vaccines for leishmaniasis in the fore coming 25 years

Vaccine (2008) 26, 17091724

avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate /vacc ine

REVIEW

Vacccomi

Clarisa B. Palatnik-de-Sousa

Instituto de Microbiologia Prof. Paulo de Goes, Centro de Ciencias da Saude, Universidade Federal do Rio de Janeiro, PO Box68040 CEP 2

Received 3Available on

KEYWOHuman vMice andvaccinesLeishmaFirst-, sethird-gePhase I,Adjuvan

Contents

IntroduImmunFirst-g

CorrespoUniversitaria

E-mail a

0264-410X/$doi:10.10161941-590 Rio de Janeiro, Brazil

November 2007; received in revised form 28 December 2007; accepted 11 January 2008line 30 January 2008

RDSaccines;dog

;niasis;cond- andneration;II, III trials;t

Summary Human vaccination against leishmaniasis using live Leishmania was used in MiddleEast and Russia (19411980). First-generation vaccines, composed by killed parasites inducelow efcacies (54%) and were tested in humans and dogs Phase III trials in Asia and SouthAmerica since 1940. Second-generation vaccines using live genetically modied parasites, orbacteria or viruses containing Leishmania genes, recombinant or native fractions are knownsince the 1990s. Due to the loss of PAMPs, the use of adjuvants increased vaccine efcacies ofthe puried antigens to 82%, in Phase III dog trials. Recombinant second-generation vaccinesand third-generation DNA vaccines showed average values of parasite load reduction of 68% and59% in laboratory animal models, respectively, but their success in eld trials had not yet beenreported. This review is focused on vaccine candidates that show any efcacy against leishma-niasis and that are already in different phase trials. A lot of interest though was generated inrecent years, by the studies going on in experimental models. The promising candidates maynd a place in the forth coming years. Among them most probably are the multiple-gene DNAvaccines that are stable and do not require cold-chain transportation. In the mean time, second-generation vaccines with native antigens and effective adjuvants are likely to be licensed andused in Public Health control programs in the fore coming 25 years. To date, only three vaccineshave been licensed for use: one live vaccine for humans in Uzbekistan, one killed vaccine forhuman immunotherapy in Brazil and a second-generation vaccine for dog prophylaxis in Brazil. 2008 Published by Elsevier Ltd.

ction ............................................................................................................ 1710ology of leishmaniasis ............................................................................................ 1710eneration vaccines that arrived to clinical assays................................................................. 1712

ndence address: Instituto de Microbiologia, CCS, UFRJ, Avda Carlos Chagas 373, Caixa Postal 68040, 21941-590 Cidade, Ilha do Fundao, Rio de Janeiro, Brazil. Tel.: +55 21 25626742; fax: +55 21 2560 8344x145.

ddress: [email protected].

see front matter 2008 Published by Elsevier Ltd./j.vaccine.2008.01.023ines for leishmaniasis in the foreng 25 years

1710 C.B. Palatnik-de-Sousa

Candidates for second-generation vaccines ............................................................................. 1712Live vaccines....................................................................................................... 1712Vaccines using recombinant viruses and bacteria as delivery vehicles .............................................. 1713

Vaccines based on puried Leishmania antigens......................................................................... 1713Recombinant antigens................................................................................................... 1714Candid .....Vaccine .....Synthe .....Conclu .....Ackn .....Refer .....

Introduc

Leishmaniaby the bitin 88 counthreatenincaused humneous lesioleishmanianeous leisdisease wivoirs. ThepopulationOn the otantroponootransmitteAmerica [1vaccines. Tand dogs isof chemothimmunocomdemiologicdogs [2]. VLAsia), Leishtum (Meditand L. braL. tropicaleishmaniaLeishmanialent vaccinfor prophyl

The rsby ProfessoIsrael, whotheir childrintuitivelygle rst lesin future [ulate uninflesions, inhidden [4]asites wasIsrael andgenerationcrude extrdue to uncHIV and th

paraum credialses ced lee beishmecomn geande offoreuk].ordisignts toe [1e-indAlthccinheyallent thas d

ts ofccessdedIV trparts, gils arringe tre

nolates for third-generation vaccines .............................s based on sand y salivary antigens ..........................tic vaccines ....................................................sions and perspectives .........................................owledgements..................................................ences..........................................................

tion

sis is caused by parasitic protozoa transmittede of female sand y and is currently endemictries, affecting 12 million people worldwide andg 350 million more. Several species of Leishmaniaan diseases that range form self-healing cuta-ns to fatal visceral leishmaniasis (VL), mucosalsis and diffuse cutaneous leishmaniasis. Cuta-hmaniasis (CL) is an antroponotic or zoonoticth wild rodent, canids and marsupials as reser-difculties in control of the wild reservoir

led to the development of human vaccines only.her hand, VL, the most severe disease, is anse in India and Central Africa, and a zoonosisd by domestic dogs in the Mediterranean and]; leading to the search of both, human and doghe need for safe prophylactic vaccines for humanmade greater by the drug resistance and toxicityerapy, the increase of the disease incidence inpromised subjects, and the difculties of epi-

al control based upon sacrice of seropositiveis caused by Leishmania donovani (Africa, India,mania chagasi (America) and Leishmania infan-erranean basin). CL is produced by L. mexicanaziliensis complexes in Americas and L. major,and L. aethiopica in the Old World [1,3]. As allsis are caused by closely phylogenetically relateds species, the development of a unique polyva-e is expected and will be very valuable not onlyaxis but for treatment as well.t vaccine against leishmaniasis was developedr Adler at the Hebrew University of Jerusalem,had observed that mothers of Lebanon exposedens arms to the bite of sand ies because theyknew that the development of a self-healing sin-ion would protect them from the severe disease4]. Therefore, the ancient practice was to inoc-

sons,inoculregistecacy trvaccinreplacfar haved Leman, rantige[79]The uscodingtaining[1316

Accare desubjecvaccinvaccinlenge.the vatest, tcial chagainsformedamounhas suforwarPhaseals) asreasoncontrocompavaccin

Immuected individuals with infectious material fromregions of the body where the scar would be

. After a method for axenic culture of the par-established, leishmanization became usual inRussia and further evoluted to the use of rst-vaccines composed of whole killed parasites oracts. Leishmanization process was discontinuedontrolled long-lasting skin lesions, the spread ofe use of immunosuppressive drugs, ethical rea-

Studies oadvancedcell-mediainfection.experimendogs or hum

Acquirecells [18].CD8+ are m............................................. 1717

............................................. 1718

............................................. 1718

............................................. 1718............................................. 1719............................................. 1719

site persistence, and difculties in the qualityontrol. Its use at present is limited to one vaccinein Uzbekistan and to live challenge in vaccine ef-in humans in Iran [5]. First-generation Leishmaniaomposed of killed parasites [6] have graduallyishmanization. Second-generation vaccines thusen based on the following: live, genetically modi-ania spp. designed to cause abortive infection inbinant bacteria or viruses carrying Leishmania

nes, dened synthetic or recombinant subunitsnative fractions puried from parasites [1012].third-generation vaccines [6] that include genesa protective antigen, cloned into a vector con-aryotic promoter, is the more recent approach

ng to the WHO recommendations, Phase I trialsed to compare vaccinated to placebo treatedassess the immunogenicity and safety of the

7]. Phase IIa trials are designed to check theuced protection against an experimental chal-ough they are needed in order to standardizee dosage, route and schedule prior to the eldare limited by the questionable value of arti-ge. Phase III trials analyse the vaccine efcacy

e natural infection. All these assays are per-ouble-blind randomized control trials with smallindividuals (dozens to thousand). If the vaccinefully passed these steps, the formulation can beto registration and industrialization and used inials larger populations (10,000100,000 individu-of national immunization campaigns. For ethical

ven the benet of the vaccine, no placebo treatede used and the vaccine efcacy is measured bythe incidence of the disease before and after theatment [17].

ogy of leishmaniasisf anti-Leishmania vaccine candidates havein recent years due to the understanding ofted immunological mechanisms for controllingMost current knowledge, however, is based ontal mouse models and cannot be extrapolated toans.

d resistance to leishmaniasis is mediated by TCD4+ lymphocytes are crucial for resistance andore involved in memory than as effector cells.

Vaccines for leishmaniasis in the fore coming 25 years 1711

CD4+ CD25+ regulatory T cells are involved in persistence ofL. major infection [19].

In humans, a correlation between TH1 cell responsesand resistance and healing of cutaneous leishmaniasis wasdescribed with a predominance of cells producing IFN-,while a mixterized muOther studleishmaniapredominale in patieTH1 responpatients [2ease was ddirect correase by LeTNF- wasmixed cytoease by L. cnave andskin test tlevels [27]lead to misis illustratehallmark ono skin tes

Innateral killer88 [29] hainfection ainnate immrecently duninfectedtest.

In the mand antigecell respondogs, andmediated sden and aantigen preclass II maImmunosup expressiCD4+ CD25B-7 molecthe CTLA-4CTLA-4 leaexpressionunresponsimouse modsecretion oasites insidthe role ofT CD4+ cel. In additin liver anSoluble facinvolved inis delipidat

In miceincreases d

because they fail to localize the periarteriolar lymphoidsheath (PALS) [38]. CCL21 and CCL19 expression by gp38+PALS stromal cells is decreased during infection. For thesereasons DC do not migrate properly in spleen nor do theydestroy the amastigotes. The major mechanism underlying

iveted imustu

d TH/6, Cted b- ah th, whst, tse th. In

].ananiad bealitor proba2 cyvelsLeishientbclastedof Ig]. A santlineveryE, atheed aG1an

dogsniner Echtingof t72]redbe date,thantudiatedaccigatiouse

ntiatheb cyhat hcombum led TH1/TH2 response with IL-4 and IL-10 charac-cocutaneous and chronic cutaneous lesions [20].ies show this mixed cytokine prole in cutaneoussis and in natural resistance, with IL-4 and IL-10ting in early infection and a Type-1 immune pro-nts with older lesions [21], or a main exacerbatedse with high levels of IFN- and TNF- in mucosal2]. In VL, no association of IL-4 with active dis-escribed; however sustained levels of IFN- and aelation between increase in IL-10 and active dis-ishmania donovani were detected [23,24] whilesignicantly elevated in cases of PDKL [25]. Thekine pattern has been conrmed in active dis-hagasi [26], though this study used non-adequateasymptomatic controls selected by a Montenegrohat could itself determine an increase in IFN-or, together with the immunouorescent assay,diagnosis of CL. The problem with this approachd by the identication of higher levels of IL-4, thef CL [28], in control individuals [26]. Furthermore,t was performed on the patient group [26].immunity, toll like receptors, as well as natu-cells, IL-1 and myeloid differentiation factorve been identied in early resistance to thend acquired immunity [30]. The contribution ofunity in human visceral leishmaniasis was also

escribed [31]. However, this study also selectedand asymptomatic controls by Montenegro skin

ouse model for VL, Leishmania specic TH2 cellsn presenting cells are involved in suppression of Tses [32]. The DTH response is suppressed in mice,humans and is recovered after cure. Macrophageuppression leads to the increase in parasite bur-ntigen energy, and is linked to either defectivesentation or inhibition of expression of class I andjor histocompatibility complex molecules [28].pression in mice is related to enhanced TGF-

on [33], IL-10, and the possible participation of+ regulatory cells. There are two receptors forules: the CD28 for T cell activation (TH1) andfor termination of T cell activation. Blockade ofds to resistance to infection, suggesting that theof CTLA-4 plays an important role in maintainingveness in CD4+ T cells during chronical VL in theel [34]. The expression of CTLA-4 results in thef TGF-, which promotes the growing of the par-e the macrophage. In tegumentar leishmaniasis,CTLA-4 is paradoxal, dual [35]. In VL, apoptosis ofls is accompanied by a decrease in IL-2 and IFN-ion, apoptosis is detected in inammatory cellsd spleen during infection with L. donovani [36].tors in serum, such as triglycerides, may also beimmunosuppression. These disappeared if serumed [37].infected with L. donovani, the number of DCuring infection but their distribution is altered

defectmedia

Thefor theTH1 an(C57Blmedia12. IFNthroug(iNOS)contrarespondisease[42,43

HumLeishmtion anabnormolutionIFN- pthe THhigh leof theVL patIgG suassociavation[48,49signic

CanshowsIgA, Igers ofobservwith Igdiseasenatedthe ca[67] oconicnance[53,69ufactucouldconjugtitresmost svaccinnant vinvestidiseasedifferetor ofand CPshow tthe reinfantlocalization of DCs is TNF- dependent, IL-10-nhibition of CCR7 expression [38].rine model for CL is considered one of the bestdy of the mechanisms controlling the T helper2 cell balance [3941]. In the resistant strains3H, CBA) resolution of the limited infection isy TH1 cells secreting IFN- in response to IL-ctivates the parasiticide activity of macrophagese synthesis of inducible nitric oxide synthaseich leads to production of nitrogen radicals. Inhe susceptible Balb/c mice develop a main TH2at results in progression of lesions and systemicdraining lymph nodes, CD4+ cells secrete IL-4

kala-azar is characterized by high titers of-specic antibodies appearing soon after infec-fore the development of cellular immunologicalies. The role of these antibodies in disease res-rotection is largely unknown. The TH1 cytokinebly upregulates IgG1 and IgG3 in humans, whiletokines IL-4 and IL-5 stimulate the production ofof IgM, IgE, and IgG isotypes such as IgG4. Analysismania specic Ig isotypes in and IgG subclasses insera revealed elevated levels of IgG, IgM, IgE andses during disease [4447]. Drug resistance waswith a reduction in IgG2 and IgG3. A marked ele-G1, however, was observed in all these patientsuccessful cure corresponded with a decline, mosty, in the levels of IgE, IgG4, and IgG1 [45,46,48].visceral leishmaniasis (CVL), on the other hand,similar characteristics to the human disease [50].nd IgM responses have been shown to be mark-disease [5153]. IgG antibody increase is alsond correlated with symptomatology [50,54,55]subtype associated to susceptibility and severed IgG2 increased in natural resistant or vacci-[15,5666]. IgG2 subtype also predominates inresponse to vaccination against Lyme diseaseinococcus granulosus infection [68]. However,results have been reported, with the predomi-he IgG2 subtype observed in symptomatic dogs. Since all investigations used the same man-anti-dog conjugates, the discrepancy in resultsue to difference in titration mainly of the IgG2which systematically shows signicantly higherthe IgG1 sera [59]. On the other hand, while

es defending the IgG2 predominance deal withdogs and used puried [59,63,66] or recombi-

ne antigens for diagnosis [15,57,62,64,65], thens that associate IgG2 to symptomatology andd promastigote crude antigens [15,6971]. Thel afnity of the antigen could be another fac-discrepancy. In dogs vaccinated with the CPastein proteinases, the work of Rafati et al. [15]igher IgG2 than IgG1 titers are detected againstinant antigens but not against the Leishmania

ysate. While the recombinant antigen interacts


with a dened fraction of sera antibodies, the total lysateinteracts with the whole plethora of antibodies directedagainst the total parasite, masking or diluting the responseagainst the recombinant protein, which is not a majorLeishmania antigen. Another factor of discrepancy couldbe the useand anti-Igexperiencedifcultiesantibodies[72]. Unforavailable.

When ceasymptomaand TNF-marily TH1asymptomaamastigoteCD8+ incremolecules[75]. In coresponse, wfollowing adecrease inLeishmaniathe co-stimexpressionmal more sand IL-4 haasymptomaease is relagene NRAMof Leishmato the infeMHCII allelinfection wciated to s

First-genclinical a

The humanvaccines,started inin Table 1.with no prwere develdirected ag

Since thtively lowlarge. In Aautoclavedspecies [86claved L. mkilled paraform of stetries thatwhere a coof de Luca eprotozoa pof the para

ertheless, the LPG complex resists autoclaving [94] and hasbeen implicated in immunogenicity and immunosuppression[95] in the mice model.

While some protocols have used no adjuvant [86,87,96](Table 1), most prophylactic vaccines used BCG for human

] anmo

es isate6,89ed a91]t incadorly d1). Oof Vity arsvisc

7] buse I tle [ed athest humTablnd inthatt inso re0]. Ben re

idat

acc

ategknosuc[101orter

tove inntro insingpes Ieamthorleconpartparaive tnt tochale [luabof different batches of the polyclonal anti-IgG2G1 antisera, which show low repeatibility (ourand Dr MJ Day personal communication). Thesestimulated Dr MJ Day to obtain dog monoclonalthat recognize four different IgG subfractionstunately, these antibodies are not commercially

llular immune response is present, dogs appeartic and IDR positive, with higher levels of IL-2and a mixed TH1/TH2 response involving pri-mediated by IL-12, IL-18 and IFN-Y [73]. In

tic dogs, macrophages are capable of killings through the nitric oxide route [74], and aase in peripheral lymphocytes, MHC class IIand receptors CD45RA e CD45RB are also foundntrast, symptomatic dogs show a failed cellularith no IDR or lymphocyte proliferation and the

dditional characteristics: decrease in monocytes;CD8+ [75] CD4+ and CD21+ B lymphocytes, either-specic or not; antibody increase; deciency ofulatory response; decrease of IFN-; and reducedof MHC class II molecules, which renders the ani-usceptible [66,7580]. The involvement of IL-10s been shown in both symptomatic [81,82] andtic dogs [73]. The susceptibility of the canine dis-ted to a genetic polymorphism. Mutations on theP1, which controls the intralisossomal replicationnia, determine the susceptibility or resistancection [83]. Moreover, a relationship between thees DLA (DRB1, DQA1, DQB1) and the course ofas described with the genotype DLA-DRB1 asso-evere disease [84].

eration vaccines that arrived tossays

efcacy trials involving the so called killed-composed of crude total parasite antigens,Brazil in the 1940s [85] and are summarizedThese Phase III efcacy trials were performedevious Phase I or Phase IIa assays. Most trialsoped against CL [8690] while only one [91] wasainst VL.e average vaccine efcacy value (VE) was rela-(54.38%), the number of individuals tested wasmerica, most vaccines used the L. amazonensislysate (ALA) [87,88] or a mixture of autochtonous], while all studies in the Old World used the auto-ajor antigen (ALM) (Table 1). Autoclaving of thesite vaccine was introduced [8992] as the bestrilization and preservation of vaccines in coun-have a rudimentary Biotechnology industry andld-chain for distribution is not feasible. The workt al. [93] proved however, that, as expected for a

arasite, autoclaving destroys most of the proteinssite and the vaccine loose immunogenicity. Nev-

[8891The

vaccincandidity [8obtain[86,89did noin Ecuprobab(Table43.3%mortal

ThecanineIran [9of Phaavailabobtainmore,agains[100] (vant aagreeapy buorder t[96,10has be[96].

Cand

Live v

This cied,genes,thasetranspenoughabortiis to isettesexpresof Hersine dThe auwhereensureber ofuse ofeffectresistaof liveceptabthe vad dog assays [97,98] (Table 1).st striking aspect of the rst-generation humanthat a leishmanin skin test (LST) is used for

selection and for conrmation of immunogenic-91]. Whenever the LST is performed, VE is

mong the individuals whose skin tested positive, whereas no efcacy is detected in assays thatlude an LST [87]. One exception is a study done[88] that showed positive LST but not efcacy,

ue to the use of a lower number of vaccine dosesn the other hand, a study in Sudan that achievedE against VL is impressive considering the highnd virulence of kala-azar there [91].t-generation vaccines were also used againsteral leishmaniasis (CVL) inducing protection int failing to do so in Brazil [98] (Table 1). Resultsrials of rst-generation human vaccines are also92,99], and they show that immunogenicity isfter a single vaccine dose [92] (Table 1). Further-e vaccines have been used for immunotherapyan CL with success in Brazil [96] and Venezuela

e 1). Although differing in the use of BCG adju-the number of doses, the authors of both studiesthe vaccine should not be used as monother-tead, combined with the usual chemotherapy, induce toxic and very painful antimonial treatmentased on these results, a rst-generation vaccinegistered as adjunct to antimony therapy in Brazil

es for second-generation vaccines

ines

ory includes vaccines made of genetically mod-ck-out Leishmania spp., which lack essentialh as dyhydrofolate-reductase thymidilate syn-], cystein-proteinase [102,103] or biopterin[104]. These parasites undergo a short life cycle,generate a specic immune response causingfection and no disease in man. Another approachduce in the Leishmania genome suicidal cas-cluding drug sensitive genes, such as L. majorthe ganciclovir-sensitive thymidine kinase genevirus [105] or the Saccharomyces cerevisae cyto-inase gene sensitive to 5-uorocytosine [106].s of these studies have suggested that in Iran,ishmanization is used as vaccine challenge tosistency of infection rates and reduce the num-icipants and duration of the assay [5,106], thesites with suicidal cassettes would guaranteereatment of non-resolving lesions or of infectionsthe usual chemotherapy [106]. However, the usellenge for humans is considered ethically unac-107], and an articial challenge cannot providele information obtained by exposure to natural


Table 1 Vaccine efcacies of rst-generation vaccines (since 1941)

Disease N Leishmania Country Adjuvant Positive LST (%) VE (%) Treatment Doses Ref.

CL 1312 Five killed Brazil none 51.5 67.3 Prophylaxis 3 [86]CLCLCLCLVL

CVLCVL

CLCL

CLCL

infection,saliva [108ticipants nvaccine efcines withones formu

Vaccinesdelivery v

Another aplive recomparasite ancarrier andpractical aL. major Ggen, clonedBCG [110];agellar prtid) antige[112]. Examexpressingwhich protexpressingto the recin prime b[114] and d

Vaccinesantigens

A further athe puriephoglycanbut becausnever advrst PhaseDunan et apreparatioachieved acinated gro

ffect candog

ls: t20] vonov[12

rmulIIIIil [1mun

rialsand3%)R. Non wpondn theyearlaceng toThisitan

easeL-saandoth2597 ALA Columbia none Nd1506 ALA Ecuador BCG 74.43637 ALM Iran BCG 9.852543 ALM Iran BCG 36.22306 ALM Sudan BCG 30

349 ALM Iran AlOH+BCG Nd1763 Lb Brazil BCG Nd

900 ALA Venezuela BCG +/ 36.536 ALM Sudan BCG 61.6

542 Five killed Brazil Nd94 Live and killed Venezuela BCG Nd

which is modulated by components of sand y]. On the other hand, the large number of par-eeded for a eld trial, due to the low expectedcacy [17], can be reduced only by the use of vac-higher efcacies [17], such as second-generationlated with potent adjuvants.

using recombinant viruses and bacteria asehicles

proach to second-generation vaccines is to usebinant bacteria or virus expressing Leishmaniatigen; the bacteria and virus serve as expressionadjuvant system. These vaccines have limited

pplication. Examples of bacteria vaccines are:P63 surface protease, a major Leishmania anti-in Salmonella thypymurium mutant [7,109] or inthe LCR1 L. chagasi antigen (similar to a T. cruziotein) in BCG [111] and the KMP-11 (kinetoplas-n in attenuated tachyzoites of Toxoplasma gondiiples of vaccines based on virus are Vaccinia virusthe G46/M-2/PSA-2 promastigote surface proteinects against L. amazonensis [113]; or Vacciniathe L infantum LACK antigen (parasite analogueeptor for activated mammalian kinase C) whichoost vaccination, protects mice against L. major

while eagains

TwoIII tria[119,1of L. dligand)was foPhasein Brazand imeld t4 obitsdogs (3and PCinfecticorres[11]. Iand 2(25%) pspondi[118].concomthe disthe FMdisease

The

ogs against L. infantum infection [64].

based on puried Leishmania

pproach to second-generation vaccines includesd Leishmania sub-fractions. Proteins or lipophos-have been used to assess their immunogenicity,e of difculties in their mass production, theyanced to Phase IIa or Phase III trials. In theIII trial with a second-generation dog vaccine,l. [115], using a semi-puried lyophilized proteinn from L. infantum (9467 kDa), paradoxicallysignicantly higher rate of infection in the vac-up than in the control group. This vaccine then,

of the 54 kprotected dtion. Parasplacebo trvaccinatedfurther perdogs in Souof infectio(12/175) indog showinwas conrmaspirates csis [120]. I[117,118],tion [120],0 Prophylaxis 3 [87]0 Prophylaxis 2 [88]56.7 Prophylaxis 1 [89]35.5 Prophylaxis 1 [90]43.3 Prophylaxis 2 [91]

69.3 Prophylaxis 1 [97]0 Prophylaxis 3 [98]

nd Phase I 3 [99]nd Phase I 1 [92]

76.0 Immunotherapy Monthly [96]95.7 Immunotherapy 3 [100]

tive in murine models, did not induce protectionine kala-azar in the eld [115].vaccines achieved successful results in Phase

he FML-saponin [116118] and the LiESAp-MDPaccines. The glycoproteic enriched preparationani promastigotes, named FML (FucoseMannose], antigenic for human [121] and dogs [122],ated with Quillaja saponaria saponin and passedtrials to became the Leishmune licensed vaccine23]. FML was immunogenic, immunoprophylacticotherapeutic, in mice and hamsters, and dogs[116118,124126]. In the rst Phase III assay,6 symptomatic cases among 30 placebo treated

were detected and conrmed by parasite analysiso obits were detected among vaccines (n = 36) andas conrmed in 3 olygosymptomatic dogs (8.33%)ing to 92% of protection and 76% vaccine efcacysecond assay, the infective pressure was higher

s after vaccination, obits were detected in 8/33bo treated and 1/20 (5%) vaccinated dogs, corre-95% of protective effect and 80% vaccine efcacyprotection lasted for at least 3.5 years and wast with the reduction of the human incidence ofin the area [118]. Noteworthy, the VE values forponin vaccine showed protection against severeobits due to visceral leishmaniasis.

er second-generation vaccine, LiESAp, composed

Da excreted protein of L. infantum plus MDP,ogs in a kennel assay against L. infantum infec-ites were detected in the bone marrow of 3/3eated controls, while they were absent in 0/3dogs [119]. A double-blind randomized trial wasformed with LiESAp +MDP in naturally exposedthern France [120]. After 2 years, the incidencen was 0.61% (1/165) in vaccinees versus 6.86%control dogs, corresponding to a 92% VE. In anyg clinical and/or serological evidence, infectioned by the presence of parasites in bone marrowultured in NNN media and also by PCR analy-n contrast to results of the FML-saponin vaccineLiESAp vaccine induced protection against infec-but not against severe disease or death by VL.


No obits at all were described in the 2 years LiESAp assay[120], reecting the lower infective pressure of the endemicregion. As explained by WHO guidelines [17], conrmationof infection by very sensitive method such as PCR or culture[119,120] represents a very early end-point of infection,while kala-distant endcacies of twinfective pspite of thoage of VE o(IC 95%, 71

The FMLcine candion Second-in May 20centration,[66] or expgasi. The sulevels inthat the FMThe adjuvand aldehysaponariarst seconveterinarydogs vaccitious, as inof skin-parand lymphblocking vaprotection,80% inhibitmastigote bof in vivo sthe epidemerable, safdogs assays

Recentlwas elucidattached ta hydrophattached rto the indresponse,C4 is invoicking theprotectiveprotectionto the actresponse atdendritic ctors therebchagasi [13

Recombi

The lastthe usesively testrecombina

[7,16,109,110,133142], in combination [143145], or aspolyproteins or chimeras [65,144,146151]. In order todevelop protection, most of them needed to be formulatedwith adjuvants [17,65,135,137141,143145,147151], ordelivered by bacteria [7,109,110,142,146], with the

ionmosogen09,1

advaaysnicas Phseds (Tacondof pin mosursideor D

itedted csultssizeredtimele.

says8.32inteity bA (tindugainnt ILromormproeishith M[14

ime,gasit Lessiondogsith 1d toeers

Peish1hsp8icalts wted1 (hbinaericntse hiotecazar obits and severe clinical cases [117,118] are-points of infection. A comparison between ef-o vaccines should only be made using the same

ressure and the same end-point targets [17]. Inse differences, it is worth to note that the aver-f second-generation vaccines together is 82.67%.1394.21).-vaccine is considered a second-generation vac-date and it was featured at the fourth MeetingGeneration Leishmania vaccines held in Merida01 [127]. When used at double adjuvant con-it is also immunotherapeutic for dogs naturallyerimentally infected [63] with Leishmania cha-stained proportions of CD4 and CD21 lymphocyteblood of vaccinated animals [63,66] indicateL-vaccine reduces dog infectivity to sand ies.

ants used in the FML-vaccine are the QS21de-containing deacylated saponins of Quillaja

[128,129]. In 2004, the FML-vaccine became thed-generation vaccine licensed for prophylacticuse, under the name of Leishmune [55]. Exposednated with Leishmune proved to be not infec-dicated by the complete absence of clinical signs,asites based on the negative PCR results of bloodnode samples [55]. Leishmune is a transmissionccine [130] for the IgG2 antibodies associated togenerated in vaccinated dogs, [59] caused an

ion of the in vitro L. donovani and L. chagasi pro-inding to sand y midguts and a 79.3% inhibitionand y infection, helping in the interruption ofics [130]. Leishmune vaccine proved to be tol-e, and highly immunogenic [123] in a recent 600in the eld.

y, the mechanism of action of the QS21 saponinated. QS21 contains two carbohydrate chainso a triterpene nucleus C3 and C28, besidesobic moiety which is acylated to a C28 sugaresidue. The QS21 hydrophobic moiety is relateduction of the CTL CD8+ protective lymphocytewhile the aldehyde group present in triterpenelved in direct T lymphocyte stimulation, mim-B7-1 co-stimulatory molecule to induce the TH1response [131]. Preliminary results suggest thatinduced by the FML-QS21 vaccine is also relatedivation of a bradikinin mediated inammatorythe site of injection, which stimulates immatureells through their B1R and B2R surface recep-y triggering a TH1 response against Leishmania2].

nant antigens

approach in second-generation vaccines isof recombinant proteins that were inten-ed since the 1990s (Table 2). The Leishmaniant vaccine candidates were assayed alone

exceptWhileimmun[7,17,1themnel asspre-clito dogwere uspecieand seresultslationno expwe conbinantof limuntreathe reof thecompalatestavailabthe as95%, 5

AnimmunThe TSstresskeys aadjuvamay plong-tefusionLeIF (Ltion wand VLAdjuPrL. chaprevenprogreThesecine wshowevoluntand inthe Le83 (Lbto clinpatienprotecHASPBin coma chimfragmeand thtial prof LeIF [134] and HASPB1 proteins [133].t recombinant proteins were assayed for theiricity and protective potential in mice models10,133137,139143,149,150], only a few ofnced to monkey trials for CL [143,138], to ken-in dog model against VL [65,144146,151], or tol studies in humans [147,148]. None has advancedase III trials (Table 2). The recombinant proteinsagainst all forms of the disease and all parasiteble 2). In contrast to the results for rst (Table 1)-generation vaccines with native antigens, therotection developed by each recombinant formu-ice do not allow determination of VE values, sincee to natural infection occurred. In this review,red for comparison of protection due to recom-NA vaccines, the reported reduction of LDU ordilution values in vaccinated mice compared toontrols. In the specic case of CL mouse models,were obtained from each report as the percentof the footpad lesion of the vaccinated animalsto that of the untreated saline control, at thepoint when data for the saline control was still

The mean average of parasite reduction for allwith recombinant antigens (Table 2) is 68.02% (IC77.71).resting approach is the induction of protectivey a polyprotein vaccine formulation (Table 2).hiol-specic antioxidant) and LmSTI1 (L. majorcible protein 1) are protective for mice and mon-st CL [143], although the use of recombinant-12 is not recommended at present because itte immune disorders [152] and fail to induce

immunity [135]. The multicomponent Leish-111ftein containing the antigens TSA, LmSTI1 andmania elongation initiation factor), in formula-PL-SE9 and squalene, protect mice against CL9,150,153] but, in combination with MPL-SE orwas only immunogenic in dogs challenged with

[65] and L. infantum (MML) [144], and failed toishmania infantum natural infection, or diseasein dogs in an open kennel trial [151] (Table 2).[151] received two courses of three-dose vac-year interval. Nevertheless, Leish111f +MPL-SE

be safe, immunogenic, and reactogenic in healthyin the US and in patients of CL and ML in Brazilru, respectively [148], while vaccination with11f components, Leishmania heat shock protein3) and GM-CSF, combined to chemotherapy, ledimprovement and complete cure of six humanith MCL [147]. Furthermore, the H1 histone thatmice [137] and monkeys against CL [138], theydrophilic acylated surface protein B1) or bothtion with Montanide [144], and the protein Q,antigen composed of the genetic fusion of veof the acidic ribosomal protein Lip2a, Lip2b, P0stone H2A used with BCG [146] developed par-tion against CVL in dogs against infection and at

Vaccinesfor

leishmaniasis

inthe

forecom

ing25

years1715

Table 2 Second-generation recombinant antigens (1990s)

Antigen Adj./delivery system Model Disease Leishmania Parasite reduction (%) Phase Ref.

GP63 S. thyphimurium Mice CL L major 6778 IIIa [7,109]GP63 BCG Mice CL L. mexicana 6883 IIIa [110]GP63 BCG Mice CL L. major 42 IIIa [110]HASPB1 Mice CL L. donovani 60 IIIa [133]LeIF Mice CL L. major 57 IIIa [134]TSA and LmSTI1 IL12 Mice CL L. major 9899 IIIa [143]

IL12 and alum Monkey CL L. major 97 IIa [143]Leish 111 = LeIF + TSA + LmSTI1 MPL-SE/RIBI 529-SE Mice CL L. major 5367 IIIa [149]Leish 111 = LeIF + TSA + LmSTI1 MPL-SE/Adjuprime Mice VL L. infantum 99.6 IIIa [150]Leish 111 = LeIF + TSA + LmSTI1 MPL-SE/Adjuprime Dog CVL L. chagasi I [65]Leish 111 = LeIF + TSA + LmSTI1 MPL-SE/Adjuprime Dog CVL L. infantum Open kennel [151]MML MPL-SE Dog CVL L. infantum IIa [144]LeIF + TSA + LmSTI1 + Lbhsp83 GM-CSF Human MCL L brasiliensis Cure ita [147]Leish 111 = LeIF + TSA + LmSTI1 MPL-SE Human CL Safe I [148]LACK IL-12 Mice CL L. major 53 IIIa [135]LACK Mice CL L. amazonensis 0 IIIa [136]H1 IFA and IL-12 Mice CL L. major 6453 IIIa [137]H1 Montanide Monkey CL L. major 37.5 IIa [138]H1, HASPB1, H1 +HASPB1 Montanide Dog CVL L. infantum 5/84/8 IIa [144]CPb Poloxamer 407 Mice CL L. major. 41 IIIa [139]CPb +CPa QuilA + IL-12 Dog CVL L. infantum 0 IIIa [145]PSA-2 C. parvum/ISCOMs Mice CL L. major 0 IIIa [140]LCR1 CFA Mice VL L. chagasi 67 IIIa [141]NH36 Q. saponaria saponin Mice VL L. chagasi 79 IIIa [17]A2 P. acnes Mice VL L. donovani 88 IIIa [142]Protein Q chimera BCG Dog CVL L. infantum 5090 IIIa [146]a it = Immunotherapy.

1716C.B.

Palatnik-de-Sousa

Table 3 Candidates for third-generation-DNA vaccines against leishmaniasis (1990s)

Antigen Adj./delivery system Model Disease Leishmania g Virulence Challenge Parasitereduction (%)

Phase Ref.

GP63 pCMV Mice CL L. major 100 NV 1 106 pro 57 IIIa [14]LACK pcDNA3 Mice CL L. major 100 NV 1 105 pro 42 IIIa [135]LACK pcDNA3 Mice CL L. major 100 NV 1 105 pro 56 IIIa [9]LACK pcDNA3 IL-12 Mice VL L. donovani 100 NV 1 106 pro 0 IIIa [162]HPB-LACKa pcDNA3-Vaccinia Virus Mice CL L. major 100 NV 5 104 pro 68 IIIa [114]HPB-LACKa pCIneo-Vaccinia Virus Dogs CVL L. infantum 100 V 1 108 pro 3/5 IIIa [64]LACK pCMV3ISS Mice CL L. major 50 V 5 103 ama 26 IIIa [156]GP63 pCMV3ISS Mice CL L. major 50 V 5 103 ama 21 IIIaP20 pCMV3ISS Mice CL L. major 50 V 5 103 ama 20 IIIaPSA2 pCMV3ISS Mice CL L. major 50 V 5 103 ama 26 IIIaTSA pcDNA3 Mice CL L. major 100 V 1 104 ama 84 IIIa [157]LmSTI1 pcDNA3 Mice CL L. major 100 V 1 104 ama 32 IIIaTSA + LmSTI1 pcDNA3 Mice CL L. major 100 V 1 104 ama 90 IIIaH2A +H2B +H3 +H4 pcDNA3 Mice CL L. major 200 NV 5 104 pro 74 IIIa [158]Cpa +CPb pCB6 Mice CL L. major 200 NV 1 106 pro 38 IIIa [159]HPB CPa and CPba pCB6+ Montanide 720

and CPGDog CVL L. infantum 200 NV 5 106 pro 8/10 IIIa [15]

KMPII, TRYP, LACK and GP63 pMOK Dog CVL L. infantum 800 V 5 107 pro 0/12 IIIa [51]P4 pcDNA3 Mice CL L. amazonensis 100 V 2 105 pro 25 IIIa [168]P4 pcDNA3 +HSP70 Mice CL L. amazonensis 100 V 2 105 pro 60 IIIaP4 pcDNA3 + IL-12 Mice CL L. amazonensis 100 V 2 105 pro 99 IIIaA2 pcDNA3 Mice VL L. donovani 100 NV 2 108 pro 97 IIIa [160]ORFF pcDNA3 Mice VL L. donovani 100 NV 2 108 pro 59 IIIa [163]NH36 VR1012 Mice VL L. chagasi 100 V 2 108 ama 88 IIIa [16]NH36 VR1012 Mice CL L. mexicana 100 V 1 106 pro 65 IIIa [16]NH36 VR1012 Mice CL L. amazonensis 100 V 1 106 pro 81 IIIa [164]NH36 VR1012 Dog VL L. chagasi 750 CVL 7 108 ama 4/6 IIIa [165]KMP11 pCMV-LICK Hamster VL L. donovani 100 V 1 106 pro 85 IIIa [166]papLe22 pcDNA3.1 Hamster VL L. infantum 100 V 1 107 ama 50 IIIa [161]NH36 VR1012 Mice VL L. chagasi 100 V 2 108 ama 91 IIIa [167]a HPB =Heterologous prime-boost.


the clinical level. Finally, poloxamer 407 adjuvant CPb, butnot CPa was able to protect mice against CL [139] whereasboth in combination with IL-12 and QuilA, failed to protectdogs from L. infantum infection, probably due to the lowconcentration of the adjuvants (50g in each vaccine dose)[145].

As singlethe L. maplasmid DNnant PSA2or with C.tion againsthe amastition to VL.an essentianucleoside[16], sincebiosynthescomplex [1CVL [55]. Tlaja saponchagasi anresponse [1

In 1996studies ofthat time.Leishmaniatrends in Leantigens intion in navprotectedNo assays wof reductioproblem wtransportasevere procine produ

Candidat

Comparedare much mcost of proand exibistruct. Thepotent immtion of innasequencesthe host, leteins for lo

The moviously as137,1561[9,14,16,13of genes(HPB), whfollowed[15] or atein [64,1in only twobserved in

[14,16,64,135,156,159,161,164167], with the exceptionof pMOK in a dog assay [51], and of pcDNA3 in a miceassay [162]. Most trials were performed in mice againstCL and VL [9,14,16,114,135,156160,162168], and somein the hamster against VL [166,161] and in dogs against

5,51,64,165]. All the studies involved articial chal-(Phase IIIa) with agents of the New and Old Worldaniasis (Table 3). The most striking characteristic ofA vaccine studies is that about a half of them usedallenge non-virulent (NV) Leishmania strains, grownid culture media [9,14,15,114,135,158160,162,163]a low number of parasites [9,114,135,158], while

st used virulent [V] parasites isolated from infecteds [16,51,64,156,157,161,164168] (Table 3). Thisgeneity of protocols determined a high variation ofts of reduction of parasite load, with a mean average4% (IC 95% 47.7570.73).diffdifresuthis

pe of2 [16isogusedue

otecof Ne ney.vac

se Isedy, avelo

s goiLeIF,mo

rth cesteice,

tedni [A2 ginfe3) p4]. TIL-12o Ig

4nes

idate

TSA,

CPb1candidate recombinant vaccines tested in mice,jor LACK with IL-12 was less efcient than theA encoding LACK [135,136,154], the recombi-antigen (parasite surface antigen 2) in ISCOMsparvum, induced a TH1 response but not protec-t CL [136,140], while the LCR1 protein [141] andgote-specic A2 antigen [142] conferred protec-The nucleoside hydrolase of L. donovani (NH36) isl enzyme that releases DNA bases from importeds, allowing the parasite to construct its own DNALeishmania parasites lack the de novo purine

is pathway. NH36 is the main antigen of the FML55] that constitutes the Leishmune vaccine forhe recombinant NH36 in combination with Quil-aria Molina saponin protected mice against L.d L. mexicana infection (Table 2) through a TH16]., the WHO-TDR program organized comparativeseveral leading recombinant proteins known atIn 2001, the fourth Meeting on Second-Generationvaccines evaluated these studies and discussedishmania vaccine development [127]. While mostduced lymphocyte proliferation and IFN secre-emice and human patients, the only antigens thatmice against CL were the MIX, LACK, 4H6 and FPA.ere performed against VL and no quanticationn of parasitic load was done. The main detectedas the lack of stability and potency during thetion of the antigens [127], suggesting potentiallyblems with future scale-up of recombinant vac-ction.

es for third-generation vaccines

to recombinant protein vaccines, DNA vaccinesore stable and have the advantage of their lowduction, no need of cold chain for distribution,lity of combining multiple genes in a simple con-mechanism by which DNA vaccination generatesune responses appears to be through the activa-te immune responses by the non-methylated CpGof bacteria and to the intense replication withinading to the expression of the recombinant pro-nger periods.st-studied antigens (Table 3) were those pre-sayed as recombinant proteins [14,16,51,135,61]. Most of them were tested as single vaccines5,156,157,160167], and some, as combination

[156159,168] or as heterologous prime-boostich involves an injection of the DNA vaccineby an injection of the recombinant proteinVaccinia virus expressing the recombinant pro-14]. Adjuvants were added to formulationso studies [15,162] (Table 3). Protection wasvaccines using all the tested plasmids (Table 3)

CVL [1lengeleishmthe DNfor chin liquand atthe reanimalheteropercenof 59.2

Thefor theversialrefutethe tyof IL-1tion inthe motectionand pr750ggest thefcac

DNAno Phais focuefcaccine destudieLACK,are thethe fobeen t

In mprotecdonovaand PSmajor(TableCVL [6 andIgG2 t

Tablevacci

Cand

LACKLeIF,H1Cpa +KMP1NH36erent codied antigens could be the responsibleferent degrees of protection. However, contro-lts for the same antigen [9,64,114,135,156,162]idea (Table 3). Protection did not correlate toexpression vector (p > 0.05), or to the addition2,168]. It seems to be easier to achieve protec-enic mice than in dogs [15,51,64,165]. Most ofinvestigations used 100g of plasmid, while pro-to CP proteinase vaccine required 200g [159],

tion of dogs against CVL was achieved by usingH36 plasmid DNA vaccine [165]. These results sug-ed for higher plasmid concentrations to achieve

cines are indeed protective, although to dateII trial data are available. Although this reviewon vaccine candidates that already show anylot of interest was though generated in vac-pment against leishmaniasis in recent years, withng on in the labs on experimental models. TheTSA, LmSTI1, H1, CpA +CpB, KMP11 and NH36

st promising candidates that may nd a place inoming years (Table 4), since they have alreadyd in more animal models.LACK DNA induced a TH1 response [9,156] that

against infection by L. major [9,135], but not L.162]. Even truncated portions of the LACK geneene were superior to GP63 and p20 against L.ction [156], and immunization with HPB-LACKrotected mice against VL [114] and dogs fromhe immunization resulted in an increase in IFN-expression, lymphocyte proliferative response,

G1 ratio while it led to decreases in clinical

Promising candidates for third-generation

antigen Tested animal model

Mice and dogsLmSTI1 Mice, monkey, human

Mice, monkeyMice, dogsMice, hamsters, dogsMice, dogs


Table 5 Efcacy of vaccines for leishmaniasis developed so far (19412007)

Generation Adjuvant Phase Average protection IC 95% Evaluation Model

1st Yes/no III 54.38 39.8468.92 VE Human, dog2nd native 71.1294.20 VE Dog2nd recom 58.3277.71 Parasite load Mice, monkey, dog3rd 47.7

symptoms,expression

VaccinaDNA vaccin[157], protThe digenethe latter i

InjectioH3 and H4)infection t

While pcombinatiomajor [159720 and CPincrease inliferation,or clinical slow infectivaccinatedPCR negatision couldtwo untrea

Vaccinaagainst VL[166], whilLACK, anddogs agains

The NH3by L. chagaindicatingprophylactProtectionby the recoand it is rare characdog Phasethe NH36 vobserved soapeutic PSAskin lesionparasite lochagasi [16

The comferent gensummarizewith or wisecond-gensignicantthese twoarise fromthe target

the-genbinam t

sultsion oudie

nes

onces tesigns thbilitcase15 ad su[175

eti

repplayy epsyntas sspechrouproaes ag

lusi

e ofw knP ree pof scieies,creasaniaYes III 82.66binant Yes IIa 68.02

No IIa 59.24

number of parasites in target tissues, and IL-4.tion of mice either with the TSA or the LmSTI1es, or with both as a tandem digene constructected against CL through a CD4 +TH1 response.and the TSA gene were the most protective, withnvolving a CD8+ response [157].n of a mixture of four histone plasmids (H2A, H2B,in Balb/c mice also protected against L. major

hrough a TH1 response [158].lasmid CPa and CPb on their own did not, then of both conferred protection on mice against L.], and a prime boost vaccination with MontanideG protected dogs against CVL, as evidenced by theIgG2 specic antibody synthesis, lymphocyte pro-IFN/IL10 secretion, and DTH response. No deathigns were reported [15], probably due to the veryve challenge (5 106 promastigotes). Eight of 10dogs were considered protected based on theirve results [15]; however, no signicant conclu-be drawn from the study, because it used onlyted control dogs.tion with 200g of KMP11 protected hamstersthrough a mixed cytokine TH1/TH2 response

e a cocktail of plasmid DNA encoding KMPII, TRYP,GP63 (200g of each plasmid) did not protectt L. infantum virulent challenge [51].6 DNA vaccine protected mice against infectionsi, L. mexicana [16], and L. amazonensis [164],its potential usefulness in a bivalent immuno-ic vaccine for the control of both endemics.by the DNA vaccine was higher than that inducedmbinant NH36 or the FML antigen plus saponin,elated to IFN--producing CD4+ T cells, whichteristic of a TH1 type immune response [16]. AI-IIa study on the immunoprophylactic effect ofaccine is in progress, and protection has beenfar in 4/6 vaccinated dogs [165]. While the ther-/GP46 DNA vaccine caused reduction of L. major

s in mice [169], the NH36 DNA vaccine reducesad and increases survival of mice infected with L.7].parison of the efcacies achieved by the dif-erations of vaccines against leishmaniasis isd in Table 5. While the rst-generation vaccines,

whilesecondrecoment frothe rereducttory st

Vacci

Basedenhanbeen dantigethe viais thethe SPinducemajor

Synth

Recentmightidentiffuturetein wwhichcells tthis apvaccin

Conc

In spitthe slothe GMest, thlack ocountrthe inleishmthout adjuvant, display a relatively low VE, theeration vaccines with native antigens show aincrease in VE. The VE results derived fromgroups of vaccines are very robust, since theyeld trials with exposure to natural challenge inspecies (human and dogs). On the other hand,

ous use ofdelay in thHealth. Ththe fore cocomposedadjuvants.570.73 Parasite load Mice, dog

protection induced by the recombinant proteineration vaccines are slightly greater, neither thent nor the DNA vaccines are signicantly differ-he rst-generation ones. It is worth noting thatof these last two groups are calculated based onf parasite load achieved mainly in mice labora-s.

based on sand y salivary antigens

the observations that the saliva of sand ieshe infectivity of pathogens [170], vaccines havened against components of saliva or insect gutat can protect from infection [171] and decreasey and reproducibility of the insect [172,173]. Thisof the protein MAX or MAXADILAN [174] and of

ntigen obtained from Phlebotomus papatasi thatbstantial resistance in mice to infection by L.].

c vaccines

orts suggest that CD8+ in addition to CD4+ T cellsa role in defence and cure of leishmaniasis. Toitopes recognized by CD8+ T cells to be used inhetic vaccines, the sequence of the KMP11 pro-canned and thirty nonapeptides were identiedically trigger IFN- secretion by human CD8+gh the MHC class system [176]. In the future,ch will allow the development of synthetic T cellainst leishmaniasis.

ons and perspectives

the many genes identied as vaccine candidates,owledge transfer from the laboratory to industry,gulations that dampen down the industrial inter-orly developed biotechnology industry and thentists in regulatory agencies of underdevelopedthe ethical constraints on research in animals anding dog-chemotherapy in Europe, where humansis is less frequent, contribute to the continu-

rst-generation and live vaccines, and to thee arrival of combined DNA vaccines to Publice prediction is that vaccines for leishmaniasis inming 25 years will be of second-generation type,of complex native antigens and well developed


Acknowledgements

This work was nancially supported by Conselho Nacionalde Desenvolvimento Cientco e Tecnologico (CNPq) (Edi-tal Universal and productitivity fellowship), Fundacaode Ampar(FAPERJ-PRFAPERJ-INF

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[148] Gdddc

[149] CncLnIn

[150] C1vIm

[151] GRlenin

[152] NHt2

[153] SCmf

[154] MeS

[155] Pvc1

[156] Acd2

[157] CRlemIm

[158] IbVna2

[159] RtwV

[160] GLDr

[161] FJtin

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