) 641 23 00.Fax: 0 (262) 641 23 09; E-mail: [email protected]

Hepatitis B Virus (HBV) infection is a major causeof acute and chronic hepatitis, cirrhosis and hepato-cellular carcinoma. Approximately 2 billion peoplehave evidence of current or past HBV infection, thusrepresents a serious worldwide problem. Commer-cially available HBV vaccines prepared with alumi-num hydroxide are safe and effective but multipleimmunization schedule is required to elicit a protec-tive immune response. Besides, it has less effect inthe elderly and the immuno compromised and also thetiters of antibodies to HBV may decline in the courseof 6 months following third immunization. Thus therisk of acquiring HBV infection increases as anti-HBVlevels become undetectable. While alum has a goodsafety record, it mainly augments the humoral immu-nity and not effective in raising the cellular immunityand there is still a growing need for an appropriatevaccine systems which effectively potentiate the im-mune response to HBV with fewer immunizations. Inthe present study, both IgM and IgG type anti- HBVmonoclonal antibodies were complexed to commer-cially available HBV vaccine (GenHevac B Pasteur,France). BALB/c mice were immunized with HBVvaccine complexed to IgM and IgG antibodies at vary-ing concentrations of antibodies. An enhanced immuneresponse was observed when HBV vaccine wascomplexed to IgM in the dose range of 1 to 5 µg. Onthe contrary, increased antibody levels were detectedwhen mice were immunized with HBV vaccinecomplexed to IgG in excess of antibody (5, 10, 20 µg).When IgG antibody is used at a dose near or equivalancewith HBV vaccine (0.5, 1, 1.5 µg) an enhanced anti-body response was obtained especially after second-ary immunization compared to those immunized byvaccine alone. The results indicate that complexedmonoclonal antibodies to HBV vaccine may induce animmunopotentiating effect against HBsAg.


A comparison of methods for measuringantibody responses in mice and cattlefollowing vaccination against BHV-1(use of adjuvants in vaccines)Escalada J, Zamorano P, Romera A, Puntel M,Dominguez M, Sadir, A. Instituto de Virología, C.I.C.V-INTA, Castelar, C.C. 77, Morón. Prov. Bs. As. Argen-tina. [email protected]

Infectious Bovine Rhinotracheitis is a disease whichcauses severe economical losses in Argentina due torespiratory and reproductive disorders. Vaccinationis a common preventive measure. Vaccines usuallyprevent the development of clinical signs and reducethe shedding of virus after infection. Inactivated vac-cines against BHV-1 are usually poor immunogensand require adjuvants to induce a significant immuneresponse. To assess the possibility of using the mu-rine system as an indicator of the vaccine´s immuno-genicity, antibody levels induced by different vaccinesin mice were compared with those in cattle. We used amurine system because mice are genetically identicaland less expensive than cattle. The study was con-ducted by the development of an ELISA test using a

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recombinant gD protein from BHV-1. Mice and bo-vine were immunized with inactivated vaccines againstBHV-1 containing different immunomodulators (SL-CD/S/W-non mineral oil and biodregradable, Avridine,RN-205, oleous adjuvant) and viral concentrations. Inthis report we demonstrate a close similarity betweenmice and cattle in terms of humoral immune response(similar antibodies profile and similar levels). In addi-tion, we were able to detect differences between vac-cines with different viral amounts. In summary, wepropose the murine model for vaccine screening be-fore the vaccination in the natural host.

The effect of high hydrostatic pressure onherpes simpex virus type 1 (HSV-1) evidencesfor changes on immuneGiongo V, Silva JL. Laboratorio de Termodinâmicade Proteínas e estruturas virais, departamento deBioquímica Médica, ICB, CCS, universidade do Brasil,RJ, Brazil. [email protected]

Herpes Simplex Virus Type I belongs to Herpes-viridae family and was the first virus associatedwith latent or innaparent infections primarily insensory ganglia. Their lesions are limited to orophar-ynx and are transmitted by direct contact of a sus-ceptible individual with infected secretions,i.e.vesicular fluids. In animal models, Balb/c, Natu-ral Killer cells and lymphokine responses (IFN-γ ,IL-6) have been implicated as important host de-fenses against HSV infections. Although humoralimmune response exist, the development of vaccineis difficult due to recurrences in HSV-1 infections.Inactivated (or killed) virus appear to show weakprotection to the animal host. In our lab we demon-strated in other works that high hydrostatic pres-sure is a tool for inactivation of virus particle [1, 2].The use of this method alters the conformationalstate of the proteins in the capside. When returnedto atmospheric pressure this new conformationalstate enhances its immunogenicity. It was also suc-cessful with Leptospira bovis [3]. In this work wedemonstrated that HSV-1 submitted to pressure(41Kpsi) for one, 4 and 8 hours reduces the infec-tivity titer by at least five order of magnitude. Fur-thermore analises with Natural Killer cells, IFN andNO demonstrate that this method alters the anti-genic presentation.1. Chebble, et al. 2000.2. Pontes L. 20013. Giongo. 2001.

Algammulin as adjuvant of the HBsAgin BAlB/c miceZ Cinza, MJ González Griego, A Ortega, G Guillén, GGarcía, M Hechevarría. Div. Vaccines Division, Cen-ter for Genetic Engineering and Biotechnology. PO Box6162, Havana 10600, Cuba. Tel: (53-7) 2716221; Fax:(53-7) 2714764; E-mail: [email protected]

Algammulin is a vaccine adjuvant that is formedwhen inulin is crystallized in a suspension of alumand the two are enmeshed to form similar ovoidsparticles that are electron-dense. These both par-ticles carry antigen and activate the AlternativeComplement Pathway. Inulin strongly enhancesmurine Th1 and to some extent Th2 immune response

pathways. Alum is a potent immune modulator em-phasizing Th2 response. Thus the type of responseto Algammulin (Th1 or Th2) may be predicted todepend on the proportion of the Algammulin sur-face occupied by inuline or alum [1, 2]. Here it isshown the immunogenicity when are applied severalformulations of Algammulin, as adjuvant, and theCuban HBsAg produced in yeast P. pastori [3], asantigen, in BALB/c mice. In conclusion, some for-mulations of Algammulin can produce a superior re-sponse of anti-HBs when they are applied withHBsAg in BALB/c mice using this antigen adjuvatedwith alum as control.1. Cooper PD, Steele EJ. Vaccine 1991;9(5):351-7.2. Cooper PD, et al. Immunology Letters 1991;27(3):

131-4.3. Pentón E, Muzio V, González-Griego M. Biotec-

nología Aplicada 1994;11(1):1-11.

Additives modulate the antibody responseelicited by a hepatitis C virus core-encodingplasmid in miceL Álvarez-Lajonchere, S Dueñas-Carrera, A Viña, TRamos, D Pichardo, J Morales. HCV Department,Vaccine Division, Centro de Ingeniería Genética y Bio-tecnología. PO Box 6162, Havana City, [email protected]

Humoral and cellular immune responses are cur-rently induced against hepatitis C virus core fol-lowing vaccination with core-encoding plasmids [1].However, the anti-core antibody response is fre-quently weak or transient. In this work, we evaluatedthe effect of different additives and DNA-proteincombinations on the anti-core antibody response.BALB/c mice were intramuscularly injected with anexpression plasmid (pIDKCo), encoding a C-terminaltruncated variant of the HCV core protein, alone orcombined with CaCl2, PEG-6000, Freund´s adjuvant,sonicated calf thymus DNA and Co.120. Mixture ofpIDKCo with PEG 6000 and Freund´s adjuvant ac-celerated the development of the anti-core Ab re-sponse. Combination with PEG 6000 also induced abias to IgG2a subclass predominance among anti-coreantibodies. The kinetics, IgG2a/IgG1 ratio and epitopespecificity of the anti-core antibody response elicitedby Co.120 alone or combined with pIDKCo was dif-ferent regarding that induced by the pIDKCo alone.Our data indicate that the antibody response inducedfollowing DNA immunization can be modified by for-mulation strategies.1. Lechmann M, Liang TJ. Semin Liver Dis 2000;

20(2):211-26.

Evaluation of the recombinant envelopeprotein from dengue-4 virus formulatedin two different adjuvantsL Hermida,1 R Rodríguez,2 C López,1 L Lazo,1 GMárquez,1 J García,1 R Espinosa,1 R Páez,1 MGGuzmán,2 G Guillén.1 1Centro de Ingeniería Genéticay Biotecnología. PO Box 6162, Havana, [email protected] 2Instituto de MedicinaTropical Pedro Kourí. PO Box 601, Marianao 13,Havana, Cuba.

Envelope protein (E) of Dengue virus (DEN) is animportant target of immunological response in humans

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[1]. For this reason, is considered a suitable componentof a recombinant subunit Dengue vaccine. Several labo-ratories have reported the obtention of the E protein indifferent hosts like: insect cells [2], E. coli and andyeast [3]; all of them were capable to induce functionalantibodies and protection in mice. In our work, weobtained the E protein of DEN-4 virus (E4 rec) in Pichiapastoris yeast associated to the host cell membrane.The immunological evaluation was carried out usingtwo different adjuvants: Freund’s and aluminium hy-droxide. The E4rec formulated in both adjuvants wascapable to induce a functional immune response as wellas a partial protection against lethal virus challenge.1. Halstead, SB. Bull WHO 1980;58:1-21.2. Feighny R, Borrous J, Putnak R. Am J Trop Med

Hyg 1994;50(3):322-8.3. Sugrue RJ, et al. J Virol 1997;78:1861-6.

Enhanced anti-HCVE2 antibody responseis elicited in mice by mixing or conjugationwith a truncated variant of the HCV coreproteinG Martínez, L Álvarez-Lajonchere, LJ Cruz, JCÁlvarez, JC Aguilar, J Morales, S Dueñ[email protected]

Hepatitis C virus (HCV) remains persistently inabout 85% of infected individuals, and chronic HCVinfection is associated with cirrhosis and hepatocel-lular carcinoma. The structural E2 glycoprotein hasbeen considered a potential antigen for use in a vac-cine against Hepatitis C. Both humoral and cellularimmune response against E2 protein are likely to beimportant for controlling HCV infection and are in-dispensable requirements to take into account for de-velop an effective HCV vaccine. The HCV genomeencodes structural proteins (core, E1, E2) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A,NS5B). A truncated variant of HCV core protein(Co.120), has previously elicited a strong immuneresponse in mice. In the present work, two C-termi-nal truncated forms of hepatitis C virus E2 glycopro-tein (384-605) and (384-680) produced in E. coli andyeast (Pichia pastoris), respectively, were mixed orconjugated to Co.120 protein with the purpose toenhance the immune response against E2 protein. Thehumoral immune response was substantially in-creased in mice immunized with the mixtureE2(680):Co and the best titers against E2 protein wereobtained with conjugated E2(680)-Co. While usingE2(384-605) protein, the highest humoral responsewere obtained in mice immunized with E2(605) onlyin PBS. The predominant antibody subclass observedfor both proteins was IgG1. Additionally, we studiedthe specific cellular immune response induced byE2(680) protein by in vitro lymphoproliferative as-says, no significant difference was observed betweenimmunized groups. These results indicate that im-mune response to E2(680) B-cell epitopes could besignificantly enhanced by mixture or conjugation ofE2(680) to Co120 protein.

Use of the immunomodulators and variousschemes of vaccination for increasingof efficiency of vaccination against influenzain the persons with immunodeficiency

Koltsova IG,1 Zubkova IN,2 Trubina LM,2 TishechkinaVA.2 1Odessa Medical State University, 1, Knyazeskayastr. 2I. I. Mechnikov Research Institute of EmergingInfectious Diseases, 6,Yadova str., Odessa, Ukraine.E-mail: [email protected]

Peculiarities of anti-influenza immunity forma-tion after alive (AIV) and inactivated influenzavaccination (IIV) in professional collectives of poul-try farms were studied. In such collectives infectionof T. gondii is an occupational harm and seroposi-tivity reached 73,8% (30,6% in control). Differentirregularities of immune state (such as decreasing ofrates of T-lymphocytes, T-helpers, antihemagglu-tinin (AGA) levels to actual influenza virus strainsand different classes of immunoglobulines) weredetermined in infected people. The highest sicknessrates of influenza and acute respiratory diseases(ARD) with broncho-pulmonary complication and longtime disability (up to 44%) were registered. Inocula-tion of the AIV and IIV was not effective and did notlead to the valid humoral response in seropositive per-sons with the immunodeficiency. Earlier we presentedthe results of the use of different immunomodulators(antiparasitic drugs Piremetamin and Delagil, whichgives an effect of immunostimulation in low dosage,Levamisol, Tactivin, Timogen and Natrii nucleinat) inanimal models (mice and rabbits) with experimentalchronic T.gondii infection in complex with AIV andIIV. On the base of these experiments we selected someimmunomodulators with the best stimulating effect andused them in infected persons. Materials and methods:intranasal inoculation of AIV-A(H1N1), parenteral in-oculation of IIV – A(H3N2), A(H1N1), ELISA, RIFwith Toxoplasma antigens, RHI with the commercialInfluenza antigens: A(H1N1), A(H3N2), B. The pri-mary AIV vaccination gave a low levels of AGA andprotection against Influenza, but treatment of pharynxand glands with levamisol fresh solution in 0,05-0,1-0,2% concentration for 6 - 8 days before AIV resultedin 2 times decreasing of morbidity of toxoplasma in-fected people. After repeated AIV inoculation high pro-phylactic effect and decreased morbidity of toxoplasmaseropositive people in 3,3 times in comparison withnot vaccinated were observed. The usage ofPiremetamin by proposed schedule (half of day’s dose(12,5 mg) during 6-7 days after inoculation of IIV) inseropositive persons led to the significant rise of theAHA titers for the both vaccine strains and reductionof Influenza and ARD morbidity. Use of the abovementioned immunoimprovers is recommended for in-creasing of immune response against Influenza inimmunocompromised persons and decreasing of ARDmorbidity.

Mechanisms of virus clearance from thecentral nervous system of mice persistentlyinfected with measles virusMcPeake SJW1 and Cosby SL1,2. School of Biologyand Biochemistry 1, Centre for Infection, Inflamma-tion and Repair, School of Medicine and 2 The Queen’sUniversity of Belfast, Medical Biology Centre, 97Lisburn Road, Belfast, BT9 7BL, UK, Tel. 44 2890272127, Fax 44 28 90 236505. [email protected]

Measles virus (MV) is the causative agent of a va-riety of central nervous system (CNS) disorders i.e.

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acute measles encephalitis (AME), measles body in-clusion encephalitis (MIBE) and subacute sclerosingpanencephalitis (SSPE). In SSPE virus can persist forlong periods in the CNS and is not cleared by the im-mune response. To investigate whether it is possible toclear virus from the CNS, by enhancing the immuneresponse, a persistent MV infection of the CNS inmice was established where virus can be detected up to90 days after infection 1. Clearance of virus from theCNS was observed over a 5 day period following re-exposure to virus in the periphery (intraperitonealboost). Expression of Interferon gamma (IFNγ), Inter-leukin 10 (IL10) and Interleukin 6 (IL6) mRNA were

Workshop on Proteoliposomes, Liposomes, and Virosomes

Chairpersons: Mark Chambers,1 Gustavo Sierra2

1TB Research Group, Department of Bacterial Diseases, Veterinary Laboratories Agency Weybridge, UK.E-mail: [email protected] 2Finlay Institute, Havana, Cuba. E-mail: [email protected]

IntroductionThis session was on liposomes, which are artificialvesicles typically formed from phospholipids. Lipo-somes have been used successfully for a number ofyears to deliver encapsulated enzymes and drugs tothe body (Koff & Fidler, 1985), sometimes in a man-ner that directs them to specific cellular targets (Norleyet al., 1986). The use of liposomes as adjuvants anddelivery systems for vaccines is a relatively new, ex-citing and expanding field of research (O’Hagan et al.,2001). Proteoliposomes and virosomes are liposomesthat additionally contain proteins, usually proteinsfrom the viral envelope in the case of virosomes(Kaneda, 2000).

Dr. Lockhoff from Bayer AG, Germany presentedhis work using a new class of synthetic glycolipidadjuvants (GLA) of which BAY R1005 is an ex-ample. These GLA are not liposomes, in that theyresemble the structure of ceramide, rather than form-ing vesicles. The GLA BAY R1005 is of particularinterest since it acts as an adjuvant even when ad-ministered to the antigen by a different route. In thissense it acts like a drug. It is relatively easy to chemi-cally synthesise and to produce to high purity sinceit forms crystals: an unusual property for a lipid-containing molecule. In relation to safety, BAY R1005produces little to no local irritation, has low acuteand sub-chronic toxicity and is free of T-cell mito-genicity. BAY R1005 has been evaluated in mice,sheep and cattle with a variety of particulate andsoluble antigens of viruses and bacteria, as well aspurified virus vaccines. The use of BAY R1005 asthe adjuvant in an attenuated trivalent bovine respi-ratory viral marker vaccine (IBR-Marker+BRSV+PI3) and an attenuated bivalent bovine respiratoryviral vaccine (BRSV + PI3) looks particularly en-couraging.

As mentioned previously, liposomes are usuallymade from phospholipids. However, a series of pauci-lamellar liposomes composed of non-phospholipids,sterols, oils and buffers (NovasomesTM) (Wallach &Philippot, 1993) have been made by Novavax, Inc.,

USA and show considerably promising as adjuvants.Dr. Chambers from the Veterinary LaboratoriesAgency, England presented data using formalin-inac-tivated whole mycobacterial cell preparations mixedwith a variety of NovasomesTM as part of theirprogramme to develop improved and safer tuberculo-sis vaccines. The vaccines produced no adverse reac-tion in guinea pigs and a number of the preparationsprotected guinea pigs from challenge with a low doseaerosol of viable Mycobacterium bovis. In some cases,the levels of protection were equivalent to thatachieved with the current live TB vaccine, BCG. Theformalin-inactivated whole mycobacterial cell prepa-rations were ineffective in the absence of the adju-vant. NovasomesTM can be produced rapidly in litrequantities with little batch to batch variation, theycan be manufactured to possess different surface chargeand membrane fusogenicity, and incorporate other mol-ecules such as proteins. These properties makeNovasomesTM an attractive alternative to conventionalphospholipid-based liposomes.

Dr. Sierra from the Finlay Institute presented stud-ies on the adjuvant activity of a proteoliposome de-rived from N. meningitidis. The highly efficaciousvaccine VA-MENGOC-BC was the first effective vac-cine against N. meningitidis B and the first proteoli-posome vaccine licensed for humans and clinicallyapplied. Over 45 million doses of the vaccine havebeen administered in Latin America and the first Eu-ropean trial is scheduled to begin in Belgium. Thevaccine consists of purified Outer Membrane Pro-teins (OMP) from N. meningitidis B and purified Cpolysaccharide from N. meningitidis C. The OMP self-assemble into vesicular proteoliposomes that arestabilised with alum to form one of the major compo-nents of the VA-MENGOC-BC vaccine. The OMPproteoliposome adjuvant has been demonstrated tobe a broad-spectrum Th1 adjuvant and is currentlybeing evaluated for other vaccine targets as diverse ascholera and salmonella, parasites, cancer, and allergy.The technology is patented in 25 countries.

detected in the CNS of boosted and non-boosted ani-mals (concentrations to be determined). An highly, sig-nificant increase in the titre of neutralizing antibodywas recorded up to 10 days after the animals receivedthe boost. Initial results also show that virus clearanceoccurs following treatment with immune serum. Theresults suggest that induction of an increased antibodyand/or cytokine response may be sufficient to clearvirus from the brain and raises the possibility of vacci-nation therapy in persistent CNS infections.1. Gailbraith. Rinderpest and peste de petits rumi-

nants viruses exhibit neurovirulence in mice. JNeurovirol 8 (2002, in press).

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This report was expanded by Dr. Bracho, also ofthe Finlay Institute, Cuba with a thorough descrip-tion of the immune response elicited to injections withVA-MENGOC-BC. The vaccine induced delayed typehypersensitivity, the opsonophagocytic killing of bac-teria and nitric oxide. There was rapid (in 2 hours)production of tumour necrosis factor-alpha (TNF-α)by polymorphonuclear cells, as well as monocytesafter the injection of the vaccine. The production ofinterferon-gamma (IFN-γ ) followed 8 hours post-in-jection. Bactericidal antibodies were stimulated afterthe injection of the vaccine, but also when given intra-nasally and gastrically. Salivary IgA was additionallyproduced following intransal vaccination. The cytokinepattern elicited by in vitro re-stimulation of periph-eral blood mononuclear cells from vaccinated volun-teers was also examined. Messenger RNA for IFN-γand IL-2 was demonstrated, reaching a peak at 14hours and 24 hours, respectively and remaining highuntil 72 hours. Bioactive IFN-γ was detected in theculture media. In contrast, IL-10 and IL-4 were notdetected. Since OMV proteoliposomes have a goodsafety profile and generate a Th1-biased response inhumans, they may be applicable as adjuvants to anumber of vaccines.

In the final presentation, Dr. Alonso from the Uni-versity of Havana, Cuba presented his finding on theuse of DPPC:Cholesterol (Cho) liposomes to deliverrecombinant human epidermal growth factor (rhEGF).Whilst principally used by these workers as a modelantigen with which to study the modulating ability ofthese vesicles upon the immune response in mice,hEGF and its receptor are over-expressed in certaintumours, making hEGF and its receptor targets forcancer immunotherapy. A vaccine comprised of hEGFconjugated to carrier protein (P64K protein from Neis-seria meningitidis) plus the conventional adjuvant alu-minium hydroxide (AlOH) is currently undergoingpre-clinical trial. Using liposomes as the adjuvant, theseworkers have demonstrated in mice that it was pos-sible to simplify the immunisation protocol from fourto two doses without changes in the total anti-EGFantibody titres. Liposomes of saturated phospholip-ids (DPPC:Cho and DSPC:Cho) were more immuno-stimulatory than unsaturated phosphatidylcholinefrom soybean (sPC:Cho) or AlOH, in terms of thetitre of IgG generated and their ability to block theinteraction between rhEGF and its receptor. Theseworkers concluded that DPPC:Cho and DSPC:Choliposomes are better adjuvants for rhEGF than AlOHbecause they enhance the levels of antibody, and in-duce DTH responses and lymphocyte proliferation,even in the absence of carrier proteins, at least in anexperimental system.1. Kaneda Y. Virosomes: evolution of the liposome as

a targeted drug delivery system. Adv Drug DelivRev 2000;43(2-3):197-205.

2. Koff WC, Fidler IJ. The potential use of liposome-mediated antiviral therapy. Antiviral Res 1985;5(3):179-90.

3. Norley SG, Huang L, Rouse BT. Targeting of drugloaded immunoliposomes to herpes simplex virusinfected corneal cells: an effective means of inhibit-ing virus replication in vitro. J Immunol 1986;136(2):681-5.

4. O’Hagan DT, MacKichan ML, Singh M. Recentdevelopments in adjuvants for vaccines against in-fectious diseases. Biomol Eng 2001;18(3):69-85.

5. Wallach DFH, Philippot JR. In: Liposome technol-ogy: liposome preparation and related techniques.Florida: CRC Press; 1993 Volume I, Chapter 9.p.141-6.

Oral Presentations

BAY R1005, a new specific adjuvantfor veterinary vaccinesO Lockhoff. Bayer AG, Central Research, ZF-HWQ18, D-51368 Leverkusen, Germany. Phone: +49-(0)214-30-57958 / Fax: +49-(0)214-30-30896 /E-mail: [email protected]

The identification of new potent adjuvants plays amajor role in modern vaccine development. An adju-vant must not only enhance the immune response butshould also drive this response to achieve protectiveimmunity. Additionally, adjuvants must fulfil increasedsafety standards. We have identified a new class ofsynthetic glycolipid adjuvants (GLA) which structur-ally resemble the lipid second messenger ceramide. BAYR1005 is the most prominent GLA representativewhich has been developed as adjuvant for veterinaryvaccines (Figure).

In mice BAY R1005 elicits humoral immune re-sponses in vitro and in vivo against a variety of par-ticulate and soluble antigens in a dose dependentmanner. The increase in antibody synthesis is spe-cifically dependent on the antigen and is not the re-sult of a polyclonal stimulation. BAY R1005 incombination with purified virus vaccines or subunitvaccines led to increased protection of virus-chal-lenged mice. Parenteral immunization with recombi-nant urease mixed with BAY R1005 induced strongTh1 and Th2 response and thereby elicited betterprotection against H. pylori infection than adjuvantswhich induced a Th2 type response only. In cattlean attenuated trivalent bovine respiratory viralmarker vaccine (IBR-Marker+ +BRSV+PI3) and anattenuated bivalent bovine respiratory viral vaccine(BRSV + PI3) both adjuvanted with BAY R1005have been developed that are exceptionally effica-cious even when applied parenterally to very youngcalves in the presence of maternally derived antibod-ies. The vaccines are safe in all categories of cattle,including the most susceptible seronegative calvesand pregnant animals. Due to the gE-deletion of theBHV1 vaccine strain, the trivalent vaccine can beused in modern BHV1 control programs.

Figure. Chemical structure of BAY R1005.

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Adjuvant activity of proteoliposomefrom N. meningitidis BG Sierra. Finlay Institute. PO Box 16017, Havana,Cuba, 11600. E-mail: [email protected]

Vesicles of lipid bilayers have been investigatedas drug delivery vehicles for more than two decades.More recently vesicles of protein + lipid bilayerswith self-assembly capacity have been incorporatedto this investigation tendency, more specificallyproteo-lipidic bilayers vesicles (Proteoliposomes)self-assembled after the purification of Outer Mem-brane Proteins (OMP) of different bacteria, withthe aim to obtain effective vaccines against this bac-teria. A purified vaccine VA-MENGOC-BC consist-ing of purified OMP(s) from N. meningitidis B andpurified C polysaccharide from N. meningitidis Cpresented as a protein-polysaccharide complexbased on a Proteoliposome model has been devel-oped and carefully tested. In a well controlled, ran-domized, stratified, prospective, double blindedplacebo-vaccine trial this vaccine has shown an ef-ficacy of 83% ; 95% Cl (42%-95%). After ten years(1987-97) of massive application in Cuba and manyother countries of more than 40 millions of VA-MENGOC-BC doses under epidemic and endemicsituations an effectivity ranging from 75 to 98%have been proved under different trial conditions(cohort studies, case-control studies, etc..). Veryimportant results have been obtained in laboratoryas well as field clinical-epidemiological studies whenmeasuring the effectivity of the vaccine against theC meningococcus. A clear difference has been shownwhen VA-MENGOC-BC was compared with theclassical C polysaccharide vaccine. VA-MENGOC-BC was more efficacious against C Meningococcusthan the C polysaccharide vaccine. Monosialogan-glioside GM3 and its derivaties seen to be potentialmolecular targets for experimental therapeutic can-cer vaccines for example in human breast tumors inwhich this molecule is relatively highly expressed.The main problem is the low immunogenicity ofthis molecule in human, as well as many experimen-tal animal models. This problem has been avoidedpresenting to the immune system an OMP-GM3complex. At least in the murine animal model, theOMP-GM3 complex was able to induce a potentIgG response. Neoglicoconjugates of oligosaccha-rides from cholera LPS conjugated with OMP fromN. meningitidis has also shown its capacity to de-stroy target cholera strains in vibriocidal assays.Synthetic as well as natural oligosaccharides (PRP)nfrom Haemophilus b conjugated with N. meningitidisOMP (s) presented in a proteoliposome complexhave shown equivalent or higher specific immuneresponse when tested against Hib target strains incomparison with commercially available anti Hibvaccines.

Examples of the use of proteoliposome-based vac-cine formulations.

PLS ————— OMP (NMB) + Poly CPLS ————— OMP (NMB) + PRP(n) naturalPLS ————— OMP (NMB) + PRP(n) SyntherPLS ————— OMP (Hib) + PRP(n) naturalPLS ————— OMP (NMB + GM3)PLS ————— OMP (NMB + Oligo cholera)

The proteoliposome basis for the presentation ofoligosaccharidic antigens as well as other antigentypes to the immune system has shown very ad-equate properties for new generation vaccines in chil-dren and in young animal models. A well definedTh-1 pattern have been demonstrated to be inducedby the proteoliposome and the proteoliposome con-taining formulations.

NovasomeTM adjuvanted killed mycobacterialwhole cell vaccines that protect guinea pigsagainst aerogenic Mycobacterium bovischallengeMA Chambers,1 DC Wright,2 J Brisker,2 A Williams,3 GHatch,3 D Gavier-Widén,4 G Hall,3 PD Marsh,3 RGHewinson.1 1TB Research Group, Department of Bacte-rial Diseases, and 4Department of Pathology, VeterinaryLaboratories Agency Weybridge, New Haw, Addlestone,Surrey, KT15 3NB, UK, Tel: 44 1932 357884; Fax: 441932 357684.2Novavax, Inc., 12111 Parklawn Drive,Rockville, MD 20852. 3CAMR, Salisbury, SP4 0JG, [email protected]

Tuberculosis has been the scourge of man and ani-mals for centuries and continues to inflict a huge cost,both in terms of human and animal health and finan-cially. This is particularly disappointing since for over70 years a vaccine has been available for tuberculosis:bacille Calmette-Guerin (BCG). BCG possesses manyof the qualities of an ideal vaccine: it is cheap to pro-duce and administer, it is safe and has been shown tobe efficacious in many circumstances [1]. Unfortu-nately, administration of live BCG to HIV infectedpatients has resulted in disseminated BCG infection.This and other problems surrounding the lack of uni-versal efficacy and safety of BCG have resulted inincreased efforts to develop a new generation of tu-berculosis vaccines. Vaccines based on killed wholecell preparations of mycobacteria should be safe andwould present a wide repertoire of antigens to therecipient. However, such killed vaccines have classi-cally conferred little to no specific protection to sub-sequent challenge with virulent mycobacteria [2]. Inorder to re-evaluate the vaccine potential of killed wholemycobacterial cell preparations, we inactivated cul-tures of M. bovis and BCG strains Pasteur and Tokyousing formalin. The cultures inactivated in this waywere found to be completely non-viable as determinedby culture on growth medium and inoculation intosevere combined immunodeficient (SCID) mice. Theformalin-inactivated preparations were mixed with arange of NovasomeTM adjuvants (paucilamellar lipo-somes composed of non-phospholipids, sterols, oilsand buffer) [3] and were used to vaccinate guinea pigs.The vaccines produced no adverse reaction in the ani-mals. Significantly, a number of the killed vaccinesprotected guinea pigs from challenge with a low doseaerosol of viable M. bovis. In some cases, the levels ofprotection were equivalent to that achieved with thegold-standard vaccine, live BCG Pasteur. These datagive hope for the development of a safe, efficaciousvaccine for tuberculosis based on NovasomeTM

adjuvanted, inactivated whole cell cultures of myco-bacteria.1. Bloom BR, Fine. In: Bloom BR, editor. Tubercu-

losis: pathogenicity, protection, and control.

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Washington: American Society for Microbiology;1994. p.531-7.

2. Orme. Infect Immun 1988;56(12):3310-2.3. Wallach, Philippot. In: Liposome technology: li-

posome preparation and related techniques. Florida:CRC Press; 1993. p.141-6.

Th1 cytokines profile inducedby VA-MENGOC-BCTM

Bracho G, del Campo J, Lastre M, Taboada C, DíazM, Zayas C, Lapinet J, Sierra G, Pérez O. FinlayInstitute. Ave 27 No 19805, La Lisa, AP 16017, Ciudadde La Habana, Cuba, 11600. [email protected]

An effective vaccine should stimulate the T cellssubset (Th1 or Th2) that efficiently activate the ap-propriated defense mechanisms. Cytokine pattern in-duced after antigen challenge direct T cells todifferentiate into Th1 or Th2 response. MeningoccocalMeningitis is a widely spread health problem either indeveloping and developed countries. Meningoccocoserogroup B and C constitute the main causes of men-ingitis. VA-MENGOC-BC is an anti B and Cmenigoccocal vaccine that had demonstrated high effi-cacy and safety in masive vaccination trials and is com-posed by outer membrane vesicles (OMV) fromserogroup B and polysaccharide from serogroup C.The defense mechanisms induced by this vaccine in-clude delayed type hipersensitivity, bactericidal anti-bodies, opsonophagocitic killing of bacteria and nitricoxide. Additionally we determine the cytokine pat-tern elicit by in vitro re-stimulation of peripheral bloodmononuclear cells from vaccinated volunteers. Theinduction of mRNA of IFNγ and IL-2 was demon-strated, raising a peak at 14 h and 24 h remaining athigh levels until 72 h. IFNγ mRNA was translated andsecreted to the culture media eliciting IFNγ elicit bio-logical. In contrast, IL-10 and IL-4 was not detectedindicating a prevalence of a Th1 activation. Ours re-sults demonstrate the preferent induction of a Th1immune response by vaccination with VA-MENGOC-BCÒ. Finally, our result support the possibility touse the main component of this vaccine as adjuvantsor immune modulators. The availability and easy modi-fication for new antigens inclusion on OMV make issuitable for this purposes

Adjuvant effect of liposomes encapsulatinghuman recombinant epidermal growth factorMC Luzardo*, L Calderón*, Y Martínez*, Y Ramos*,Y De León**, I.F Pazos*, ME Alonso* y ME Lanio*.*Center for Protein Study, Faculty of Biology, Univer-sity of Havana and **Center for Molecular Immunol-ogy. [email protected]

For the last few years, we have been using hrEGF, asa model antigen, encapsulated into DPPC:Colesterol(Cho) Liposomes (LP) in order to study the modulat-ing ability of these vesicles upon the immune responsein mice. Previously, it was demonstrated that it is pos-sible to induce anti-hrEGF antibodies by immuniza-tion with this antigen conjugated with P64K proteinfrom Neisseria meningitidis, included in conventionaladjuvants. In this work we demonstrate that it is pos-sible to simplify the immunization protocol from fourto two doses without changes in the total anti-EGFantibody titers. The sensibilization of animals with LP/hrEGF, in the absence of P64K protein, and challengedwith this antigen in Aluminium hydroxide (Al), showedantibody levels similar to those observed in the groupsimmunized with the conjugate (hrEGF-P64K) encap-sulated into LP or included in Al, after two immuniza-tion. The isotype levels (IgG2a and IgG2b) in micetreated with LP containing only hrEGF was similar tothose observed in the groups immunized with hrEGF-P64K in Al or in PBS, and higher than that treatedwith Al/hrEGF. Liposomes of saturated phospholip-ids (DPPC:Cho and DSPC:Cho) potentiate more effi-ciently the immune response against hrEGF thanunsaturated phosphatidylcholine from soybean(sPC:Cho) or Al in terms of: IgG titers, the inductionof IgG2a and IgG2b isotypes and the antibody abilityto block the hrEGF and its receptor (R-EGF) interac-tion. Liposomes are better adjuvant for the hrEGF thanAluminium hydroxide because they enhance IgG2a andIgG2b levels, induce DTH response and lymphocyteproliferation, even in the absence of P64K. This mightbe indicating a switch in the anti-EGF immune responsetoward a Th1 pattern associated with a cellular re-sponse. Taking into account these results, it is possibleto suggest that it is not necessary the use of P64K as acarrier protein when hrEGF is encapsulated into Lipo-somes, at least in experimental models.

Workshop on Adjuvants for Parasitic Vaccines

Chairpersons: José Alejandro López,1 Oliver Pérez2

1Mater Medical Research Institute, Brisbane, Australia. E-mail: [email protected] Institute, Havana, Cuba. E-mail: [email protected]

ABSTRACTA number of vaccine candidates have been tested in animal models and they have been able to successfullyprevent the disease and provide efficient long-lasting immunity. However, it has not been possible to translate thisknowledge into clinical use, due to various technical hindrances. The most important one is the limitation of usingadjuvants strong enough to elicit the level of the immune response required to prevent the disease. Recently, somepotent adjuvants have been tested and the results are very promising. In this workshop a successful case ofimmunization in humans, a novel methodology for delivering antigens and an innovative diagnostic assay werediscussed. The use of Montanide ISA-720 was shown to generate a comprehensive and efficient immune responsein humans immunized with a synthetic peptide comprising the C-terminal region of the circumsporozoite protein of

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New vehicles for the delivery of antigenic se-quences have been explored in many systems. In thecase of malaria antigens, a model for the insertion ofvarious sequences into BCG was presented. A frag-ment of region II of EBA-175 and the repeated (NANP)region of the circumsporozoite protein of P. falciparumwere inserted by assembling PCR into BCG. The datashowed that the inserts were present and that specificantibodies could be generated against the deliveredproteins. Data on the immunogenicity of the con-structs in animal models are underway (Norazmi,Malaysia).

A sensitive antigen detection system has been de-veloped utilizing peptides from various parasite pro-teins. In the case of malaria parasites, a combination ofsynthetic peptides present in the histidine rich proteinII (HRPII), glutamate rich protein (GLURP), falciparuminterspersed repetitive antigen (FIRA) was used to gen-erate specific antibodies in rabbits capable of detectingvery low levels of circulating antigen. A similar strat-egy was employed for schistosoma antigens cathepsinB (Sm31) and asparaginil endopeptidase (Sm32) to gen-erate an agglutination-based antigen detection system(Noya, Venezuela).

SummaryMajor advances in the field of adjuvant developmenthave now reached the development of parasitic vac-cines. For the first time, a malaria vaccine success-fully elicited strong responses from the three armsof the immune response and promise to convey clini-cal protection. The use of lower peptide concentra-tions in the presence of potent adjuvants appeals asan optimal combination. Other interesting advancesin the field include the use of cellular forms for deliv-ery systems such as BCG and the utilization of moreefficient diagnostic kits for the early and accurateidentification of the disease; in the case of the latertechnology, an additional product from research ledto the identification of promising proteins, potentialvaccine candidates against schistosomiasis.

Oral Presentations

Phase I clinical trials of long synthetic malariapeptides using different adjuvantsLópez JA,1 Weilenmann C,2 Audran R,1 Roggero MA,1

Bonelo A,1 Tiercy JM,3 Spertini F,2 Corradin G.1 1Insti-tute of Biochemistry, University of Lausanne, 1066Epalinges, Switzerland. 2Immunology and AllergologyDivision, CHUV, Lausanne, Switzerland. 3Transplan-tation Immunology Unit, CMU, Geneva, [email protected]

Objective: Evaluate the safety and immunogenic-ity of synthetic peptide Pf CS 282-383 with different

P. falciparum. In this Phase I/II human trial, naïve individuals reached levels of antibodies, CD4+ and CD8+ lympho-cyte responses comparable to those observed in individuals from endemic areas. Most strikingly, this is the firstreport of a protein vaccine generating high levels of IFN-? producing CD8+ lymphocyte responses, known to beessential in the eradication of the disease. A model for the introduction of P. falciparum sequences into BCG inwhich two sequences present in proteins of the parasite were successfully introduced into BCG to be used in humanvaccination was discussed. Finally, the use of an immunodiagnostic method utilizing an antigen-based agglutina-tion test for schistosomiasis and malaria that may detect very low levels of circulating antigen was described.

IntroductionThree talks covered the scope of this workshop andthey were presented by:

José Alejandro López from the Mater MedicalResearch Institute, South Brisbane, 4101, (Australia)with the title: “Phase I clinical trial of long syntheticmalaria peptides using different adjuvants”.

Mohd Norazmi from the School of Health Sciences,University Sains Malaysia, Kelantan (Malaysia) withthe title: “Recombinant BCG containing selectedepitopes of Plasmodium falciparum”.

Oscar Noya from the Instituto de Medicina Tropi-cal, Facultad de Medicina, Universidad Central deVenezuela, Caracas (Venezuela) with the title: “Im-munogenicity of schistosoma synthetic peptides”.

Main BodyThe use of natural sources for immunization has beenapplied to malaria research quite successfully. In fact,the complete prevention of the disease has been achievedwith the use of irradiated sporozoites. However, due totechnical limitations in the production of large quanti-ties of sporozoites (manually dissected from mosquitoglands) an alternative option needs to be found. For thefirst time in humans, a vaccine has been developed whichis capable of inducing strong responses from the threearms of the immune system (López et al, 2001. Eur. J.Immunol. 31:1989). The vaccine preparation included a100 amino acid long synthetic peptide representing theC-terminal region of the circumsporozoite protein de-livered in two immunizing doses (100 and 300 mg) at 0,1, and 6 months in the presence of two adjuvants (Alumand Montanide ISO 720). It elicited antibody titers higherthan those observed in individuals from endemic areas,similar to those reached with the classical tetanus toxoidvaccine, and most importantly, they recognized (byIFAT) the infective form of the parasite, the sporozoitewith titers comparable to those of “immune” adultsfrom endemic areas. Additionally, the vaccine also elic-ited strong proliferative responses that produced highlevels of IFN-γ secretion. Perhaps the most strikingfeature of this preparation was the successful genera-tion of CD8+ lymphocyte responses upon immuniza-tion; as measured by antigen specific IFN-γ ELISPOT.The levels of response obtained were comparable tothose observed in individuals from endemic areas. Twoadjuvants were evaluated, Montanide IS0-720 and Alumand both appeared to generate CD8 responses. How-ever, Montanide appeared to be superior in the genera-tion of antibodies. Proliferative responses were observedsimilarly in both groups. An interesting finding of dose-scaling study was that the injection of 300 ?g peptideappeared to have a deleterious effect on the magnitudeof the immune response. The one hundred micrograminjection appeared to be the level necessary for an opti-mal response (López, Australia).

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adjuvants.Methods: This peptide, produced accord-ing to good laboratory practice and spanning the C-terminal region of the circumsporozoite protein of P.falciparum strain NF-54 was evaluated as a vaccinecandidate in an open, non-randomized, phase I trial.Sixteen volunteers in 4 groups received 3 injections(at months 0, 1 and 6) of peptide (100 or 300µg)combined with Montanide ISA-720 or Alum as adju-vants. Immunogenicity was evaluated by 1) antibodytiters (ELISA) and lymphocyte proliferation re-sponses to peptide Pf CS 282–383, 2) antibody re-sponse to the Pf NF-54 (IFAT) 3) inhibition ofsporozoite invasion (ISI) and 4) CTL response to thetwo epitopes Pf CS 327-335 and 299-308) (ELISPOT).Results: In only 11 of 48 injections mild local painwere reported. In Montanide groups, particularly withthe 100µg dose of peptide, antibody responses to thepeptide and to the Pf NF-54 sporozoite were similaror higher than those observed in adults from endemicarea in Burkina Faso. In contrast, immunization withAlum resulted in weaker responses. A vigorous cellu-lar response was observed in all volunteers which didnot correlate with the humoral response. In 6 out of 8HLA-A*0201 volunteers, a specific CTL responseagainst one or both mentioned epitopes was detectedwith a frequency of 9x102 and 7.5x103 specific CTLper million CD8+ cells. The results concerning the in-hibition of sporozoite invasion are currently analyzed.Conclusions: The malaria vaccine formulation usingPf CS 282-383 with Montanide ISA-720 or Alum asadjuvants was safe, well tolerated and immunogenic.

Recombinant BCG containing selectedepitopes of Plasmodium falciparumN Mohd, R Suppian. School of Health Sciences,Universiti Sains Malaysia 16150 Kubang Kerian,Kelantan, Malaysia, Tel: 609-7601801; Fax: 609-7647884. [email protected]

Mycobacterium bovis bacille Calmette Guerin(BCG) has been suggested to be an attractive vehiclefor the delivery of various vaccine candidates, includ-ing those for malaria [1, 2]. We have previously shownthat cloning of a synthetic malarial epitope constructedwith mycobacterium codon bias into M. smegmatisincreased the transformation efficiency and growth ofthe transformants, as well as the expression levels ofthe fusion protein [3]. The objective of this study isto employ the same strategy, known as assembly PCR,to clone two malarial epitopes; a fragment from theso-called region II of EBA-175 (F2RII-EBA) and threeNANP repeat sequence of CSP, generated from a se-ries of oligonucleotides, into BCG. In addition, weincorporated the 65 kDa hsp promoter of M. tubercu-losis, a signal peptide, a 6 His tag and 2 T-cell epitopesfrom M. tuberculosis ESAT-6. The technique involvedthe generation of the full 1,413bp fragment of thecomposite sequence from 36 overlapping, 25 to 45bpoligonucleotides, designed in favour of mycobacte-rium codon usage. The fragment was cloned into thecloning vector pCR2.1 TOPO (Invitrogen) and theresultant clone converted into a shuttle vector by in-sertion of the mycobacterial replicon. Cloning suc-cess was verified by sequencing. Transformation ofthe shuttle vector into BCG was achieved by elec-troporation. Western blot analyses using a polyclonal

anti-F2RII-EBA antibody raised against the nativeEBA epitope, confirmed the expression of the fusionprotein in the culture supernatant with minimal reac-tivity in the cell pellet - suggesting that the epitope ofinterest could be expressed in its native form and thatthe signal peptide is functional. Immunogenicity stud-ies are currently being performed in mice. AssemblyPCR is a flexible technique which would facilitate thecloning of composite candidate molecules coded forby genes located in different chromosomes or fromdifferent organisms.1. Haeseleer et al. Mol Biochem Parasitol 1993;57:117.2. Matsumoto et al. J Exp Med 1998;188:845-54.1. Norazmi et al. Biotech Tech 1999;13:458-89.

Antigenicity and immunogenicity of syntheticpeptides derived from parasite proteinsNoya O,1,2 Bermúdez H,1 Zerpa N,2 Noda A,2 GuzmánC,1 Chacón N,1 Losada S,1 Colmenares C,1 LorenzoMA,1 Alarcón de Noya B.1 1Instituto de Medicina Tropi-cal, Facultad de Medicina, U.C.V. 2Laboratorio paraEstudios sobre Malaria, Malariología – INH-MSDS.Caracas, Venezuela. [email protected]

Synthetic peptides have been recently used as can-didate antigens for immunodiagnosis and vaccines.Two mayor parasitic diseases, schistosomiasis andmalaria lack effective vaccines and ideal diagnostictests. In the case of malaria, 29 peptides derivedfrom ten different excretory-secretory molecules fromPlasmodium falciparum have been synthesized inorder to use them in antibody and antigen-based di-agnostic agglutination tests. Peptides derived fromthe Glutamate Rich Protein (GLURP) have shownthe highest sensitivity and specificity when evalu-ated with human sera, by ELISA. Antibodies raisedin rabbits against HRPII (histidine rich protein II),GLURP and FIRA (falciparum interpersed repeti-tive antigen), were able to detect circulating antigensdown to 1 ng of protein in the plasma of infectedpatients. For the diagnosis of Schistosoma mansoni,we have basically worked with the Sm31-cathepsinB and the Sm32 – asparaginil endopeptidase; twoprominent proteases, from the gut of this trematode.Three peptides from the Sm31 were antigenic whenevaluated with patients sera (range 49 – 86% sensi-tivity and 100% specificity). Antibodies raisedagainst two peptides from the Sm31 (IMT 172 and180) and three from the Sm32 molecules (IMT12, 14and 64) where highly immunogenic, eliciting anti-bodies against these two enzymes. These antibodiesare currently evaluated in an antigen-capture assay.Based on previous works and also on the preferen-tial recognition of antigenic enzymes from non per-missive schistosome hosts, we have identifiedpotential protective epitopes from the GST-28 (glu-tathione-S-trasferase), Sm32 and TPI (triose phos-phate isomerase). Preliminary results have shownthe protective effect of GST-28 and Sm32 derivedpeptides. All this work has been carried out immu-nizing the animals subcutaneously and using com-plete and incomplete Freund´s Adjuvant. However,based on the interest to primarily elicit a potent Th2response in both experimental models, other adju-vants, like QS-21 and other routes of inoculation,need to be evaluated.

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Th1 immune response induced by KM+lectin: a consequence of IL-12 productionA Panunto-Castelo,1 MA Souza,2 JS Silva,1 MC Roque-Barreira.1 1FMRP, USP - Ribeirão Preto - SP. 2ICBIM,UFU - Uberlândia - MG. [email protected]

The outcome and severity of some diseases is di-rectly correlated with the dominance of either the Thelper 1 (Th1) or Th2 immune response, which isstimulated by IL-12 or IL-4, respectively. In thepresent study we demonstrate that spleen cells fromBALB/c mice stimulated with the lectin KM+, a man-nose-binding lectin from Artocarpus integrifolia, pro-duced interferon gamma (IFN-γ ) in dose-responsemanner. In order to determine if the IFN-γ productionby spleen cells was dependent on a soluble mediatorinduced in macrophages, the supernatant of J774 cellsstimulated with KM+ was depleted of lectin by ad-sorption to immobilized mannose and added to a non-stimulated culture of mouse spleen cells. Thissupernatant induced expressive production of IFN-γ ,an effect blocked by anti-IL-12 monoclonal antibody.In fact, when macrophages from several sources, in-cluding cell lines, spleen and peritoneal cavity, werestimulated with KM+ they produced IL-12. Further-more, this KM+-induced IL-12 production by mac-rophages was remarkably inhibited by D-mannose,but not by D-galactose. Additionally, IFN-γ produc-tion by spleen cells stimulated with KM+ in the pres-ence of either D-mannose or mannotriose were inhibitedin a dose-dependent manner, whereas no inhibitionwas determined by carbohydrate when spleen cellswere directly stimulated with IL-12. These observa-tions indicate that KM+ induces IL-12 production bymacrophages via its carbohydrate recognition domain.

The FML-vaccine: a second generationcandidate for vaccination against murineand canine leishmaniasisPalatnik de Sousa CB, Santos WR, Paraguai de SouzaE1, Borja-Cabrera GP, Gomes EM, Bernardo RR, daSilva VO, Correia Pontes NN, Luz KG, Parente JP,Palatnik M. Federal University of Rio de Janeiro.Cidade Universitária, Ilha do Fundão. CP 68040. CEP21941-590. Rio de Janeiro, RJ, Brazil. Phone: 005521-25626742; E-mail: [email protected].

The FML antigen is a complex glycoprotein frac-tion isolated from L. donovani promastigotes. Thisantigen was used to immunize mice using the adju-vants: IFA, alum, BCG, IL-12, saponin, QS21 likefraction and QuilA. Antibody responses were pri-marily IgG1, IgG2a and IgG2b. Highest titers werefound in mice receiving FML+QS-21-like, followedby FML+QuilA, FML+saponin, FML+IL-12, andFML+BCG and FML+alum; no Ig2a response wereobserved for mice receiving FML with either alum orBCG. All vaccinees showed a significantly enhancedDTH response. Significant protection was observedin FML+QuilA (93%) and FML+saponin (73%)mice. Sapogenins were obtained by chemical removalof the oligossacharides from saponin and QuilA.Despite of the reduction of toxicity in both sapo-genins, only QuilA fraction maintained protectivepotential (85%) and significantly enhanced DTH andantibody response. Recent studies employing sapo-nin and the GP36 component of FML antigen com-

plex indicate that this molecule can provide signifi-cant protection in the murine model (BALB/c) againstL. donovani infection (reduction of parasite burdensof 68%). The protection provided by vaccinationwith FML-vaccine was examined in dogs naturallyexposed in an endemic area in Natal, Brazil. After 2years of monitoring the dogs, data indicated that theFML vaccine induced a 92% significant protection(76% of vaccine eficacy). With the FML-QuilA for-mulation a 95% protection (80% of vaccine efficacy)against canine kala-azar was achieved. No significantdifference between the two formulations was found.3.5 years after vaccination, saline controls showedpositive PCR for Leishmania DNA and FML-serol-ogy with no DTH reaction. Higher seropositivitiesand DTH with no Leishmanial DNA were detectedin vaccinees. Both FML-vaccines induced a signifi-cant, long-lasting and strong protective effect againstcanine kala-azar in the field. Support: PCDEN andPNUD- FNS; FINEP; CNPQ; CAPES; MCT/PRONEX; CNPQ; RHAE-CNPQ; FUJB-UFRJ;FAPERJ; CEPG-UFRJ, Brazil.

QS-21 saponin adjuvant in the FML-vaccineagainst experimental visceral leishmaniasisSantos WR, Paraguai de Souza E, Bernardo RR, Borja-Cabrera GP, Gomes EM, Palatnik de Sousa, CB. In-stitute of Microbiology “Prof. Paulo de Góes”, FederalUniversity of Rio de Janeiro. Cidade Universitária,Ilha do Fundão. CP 68040. CEP 21941-590. Rio deJaneiro, RJ, Brazil. Phone: 005521-25626742. Fax:005521-5608344; E-mail: [email protected]

The development of a successful vaccine formula-tion for subunit antigens requires the use of a potentadjuvant that induces both humoral and cellular re-sponse. In the present work, we analyzed the protec-tive potential of the FML antigen formulated withQS-21 fraction obtained from Quil-A saponin(Superfos), according to Kensil et al., 1991 (J. Immunol146: 431-7). Briefly, the QuilA was applied on asemipreparative HPLC column (Alltima-Altech C185U, 5mm particle size, 3000 nm pore size, 10 mmI.D. x 25 cm length). The gradient was 30 to 40%0.1% TFA/acetonitrile/ 30min, 40% /15 min at a flowrate of 4 mL/min and saponins peaks were monitoredby absorbance at 214nm. As described by Kensil etal., four different peaks were detected. The major peakshowed retention time and sapogenin polarimetricvalues ([a]20D= + 55,6) compatible with QS-21([a]20D= + 56,1). Swiss Albino females were immu-nized with three weekly doses of the FML150 µg andQS-21100 µg by s.c. route. Saline and adjuvant treatedanimals were included as controls. The parametersanalyzed were: anti-FML antibody levels (types andsubtypes) and delayed type of hypersensitivity(DTH) against LD1S f/t promastigote lysate antigen.The anti-FML antibody response (titre log2: IgM/total IgG/ IgG1/ IgG2a/ IgG2b/ IgG3) before and afterinfeccion was: 5/8/6/9/9/7 and 10/5/8/12/9/4 for sa-line, 6/5/7/6/7/7 and 8/4/5/7/6/4 for adjuvant and 11/18/19/19/19/17 and 10/16/18/22/16/16 for FML+QS-21, respectively. The DTH response was specific andsignificantly higher than saline controls (p<0.005) inFML-vaccinees both before and after infection (0.580/0.042mm and 0.384/0117, respectively). Compared

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to previous work with IL12, QuilA and Riedel Dehaen saponin, these results show the success in puri-fication of the active component of the QuilA mixtureand the remarkable potency of QS-21 adjuvant againstvisceral leishmaniasis. Support: PCDEN and PNUD-FNS; FINEP; CNPQ; CAPES; MCT/PRONEX;CNPQ; RHAE-CNPQ; FUJB-UFRJ; FAPERJ;CEPG-UFRJ, Brazil.


The adjuvant effect of KM+, a mannose-binding lectin, in experimental toxoplasmosisGenari AB,1,2 Souza MA,1 Silva DAO,1 Ferro EAV,1

Mineo JR,1,2 Panunto-Castelo A,2 Roque-BarreiraMC.2 1Lab. Imunologia - UFU. 2Lab. Glicobiologia eImunoquímica - FMRP – USP. [email protected]

In order to determine if KM+, a mannose bindinglectin extracted from Artocarpus integrifolia, couldinduce a protective immune response in an experi-mental model of murine toxoplasmosis, a total of fiftyBalb/c mice was divided into four groups and immu-nized with: Group 1- PBS (n=12); Group 2- Solubleantigen of T. gondii, STAg (n=12); Group 3- KM+(n=13); Group 4- STAg plus KM+ (n=13). The ex-perimental groups of animals received a total of threeinjections, with doses of 25 micrograms of STAg and/or 0.5 mg of KM+ per inoculation, in weekly inter-vals. Animals from control group received three injec-tions of PBS only. Ten days after the last injection,part of the animals from each group was challengedwith 1,000 tachyzoites from RH strain of T. gondii,via intraperitoneal. The remaining animals were per-orally challenged with 50 cysts from ME-49 strain orsacrificed in order to count the number of cells fromspleen, mesenteric and popliteal lymph nodes, as wellas, to analyze the secretion of nitric oxide under stimu-lation with Con-A, STAg, KM+ or medium only. Itwas observed that, when challenged with RH strain,mice from Groups 3 and 4 presented higher percent-age of survival than animals from Groups 1 and 2.However, when challenged with ME-49 strain, ani-mals from Group 3 presented the highest number ofbrain cysts and only those from Group 1 presentedsignificant rate of mortality. In addition, animals fromGroup 3 showed the highest number of spleen andlymph node cells when compared with other groups.The nitric oxide production was detected in all groups,mainly under KM+ stimulation. It can be concludedthat KM+ modified the profile of immune responsefor both strains of T. gondii and therefore further stud-ies should be done to determine its role as immuno-logic adjuvant in this system. Support: FAPESP,FAPEMIG, CNPq and CAPES

Th1 immune response induced by KM+lectin: protection against leishmania infectionMA Souza, A Panunto-Castelo, JS Silva, MC Roque-Barreira. FMRP, USP - Ribeirão Preto - SP. ICBIM,UFU - Uberlândia - MG. [email protected]

When experimentally infected with Leishmaniamajor, an obligate intracellular parasite of macroph-ages in mammalian hosts, most of inbred mice strainsare resistant to leishmaniasis. In contrast, BALB/cmice are susceptible, develop severe lesions and do

not become immune to reinfection. Murine resistanceand susceptibility are clearly related to the develop-ment of the polarized CD4+ T helper 1 (Th1) andTh2 response, respectively. The differentiation of Tcells to Th1 and Th2 cells has been associated withproduction of interleukin-12 (IL-12) and IL-4, respec-tively. Since KM+, a mannose-binding lectin fromArtocarpus integrifolia, induces IL-12 production invivo, we aim here to verify if KM+ could switchBALB/c mice susceptibility to L. major infection.When BALB/c mice were immunized with solubleleishmanial antigen (SLA) in combination with KM+,they became resistant to L. major infection. Draininglymph node cells from these immunized BALB/c mice,in contrast to cells from animals immunized only withSLA, secreted high levels of IFN-γ and low levels ofIL-4, which characterized a Th1 rather than a Th2response pattern. The footpad thickness of BALB/cmice immunized with SLA plus KM+ and challengedwith L. major was similar to that of uninfected mice.In contrast, control mice immunized only with SLAand challenged with L. major showed footpad thick-ness as large as of infected mice treated only withPBS. The beneficial effect induced by KM+ againstleishmanial infection was blocked by pretreatment ofthese mice with anti-IL-12 antibody. These observa-tions indicate that KM+ has an adjuvant effect on theinduction of a protective immune response to L. ma-jor infection.

Role of IL-4 in experimental infectionby the helminth Echinococcus granulosusFerragut G,1 Dematteis S,2 Baz A.2 1Laboratorio deInmunología, Regional Norte-Sede Salto. 2Cátedrade Inmunología, Facultad de Química-Facultad deCiencias Montevideo. Universidad de la Repú[email protected]

Type-2 cytokine responses have been proposed asprotective in infections by helminth. The experimen-tal infection by E. granulosus induces an early type-2response which is unable to impair parasite establish-ment. The aim of this work was to evaluate in vivo therole of IL-4 in the susceptibility to E. granulosusinfection. Cytokine production by spleen cells, ni-trite production by peritoneal adherent cells and ma-jor spleen cellular populations were analysed in:infected and treated with anti-IL-4 MoAb mice, in-fected and treated with homologous antibodies non-specific for IL-4 mice and normal non-treated mice,on days 3, 7 and 14 post-infection. Mice treated withanti-IL-4 MoAb showed reduced levels of IL-4, IL-10 and IL-5 compared with control infected mice.Blocking of IL-4 did not induce IFN-γ production.Treatment with anti-IL-4 MoAb led to an increase innitrite production on day 7 post-infection in bothperitoneal adherent and spleen cells respect to controlinfected mice. No significative differences were ob-served at chronic infection in the levels of cytokinesand in the percentages of the major cellular popula-tions in spleens between mice infected and treatedwith anti-IL-4 MoAb and control infected mice. Com-parison of the percentage of infection and size of thecysts developed in mice treated with anti-IL-4 MoAband infected controls mice, showed no significativedifferences. In conclusion, in vivo blocking of IL-4

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produced early in experimental infection by the helm-inth E. granulosus seemed not to affect the establish-ment of infection and parasite growth. Supported by:CONICYT Fondo Clemente Estable (grant 4002); IFS(grant F/2930-1); CSIC; Regional Norte-Sede SaltoUniversidad de la República

Posters on Vaccines and Others

Comparative study of the immunogenicityand protective capacity of adsorbed and nonadsorbed vaccines against Leptospirosis inhamstersM González, I González, L Estévez, R Yi, C Hidalgo,N Batista, Y Rodríguez, JF Infante, R Oliva, G Sierra.Finlay Institute. PO Box 16017, Havana, [email protected]

The increasing incidence of Leptospirosis in Cubaat the beginning of the 80’s, conditioned the produc-tion of the first Cuban leptospiral vaccine for use inman. Two experimental vaccines based on inactivatedwhole cells were formulated: non adsorbed (NAV) andadsorbed in aluminium hydroxide (AV), containing 50-80 x 106 cells of serovars canicola, copenhageni andmozdok per 0.5-mL dose. The immunogenicity andprotection were evaluated in hamsters. Two assayswere developed; in the first one 120 hamsters wereinoculated by intramuscular (IM) route with two doseof 0.5 mL each, six weeks apart. Blood samples weretaken at 0 (T0), 6 (T6) and 9 (T9) weeks. The IgGimmune response was evaluated by ELISA and theprotection induced, by challenge against virulent strainsof canicola, copenhageni and mozdok. In the secondassay 180 hamsters were inoculated with a single doseand challenge 1.5, 3 and 6 months later. The resultsshowed that IgG response of hamsters vaccinated withAV was significantly higher than the response of theones vaccinated with NAV. Both vaccines protectedagainst challenge with 1000-10 000 LD50 of the strains,but AV induced a longer immunity and protected alsoagainst renal infection.

Comparison of three types of oily emulsionsused in the formulation of a vaccine for pigsN González, J Zamora, I Wong, E Bover, R Hernández,M Domingo. Center for Genetic Engineering and Bio-technology. PO Box 387, Camagüey 70100, Cuba.Phone (53-322) 61014; Fax (53-322) 61587;E-mail: [email protected]

Three oily formulations (oil in water, water in oiland double emulsion) of several compositions andphase distribution are evaluated as vehicles for vacci-nation in pigs using K99 and K88ab enterotoxigenicE. coli fimbriae as antigens. The physical propertiesand immunological properties in rabbits were appro-priate for each emulsion. The three variants were in-oculated to sows at eight and fourteen weeks ofgestation, and to piglets at the ages of one and threeweeks old. The double emulsion was the most attrac-tive for vaccine formulation, because the immune re-sponse is greater or equal to the other two emulsionsused. Moreover, the low viscosity makes the doubleemulsion easier to inoculate and more fluid into thetissues. The biological stability of both double andwater in oil emulsions is 2 years.

Cox JC, Coulter AR. Vaccine 1997;15(3):248-56.Woodard LF. Surface chemistry and classificationof vaccine adjuvants and vehicles. Bacterial Vac-cines 1990:281-306.Wong I, et al. Biotecnología Aplicada 1995;12(1):9-15.

Immunomodulating effects of polysaccharidefractions from mycelium of Pleurotusostreatus. I. In vitro macrophage-stimulationactivityH Morris, G Llauradó, J Marcos, S Rodríguez, NGarcía, RC Bermúdez. Centro de Estudios de Biotec-nología Industrial, Universidad de Oriente. PO Box4011. Santiago de Cuba CP 90 400. Cuba. Phone:(53-226) 632095; E-mail: [email protected]

Water soluble, complex carbohydrate derived fromnatural sources have been shown to augment variousfacets of immune responsiveness in human and ani-mals. These natural product carbohydrate polymersbelong to the class of drugs known as biological re-sponse modifiers (BRMs) [1]. The ability of BRMs,including polysaccharides isolated from the mycelium,fruiting bodies, and culture media of fungi, to exertbeneficial immunomodulating and antitumor effectshas stimulated research into their potential biomedi-cal applications [2]. Since macrophages have been sug-gested to play important roles in immunologicalsurveillance, the purpose of this study was to assessthe in vitro effects on macrophage activation of fivewater soluble polysaccharide fractions (F-I to F-V)from mycelium of Pleurotus ostreatus. Murine peri-toneal macrophages were obtained as adherent cellsfrom peritoneal exudate collected with Hanks´ solu-tion. After incubation of macrophages with polysac-charides (500 mg/mL) or LPS used as a positive control(100 mg/mL) for 48 h, the glucose concentration wasmeasured in the supernatant using the Rapi GlucoTestkit (EPB Carlos J. Finlay). The glucose consumptionof the macrophages significantly increased to 1.8-2.9times that of the control without polysaccharides. Alarge quantity of glucose is known to be consumed bymacrophages to synthesise the ATP and NADPH re-quired for phagocytosis and to produce active oxygenspecies [3]. The lysosomal enzyme acid phosphataseactivity was determined in cell lysate by the amountof nitrophenol released (mmoles) per 1x105 macroph-ages per 60 min. The enzyme activity was also en-hanced positively compared with physiological salinecontrol group. This evidence could be of interest, sinceit has been reported that the lysosomal enzyme inmacrophages participates in the antitumor activity. Inconclusion, these results suggested that resident mac-rophages are stimulated by Pleurotus polysaccharidesin vitro.1. Müller A, et al. J Chromatogr B 1995;666:283-90.2. Chihara G. Develop Biol Standard 1992;77:191-7.3. Yoshida I, et al. Biol Pharm Bull 1996;19:114-21.

Characterization of the antibody responseelicited after immunization of human healthyvolunteers with recombinant p64kmeningococcal proteinS González, G Sardiñas, E Caballero, A Musacchio,C Nazábal, O Reyes, E Rodríguez, Z Cinza, R Silva.

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Center for Genetic Engineering and Biotechnology,PO Box 6162, Havana 10600, Cuba. Tel: (53-7)2716221; Fax: (53-7) 2714764; E-mail:[email protected]

Recombinant P64k, expressed in Escherichia coli,acts as an efficient carrier protein [1] and is safe andimmunogenic in human healthy volunteers, as ascer-tained in a Phase I clinical trial [2]. The volunteersreceived three doses of recombinant P64k and wereboosted 9 months after the third dose. In the presentstudy, we further characterized the human antibodyresponse against this protein, by using volunteer seracollected during the trial. As expected, IgG1 was themain subclass of anti-P64k antibodies all over the study.However, after the booster dose, a statistically signifi-cant amount of anti-P64k IgG4 was detected in thesera. A maturation of the antibody affinity was foundin most of the sera. The presence of antinuclear andantimitochondrial antibodies in paired volunteer serawas examined by immunofluorescence. None of thesera contained such autoantobodies. Additionally, 84overlapping synthetic peptides spanning the entire se-quence of P64k were probed by SPOTscan [3], wherethe assayed sera recognized more frequently 12 pep-tides. We can conclude that IgG1 and IgG4 are the mainsubclasses of anti-P64k antibodies developed after im-munization, the antibodies do not react with mamma-lian tissue and the continuous B-cell epitopes theyrecognized are mainly exposed in the protein.1. González S, et al. Scand J Immunol 2000;52:113-6.2. Silva R, et al. In: Gavilondo JV, Ayala M, Acevedo

B, editors. Medical Applications of Biotechnol-ogy. Havana: Elfos Scientiae; 1999. p.O18.

3. Frank R, Overwin H. Methods in molecular biol-ogy: epitope mapping protocols. Totowa: HumanaPress Inc.; 1996. p.149-68.

Histopathological study of the inoculation siteof rats inoculated with adjuved vaccinesJF Infante, S Sifontes, V Pérez, P González. FinlayInstitute. PO Box 16017, Havana, Cuba. E-mail:[email protected]

Lesions at the inoculation site caused by differentkinds of adjuvants included in vaccines have been thor-oughly studied. However, regulatory agencies demandfurther in-depth research on this topic for new candidatevaccines. In the present work, the microscopic lesionscaused at the inoculation site by three different adjuvedvaccines were studied. Tissue samples were taken fromthe inoculation site of young adult male/female rats en-rolled in the studies of local irritation of DPT, VA-MEN-GOC-BC + DPT and combined MEMB vaccines.Samples were fixed in 10% neutral formaldehyde, pro-cessed by the technique of inclusion and cut in paraffinand stained with haematoxiline-eosin. Those rats inocu-lated with either Aluminum hydroxide or pertusiss anti-gen had granulomatous macrophagic processes in theinoculation site. Both substances are recognized as re-sponsible for eliciting that kind of local lesions, whichare part of the immune response developed.

Effect of Aloe barbadensis on opsonophagociticrate in burnt patientRodríguez M, Castellano E, Vázquez T, Rojas A,Jonhston N. Instituto Superior de Medicina Militar

“Dr Luis Díaz Soto”. Laboratorio de Medicina Her-baria. PO Box 11700, Ciudad de La Habana, [email protected]

Behavioral results of opsonophagocytic rate in 40severe, highly severe and critical burned patients dis-tributed in two groups is presented: group I (treatedwith Aloe barbadensis Miller extract and usual treat-ment) and group II (usual treatment). Blood sampleswere taken in different times (24 h and 10 and 21days) were analysed and phagocyte parameters weredetermined at 15-60 min. First, during a few hours,behaviour between groups was similar, but at the 10th

days a significant difference in opsonophagocytic rateof Aloe group at 60 min was noticed and neutrophilesincreased their possibility of carrying microorganism.

Some toxicity mechanisms of immunologicaladjuvantsA Batista. Biomedicine and Toxicology Center(TOXIMED), Autopista Nacional Km 1 1/2. PO Box4033 Santiago de Cuba 90400, [email protected]

The search of a potent and safe immunological ad-juvant for routine human use is a scientific challenge.In this way the most important issue is the safety,specially for preventive vaccines, for this reason, onlyalum has been licensed by regulatory organisms likeFDA since 1926 as adjuvant for human vaccine. Theadverse effects of adjuvants can be a direct conse-quence of the inclusion of toxic or non metabolizablecomponents in the formulation, or use of elementsoverstimulating of the immune or inflammatory re-sponse. These effects are differentiated as local (sincelocal and erythema until delay hypersensibility, ab-scess, ulcers, granulomes and cysts) or general effects(flu like symptoms, autoimmune disorders and changesof the hepatic metabolism). Also extrinsic factors candeterminate apparitions of side effects, like: doses,frequency and routes of application, age, genetic fac-tors and health history. In this work are consideredsome mechanisms that can explain many of the re-ported side effects in preclinical and clinical assays ofadjuvants.

A repeated doses toxicity study of magnetizedCM 95A Batista,1 O Fong,1 J Betancourt,1 R Laria,1 IUrdaneta,1 M Colón,1 C Martínez.2 1Biomedicine andToxicology Center (TOXIMED), Autopista NacionalKm 1 1/2 PO Box 4033 Santiago de Cuba 90400,Cuba. 2Biotechnology Studies Center (CEBI), Univer-sity of East, Santiago de Cuba, Cuba. E-mail:[email protected]

Was performed a repeated doses toxicity assay ofmagnetized CM 95, immunomodulator developed inBiotechnology Studies Center (CEBI), using SpragueDawley rat. According to Guidelines 407, 1995 Orga-nization for Economic Cooperation and Development(OECD), using three levels of magnetization of theproduct, via intraperitoneal routes and two controlgroup: non magnetized CM 95 and physiologic salinesolution. The endpoints evaluated were: clinical signsof toxicity (weight loss, piloerection, conductual dis-turbs), macro and microscopic anatomopathology,relative weight of thymus and spleen, bone marrow

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cellularity, global and differential leucocytes, glyce-mia, total proteins, alaninaminotransferasa enzyme,creatinine and serum immunocomplex. Not was de-tected significant modifications on the endpointsanalized in treated and control rats, except a lightlydiminutions of glycemia on the highest level of mag-netization group, reported before in previous preclini-cal studies. Looking together all these results, there isnot evidence of general toxicity under the experimen-tal conditions employed.

Immune response to house dust miteallergens in a mouse modelA Labrada, E Facenda, N Martínez, RE Aranda. Dpto.Alergenos. Centro Nacional de Biopreparados(BIOCEN). PO Box 6048, Havana 10600, Cuba. Fax:(53-7) 331144; E-mail: [email protected]

Allergic sensitisation to house dust mite allergenshave been described as a major cause of asthma andother allergic diseases worldwide. Immune responseto these inhaled allergens in allergic patients ischaracterised by IgE production and a Th2 bias. Al-lergen Immunotherapy is an effective treatment againstrespiratory allergy, which consists in injecting increas-ing amounts of the allergen, achieving a decrease ofTh2 cytokines. The aim of this work was to study theantibody (Ab) response to allergens of Dermatopha-goides pteronyssinus (DP), D. siboney (DS) and Blomiatropicalis (BT) mites, either in aqueous (PBS) or alum-adsorbed (Al) form, in a mouse animal model. Freeze-dried VALERGEN Allergen extracts were obtainedfrom BIOCEN, Cuba. The strength of the products isexpressed in terms of the major allergen content (Derp1/Der s1) or biological activity (Biological Units,BU). Reconstituted allergen extracts were adsorbedon aluminium hydroxide gel at a final concentration of10 mg/mL Der p1/Der s1 for DP and DS, and 2000BU/mL for BT. Immunisation was carried out at theweeks 0 and 3. Each mouse (Balb/c) received a 0.5 mLdose of either individual or mixed preparations. Bleed-ing was performed at the weeks 5 and 7. Serum IgG,IgG1, IgG2a and IgG2b specific to each antigen andtotal IgE were measured using ELISA (Pharmingen,USA). It was observed a predominant IgG1 responseto DS, while the response to DP was more balancedbetween IgG1 and IgG2a. On the contrary, the Abs toBT were predominantly of the IgG2a subclass (Fig-ure). The adsorption on Alum did not affect impor-tantly the IgG2a/IgG1 ratio, but certainly increased IgGand total IgE Abs. It produced also a time shift in thepeak values. However, the effect of mixing the threeallergens (Tri), did produce a significant decrease of theIgG2a/IgG1 ratio to BT, suggesting a non-specific in-

fluence (Figure). It is concluded that the IgG2a/IgG1ratio (and presumably the Th1/Th2 balance) to com-mon mite allergens can be affected by the allergen na-ture and concomitant administration of other antigens.

Adjuvanticity of specific immunocomplexof surface system (HbsAg�anti HBsAg)of hepatitis virusA González-Griego,1 R Caro,2 VE González-Ramírez,1

E García,1 A Santiesteban,1 A Alerm,1 O Pérez,3 NBenítez,2 G Sierra,3 A Cadiz,3 V Ramírez-Albajés.1

1ICBP “Victoria de Girón” Inst. Superior de CienciasMédicas, Havana, Cuba. 2Centro de Hemoderivados,Havana, Cuba. 3Finlay Institute, Havana, [email protected]

With the objective to increase the immunogenicityof the HbsAg through the use of biological adjuvants,we have developed a procedure for obtaining immu-nocomplex in an excess of antibodies by means ofimmunological methods in solid phase, from alumadjuvanted vaccines and in liquid phase. They weregiven to experimental animals (sheep); susceptiblehumans with high risk of exposition and personschronically infected (reservoirs of hepatitis B virus).The sheep antibody titers obtained post vaccinationwere higher than 106 IU/L, and the short period oflatency was an outstanding subject by using this pro-cedure. This fact, allows us to early select the hyper-responders animals. In non reservoir human, it wasobserved in only seven days after the booster dose;and in the reservoirs humans it has not been obtainedany type of side effects or undesired response. Forthese reasons, the possibility to use this procedure asa form of therapy can not be discarded, then, as it isknown, it does not exist an effective therapy for thisdisease at the present time.

IgG2a/IgG1 ratio

Immunogens

3.5

2.5

1.5

0.5

Tri Al DS Al Dp Al BT Al BT PBS

DS

DP

BT

Figure. Antibody response to allergens.

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