VACCINES (VC)

A PUTATIVE LEPROSY VACCINE INDUCES CYTOKINES SECRETION ONLY IN PAUCIBACILLARY PATIENTS

AND HOUSEHOLD CONTACTS

MAYARA INGRID SOUSA LIMA1; ALINE GOMES DE SOUZA

2; PATRÍCIA TERRA ALVES

3; ISABELA MARIA

BERNARDES GOULART4; MALCOLM S. DUTHIE

5; STEVE REED

6; LUIZ RICARDO GOULART

7.

1,2,3,4,7.FEDERAL UNIVERSITY OF UBERLANDIA, UBERLANDIA - MG - BRASIL; 5,6.INFECTIOUS DISEASE

RESEARCH INSTITUTE, SEATTLE - ESTADOS UNIDOS.

Introduction: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, with variable clinical,

immunological, and pathological manifestations. These variations reflect differences that span from a strong cellular

mediated immunity in the tuberculoid pole (TT) to a poor T cell immunity in the lepromatous (LL) pole. Survival or

death of the M. leprae, innate and acquired immune responses (Th1, Th2 or Th17) variations in the host are

generated by interactions among host proteins and bacterial antigens. A number of antigens have been reported to

induce T-cell responses from tuberculoid leprosy patients and household contacts in vitro. Thus, we evaluated the

antigenic potential of a recombinant leprosy antigen ML89 (IDRI) to produce cytokines involved in protective immunity

and infection in patients and contacts. Methods and Results: PBMC patients (n=15) and household contacts (n=6)

were isolated on Ficoll-Paque gradients and cultured (1×105 cells/mL by well) for 6 days. Each sample was stimulated

by ML89 (10μg/mL). Stimuli controls consisted of Concanavalin A (10μg/mL, positive) or cell without stimuli (negative).

Cytokine levels were measured by flow cytometry (BDTM

Human CBA, Th1/Th2/Th17 kit). The ML89 induces IFNγ

production in paucibacillary (PB) patients (p<0, 01) and household contacts (HC) that presented positive (HC+) and

negative (HC-) serology anti-PGL1 (p<0,001). IL-10 and IL-17a secretion were induced in BT patients (p<0,001) and

HC- (p<0, 01). TNF-α secretion was higher in BT patients, HC+, and HC- when compared to the other clinical

forms. Conclusion: The putative leprosy vaccine, ML89, induces Th1 and Th17 responses in PB patients and their

HC, with a balance of IFN-γ and IL10 production, different from PGL-1, known to produce humoral response. The

cytokine profile induced by the putative vaccine suggest a possible role inn protective immunity against M. leprae.

Financial Support: CNPq, FAPEMIG, CREDESH-UFU

ANALYSIS OF THE COVERAGE CAPACITY OF THE STREPTINCOR CANDIDATE VACCINE AGAINST

STREPTOCOCCUS PYOGENES

KARINE MARAFIGO DE AMICIS1; SAMAR FRESCHI DE BARROS

2; RAQUEL ELAINE ALENCAR

3; EDILBERTO

POSTÓL4; CARLO OLIVEIRA MARTINS

5; HELEN ANDRADE ARCURI

6; CIBELLY GOULART

7; JORGE KALIL

8;

LUIZA GUILHERME9.

1,2,3,4,5,6,9.HEART INSTITUTE, IMMUNOLOGY INVESTIGATION INSTITUTE, FMUSP, SAO PAULO - SP -

BRASIL; 7.BIOTECHNOLOGY CENTER, BUTANTAN INSTITUTE, SAO PAULO - SP - BRASIL; 8.HEART

INSTITUTE, IMMUNOLOGY INVESTIGATION INSTITUTE, CLINICAL AND ALLERGY DIVISION, FMUSP, SAO

PAULO - SP - BRASIL.

Introduction: Streptococcus pyogenes causes infections as pharyngitis and necrotizing fasciitis. Susceptible

individuals may develop post-streptococcal autoimmune sequelae as rheumatic fever (RF) and rheumatic heart

disease (RHD). According to the World Health organization, there was 15,6 million of RHD cases around the world in

2005 (Lancet Infect Dis.11:685-694, 2005). M protein, the major bacterial antigen, is composed by a conserved and a

polymorphic region. There are currently over 250 known M-types worldwide (Trends Microbiol.6:275-282, 2010).

Despite the scientist’s efforts, there is no anti-streptococcal vaccine available. A vaccine against S. pyogenes, based

on the M5 protein conserved region, is being developed by our group. Potentially protective epitopes were defined by

humoral and cellular reactivity analysis. A peptide was synthesized (StreptInCor - medical ID), containing both T and B

epitopes (Clin Dev Immunol.2-4:125-132, 2006). Challenge with M1 strain in immunized mice showed a survival rate

of 100% for up to 21 days, compared to the controls (40%) (PLoS One.4:e60969, 2013). HLA class II transgenic mice

produced high titers of IgG1 and IgG2a, without autoimmune lesions in several organs after one year period post

vaccination (Vaccine.46:8250-8256, 2011).

Methods and results: We analyzed the in vitro ability of anti-StreptInCor antibodies to neutralize/opsonize the most

common S. pyogenes strains in Sao Paulo. Anti-StreptInCor antibodies from a sera pool of 6 BALB/c mice recognized

both the M5 and M1 proteins by Western blotting (WB). Specific antibodies from 9 Swiss mice sera were able to

neutralize the M1, M5, M12, M22 and M87 strains with a Median Fluorescence Intensity 2 or 3 times greater

compared with controls, analyzed by flow cytometry (p<0,0436 - Mann-Whitney test). Anti-StreptInCor antibodies from

BALB/c or Swiss mice were able to promote opsonophagocytosis and death of the M1, M5, M12, M22 and M87

strains by murine peritoneal cells (p<0,035 - Kruskal-Wallis test) compared with preimmune sera. No autoreactive

antibodies against human heart valve protein extracts were observed by WB.

Conclusion: Anti-StreptInCor antibodies were able to neutralize/opsonize diverse S. pyogenes strains by recognize

conserved epitopes on different M-types, indicating that StreptInCor have the potential of prevents streptococcal

infections and sequelae, without causing autoimmunity.

Financial support: FAPESP, CNPq and CAPES.

ANTIGENICITY AND IMMUNOGENICITY OF AN AMASTIGOTE-SPECIFIC HYPOTHETICAL LEISHMANIA

PROTEIN IN VISCERAL LEISHMANIASIS

VIVIAN TAMIETTI MARTINS1; MIGUEL ANGEL CHÁVEZ-FUMAGALLI

2; LOURENA EMANUELE COSTA

3; PAULA

SOUZA LAGE4; DANIELA PAGLIARA LAGE

5; MARIANA COSTA DUARTE

6; RUBENS DANIEL MISERANI

MAGALHÃES7; TATIANA GOMES RIBEIRO

8; WANDERSON DUARTE DAROCHA

9; EDUARDO ANTONIO FERRAZ

COELHO10

; CARLOS ALBERTO PEREIRA TAVARES11

.

1,2,3,4,5,6,7,8,10,11.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL;

9.UNIVERSIDADE FEDERAL DO PARANÁ, CURITIBA - PR - BRASIL.

Introduction: The present study aims to evaluate an amastigote-specific hypothetical Leishmania protein (LiHyp1) in

an attempt to select a new candidate antigen for a serodiagnosis, as well as to compose a vaccine against visceral

leishmaniasis (VL).

Methods and results: The rLiHyp1protein was recognizable by antibodies from sera of asymptomatic and

symptomatic canine visceral leishmaniasis (CVL) and did not presente no cross-reactivity to sera of dogs vaccinated

with Leishtecâ or infected with Trypanossoma cruzi. The immunogenicity and protective efficacy of rLiHyp1 plus

saponin was evaluated in BALB/c mice challenged with L. infantum. Spleen cells of rLiHyp1 vaccinated mice showed

a high production of IFN-g, IL-12 and GM-CSF and, as compared to the control groups, the animals showed a

significant reduction in the number of parasites in the liver, spleen, bone marrow and in the draining lymph nodes in

the paws. Protection was associated with an IL-12-dependent IFN-g production, produced mainly by CD4 T cells,

which activate macrophages to eliminate the parasites. A decrease in the parasite-mediated IL-4 and IL-10 responses

was also observed.

Conclusion: This study showed that a new amastigote-specific Leishmania protein can be used for a more specific

serodiagnosis of CVL and, when combined with a Th1-type adjuvant, has proven to be useful in the protection against

VL.

Financial support: INCT-NanoBiofar, FAPEMIG, CNPq and PRPq/UFMG.

CLONING OF THE GENE THAT CODIFIES FOR LISTERIOLYSIN ON BCG ΔLEUD AND EVALUATION OF THE

ANTITUMORAL ACTIVITY ON BLADDER CANCER CELLS

KAREN SILVA LEAL; CAROLINE RIZZI; SIBELE BORSUK; ANA CAROLINA PEITER; SÉRGIO JORGE; KARINE

BEGNINI; ODIR ANTÔNIO DELLAGOSTIN.

UFPEL, PELOTAS - RS - BRASIL.

Introduction: Mycobacterium bovis BCG is worldwide used as a vaccine against tuberculosis for over fifty years with

high levels of security. BCG has features that make it a promising vector for production of recombinant multivalent

vaccines. Genetic modification can enhance the adjuvant and immunogenic properties of BCG. One of these

modifications would allow BCG to escape the endosome of antigen presenting cells. Therefore, the immunogens of

BCG access antigens cross priming routes, increasing their immunogenicity. Moreover, has developed a BCG ΔleuD

expression system, which allows for stable genetic transformation of exogenous genes without the use of antibiotics

as selection markers. This system employs the complementary auxotrophic selection marker, allowing the production

of safe and stable recombinant strains, in vivo, resulting in high levels of protein expression. This paper proposes to

develop a strain of BCG ΔleuD expressing listeriolysin (Hly) of Listeria monocytogenes and evaluate its cytotoxic

potential in cell line 5637 of bladder cancer.

Methods and Results: BCG ΔleuD was transformed with recombinant plasmid pUP410 containing our gene of

interest. Under these circumstances, we obtained a recombinant strain expressing listeriolysin and, on the preliminary

tests as determination cytotoxicity and flow cytometry.

Conclusion: These results showed a similar level of protection compared to BCG Pasteur strain used in conventional

treatment.

DEVELOPMENT OF AN ELISA USING THE RECOMBINANT CP0369 PROTEIN OF CORYNEBACTERIUM

PSEUDOTUBERCULOSIS FOR DIAGNOSIS OF CASEOUS LYMPHADENITIS IN SHEEP

ANDRÉA DE FÁTIMA SILVA REZENDE1; ALEXANDRE ANTUNES BRUM

2; HENRIQUE RAMOS ANGELO

3;

CARLOS GUILHERME REIS4; ALÉX RODRIGUES

5; KAREN SILVA LEAL

6; SIMONE SIMIONATTO

7; VASCO

ARISTON DE CARVALHO AZEVEDO8; SIBELE BORSUK

9.

1,2,3,4,5,6,9.UFPEL, PELOTAS - RS - BRASIL; 7.UFGD, DOURADOS - MS - BRASIL; 8.UFMG, BELO HORIZONTE

- MG - BRASIL.

Introduction: Caseous lymphadenitis (CLA) is a disease caused by Corynebacterium pseudotuberculosis which

mainly affects small ruminants and has caused serious economic losses worldwide. In Brazil, the prevalence of clinical

CLA ia around 30%. The diagnosis of CLA is not very effective, due the signs and symptoms are not noticeable

immediately. Thus, several serological tests are being developed in order to detect the disease in animals especially

in asymptomatic. The protein CP0369 was identified in an exosecretoma study and therefore has potential for use in

diagnosis. This protein was used in recombinant form for the development of a diagnostic test based on ELISA

Methods and Results: we performed the amplification of the cp1002_0369 gene using the primers F5'CGG GGA

TCC CAG CCA GTG CTT CAG GTC 3', and R5'CCC AAG CTT TTA TTT TTG TAC CGC TTG CTC 3'. The

cp1002_0369 gene was cloned in the BamHI and HindIII sites of the pAE vector. The recombinant clones (pAE/0369)

were characterized enzymatically and by DNA sequencing. The pAE/0369 vector was performed into E. coli BL21 Star

for expression of recombinant protein CP0369. Purification was performed by affinity chromatography on a Sepharose

column charged with nickel. The purity was determined using a 12% SDS-PAGE, and the concentration determined

by BCA kit. The rCP0369 purified protein was evaluated in an indirect ELISA using 182 sera from sheep. ROC

analysis showed a sensitivity and specificity of 90% and 75.6%, respectively. Using the cutoff (OD600nm = 0.076),

positive predictive value varied from 29% (for 10% CLA prevalence) to 97% (for 90% CLA prevalence), while the

negative predictive value ranged from 98. 5% to 45.6%.

Conclusion: the format ELISA assay (ELISA-rCP0369) described here can be used in the diagnosis of CLA sheep

herds with a good level of sensitivity.

DNA VACCINES ENCODING GLIOBLASTOMA ANTIGENS AND IMMUNOMODULATORY PROTEINS:

CONSTRUCTION AND IMMUNOGENICITY EVALUATION

WENDY MARTIN RIOS1; JEANNE BLANCO DE MOLFETTA

2; IZAÍRA TINCANI BRANDÃO

3; ANA PAULA

MASSON4; ROSIANE PERIPATO

5; IDALETE DA SILVA

6; RODRIGO FERRACINE RODRIGUES

7; ROGÉRIO SILVA

ROSADA8; LUIS CARLOS DE SOUZA FERREIRA

9; CELIO LOPES SILVA

10.

1,3,4,7,8,10.SCHOOL OF MEDICINE OF RIBEIRAO PRETO - UNIVERSITY OF SAO PAULO, RIBEIRAO PRETO -

SP - BRASIL; 2,5,6.FARMACORE BIOTECNOLOGIA LTDA, RIBEIRAO PRETO - SP - BRASIL; 9.INSTITUTE OF

BIOMEDICAL SCIENCES - UNIVERSITY OF SAO PAULO, SAO PAULO - SP - BRASIL.

Introduction: Glioblastoma (GBM) is the most common form of primary brain cancer and the most severe tumour

affecting glia cells. GBM is a grade IV astrocytoma known by uncontrolled proliferation, diffused infiltrate, necrosis

tendency, angiogenesis, apoptosis resistance and a wide genetic heterogeneity. The standard of care consists of

maximal surgical resection, followed by a combination of radiation and chemotherapy. Despite that, tumour becomes

resistant to drugs used to treatment, and the patient experiences recurrence followed by death in less than 15 months

after diagnosis. An alternative in GBM treatment could be immunotherapy which aims to stimulate patient’s

immunological system in order to obtain a specific and long-term response that can protect against recurrence. One of

these alternatives involves the use of DNA vaccines codifying tumoral antigens and immunomodulatory proteins that

can effectively activate tumour antigen specific B and T lymphocytes. In this context, the objective of this work was the

construction of DNA vaccines using GBM antigen genes (EGFRvIII, cERBB2, MAGE e GLEA) and immunomodulatory

proteins (hsp65, hsp70, gp96 e gD), followed by their immunogenicity evaluation. Methods: Genes were evaluated in

silico, synthesized in vitro and used in DNA vaccines construction. Molecular biology tools and the pVAX vector were

used to obtain the vaccine. They were characterized by sequencing, western blot and were used in the immunization

of C57BL/6 mice. Immunizations were performed in 3 doses of a DNA vaccine combining a tumoral antigen and an

immunomodulatory protein at each 12 days. Immunogenicity was evaluated 20 days after the last dose. The ex vivo

assays were performed with the serum of immunized animals for antibody evaluation and spleen cells were stimulated

with EGFRvIII, cERBB2, MAGE e GLEA proteins to assess tumoral antigen specific cells. Results: The

pVAXgDGLEA vaccine was the only able to induce IgG2a subtype anti-GLEA antibodies. Vaccines pVAXgDGLEA,

pVAXgDEGFRvIII e pVAXgDMAGE were able to activate antigen-specific cells that produced IFN-γ e IL-10 quickly

after reestimulation. Conclusion: The gD immunomodulatory protein was able to induce a Th1 immune response,

specific to GBM antigens, which is important in tumor combat while IL-10 could favor and/or balance the response in

brain, which should be effective but not exacerbated. Financial support: FAPESP, FAEPA and CNPq

EFFECT OF SCHISTOSOMA MANSONI ENZYMES ADENYLATE KINASE (ADK) AND ADENOSINE KINASE (AK)

ON WORM BURDEN IN EXPERIMENTAL SCHISTOSOMIASIS

ANA CAROLINA MARAGNO FATTORI1; ELISANDRA DE ALMEIDA MONTIJA

2; SANDRA REGINA PEREIRA DE

OLIVEIRA3; RICARDO DE OLIVEIRA CORREIA

4; JOICE MARGARETH DE ALMEIDA RODOLPHO

5; LUCIANA

CAMILLO6; RICHARD CHARLES GARRATT

7; FERNANDA DE FREITAS ANIBAL

8.

1,2,3,4,5,6.FEDERAL UNIVERSITY OF SÃO CARLOS – UFSCAR, SÃO CARLOS - SP - BRASIL; 7,8.UNIVERSITY

OF SÃO PAULO – USP, SÃO CARLOS - SP - BRASIL.

Introduction: Schistosomiasis mansoni is considered the most important of the human helminthiasis in terms of

morbidity and mortality. In Brazil, an estimated six million people are infected. However, limited options are available

for the treatment of schistosomiasis and the drug of choice remains praziquantel. Thus, several studies have been

proposed to find vaccines to schistosomiasis control. The interference in the nucleotide metabolism of the parasite is

an interesting way to try to infection modulate, so this interference in the production of DNA, RNA and ATP, could

affect the high biosynthetic capacity of Schistosoma mansoni. Even with the existence of an effective drug, it does not

prevent reinfection. The enzymes are of great interest because they play an important role in purine nucleoside

reconstitution in a number of parasites. To this end, study new options of treatment or vaccine based on the

investigation of enzymes in metabolic pathways of S. mansoni become an interesting research path. The objective of

this study was verify the antiparasitic effect of immunization with the S. mansoni enzymes Adenylate Kinase (ADK)

and Adenosine Kinase (AK), through the recovery of adult worms. Methods and Results: The animals used were

female mice (BALB/c), and the experimental design of this work was approved by the Ethics Committee on Animal

Experimentation of UFSCar. The mice were divided into 5 experimental groups: control, without infection (n = 7);

infected, without immunization (n = 7); ADK, immunized with 100 µg of the enzyme ADK (n = 6); AK, immunized with

100 µg of the enzyme AK (n = 5) and ADK + AK, immunized with 50 µg of the enzyme ADK and 50 µg of the enzyme

AK (n = 6). The groups were immunized with three doses at intervals of 15 days. After 15 days the last immunization

the animals were infected with 80 cercariae of S. mansoni. In 48 days of infection the animals were performed

euthanasia for the recovery of adult worms by perfusion of the portal system. The statistical test used was the

nonparametric test One-way ANOVA. Significance was established for p < 0.05. The recovery of the couples of adult

worms in the group AK showed degree of protection of 27.32%, in group ADK + AK, 6.98% and in group ADK, 6.39%,

compared with the infected group, without immunization. Conclusion: The results suggest that the enzyme AK was

able to reduce the worm burden and it can modulate the parasite’s biological cycle.

Financial support: CNPq and FAPESP

HIV CD4 BASED VACCINE PRE-IMMUNIZATION INCREASES CD8 T CELL RESPONSES AND CONFERS

PROTECTION AGAINST CHALLENGE WITH A RECOMBINANT VACCINIA VIRUS ENCODING HIV GAG-POL

PROTEINS

SUSAN PEREIRA RIBEIRO; ADRIANA DALBEN FARIA; RAFAEL RIBEIRO ALMEIDA; VINICIUS CANATO

SANTANA; DANIELA SANTORO ROSA; ESPER GEORGES KALLAS; JORGE KALIL; EDECIO CUNHA-NETO.

FMUSP, SAO PAULO - SP - BRASIL.

Generation of competent and long-lived memory CD8+ T cells is highly dependent on CD4+ T cell help. A vaccine

able to provide cognate help may efficiently prime CD8+ T cell responses establishing an appropriate immune control

of HIV. Furthermore CD4 T cells responses also have been correlated with HIV infection control. Our group has

identified a set of 27 conserved and promiscuous CD4 epitopes from 8 HIV-1 proteins. HIVBr27 encoding such

epitopes was highly immunogenic in BALB/c mice inducing strong and broad CD4+ T cell responses. Here we tested

whether pre-immunization with HIVBr27 could provide cognate help and improve CD4 and CD8 T cell responses

against other HIV proteins (Gag, Pol and Vif) correlated with HIV protection/disease control. The pre-immunization

with the CD4 based vaccine increased and broadened the CD8 T cell response against Gag, Pol and Vif. Higher

proliferation and cytokine secretion were observed in pre-immunized animals when compared to control pre-

immunized groups (empty vector). In relation to the CD8 T cells, we observed an overall increase in 57% and 40% of

the breadth and magnitude of the responses, respectively, when comparing the CD4 based vaccine pre-immunized

group to the control (empty vector pre-immunization). Into the CD4 T cell compartment we noticed an increase of 3

and 2 times in the breadth and magnitude of the responses, respectively, comparing CD4 versus control primed

group. We also observed a 10 times reduction on the PFUs after recombinant Gag-Pol vaccinia virus challenge as

well as a 32 fold increase in the anti-p24 antibody levels in the HIVBr27 pre-immunized group when compared to the

control one. These results highlight the importance of having a CD4 inducer based component as a primer for the

global responses, impacting in both cellular and humoral immune responses.

IMMUNE SELECTION OF EPITOPES FROM THE RECOMBINANT PROTEIN PB27 OF PARACOCCIDIOIDES

BRASILIENSIS FOR A POTENTIAL VACCINE.

ELIZA MATHIAS MELO1; ELIS ARAUJO MORAIS

2; DANIELA FERREIRA CHAME

3; CINTIA MARTINS REZENDE

4;

RICARDO ANDREZ MACHADO DE AVILA5; CARLOS CHAVEZ OLORTEGUI

6; DAWIDSON ASSIS GOMES

7;

ALFREDO MIRANDA GOES8.

1.UNIVERSITARY CENTER UNA, BELO HORIZONTE - MG - BRASIL; 2,3,4,5,6,7,8.FEDERAL UNIVERSITY OF

MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Introduction: Paracoccidioides brasiliensis is a thermo-dimorphic fungus, endemic in Latin America where cause the

main systemic mycosis called paracoccidioidomycosis (PCM). Conventional therapy for the mycosis involves chemical

treatment resulting in relapse and often sequelae and long period of treatments. The antigenic protein Pb27 of P.

brasiliensis showed a significant protective activity as a vaccine for PCM and positively modulates the inflammatory

response against P. brasiliensis infection in experimental models. Thus, the aim of this work was to select

immunodominant epitopes from the recombinant protein rPb27 for use as potential vaccine.

Methods and results: For this purpose, the ELISA assays was used to evaluate sera of patients with PCM that were

used to select immunodominant B and T epitopes. The epitopes were predicted from the sequence of recombinant

Pb27 (rPB27) using bioinformatic tools and synthesized on a membrane using the spot method. To select the

immunodominants peptides we evaluated the reaction, performing an immunoblot test, of the human sera with PCM.

These results showed that among 20 B epitopes and 105 T epitopes tested were selected 4 promising epitopes to

evaluate a vaccine against PCM.

Conclusion: We believe that these four peptides of rPb27 are promising candidates for the vaccine against PCM

because the use of the peptides for vaccine can increase the specificity of the immune response, increase the purity

of the vaccine, decrease the adverse effect against PCM and lower costs in vaccine production.

Financial support: FAPEMIG, CAPES and CNPq.

IMMUNIZATION AGAINST INFLUENZA VIRUS (MYXOVIRUS INFLUENZA) AND ABSENTEEISM IN SCHOOLS IN

MUZAMBINHO CITY -MG

ANDRÉA DE CARVALHO CARNEVALI; DEBORA VALIM DA SILVA; AMANDA GOULART; RAPHAEL PRADO DIAS;

JAQUELINE CARLOS FUNAYAMA.

IFSULDEMINAS, MUZAMBINHO - MG - BRASIL.

IMMUNIZATION AGAINST INFLUENZA VIRUS (Myxovirus influenza) AND ABSENTEEISM IN SCHOOLS IN

MUZAMBINHO CITY -MG.

ANDREA DE CARVALHO CARNEVALI (IC) (1)

; DEBORA VALIM DA SILVA(IC)(1)

; AMANDA GOULART (IC)(1)

;

RAPHAEL ANTÔNIO PRADO DIAS (MS) (1)

; JAQUELINE CARLOS FUNAYAMA (PhD) (1)

1 - Federal Institute of Education, Science and Technology of the South of Minas Gerais (IFSULDEMINAS)

Introduction: Biosecurity in the workplace includes the vaccination of employees, which contributes to the quality of

life of the workers, increases productivity, reduces the incidence of absenteeism and provides savings for the

company. With universal importance, influenza is a matter of continuing concern to the World Health Organization, is

the primary cause of absenteeism amongst employees, which may cause even greater problems when the person

infected is a professional teacher, which in turn can affect student learning. The objective of this study is to establish

the relationship between immunization against influenza virus (Myxovirus influenza) and absenteeism of employees in

schools in Muzambinho city -MG. Methods and results: Teachers and employees of four city schools were

interviewed and their medical certificates were analyzed looking specifically at the CIDs J10 and J11. Of the 82

interviews conducted, it was found that; 37 (45%) positive responses in relation to absenteeism due to influenza, 11

(30%) said they are vaccinated individuals and 26 (70%) were not vaccinated. Of the the 45 (55%) negative

responses, 32 (71%) people claimed to be vaccinate while 13 (29%) said no. In relation to CID's certificates for the

year 2012 there was 1 (0.5%) person affected by the j10 and 3 (1.5%) people affected by J11. In 2013, until April, 4

(2%) were absent due to J11. Of the total of 265 employees examined; 45 (17%) people were absent, 220 (83%) were

not affected by the flu. Of the 45 employees that were absent, 19 (42%) of them were vaccinated and 26 (58%) were

not. Of those who were not absent due to influenza, 207 (94%) took the vaccine and 13 (6%) did not take it. Many

teachers said they were under the symptoms, and even then, were working to not harm students. It can be seen then

that vaccinated employees have lower rates of absenteeism, while those who did not receive the vaccine currently

lacking, there is a relationship between immunization against influenza virus (Myxovirus influenza) and employee

absenteeism in schools (p <0,05). Conclusions: Considering the school environment, which brings together a large

number of people in an enclosed space, a solution to decrease absences would be the inclusion of teachers and

school staff in groups to officially participate in the annual vaccination campaign against influenza virus, decreasing

also the spread of this virus within the school environment.

IMMUNOGENICITY EVALUATION OF RECOMBINANT PROTEINS FROM LEISHMANIA (VIANNIA)

BRAZILIENSIS

GRAZZIELLE GUIMARÃES DE MATOS1; JOÃO PEDRO TORRES GUIMARÃES

2; JOSÉ VITOR ALVES

3; ROSANA

MORAES BALIAN4; GERSON SALAY

5; MILTON ADRIANO PELLI OLIVEIRA

6; FATIMA RIBEIRO-DIAS

7; MAURÍCIO

MARTINS RODRIGUES8; MIRIAM LEANDRO DORTA

9.

1,2,3,4,6,7,9.INSTITUTO DE PATOLOGIA TROPICAL E SAÚDE PUBLICA, GOIÂNIA - GO - BRASIL; 5,8.CENTRO

INTERDISCIPLINAR DE TERAPIA GÊNICA, SÃO PAULO - SP - BRASIL.

Introduction: Leishmaniasis is a neglected tropical disease with an estimated annual incidence of two million and a

prevalence of twelve million cases globally (Vac. 28:329-337, 2010). This disease is caused by protozoan Leishmania,

and presents several clinical ranging from cutaneous lesions to a fatal systemic disease (J Glob Infect Dis. 2: 177–

185, 2010). Treatments for all forms of leishmaniasis consist in the use of toxic and expensive drugs (Neg. Trop. Dis.

5:943-952, 2011). The development of a safe and effective vaccine is important and has been target on studies.

Conserved antigens among the Leishmania genus, such as TSA, LbSTI1 and LeIF, have been shown to induce

excellent protection in murine (Vet. Res. 36:827–838, 2005). Investigations of the immune protection mechanism

against Leishmania spp. revealed that a Th1 type cell response interferon (IFNγ) production is important to the

resistance (Mol. Pharm. 9:59-70, 2012). Thus, this work was aimed to evaluate the immunogenicity of the TSA, LbSTI

and LeIF recombinant proteins potential antigens for development of an effective vaccine to Leishmania (Viannia)

braziliensis.Methods and Results: This study was approved by the Committee of Ethics in Human and Animal

Research of Clinical Hospital/UFG. BALB/c mice were immunized three times with TSA, LbSTI and LeIF recombinant

proteins, single or in combination, and all were administered in association with Monophosphoryl Lipid A as adjuvant.

After, each serum was collected to evaluate the production of IFNγ and IgG antibodies. These measurements were

performed by using enzymatic immunoassays. All immunized groups with recombinant proteins produced high

concentrations of IFNγ and IgG, mainly after the last immunization. There was significant difference between control

groups and immunized groups (p<0.05), as well between the immunized groups with the different recombinant

proteins. Conclusion: The TSA, LbSTI and LeIF recombinant proteins from Leishmania (V.) braziliensis were

immunogenic, inducing Th1 cytokine and IgG antibodies.

Financial support: CNPq and FAPEG/GO.

IMMUNOGENICITY INDUCED BY OMPL37 OF LEPTOSPIRA INTERROGANS IN DIFFERENT VACCINE

FORMULATIONS

THAÍS LARRÉ OLIVEIRA; ANDRÉ ALEX GRASSMANN; RODRIGO ANDRADE SCHUCH; MARIANA BRUTSCHIN

PEREIRA; JULIA LABONDE; DAIANE DRAWANZ HARTWIG; ODIR ANTÔNIO DELLAGOSTIN.

UNIVERSIDADE FEDERAL DE PELOTAS, PELOTAS - RS - BRASIL.

Introduction: Leptospirosis is an emerging and neglected zoonosis, caused by pathogenic spirochetes of Leptospira

genus, responsible for over 500,000 human cases each year. Currently available vaccines provide short-term

immunity without cross-protection against different Leptospira serovars. The protein OmpL37 represents a potential

target for vaccine development against leptospirosis for the following reasons: it is conserved and recognized by

convalescent sera, binds to extracellular matrix components, and probably plays an important role in the

pathogenesis. This study aimed to evaluate the immunoprotective potential of OmpL37 from L. interrogans serovar

Copenhageni strain Fiocruz L1-130 using prime-boost, DNA, and protein-based immunizations. Methods and

Results: The ompL37 gene was amplified by PCR and cloned into the Escherichia coli expression vector pAE and

mammalian expression vector pTargeT, to obtain a subunit and a DNA vaccine, which were characterized by Western

blot (WB) and indirect immunofluorescence (IFI) of CHO-K1 cells transfected in vitro, respectively. Two independent

experiments were conducted. Groups of 6 hamsters were immunized twice with 21 days of interval as follows:

rOmpL37-Alhydrogel (2 x 100 µg), pTargeT-ompL37 (2 x 100 µg), prime-boost pTargeT-ompL37 (100 µg) plus

rOmpL37 (100 µg), pTargeT (2 x 100 µg) and PBS-Alhydrogel. The antibody levels were evaluated by indirect

enzyme-linked immunosorbent assay (ELISA) using rOmpL37 as antigen. Both pAE/ompL37 and pTargeT/ompL37

were functional, and able to express OmpL37 in prokaryotic or eukaryotic cells in vitro. rOmpL37 induced higher

antibody levels than pTargeT/ompL37 and PBS at 21 and 42 days in both experiments (p<0.005). There was

statistical difference (p<0.005) between the first and second dose of rOmpl37 in experiment 1, but not on experiment

2. No statistical difference was observed between pTargeT/ompL37 and negative control groups. Conclusion: The

protein rOmpL37 is immunogenic, able to induce specific IgG antibodies in vaccinated animals. However, there was

no significant IgG response in the hamsters immunized using the DNA vaccine. Financial support: CNPq, CAPES

and FAPERGS.

IMMUNOLOGICAL PROFILE OF SNAK1: A SODIUM POTASSIUM ATPASES PRESENTS IN TEGUMENT OF

SCHISTOMA MANSONI

NATASHA DELAQUA RICCI1; NATAN RAIMUNDO GONÇALVES DE ASSIS

2; VICENTE DE PAULO MARTINS

3;

BÁRBARA DE CASTRO PIMENTEL FIGUEIREDO4; CARINA DA SILVA PINHEIRO

5; SUELLEN BATISTONI DE

MORAIS6; RODRIGO MARQUES BICALHO

7; SERGIO COSTA OLIVEIRA

8.

1,2,4,5,6,7,8.UFMG, BELO HORIZONTE - MG - BRASIL; 3.UNB, BRASÍLIA - DF - BRASIL.

Introduction:The flatworm Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating

disease that occurs throughout the developing world. Current schistosomiasis control strategies are mainly based on

chemotherapy, but many researchers believe that the best long-term strategy to control the disease is through

immunization with anti-schistosomiasis vaccines, alone or combined with drug treatment. Based on the advent of the

parasite genome, transcriptome and proteome, our group selected some proteins as potential target for the

development of a vaccine. In this study, we evaluated the ability of eight individual proteins including the SNaK1, a

sodium, potassium ATPases. These enzymes generate cationic gradients that are fundamental to a variety of cellular

functions such as the regulation of cell volume and pH. Proteomic analysis detected this protein the tegument of

adults worms. Physiological data show that the active transport of Na and K is particularly important to schistosomes

for the maintenance of normal ionic gradients and the specific inhibition of Na,K-ATPases using ouabain elicits a large

depolarization of the tegumental cytoplasm and a dramatic contraction of the somatic musculature. The goal of this

work was to evaluate the potential of a recombinant SNaK1 as a vaccine against S. mansoni in a murine

model.Methods and Results: Mice were immunized with SNaK1 with Freund adjuvant. The mice were infected with

100 cercariae of S. mansoni and euthanized seven weeks post-infection for histological liver and worm burden counts.

Analysis of mRNA expression by real time PCR show that SNaK1 was expressed more abundantly in adult worm

stage. The immunization induced a IgG1 and IgG2a mixed immunological profile. The cellular response elicited by

vaccination were evaluated in splenocyte cultures and we observed high levels of IFN-γ and IL-10. The worm burden

reduction was approximadely 15-20%. Flow cytometric analysis of splenocytes cultures showed a significant increase

of CD4+IL-10

+, CD8

+IL-10

+, CD4

+IFN-γ

+, CD8

+IFN-γ

+ and CD8

+IL-17

+ T between the groups. Conclusion: Our

findings showed high levels of IL-10, suggesting a potential immunoregulatory profile.

FINANCIAL SUPPORT CAPES, CNPq, FAPEMIG,INCT-DT

PROTECTION INDUCED IN BALB/C MICE BY IMMUNIZATION WITH A RECOMBINANT HYPOTHETICAL

PROTEIN AGAINST CHALLENGE INFECTION WITH LEISHMANIA INFANTUM

LOURENA EMANUELE COSTA1; VIVIAN TAMIETTI MARTINS

2; MIGUEL ANGEL CHÁVEZ-FUMAGALLI

3; PAULA

SOUZA LAGE4; DANIELLA PAGLIARA LAGE

5; MARIANA COSTA DUARTE

6; TATIANA RIBEIRO RIBEIRO

7;

WANDERSON DUARTE DAROCHA8; ANA PAULA FERNANDES

9; CARLOS ALBERTO PEREIRATAVARES

10;

EDUARDO ANTONIO FERRAZ11

.

1,2,3.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 4,5,6,7,8,9,10,11.UFMG,

BH - MG - BRASIL.

Abstract:

Introduction: Experimental vaccine models have been developed to protect against visceral leishmaniasis (VL),

however, an effective vaccine still does not exist. The present study aims to evaluate a hypothetical Leishmania-

specific protein (LiHyp4), which was identified by an immunoproteomic approach, in an attempt to select a new

candidate antigen to compose an vaccine against VL. Methods and results: The LiHyp4 DNA sequence was cloned,

the recombinant protein (rLiHyp4) was purified, and used to immunize BALB/c mice (8 animals per group). In the

results, rLiHyp4 vaccinated mice showed a specific-Th1 response, which was primed by high levels of IFN-g, IL-12,

and GM-CSF after in vitro stimulation. Immunized and infected mice, as compared to the control groups (saline and

saponin), showed significant reduction in the parasite load in the liver, spleen, bone marrow, and in the draining lymph

nodes in the paws. Protection was associated with an IL-12-dependent IFN-g production, which activate macrophages

to eliminate the parasites. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response was also

observed. The analysis of the results were performed using GraphPad Prism (version 5.0 for

Windows). Conclusion: Therefore, the present study showed that this hypothetical Leishmania-specific protein, when

combined with a Th1-type adjuvant, can be useful in the protection against VL.

Financial support: INCT NanoBiofar, FAPEMIG, CNPq and PRPq/UFMG.

PROTECTIVE IMMUNITY INDUCED BY A THYMIDINE AUXOTROPH MUTANT OF LEGIONELLA PNEUMOPHILA

IS DEPENDENT ON THE BACTERIAL DOT/ICM SECRETION SYSTEM AND MYD88 SIGNALING PATHWAY.

MARCELO DE SOUZA FERNANDES PEREIRA; DARIO SIMÕES ZAMBONI.

DEPARTMENT OF CELL BIOLOGY, SCHOOL OF MEDICINE OF RIBEIRÃO PRETO, UNIVERSITY OF SÃOPAULO

(FMRP/USP), RIBEIRÃO PRETO - SP - BRASIL.

Introduction: The importance of the priming of the lung environment by past infections is being increasingly

recognized. Exposure to any given antigen can either improve or worsen the outcome of subsequent lung infections,

depending on the immunological history of the host. Thus, an ability to impart transient alterations in the lung

environment in anticipation of future insult could provide an important novel therapy for emerging infectious

diseases.Methods and Results: To gain insight in the molecular mechanisms related to protection during previous

bacterial infection we used a mouse model of Legionella pneumophila. Mouse were infected nasally with thymidine

auxotroph mutants of Legionella pneumophila (thyA- , which fail to replicate in vivo) for 10 days (day -10) before

challenge with virulent bacteria (L.p flaA-). At day 0 mice were infected with L. p. flaA

- via intranasal (i.n) and after 2 or

4 days, lungs were harvest, crushed and plated on CYE. After 4 days CFU were evaluated. We show that C57BL/6

pre stimulated with thyA- bacteria became significantly resistant to infection. Moreover this protection response

required the presence of dot/icm functional system because pre stimulation with thyA- dotA

- mutants did not trigger

protection. Additionally we observed that mice Myd88 deficient mice do not became protected. MyD88 is a

cytoplasmic adapter protein required for signaling by TLRs and IL-1 family of receptors, thus, we evaluated TLRs

deficient mice as well as IL-1R-/-. We observed that these mice were also protected suggesting no involvement of

TLRs or IL-1 pathway. To evaluate if this protection requires neutrophils C57BL/6 mice were depleted with anti-Ly6G

before immunization (day-11) or after immunization (day 0). We found that anti-Ly6G depletion at day 0 promoted a

partially reduction on protection, but the same was not observed when depletion were performed at day -11.

Additionally, we observed a partially involvement of CD4+ T cells to due a deficient bacterial clearance MHCII

-/-

mice. Conclusion: We concluded mice pre-exposed to bacteria became more resistant for re-infection. This process

requires bacterial virulence (T4SS), MyD88 signaling, neutrophils and CD4+ T cells, which leads to generation of a

strong and durable response against subsequent infections. Financial support: CAPES, INCTV,FAEPA, CNPq and

FAPESP.

RECOMBINANT VLP PROTEIN FORMULATIONS AS PROPHYLACTIC OR THERAPEUTIC VACCINE

CANDIDATES AGAINST PARACOCCIDIOIDOMYCOSIS

RODRIGO ASSUNÇÃO HOLANDA1; JULLIANA RIBEIRO ALVES SANTOS

2; GUSTAVO JOSÉ COTA FREITAS

3;

CARLOS PELLESCHI TABORDA4; DANIEL ASSIS SANTOS

5; OSCAR BRUNA ROMERO

6.

1,2,3,5.UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL; 4.UNIVERSIDADE DE

SÃO PAULO, SÃO PAULO - SP - BRASIL; 6.UNIVERSIDADE FEDERAL DE SANTA CATARINA, FLORIANÓPOLIS

- SC - BRASIL.

Introduction: Paracoccidioidomycosis (PCM) is a systemic disease endemic in Latin America, caused by

Paracoccidioides brasiliensis. Currently, there is no effective vaccine against PCM. Proteins that self-

assemble into Virus-Like Particles (VLPs) are interesting candidates for vaccine strategies to incorporate

heterologous peptides or proteins. In this study, we developed two vaccine candidates based on a VLP

protein, both containing the main CD4+ T cell-specific-epitope of P. brasiliensis (P10) but presented to the

immune system either as a (His)6-purified protein (rPbMT) or as a recombinant human type 5 adenoviral

vector (HAd5VPbT).

Methods and Results: rPbMT was expressed by Escherichia coli BL21 (DE3) with IPTG induction and purified

using Ni2+

columns under denaturing conditions before dialysis and refolding. HAd5VPbT was generated by

transfection of a shuttle vector containing VLP protein and P10 encoding sequences together a plasmid

containing the HAd5V E1/E3-deleted genome into HEK-293 cells. RT-PCR (adenovirus only) and Western

blotting techniques were performed to verify the expression of chimeric VLP. Immunizations were performed

among 8 to 12 week-old male BALB/c mice, 8 animals per group, according to our institutional ethical

guidelines (CETEA 206/11). Intranasal (IN), subcutaneous (SC) and intraperitoneal (IP)

inoculations of HAd5VPbT (109 p.f.u) were used to determine the most immunogenic route. To determine a

suitable dosage, rPbMT 1 µg, 5 µg and 25 µg of protein in MONTANIDE ISA 720 adjuvant were administrated

subcutaneously. Control group was immunized via the SC route with 10 µg of MD2 protein based on the same

VLP platform as previously tested. The highest anti-VLP IgG titres were used as criteria to determine routes

and dosages. Serum anti-VLP IgG antibody was measured by ELISA 30 days after inoculations. All data were

statistically analyzed by ANOVA (P<0.05 considered significant). IN and SC routes of HAd5VPbT displayed the

highest IgG titre (1:800), while the IP route presented the lower (1:400) (P<0.05). The rPbMT dosage of 25 µg

induced highest IgG titre (1:800), followed by 1 µg (1:400) and 5 µg (1:200) dosages (P<0.05). MD2 control

presented anti-VLP IgG (1:1600) as expected.

Conclusion: The recombinant VLP proteins carrying CD4+ T cells P10 epitope are potentially promising to

induce protective immune response against P. brasiliensis. P10 is currently being investigated by

immunophenotyping.

Financial support: CNPq.

STREPTINCOR A STREPTOCOCCUS PYOGENES CANDIDATE VACCINE INCREASES THE NUMBER OF

NATURAL REGULATORY T CELLS IN PERIPHERAL BLOOD FROM RHEUMATIC FEVER PATIENTS

KAREN FRANCINE KÖHLER1; CAROLINE FERREIRA DE FREITAS

2; JORGE KALIL

3; LUIZA GUILHERME

4.

1,2,4.HEART INSTITUTE, IMMUNOLOGY INVESTIGATION INSTITUTE, FMUSP, SÃO PAULO - SP - BRASIL;

3.HEART INSTITUTE, IMMUNOLOGY INVESTIGATION INSTITUTE, CLINICAL IMMUNOLOGY AND ALERGY

DIVISION, FMUSP, SÃO PAULO - SP - BRASIL.

Introduction: Group A Streptococci (GAS) diseases remain as a public health problem both in industrialized and

development countries, leading to severe invasive infections, pharyngitis and pyoderma. These diseases can lead to

autoimmune post streptococcal sequelae as Rheumatic Fever (RF), Rheumatic Heart Disease (RHD) and

Glomerulonephritis, in non-treated patients. At least 517 000 people die per year due to severe GAS diseases. The

prevalence of RHD is 15.6 million cases with 282 000 new cases 233 000 deaths each year. RF affects mainly

children and young people and it is caused by molecular mimicry with human proteins in which autorreactive B and T

cells are involved and lead to different clinical manifestations. RHD is the most severe sequel being characterized by

progressive and permanent valvular lesions. We are developing a vaccine against Streptococcus pyogenes based on

protective epitopes of C-terminal portion of the M protein. A peptide with 55 amino acids named StreptInCor (medical

identity) was designed. The vaccine epitope induced systemic protective immune response in murine models. Here,

we evaluated the capacity of StreptInCor peptide to induce regulatory T cells (Treg).

Methods and Results: Specific cellular markers (CD3, CD4, CD25, CD127 and Foxp3) for natural T regulatory cell

subset of T cell were analyzed by gene expression and flow citometry. For inducible Treg, we evaluated the

expression of IL-10 and TGF-beta genes by qRT-PCR. Peripheral blood mononuclear cells (PBMC) of RHD (n=16)

and controls (n=22) were stimulated with 10μg/ml of StreptInCor synthetic peptide. Nonparametric tests (Mann

Whitney and Wilcoxon) were used for comparisons of the gene and protein expression of T regulatory cell markers in

peripheral blood. P values of <0.05 were considered statistically significant. We observed that StreptInCor induced the

increase of natural T regulatory cells (nTreg) in PBMC in both groups (P=0.0043 and 0.0006, respectively). The

number of nTreg cells in PBMC from DRC patients before the stimulus is lower than that observed in PBMC from

controls (P=0.0341).

Conclusion: These results strongly indicate that StreptInCor vaccine peptide may have therapeutic potential to

control both inflammatory and autoimmune response in RF/RHD patients.

Financial support: FAPESP and CNPq.

STUDY OF THE SPECIFIC IMMUNE RESPONSE IN IMMUNIZED MICE BY DIFFERENT ROUTES WITH

IRRADIATED TACHYZOITES OF TOXOPLASMA GONDII.

NAHIARA ESTEVES ZORGI1; ANDRÉS JIMENEZ GALISTEO JR

2; MARIA NOTOMI SATO

3; HEITOR FRANCO DE

ANDRADE JR4.

1.INSTITUTO DE CIÊNCIAS BIOMEDICAS DA UNIVERSIDADE SÃO PAULO, SÃO PAULO - SP - BRASIL;

2,3,4.INSTITUTO DE MEDICINA TROPICAL DE SÃO PAULO, SÃO PAULO - SP - BRASIL.

Introduction: The immune response after infection with T. gondii, is not sufficient for the elimination of parasites,

instead, there is a balance that the parasite survives for a long period or even throughout the life of the host in the

form of cysts (J.Eukaryot.Microbiol. 55:467-475, 2008). Our development of a vaccine for toxoplasmosis, using

irradiated tachyzoites induces response pattern similar to natural infection, allowing dissection of immunity against

disease and cysts formation (Immunol.Lett.2:187-196, 2011). In this work we evaluated the humoral and cellular

immune response in immunized BALB/c mice by intraperitoneally and orally with irradiated 255Gy (Cobalt-60) of T.

gondii tachyzoites.Methods and results: Immunized BALB/c mice with 107 irradiated tachyzoites by intraperitoneal

(i.p.) or orally, the animals received three doses biweekly. Detection of serum antibodies was performed by ELISA and

cell populations of T and B lymphocytes, cytokine production (CBA) and splenocyte proliferation (CFSE) were

analyzed by flow cytometry (BD LSRFortessa). For the CBA and CFSE assays the cells were maintained in culture in

the presence of the antigen of T. gondii. Immunized mice i.p. showed significant levels (p <0.05) specific IgG

antibodies in the serum of up to three months after 3rd dose. Immunized BALB/c mice by i.p. or orally had higher

populations of T lymphocytes, CD3+CD4

+CD44

+ and CD3

+CD4

+CD45RB

lo, B lymphocytes CD19

+B220

+CD23

+ when

compared with control mice. The immunized mice showed a Th1 type response, with high levels of IFN-gamma

produced by spleen cells. BALB/c mice i.p. showed a higher production of IFN-gamma (8000pg/mL). There was an

increase in the proliferation 10% of splenocytes and 13% of B cells in BALB/ i.p. in comparison with the control

group. Conclusion: Our preliminary data suggest that our model vaccine induces a response similar to natural

infection. Immunized mice with irradiated tachyzoites present immune response both humoral and cellular, with

specific antibody production, an important production of IFN-gamma and increased cell proliferation. The elucidation

of mechanisms of immune response can assist in the immunogen producing suitable and that can be used in future

animal use. Supported by LIMHCFMUSP & CNPq.

VACCINATION BASED ON PEPTIDE-CHITOSAN NANOPARTICLES AGAINST SCHISTOSOMA MANSONI

INFECTION

LUDMILA ALVES RODRIGUES; CÍNTIA MARTINS RESENDE; RICARDO MACHADO DE AVILA; CARLOS

CHAVEZ- OLÓRTEGUI; ALFREDO MIRANDA GOES.

UNIVERSIDADE FEDERAL DE MINAS GERAIS, BELO HORIZONTE - MG - BRASIL.

Introduction: Schistosomiasis remains a significant public health problem in tropical countries and it is recognized as

the most important human helminth infection in terms of morbidity and mortality. Despite the existence of highly

effective antischistosome drug, it does not prevent reinfection. Therefore, vaccine strategies associated to new

adjuvant represent an essential component for the control of schistosomiasis. A new approach of vaccination with

chitosan nanoparticles appears to be an interesting strategy because of its great stability, the ease of target

accessibility and its immunostimulatory properties. Here we propose a vaccine strategy based on the combination of

chitosan nanoparticles and peptides derived from rP22 and rP24, both membrane spanning proteins that are present

in different life cycle stages of Schistosoma mansoni. Both recombinant proteins are known to be protective after

challenge infection.

Methods and Results: Peptide sequences were previously predicted as linear B-cell epitopes corresponding to rP22

and rP24 S. mansoni recombinant proteins. Spot membrane containing peptides was incubated with either sera from

rP22 immunized-mice or rP24 immunized-rabbits to select peptides by immunoscreening. Among the reactive

peptides, four of them from rP22 and three from rP24 were selected and used to immunize mice as follow: 1- Peptides

conjugated with glutaraldehyde (PC) and; 2 – Peptides not conjugated with glutaraldehyde (PNC). These formulations

were combined to chitosan nanoparticles and used as vaccine delivery systems. Mice immunized with PC exhibited a

37.4% and 84% decrease in adult worm burden and in hepatic eggs, respectively. A strong protection was observed in

PNC-immunized mice with 62% reduction in adult worm burden and a striking reduction of 91% in the number of eggs

recovered from the liver.

Conclusion: These preliminary results demonstrate that chitosan nanoparticles loaded with rP22 and rP24-derived

peptides are a promising vaccine strategy against schistosomiasis.

Financial support: UFMG/ Pró-Reitoria de Graduação.

VACCINATION COVERAGE IN BRAZIL: AN EPIDEMIOLOGICAL STUDY.

FRANCIMAR KETSIA SERRA ARAUJO.

UNIVERSIDADE POTIGUAR, NATAL - RN - BRASIL.

Introduction: Vaccination is the most effective way to prevent disease immunopreventable, the national immunization

program of the Ministry of Health sets the schedule of Brazilian vaccination and also does the set of vaccines

considered of priority interest to public health in the country, which currently consist of 12 recommended products for

population, from birth to old age and are distributed free.

Methods and Results: This work is scoped to an epidemiological analysis using a retrospective descriptive study

based on data collected from the hospital information system (SUS HIH / SUS), DATASUS. According to DATASUS

between the period January 2000 to April 2013, 663.735.504 immunizations were performed in Brazil, in all age

groups. The region of lower vaccination coverage was the midwest region with only 7.35% of this value and the

southeast region with the highest percentage, 39.22%. It is noteworthy that the major vaccines in newborns and

children until 11 years are at greatest vaccine coverage at the expense of teenagers who have a considerable drop in

the number of immunizations done. This percentile rises from the third age with vaccinations against influenza and

pneumo23.

Conclusion: Despite the national immunization program has expanded so significantly vaccination coverage in Brazil,

there is still a major obstacle to total coverage of the country, as well as the major problem in underreporting, the

majority of the population, unfortunately, has not the habit of vaccination in adolescents, even being free and culture

that the calendar of immunizations should only be given in children, is still a frequent response of the population.

Moreover, advertisements incentives and more information on the subject can change this situation and extend the

coverage further vaccine. Thus in order to more effectively immunized population and a possible effect of flock in

communities that has not immunized.

VACCINATION WITH NEURAMINIDASE DEFICIENT INFLUENZA VIRUS RESULTS IN HIGHLY ATTENUATED

LUNG INFLAMMATION BUT ELICITS STRONG PROTECTIVE IMMUNITY

RAFAEL POLIDORO ALVES BARBOSA1; ANA PAULA CARNEIRO SALGADO

2; CRISTIANA COUTO GARCIA

(PG)3; BRAULIO HENRIQUE FREIRE LIMA (PG)

4; GABRIEL AUGUSTO O LOPES

5; MILENE ALVARENGA

RACHID6; DANILO BRETAS DE OLIVEIRA

7; MARCO ANTONIO ATAÍDE

8; ERICA AZEVEDO COSTA

9; GABRIEL

MAGNO DE FREITAS ALMEIDA10

; REMO CASTRO RUSSO11

; RICARDO TOSTES GAZZINELLI12

; ALEXANDRE

DE MAGALHÃES VIEIRA MACHADO13

.

1,2,3,4,5,6,7,8,9,10,11,12.UFMG, BELO HORIZONTE - MG - BRASIL; 13.CPQRR FIOCRUZ, BELO HORIZONTE -

MG - BRASIL.

Introduction: Recombinant influenza viruses are promising viral platforms to be used as antigen delivery vectors.

One of the most promising approaches generates recombinants viruses harboring partially truncated neuraminidase

(NA) segments. To date, all studies have been pointed to safety and usefulness of this viral platform. However, some

aspects of the inflammatory and immune responses triggered by those recombinant viruses and their safety to

immunocompromised hosts remained to be elucidated. Methods: In the present study, we generated a recombinant

influenza virus harboring a truncated NA segment (vNA-Δ) and evaluated the innate and inflammatory responses and

the safety of this recombinant virus to wild type or knock-out (KO) mice with impaired innate (Myd88 KO) or acquired

(RAG KO) immune responses. Results: Our results showed that recombinant influenza virus harboring truncated

neuraminidase segment abrogated lung and systemic inflammatory response in wild type mice and were completely

harmless to KO mice. We also demonstrated that vNA-Δ infection could prevent both cytokine storm and polarization

to type 1 immune response that is thought to be responsible for lung damage in wild-type infected mice. In addition,

the recombinant influenza virus was able to trigger both specific local and systemic humoral and cellular immune

responses which were able to protect the immunized mice against the challenge with a lethal dose of homologous wild

type influenza virus. Conclusion: Taking together, our findings indicate that the neuraminidase deficient virus results

in a silent infection, but, nevertheless, indices a strong protective immunity against influenza challenge.

Financial support: CNPq; FAPEMIG; CAPES and INCTV.

VACCINE EFFECT OF LAAG PLUS SAPONINA AGAINST LEISHMANIA AMAZONENSIS

MIRIAN FRANÇA MELLO1; RAQUEL PEREIRA RIBEIRO ROMÃO

2; BEATRIS LILIAN DA COSTA SILVA

3; JOYCE

CARVALHO PEREIRA4; DANIEL CLÁUDIO DE OLIVEIRA GOMES

5; EDUARDO FONCECA PINTO

6; HERBERT

LEONEL DE MATTOS GUEDES7.

1.IMPG - UFRJ, RIO DE JANEIRO - RJ - BRASIL; 2,6.IOC - FIOCRUZ, RIO DE JANEIRO - RJ - BRASIL;

3,4,7.IBCCF - UFRJ, RIO DE JANEIRO - RJ - BRASIL; 5.UFES - NDI, VITÓRIA - ES - BRASIL.

Introduction: Leishmania amazonensis is the main agent of anergic diffuse cutaneous Leishmaniasis. Our previous

studies demonstrated that LaAg- plus-saponin vaccine induced a partial protection against L. amazonensis infection

observed by partial reduction of lesion growth and parasite load. In this study, we investigated the initial immune

response after challenge to characterize the protective response. Methods and results: Prior to footpad infection with

L. amazonensis, BALB/c mice were twice vaccinated by the intramuscular route with 25 ug of LaAg containing 100 ug

of Saponin. We found that vaccinated mice developed an intense delayed hypersensitivity peaking at 15-18 hour

similar to Jones-Mote reaction. Histological studies demonstrated a lymphocyte and eosinophil infiltration and

reduction of neutrophil, macrophage and mast cells infiltration in comparison to nonvaccinated mice. Eosinophil and

lymphocyte (Th1) cells are related to parasite control and neutrophils and mast cells are associated to susceptibility

of L. amazonensis infection, suggesting that vaccine induced a cellular infiltrate to corroborate to parasite control.

Indeed, we observed by mRNA quantification in the peak of hypersensitivity an increase of Tbet and GATA-3, but not

Foxp3 on popliteal lymph node cells and demonstrated in footpad an increase of IL-4, IL-10, IFN-γ and IL-12

production and TGF-β reduction demonstrating a Th1 and Th2 profile. Though, we observed an increase of iNOS

levels in the footpad of vaccinated mice. The increase of iNOS in infected footpad is related to Th1 profile and

associate with parasite elimination. However, LaAg-plus-saponin vaccine did not induce protection in C57Bl6. The

vaccine induced a weak delayed hypersensitivity in C57BL6, without an increase of eosinophils and lymphocytes

recruitment as observed on BALB/c. Conclusion: These results suggest the role of a new-type of delayed

hypersensitivity in the mechanism of protection observed on BALB/c. Financial support: FAPERJ, CAPES, CNPq.