FML vaccine against canine visceral leishmaniasis: from second-generation to synthetic vaccine

Review

10.1586/14760584.7.6.833 © 2008 Expert Reviews Ltd ISSN 1476-0584 833www.expert-reviews.com

FML vaccine against canine visceral leishmaniasis: from second-generation to synthetic vaccineExpert Rev. Vaccines 7(6), 833–851 (2008)

Clarisa B Palatnik-de-Sousa†, André de Figueiredo Barbosa, Sandra Maria Oliveira, Dirlei Nico, Robson Ronney Bernardo, Wania R Santos, Mauricio M Rodrigues, Irene Soares and Gulnara P Borja-Cabrera†Author for correspondenceInstituto de Microbiologia, CCS, UFRJ, Avda Carlos Chagas 373, Caixa Postal 68040, 21941-590 Cidade Universitária, Ilha do Fundão, Rio de Janeiro, BrazilTel.: +55 21 2562 6742 ext. 145Fax: +55 21 2560 [email protected]

The Leishmania donovani glycoprotein fraction, known as FML, successfully underwentpreclinical and clinical (Phase I–III) vaccine trials against canine visceral leishmaniasis (92–95%of protection and 76–80% of vaccine efficacy) when formulated with a QS21 saponin-containing adjuvant. It became the licensed Leishmune® vaccine for canine prophylaxis inBrazil. The immune response raised by the vaccine is long lasting, immunotherapeutic andreduces dog infectivity blocking the transmission of the disease, as revealed by an in vivo assay.The preliminary epidemiological control data of vaccinated areas in Brazil indicate that, in spiteof the still low vaccine coverage, there was a significant decrease in the incidence of thehuman and canine disease. A 36-kDa glycoprotein, in the FML complex, is the human markerof the disease, which was protective in mice as native recombinant protein or DNA vaccine.The DNA vaccine is now being tested against the canine disease. This review resumes thedevelopment of the second-generation FML–saponin–Leishmune vaccine, its adjuvant and ofthe NH36 DNA vaccine, toward the identification of its major epitopes that might be includedin a possible future synthetic vaccine.

KEYWORDS: canine visceral leishmaniasis • CP05 saponin • DNA vaccine • FML antigen • human visceral leishmaniasis • Leishmania chagasi • Leishmania donovani • nucleoside hydrolase • QS21 saponin • second-generation vaccine • transmission-blocking vaccine

Need for a vaccine against visceral leishmaniasisLeishmaniasis affects 12 million people in88 countries with 350 million people at risk.Each year, 2 million new human cases are reg-istered, 500,000 of which are visceral leishma-niasis (VL). A total of 90% of these cases occurin India, Sudan, Bangladesh and Brazil [301].VL, the most severe human Leishmania infec-tion, is an anthroponose in India and CentralAfrica and a zoonosis transmitted by domesticdogs in the Mediterranean and America. Thesefactors justify the search for both human anddog vaccines to interrupt the epidemics. Leish-mania donovani is the main agent of the diseasein Africa, India and Asia, while Leishmaniachagasi causes VL in America and Leishmaniainfantum causes VL in the Mediterraneanbasin, although both parasites are consideredto be the same species [1]. The infection is

transmitted from human to human and fromdog to human through the bite of the hemato-phagous sand fly vectors of the Lutzomyiagenus in the Americas and of the Phlebotomusgenus in the Old World [2]. The epidemiologi-cal control of VL according to WHO regula-tions is accomplished by the treatment ofhuman cases, insecticide treatment of habita-tions and culling of infected dogs. The drugresistance and toxicity of chemotherapy, theincrease of infected immunocompromised sub-jects and the difficulties of epidemiologicalcontrol based upon sacrifice of seropositivedogs emphasizes the need for safe prophylacticvaccines for humans and dogs [3]. Mathemati-cal models also point out that human and dogprophylactic vaccines, rather than dog cullingor chemotherapy, are the most efficient toolsfor eradication of the disease [4]. However, anideal human vaccine is not available yet,although a first-generation killed vaccine using

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Review Palatnik-de-Sousa, de Figueiredo Barbosa, Oliveira et al.

autoclaved parasites with Bacillus Calmette–Guérin (BCG)was tested with success in Sudan, but induced a relatively lowefficacy (43.3%) [5].

Searching for a relevant Leishmania glycoconjugate candidateIn the 1980s, we started searching for glycoconjugates of Leish-mania that would be relevant in the interaction with the macro-phage host cell and with the host immune response, and thatwould show potential use in a protective vaccine. At that time,only the lipopeptidephospoglycan (LPG) [6] and the glycoproteinGP63 [7] were known as major antigens of the Leishmania genus.The LPG is a major surface component of Leishmania parasites,relevant in interactions with the vertebrate host cells. It is respon-sible for the parasite resistance in the lysosomal environment [8]

being important in metacyclogenesis within the insect vector bymediating the attachment and detachment of the parasite fromthe midgut sand fly membrane [6]. The GP63, a surface promas-tigote zinc-metalloprotease [9], is involved in the recognition ofthe promastigote, probably by macrophage CR3 complementreceptors [10,11]. We first studied the Leishmania adleri [12], a liz-ard Leishmania that gives crossprotection but not the disease inhumans, isolating a glycoproteic-enriched fraction, the glycidicmoiety of which was composed of a branched α-D-mannan andshort chain of α-D-(1–>6) and α-D-(1–>2) alternating man-nopyranosil units with terminal residues of manno- and galacto-pyranose. The lipopeptiphosphomannan (LPPM) of L. adleri[12], similar to the glycoinositolphospholipids (GIPLs) [13],exposes short chains of carbohydrates (α-D-[1–>6[ and α-D-[1–>2] mannopyranose), while a branched galactan with pre-dominant (1–>3) galactopyranose units and terminal residues ofα-D-manno- and galactopyranose was released through alkalinehydrolysis [12]. Using the same extraction procedure, we identi-fied a branched xylan backbone with (1–>4) and/or (1–>2) link-ages in Leptomonas samueli [14] similar to the glucuronoxylan ofHerpetomonas samuelpessoai [15] and discussed the adaptive valueof the hexose–glycol conjugates in mammal parasites and thepentose glycoconjugates of insect parasites [14].

When Leishmania donovani promastigotes were submitted tothe same extraction methodology [16], we isolated an enrichedglycoproteic complex, composed of multiple protein bands(9–95 kDa), two of them also stained for carbohydrates (55 and36 kDa), a lypopeptidephosphoglycan (LPPD) and a phospho-galactomannan polysaccharide (PMGL) [16]. The glycoproteicfraction contained 11% of phosphate, 44% of protein, traceamounts of glucosamine and 29% of neutral sugars (16.8 ribose,5.3 fucose, 12.6 xylose, 38.5 mannose, 3.3 galactose, 13.7 glu-cose, 7.4 NAc glucosamine and 0.2% NANA) [16,302]. The find-ing of fucose and mannose [16,302] and the observation that fucoseand α-mannan strongly inhibited the macrophages infection byL. donovani [16], justified naming this fraction as fucose–mannoseligand (FML) and was coincident to the description of the role ofthe mannose–fucose macrophage receptor (MFR) on recognition

and interiorization of L. donovani [17]. A total of 50% of the FMLglycidic moiety corresponds to N-linked oligosaccharides associ-ated to asparagine, which gave 15 distinct peaks on high-perform-ance liquid chromatography [302]. The structure analysis disclosedthe presence of a branched oligosaccharide in one of the majorpeaks, corresponding to 33.5%, and of short linear oligosaccha-rides in the second major peak, corresponding to 20.3% of theFML N-linked glycidic moiety [302]. The branched oligosac-charide is composed of mannose (52.3%), galactose (26.2%),fucose (4.1%) and NAc glucosamine (18.4%). The proposedstructure for this fraction [302] is shown in FIGURE 1. It is abranched oligosaccharide that has a glicidic core linked toasparagine, composed of a 1–>4-linked GlcNAc, also substi-tuted in C3 by a Manp terminal unit and in six by a Fucpterminal unit, and of a 1–>4-linked GlcNAc linked to abranching point of a Manp, substituted in C4 by Manp 1–>4linked to a terminal unit of GlcNAc, and in C3 and C6 byoligosaccharides containing terminal units of Galp 1–>4linked to GlcNAc and 1–>3 or 1–>4 linked to Manp units,respectively (FIGURE 1). The amino acid composition of FMLshowed enrichment in acidic (Glu and Asp) and apolar (Alaand Gli) amino acids, as previously described for L. donovanisurface glycoproteins [18].

The second FML N-linked major fraction is composed ofmannose (63.9%), galactose (32.8%) and fucose (3.2%)organized as linear oligosaccharides [302] containing the pro-posed following sequences (FIGURE 2): Galp terminal units fol-lowed by 1–>3/1–>4- or 1–>4-linked units of Manp andminor proportions of Galp terminal units linked to the C2 ofFucp followed by 1–>2/1–>4-linked Manp units [302].

Furthermore, the LPPD complex isolated from L. donovanicontained phosphate, protein carbohydrate and lipids [16]. Itsglycidic moiety was composed of mainly short oligosaccha-rides of mannose, galactose and phosphate (three to ten sugarunits) mainly of 1–>3-linked Manp, with minor proportionsof 1–>2-linked and 1–>4-linked Manp, and di-O- and3-O-hexofuranosidic units, and with stearic, palmitic andmiristic acid (14:2:1) as major fatty acids [19]. It is worth not-ing that the LPPD seems to be more closely related to theGIPLs [13] rather than to the LPG [6] of L. donovani. Finally, aphosphate galactomannan (PMGL) was also released from theL. donovani promastigotes by alkaline hydrolysis [16].

Among the glycoconjugates isolated from L. donovani, FMLwas the one that strongly inhibited both the penetration of pro-mastigotes [16] and amastigotes [20] of L. donovani in mice macro-phages in a species-specific manner [21]. In addition, and asexpected from its glycoproteic nature, it was strongly immuno-genic in mice and rabbits. The FML was present on the surfaceof the parasite throughout the life cycle (promastigotes and amas-tigotes) [16,20]. No crossreactivity between FML and the GP63surface protease was detected [20], demonstrating that we weredealing with a new and strong immunogen that, different fromGP63 [22], was very species specific [21]. This made FML a goodcandidate for the development of a future vaccine against VL.

FML vaccine against canine visceral leishmaniasis Review

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FML in diagnosis, prognosis, cure monitoring & blood-bank control of VLThe FML antigen is a sensitive, predictive and specific antigen inserodiagnosis of human [23] and canine VL [24]. Used in anELISA assay, it shows 100% sensitivity and 96% specificity, iden-tifying patients with overt VL and inhabitants in endemicregions that had subclinical infection with potential for evolutiontowards the fatal disease. The FML reactivity also decreased andnegativated during the follow-up of patients throughout thetreatment and after the parasitological cure [23,201]. No cross-reaction was observed with other Leishmania infections, Chagas’disease, Paracoccidioides brasiliensis infection, lymphoma, typhusand hemophagocytic disease [23,25].

Being an obligatory parasite of monocytes, Leishmania para-sites could, potentially, be transmitted by blood transfusion.Reports of isolated cases support this hypothesis [26–29]. In orderto analyze this possibility, the FML ELISA assay was systemati-cally used in the blood-bank control of a Brazilian endemic area[30–32]. We showed a 9% seroreactivity in blood donors, increas-ing to 25% in a VL periurban focus and to 37% in polytrans-fused hemodialysis patients from an endemic area [30]. Risk fac-tors included blood transfusion, but not the potential exposureto the sand fly bite. The prevalence significantly decreased to7% in hemodialysis patients from Rio de Janeiro (Brazil),where VL is rare and attained 0% in patients who are not sub-mitted to blood transfusion. The prospective analysis of27 FML seroreactive donors from Natal (Brazil) revealed amas-tigotes of Leishmania in the bone marrow of one subject, whilefour had clinical complaints, including splenomegaly andhepatosplenomegaly [30]. We further demonstrated a significant

correlation between the seropositivity in the FML ELISA assayand the finding of Leishmania DNA in the blood [31] and bonemarrow [32] of asymptomatic blood donors. Grogl et al.described the blood-borne transmission of leishmaniasis in sol-diers infected with Leishmania major during the Desert Stormoperation in the Iraq war [32]. Their results increased the rele-vance of blood-bank control since, in tegumentary leishmania-sis caused by L. major, parasites were thought to be restricted toskin and dermal tissues and absent from the blood [33]. We wereable to induce transfusional kala-azar in normal hamsters usingeither total blood, monocyte or plasma fraction of infectedhamsters, indicating that free amastigotes are found in fluids inthe case of severe disease [34]. We achieved ascites and hepat-osplenomegaly but not parasites in target organs of normalhamsters transfused with human blood of red blood units(RBUs) of seropositive asymptomatic donors. At that time, wewere not capable of concentrating each donor RBU that con-tained 450 ml, or to purify from them the mononuclear cells orthe free parasites, to a final volume of 0.2 ml, which is the max-imum that a hamster can tolerate. Therefore, probably due todilution problems, we never actually detected parasites in theliver or spleen of these animals. Le Fichoux and coworkers,however, proved the presence of Leishmania parasites in cultureof the buffy coat of seropositive asymptomatic blood donors [35]

and, recently, Riera and coworkers [36], using leukodepletion fil-ters, succeeded to remove parasites from red blood cell unitsefficiently and proved the infectious condition of asymptomaticseropositive blood donors in the Balearic Islands [36]. Our workwas cited by Gigger et al. [37] and Dodd [38] as the basis of therecommendations for serologic control of donated blood andorgans for VL in countries where the disease is endemic and

Figure 1. Constituents of the main branched N-linked glycosidic moiety of the FML antigen of Leishmania donovani.

O

O

OO

O

OOH

OHOH

OH

OOH

OH

NHAc

OOH

OO

O

OO

OOH

OHOH

OH

OOH

OH

NHAc

OOH

OHOH

O

OO

OH

OH

OH

NHAcOH

OH

OHOH

O

O

OH

OHO

OH

OH

NHAc

OO

OHOH

OHOH

OOH

OH

OH

NHAc

N

HO

O

NH

CO2H

NH2

Gal

Gal

GlcNAc

GlcNAc

GlcNAc GlcNAc

GlcNAc

Man

Man

Man

Man

Man

Fuc

Asn

N-glycosidic linkage



contributed to the US FDA regulations for blood quality con-trol for VL in the USA [303] since 1991, after the detection ofimported cases from the Iraq war [33] and, since 2004, in theEU [39].

Impact of the use of the FML ELISA in epidemiological controlThe FML ELISA assay showed 100% sensitivity and 100%specificity in diagnosis of canine VL, and a tenfold highersensitivity to the usual immunofluorescent (IF) assay and a100% predictive value in 21 asymptomatic naturallyinfected dogs that died of canine VL within 6 months [24].While the mathematical model described by Dye in 1996 [4]

condemned the culling of seropositive dogs as a tool for epi-demiological control of kala-azar, we [40], and others [41],demonstrated that the efficacy of the epidemiological con-trol campaign could be improved by increasing the sensitiv-ity of the serological assays used for diagnosis. Using Dye’smodel [4], we mathematically demonstrated that the controlis only not efficient at low κ values (rate at which latent andinfectious dogs are lost by the destruction program), which

match the canine seropositivity observed in the field usingthe IF assay on blood eluates. With higher κ values, corre-sponding to IF (κ = 0.07) or FML ELISA (κ = 0.25) meth-ods in sera samples, the number of infectious dogs declinesto a Ro = 1 or Ro = 0, respectively, interrupting transmissionand epidemic advancement. Results of field assays confirmedour hypothesis, since we demonstrated that the dog removalfollowing the results from IF assay of sera instead of eluatesled to a 57% (p < 0.005) decrease in canine cases and 87.5%(p < 0.005) in human cases [40]. Our mathematical and exper-imental results indicated that the control campaign becamemore efficient by enhancing the sensitivity of the diagnosticassay. As a result of these investigations [40–43], the BrazilianMinistry of Health’s present regulations for the epidemiologi-cal control of VL includes an ELISA assay for the screening ofdog seropositivity in blood eluates and encourages the use ofsera samples [44]. The IF methodology remains as a secondaryconfirmation test [44]. All these results disclosed the possibleuse of FML for human and dog diagnosis, prognosis and epi-demiological field control [25] and encouraged its use in thedevelopment of a potential protective vaccine in formulationwith a potent adjuvant.

Development of a FML second-generation vaccine: selection of routes & adjuvantsUntil the 1990s, only live or first-genera-tion Leishmania vaccines composed oftotal Leishmania-inactivated parasites wereused in human field trials, while no dogvaccine was available for the control of thezoonotic VL that occurs in the Mediterra-nean and in the Americas [45]. No vaccinewas licensed at that time and the efficaciesof the first-generation vaccines were rela-tively low (54.38, 95% confidence interval[CI]: 39.84–68.92) [45]. The first-genera-tion vaccines using Leishmania lysateswere assayed against canine VL, inducingprotection in Iran [46] but failing to do soin Brazil [47].

The formulation of a second-generationvaccine, based on purified native antigens,required a potent and specific adjuvant.Consequently, we initiated the develop-ment of a second-generation vaccinebased on the FML antigen. TABLE 1 sum-marizes the results of parasite-load reduc-tion obtained in vaccinated mice, ham-sters or dogs. As a direct correlate toprotection against VL [5,45,48–51], we alsoshow in TABLE 1 the results of delayed-typehypersensitivity (DTH) of mice and

Figure 2. Constituents of the main short linear oligossacharides of the N-linked glycosidic moiety of the FML antigen of Leishmania donovani.

O

OO O

O

OH

OHOH

OH

OHOH

OH

OH

OH

OH

O

Gal

ManMan

O

OOOH

OH

OH

OH

OH

O

OOH

OOH

OHOH

OHGal

ManMan

O

O

OO

OOOH

OH

OH

OH

O

OHOH

OH

OH O

OHOH

OH OH

Gal

ManMan

Fuc



hamsters, expressed as the ratio of the size of vaccine skin testsover that of the untreated controls and of dogs as the percentof DTH positivity in vaccines. The sustained DTH responsepoints toward a protective and active cellular immune responseagainst Leishmania.

We used 150-µg FML and 100-µg adjuvant for experimentswith mice and hamsters [52–59]. Formulating the FML with theRiedel de Haen saponin R we obtained an average strong protec-tion of 83.5% (95% CI: 89.14–77.85) [52–56]. Divergent protec-tive or immune responses were found before, in mice vaccinatedwith Leishmania glycoconjugate, depending on the immunizationroute [60–62]. The FML–saponin R vaccine, on the other hand,induced strong protection, irrespective of the use of the intraperi-toneal [52–54] or subcutaneous routes of immunization [54–56], theuse of L. donovani [52–55] or L. chagasi [56], the use of variableamounts of parasites [52–56] and the use of isogenic [52,54,56] or out-bred [54,55] rodent models for experimental challenge (TABLE 1).Protection induced by the FML–saponin vaccine against tegu-mentary leishmaniasis by L. mexicana [56] or Leishmania amazo-nensis [57] was lower (42–72%) (TABLE 1), suggesting the potentialuse as a bivalent vaccine and also the existence of some speciesspecificity. The parasite counts in mice were recorded in liversamples [63], while spleen samples of hamsters were used sincethey show less intragroup variation [53].

Aiming to develop a vaccine for field trials on heterogeneouspopulations, we initiated tests of the Swiss albino outbred micemodel (TABLE 1) [54], comparing the adjuvant potential of Alum,FIA [54], BCG, IL-12 and saponin R, Quil A and QS21 puri-fied from Quil A extract [55]. Saponin R was the only one thatdid not induce unspecific protection, while Alum was responsi-ble for 68% of the 88% of protection induced in combinationwith FML [54] and BCG alone, or BCG plus FML induced theexpected 50% protection [5,64,65]. IL-12 increased IgG2a anti-bodies and DTH but did not reduce the parasite load [55]. Thethree saponins (R, Quil A and QS21) induced the highest pro-tection (TABLE 1). QS21 induced a 43:1 increase in IFN-γ serumlevels [55].

While FML formulated with the extracts of Quillaja sapon-aria saponin (R and Quil A) induced a 6.83-fold(95% CI: 3.63–10.03) average increase of DTH (TABLE 1) inmice and hamsters [53,55–57], higher values were found in ani-mals treated with the purified QS21 saponin (12- and34-fold increase), either obtained from Quil A [55] or fromsaponin R [58], respectively. As expected, since it was theactive adjuvant component of the formulation, the purifiedQS21 saponin provoked an impressive enhancement of theDTH response in mice, while the crude extracts from whichit was purified promoted both in mice and hamsters signifi-cantly lower responses, irrespective of the animal model,species or amount of parasite challenge (TABLE 1).

While an active DTH is expected to be found in cutaneousand mucosal leishmaniasis [56,66,67], suppression of the cellularimmune and absence of DTH are expected in VL [50,51,58,68]

and in the rare cutaneous diffuse leishmaniasis [67] caused by

L. amazonensis. In spite of this, we found there was a higherDTH reaction in untreated L. amazonensis-infected animals(0.343 mm ± 0.151) than in FML saponin vaccinees(0.295 mm ± 0.198), on week 33 after infection, making a lowDTH ratio (TABLE 1) [57]. In both groups, the DTH responsesincreased with time, despite the advancment of the disease inthe untreated controls [57]. This increase of DTH with time ofinfection was also observed previously in L. amazonensis-infected Balb/c mice [69]. Furthermore, DTH was also lower inanimals treated with FML and BCG owing to its lower adju-vant capability [55]. We preserved the subcutaneous route forvaccination (TABLE 1), which proved to be efficient [54–56] andmore suitable for the development of a future canine or humanvaccine (MODDABBER F, PERS. COMM.).

We performed a kennel assay in order to standardize protocolsand dosage of the vaccine and to run further field assays. In afirst comparison, the R (50, 500 and 1000 µg) and Quil A (500and 1000 µg) saponins were used in combination with FMLantigen 1500 µg in mongrel dogs. FML seropositivity wasachieved more rapidly in the saponin R-treated animals (TABLE 2);however, the increase of anti-FML antibodies was not correlatedto the increase in concentration of the adjuvant, neither for thesaponin R (p = 0.107) nor for the Quil A (p = 814). The use of500 µg of either saponin was enough to trigger the synthesis ofantibodies. At that time, we wanted to induce a strong antibodyvaccine response to facilitate the identification of the vaccinatedanimals in field assays. However, the number of doses in theQuil A-vaccinated animals was significantly correlated to theincrease in absorbance values (p < 0.0001).

We further assayed the potential of three doses of 0.5 mg(n = 3) or 1 mg (n = 3) of Quil A in combination with 1.5 mgof FML on dogs challenged with 108 amastigotes of L. dono-vani [51,70,71]. Protection in the group treated with 1000 µg hada significantly higher antibody content (p < 0.0001) and DTHresponse (p < 0.01) than untreated controls (n = 3), which onlyshowed a positive DTH between 90–120 days after infection,losing it after 210 days. Platelet counts were significantly higher(p < 0.01) and the number of clinical signs was lower(p < 0.001) in both vaccine groups than in controls [51,67]. Theachievement of statistically significant protection by compari-son of only three dogs in each group is of note, and has notbeen shown in other kennel assays of anti-Leishmaniaprime–boost DNA vaccines [72,73]. One dog from the salinegroup and two from the FML plus Quil A 1-mg group diedfrom confirmed VL at days 297, 376 and 503 after infection,respectively [70]. They belonged to the same polysymptomaticfamily, suggesting the presence of familiar susceptibility and thehigh protective effect of the Quil A 1-mg vaccine. No obitswere detected in the more resistant dog families where controlswere olygosymptomatic and polysymptomatic and vaccineswere asymptomatic. The remaining animals were sacrificedwithin 647 days after challenge. The presence of parasites in tis-sues was confirmed in all components of the susceptible familyand in the control dog of the resistant covey [71]. Our results



Tab

le 1

. Deg

rees

of

pro

tect

ion

an

d im

mu

no

gen

icit

y o

f th

e FM

L se

con

d-g

ener

atio

n v

acci

ne

in d

iffe

ren

t an

imal

mo

del

s.A

dju

van

tM

od

elSt

rain

Dis

ease

Leis

hm

ania

Ro

ute

Sap

on

in

(µg

)C

hal

len

ge

DTH

vacc

ine/

con

tro

lPa

rtia

l re

du

ctio

n (

%)

Phas

eR

ef.

Qui

llaja

sap

onar

ia

sapo

nin

RM

ice

Balb

/cV

LL.

don

ovan

iip

.10

02

× 1

07 am

aN

D85

I–IIa

[52]

Q. s

apon

aria

sa

poni

n R

Ham

ster

sC

BV

LL.

don

ovan

iip

.10

02

× 1

07 am

a 4

90*

I–IIa

[53]

Q. s

apon

aria

sa

poni

n R

Mic

eSw

iss

albi

noV

LL.

don

ovan

iip

.10

02

× 1

07 am

aN

D85

I–IIa

[54]

Q. s

apon

aria

sa

poni

n R

Mic

eSw

iss

albi

noV

LL.

don

ovan

isc

.10

02

× 1

07 am

aN

D89

I–IIa

[54]

Q. s

apon

aria

sa

poni

n R

Mic

eSw

iss

albi

noV

LL.

don

ovan

isc

.10

02

× 1

07 am

a8

73I–

IIa[5

5]

Q. s

apon

aria

sa

poni

n R

Mic

eBa

lb/c

VL

L. c

haga

sisc

.10

02

× 1

08 am

a11

79I–

IIa[5

6]

Q. s

apon

aria

sa

poni

n R

Mic

eBa

lb/c

CL

L. m

exic

ana

sc.

100

1 ×

106 p

ro8

42I–

IIa[5

6]

Q. s

apon

aria

sa

poni

n R

Mic

eBa

lb/c

CL

L. a

maz

onen

sis

sc.

100

1 ×

106 p

ro1

49–7

2I–

IIa[5

7]

Alu

mM

ice

Swis

s al

bino

VL

L. d

onov

ani

sc.

100

2 ×

107 a

ma

ND

88I–

IIa[5

4]

FIA

Mic

eSw

iss

albi

noV

LL.

don

ovan

iip

.10

02

× 1

07 am

aN

D67

I–IIa

[54]

BCG

Mic

eSw

iss

albi

noV

LL.

don

ovan

isc

.10

02

× 1

07 am

a4

52I–

IIa[5

5]

IL-1

2M

ice

Swis

s al

bino

VL

L. d

onov

ani

sc.

100

2 ×

107 a

ma

70

I–IIa

[55]

Qui

l AM

ice

Swis

s al

bino

VL

L. d

onov

ani

sc.

100

2 ×

107 a

ma

993

I–IIa

[55]

QS2

1M

ice

Swis

s al

bino

VL

L. d

onov

ani

sc.

100

2 ×

107 a

ma

1279

I–

IIa[5

5]

QS2

1M

ice

Swis

s al

bino

VL

L. c

haga

sisc

.10

010

8 am

a34

95I–

IIa[5

8]

Qui

llaja

bra

silie

nsis

sa

poni

nM

ice

Balb

/cV

LL.

don

ovan

isc

.10

02

× 1

07 am

a3

72I–

IIa[5

9]

Q. s

apon

aria

sa

poni

n R

Dog

sM

ultip

leV

LL.

cha

gasi

sc.

500

Nat

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showed that vaccination with FML plus Quil A 1 mg inducedsignificant protection against experimental canine VL [51]. Also,a possible genetic/familiar basis of susceptibility to the diseasewas disclosed [70]. This hypothesis was supported by the furtherfindings of Atlet [74] and Quinnel [75] that described the rele-vance of the Nramp and the MHC genetic system, respectively,in canine susceptibility to VL.

The adjuvant potential increase of the Quil A vaccine in miceand dogs was, however, associated with transient asteny, pain andinflammation at the site of injection while these reactions wereabsent in the saponin R-treated animals [76]. Based on these facts,we ran two parallel field tests: one with saponin R [50] and theother with Quil A [51]. The Phase III trial of efficacy of FML(1500 µg) saponin R (500 µg) was performed in São Gonçalo doAmaranto, Brazil [50], an endemic area both for human andcanine kala-azar. A total of 97% of vaccinees were seropositive toFML and 100% showed positive DTH 7 months after vaccina-tion (TABLE 1). The absorbance values and size of DTH were bothsignificantly higher in vaccinees than in controls. After 2 years, inthe untreated group, four obits of confirmed canine VL and sixolygosymptomatic cases among 30 placebo-treated dogs surviv-ing until the end of the assay (33%) were detected and confirmedby parasite analysis and PCR. No obits were detected among vac-cinees and infection was confirmed by PCR in three out of36 olygosymptomatic dogs (8.33%), corresponding to 92%protection and 76% vaccine efficacy [45,50,51].

In this study, obits and disease were detected andrecorded [45,50,51]. These are two major end points to be consid-ered for the development of a Phase III trial, according to recom-mendations by the WHO [77]. General obits, obits due to thespecific disease, severe disease or specific symptoms (confirmedby parasitological analysis) should always be preferred to methodsof immunological or parasitological confirmation of infection,which are more earlier end points [77]. These should only be usedwhen, due to ethical impediments, one cannot wait until theonset of clinical cases or obits, whenever treatment for the diseaseis not available or when the vaccine specifically acts against the

early infection only, such as vaccines with antigens of malariasporozoites [77]. While those methods designed to evaluate pro-tection against infection are very sensitive and diagnostic, theymonitor end points far from the pathological events of the dis-ease and do not give precise information regarding protectionagainst disease or death. Recently, molecular or immunologicalmethods to evaluate protection against infection were used inorder to show protection in experimental assays with low ornonvirulent parasite challenge [72], or in field assays developedin endemic regions where the infective pressure is low andobits or severe disease are not detected at all during the assayedperiod [78]. The vaccine efficacy values of all large field assaysof human vaccines against leishmaniasis are based on therecord of obits owing to the disease and not on the proportionof infected subjects [45].

Simultaneously to the first Phase III assay of the FML vac-cine [50], we vaccinated dogs naturally exposed to a nearbyendemic area with the FML in formulation with Quil A(1000 µg) [51]. As expected, 100% of vaccinated animals wereseropositive to FML and showed DTH positivity 2 monthsafter vaccination. The absorbance values and size of DTHwere both significantly higher in vaccinated animals than incontrols over a 3.5-year period [51]. In total, 25% of the con-trol animals (eight animals) and 5% of the vaccinated animals(one dog) developed the clinical and fatal disease during theexperiment. This difference was significant (χ2 = 3.93;p < 0.05). This means that 95% protection against canine VLwas achieved in vaccinated animals, after FML–Quil A vacci-nation, representing 80% of vaccine efficacy. We were able toconfirm Leishmania infection 3.5 years after vaccination in allthe four untreated controls from the initial cohort thatremained in the area. These dogs showed positive PCR forLeishmania DNA and FML serology with no DTH response.Higher seropositivities and intradermal reactions with noleishmanial DNA were detected in vaccines. The difference inPCR results was highly significant (p < 0.005). In conclusion,the FML–Quil A vaccine induced a significant, long-lasting

Table 2. Kinetics of the anti-FML antibody production in sera of dogs vaccinated with the FML–saponin vaccine.

Saponin Dose (µg) Anti-FML antibodies in the FML ELISA assay

Preimmune After first dose After second dose After third dose

Riedel de Haen 1,000 0.184 0.659 ND ND

Riedel de Haen 500 0.338 0.520 ND ND

Riedel de Haen 50 0.321 0.453 ND ND

Quil A 1,000 0.064 ± 0.040 0.371 ± 0.123 0.862 ± 0.276 1.109 ± 0.099

Quil A 500 0.090 ± 0.065 0.350 ± 0.180 0.899 ± 0.283 1.087 ± 0.300

Adult mongrel dogs (n = 1 for the Riedel de Haen and n = 3 for the Quil A saponin experiment) received three doses, 21 days apart, of 1500 µg of FML antigen in combination with saponin. The anti-FML antibodies were assayed by the FML ELISA assay 10 days after each dose. Results are expressed as average values ± standard deviation of the individual values.ND: Not done.Data from [24].



and strong protective effect against canine VL in the field. Thevaccine efficacy (VE) and protection values of the R and theQuil A vaccine field assays were not significantly different(p > 0.05) [51].

In May 2001, we were among 17 international Leishmaniaresearchers invited by the WHO/TDR/IDRI to participate inthe Fourth Meeting on Second-Generation Leishmania Vac-cines, held in Mérida (Mexico), and to show our resultsobtained with the FML vaccine in dogs. While most presentedresults relate to mice or monkey models of cutaneous leishma-niasis, the FML vaccine was the only second-generation candi-date for vaccination against the most severe form of the disease– VL [304]. The FML–saponin vaccine was, indeed, the firstand, at that time, the only vaccine reported to give protectionagainst canine VL [50,51]. Simultaneously, the scaling up of theFML–saponin vaccine began, using the saponin R, and the for-mulation was industrialized and licensed for canine prophylaxisin Brazil in 2004, under the name of Leishmune® [79].

While our first interest was to obtain a vaccine that wouldrender the vaccines highly seropositive, facilitating their fol-low-up in a field assay, the potential wide-scale use of the vac-cine for prophylaxis in Brazil could bring a problem, since theepidemiological control of VL is performed by a serologicalsurvey of dogs [43,44]. Seropositive dogs are considered aninfected reservoir of parasites and are sacrificed [4].

Previous authors explained that the IgG antibody increase incanine VL is correlated to symptomatology [41,79,80] and that theIgG1 subtype is associated with susceptibility and severe diseasewhile the IgG2 increases in naturally resistant or vaccinateddogs [72,73,81–83]. We showed that the IgG2 anti-FML antibodiesare predominant in Leishmune-vaccinated dogs [84–86] and thismight be used to differentiate the seropositivity of infection fromthe vaccine seropositivity [84]. Discrepant results showing a pre-dominance of IgG2 subtype in symptomatic dogs were alsoreported [87–90]. As we discussed recently, the antigen-specificenhancement of the IgG2 antibodies induced by thepurified [84–86] or recombinant vaccine antigens [72,73,82,91,92] isonly seen using the same antigens for diagnosis [45]. The use ofLeishmania crude lysate for diagnosis [73,87–89] masks the detectionof the vaccine response, which is diluted in the plethora of all par-asite antigen–antibody interactions [45]. Indeed, the differentialaffinity of the antigen could be another factor of the discrepancy.In dogs vaccinated with the CPa and CPb cysteine proteinases, thework of Rafati et al. show that IgG2 is predominant against therecombinant antigens while IgG1 is predominant against theL. infantum lysate [72]. While the recombinant antigen interactswith a defined fraction of serum antibodies, the total lysate inter-acts with all the antibodies directed against the total parasite,masking or diluting the response against the recombinant protein,which is not a major Leishmania antigen. This would also explainthe results of Andrade et al., who proposed the use of fluorescent-activated cell sorting analysis against L. chagasi total promastigotesto differentiate sera of Leishmune-vaccinated dogs (n = 21) fromsera of L. chagasi naturally infected dogs (n = 29) [93]. Andrade

et al. used total promastigote antigen and found much highertitration curves (higher fluorescency and dilution titers) for IgG2antibodies in infected but not in vaccinated dogs [94], whileMendes et al., using the FML antigen in an ELISA assay, foundmuch higher absorbances and dilution titers of IgG2 antibodies inFML-vaccinated (Leishmune) dogs (n = 68) but not in theinfected dogs (n = 29) that displayed almost equivalent titrationcurves of IgG1 and IgG2 [84]. Another factor of discrepancy couldbe the use of different batches of the polyclonal anti-IgG2 andanti-IgG1 antisera, which show low reliability (MJ DAY, PERS. COMM.).These difficulties stimulated Day to obtain dog monoclonal anti-bodies that recognize the four different IgG subfractions [90].Unfortunately, these antibodies are not commercially available [45].The use of the purified antigens for diagnosis would be a solutionfor discrimination of vaccinated dogs. Another possible solution isthe recent use of a fraction of the recombinant HPS 70 protein ofL. chagasi, which, in an ELISA assay, reacts only with sera ofinfected dogs but not of Leishmune-vaccinated animals [94]. Twoother recombinant proteins of L. chagasi, rK26 [95] and rK39 [96],were reported to be serological markers of the disease. Their use ina differential ELISA assay might, potentially, contribute todiscriminate infected from Leishmune-vaccinated dogs.

An immunogenicity assay of the industrialized Leishmunewas performed on 550 healthy seronegative dogs from endemicareas [97]. After 2 years of exposure, the Leishmune vaccineproved to be highly immunogenic, tolerable [98] and protectivein 97% of the vaccinated and exposed animals [97] thatremained healthy, asymptomatic, seropositive and DTH posi-tive, while canine VL obits or clinical symptoms confirmed byparasitological assay were detected in 3% of the cohort [97].

As discussed previosuly, while the increase in anti-FML anti-body response did not correlate to the increase in saponin con-centration, the cellular immune response did [99]. The increasein saponin concentration would then result in a stronger vac-cine that not only prevents but also treats or cures infecteddogs. We then used a double saponin concentration for theimmunotherapy assays on dogs naturally [86] or experimentallyinfected [85] with L. chagasi. For that purpose, we used sero-positive, already infected but still asymptomatic dogs, since theimmunologic window for canine kala-azar was described aslasting for 90–120 days after infection [24].

We observed that the increased in the saponin concentration to1 mg/dose [85,86] resulted in no significant increase in reac-togenicity. In the first experiment, using naturally infected dogsfrom an endemic area, 90% of the infected and immunotherapy-treated dogs remained asymptomatic parasite free and DTH pos-itive for 2 years (TABLE 1) [86]. The animals showed sustained pro-portions of CD4+ and CD21+ lymphocytes and, as expected forthe Quilllaja saponaria treatment, increased proportions of CD8+

lymphocytes [86]. These dogs were revaccinated once annually,remaining healthy until present – 5 years after the first vaccina-tion. In a second experiment [83] with L. chagasi experimentallyinfected dogs, protection of vaccinated dogs was evidenced by thelower number of DTH reactions, the decreased average of CD4+



Leishmania-specific lymphocytes, the increased average of the clini-cal scores and the higher proportions of parasite finding in targetorgans or DNA evidence, of the untreated infected controls [85].

After the development of the FML vaccine, only one othersecond-generation vaccine, the 54-kDa excreted protein ofL. infantum plus MDP (LiESAp-MDP) was shown to protectdogs against experimental [100] and natural infection byL. infantum in southern France [78], showing 0.61% incidenceof infection in vaccinees versus 6.86% in control dogs(92% VE). Different from the FML vaccine [50,51], no protec-tion against obits was described [78] due to the lower infectivepressure of the endemic region, impeding the comparison ofefficacies of the two vaccines [77]. The H1, HASPB1, H1 plusHASPB1 in combination with Montanide™ [101], and arecombinant polyprotein Q chimera in combination with BCG[102], induced partial protection against L. infantum, while theLeish 111 complex in formulation with MPL–SE/adjuprimedid not protect dogs from experimental infection with L. cha-gasi [92], nor from natural infection with L. infantum [103].Finally, the prime–boost with DNA plasmid and recombinantvaccinia virus expressing the LACK antigen (rVV-LACK) fromL. infantum [73] and the prime–boost with DNA and recom-binant protein of the cistein proteinases A and B of L. infantumin combination with Montanide 720 and CPG [72] inducedpartial protection and immunogenicity, respectively, againstL. infantum experimental infection.

Potential impact of the use of Leishmune vaccine to reduce VL in endemic areasIn total, 32 exposed dogs vaccinated with Leishmune showed acomplete absence of clinical signs and of skin parasites, as bythe negative results for PCR of blood and lymph nodesamples [79]. By contrast, 11 months after the beginning of theassay, 40 untreated control dogs included 25% of symptomaticcases, 50% of FML seropositivity, 56.7% of lymph node PCR,15.7% of blood PCR and 25% of immunohistochemical posi-tive reactions. These results, supported by the literature [79],indicate the noninfectious condition of the Leishmune-vacci-nated dogs. Transmission-blocking vaccines are one of the con-trol strategies for vector-transmitted protozoan diseases [104].Antibodies raised in the vaccinated host prevent the develop-ment of the parasite in the insect vector. These antibodies,while not being involved in the individual vertebrate host pro-tection, help in the interruption of the epidemics [104].Although much work has been done in malaria TBVs, the ini-tial work on the potential Leishmania–TBV vaccines referred tomurine tegumentary leishmaniasis [105–108]. Sand flies that hadpreviously fed on mice immunized with L. major crude anti-gens, LPG alone or LPG plus recombinant GP63 showedreduced rates of infection when compared with controls [17].Reduction was significant in sand flies that fed upon LPG-immunized mice. Furthermore, sand flies fed on LPG-immu-nized mice and further on L. major-infected mice induced very

small lesion sizes [108]. These studies confirmed the presence ofL. major LPG-specific receptors on the insect midgut mem-brane and their relevance on recognition and interaction withLeishmania during its biological cycle.

We were able to characterize the TBV effect of the Leishmunevaccine on insects that fed upon sera of immunized dogs [109].FIGURE 3 shows the typical pattern of a midgut of a sandfly fedupon blood containing fresh amastigotes and preimmune dogsera. On day 4 after blood meal, the midgut is very enlarged andfull of active promastigotes [109]. On the other hand, also shownin FIGURE 3, the midgut image of a sand fly fed upon blood con-taining amastigotes and sera from dogs vaccinated with Leish-mune appears thin and empty. In each experiment, 92–100female sand flies were individually analyzed, obtaining 79.3% ofparasite reduction [109]. TABLE 3 summarizes the analysis of randomsamples of the whole insect population. While sand flies thatfed upon sera of dogs obtained before vaccination (preim-mune) showed higher percentage of infection, number of para-sites and, consequently, a higher infection index, sand flies thatfed upon sera of dogs after Leishmune vaccination (mainlyIgG2 anti-FML antibodies) showed a 74.3% reduction ininfection [109]. On the other hand, compared with the respec-tive preimmune-fed controls, the sand flies fed on sera ofinfected animals (IgG1 predominant antibodies) showed a pro-nounced enhancement of infection (331.9%) (TABLE 3). Slightvariations were found in the number of promastigote perinsect in the preimmune control group of both experiments.The difference might be due to a mild variation in time (inminutes) between the isolation of the parasites from hamsterspleens and the feeding of the insects through the membraneassay. The freshly obtained amastigotes are very sensitive andlabile and their virulence might decay with the increase of timeor temperature during the assay.

Our results indicate that the Leishmune vaccine is aTBV [77,109,104] since antibodies present in sera of dogs, even12 months after vaccination, help in the interruption of the epi-demics. Indeed, the confirmation of the blocking abilities of theantibodies of vaccinated subjects, in membrane-feeding tests, isconsidered the first level of assay required to define a TBV vaccine[104]. Preliminary results of xenodiagnosis using Lutzomyia longi-palpis reveal that two out of nine naturally infected dogs wereinfected with phlebotomines, while zero out of 19 Leishmune-vac-cinated dogs were not. The two infected dogs showing positivexenodiagnosis were the most symptomatic (more than three clini-cal signs) among the nine tested, while the other seven wereasymptomatic or oligosymptomatic (AMORIM I ET AL., PERS. COMM.).These results confirm the previous published observations thatshowed a positive correlation between increase in dog symptoma-tology and the increase in infectivity and support the TBVproperty of the Leishmune vaccine [110,111].

Confirming this possibility, the undergoing analysis of the epi-demiological control data of vaccinated areas in Brazil showsthat, in spite of the still low vaccine coverage, a significantdecrease in the human and canine incidence of this disease has



occurred since Leishmune started to be used. Indeed, while onlya 25% of decline in the number of dogs removed was noticed inthe studied period, there was a 61% severe decline in the humanincidence in Araçatuba, Brazil, probably owing to the effect ofthe prophylactic vaccination.

On the other hand, the increased infection index of sand fliesfed on sera of infected dogs might be related to the recentdescription of the infection promoter effect of sand fly salivapeptides [112]. Indeed, coinjection of some sand fly saliva pep-tides with Leishmania promastigotes renders the mice that werepreviously resistant susceptible to infection, enhancing theinfectivity of the pathogens [112,113]. It is likely that some ofthese substances that promote Leishmania replication are stillpresent in the sera of infected dogs. This would explain thehigher replication of Leishmania in these sand flies, positivelymodulating its transmission in the field.

Mechanisms of the FML-vaccine adjuvantDuring the development of the second-generation FML vac-cine, we also performed some structure–function studies of thesaponin adjuvants, since they showed the best adjuvant poten-tial. The hemolytic potential of saponins was, in the past, con-sidered as a limitation to their use in vaccinations. We [114], andothers [115], demonstrated that the hemolytic activity of sapon-ins of steroidic nuclei was higher than that of triterpenoidalsaponins and that hemolysis was related to the carbohydrate

chains attached to the nucleus, since thecarbohydrate-deprived sapogenin fractionswere less [116] or not hemolytic at all [114].

On the other hand, we extensively usedthe Quil A [51,55,86,117] and the saponin R(Riedel de Haen saponin) [50,52–56,304] invaccination studies, since both were themost potent adjuvants. While Quil A is anextract of Q. saponaria bark [118], the com-position and plant origin of the saponin Rwas not disclosed by the manufacturer,although it was very potent as well and lessreactogenic [50,52,55,117]. Our work revealedthat both saponins share the same triter-pene nucleus, the Quillaic acid [117], whichexhibits a peculiar aldehyde in the C4position of the triterpene, absent in mostdescribed saponins [119]. The H1 and C13

nuclear magnetic resonance (NMR) analy-sis revealed that the saponin R is also anextract from Q. saponaria Molina [117]. Wealso detected the C4-aldehyde of thesaponin R in the axial position to the trit-erpene, favoring the induction of astronger antibody response to the FMLwith no significant reduction of parasiteload, while the Quil A C4-aldehyde, in the

equatorial position, induced a mainly cellular immune responsewith higher DTH, IFN-γ secretion and stronger reduction ofparasite burden (77%, different from saline [p < 0.005] andfrom sapogenin [p < 0.025] controls) [117].

Furthermore, we fractionated the saponin R mixture by ionexchange chromatography to obtain one QS21 saponin frac-tion (18.0%), a mixture of two deacylsaponins (19.4%),sucrose (39.9%), sucrose and glucose (19.7%), rutin (0.8%)and quercetin (2.2%) that were identified by comparison of 1Hand 13C NMR spectroscopy [57]. The QS21 showed the typicalaldehyde group in C4 and a normonoterpene moiety acylatedin C28. The deacylsaponins showed the aldehyde group butnot the normonoterpene moiety [58].

Recently, the mechanism of the QS21 saponin waselucidated [119]. QS21 contains two carbohydrate chainsattached to a triterpene nucleus C3 and C28, besides a hydro-phobic moiety that is acylated to a C28 sugar-attached residue.The QS21 hydrophobic moiety is related to the induction ofthe CD8+ T-cell-protective lymphocyte response, while thealdehyde group present in triterpene C4 is involved in directT-lymphocyte stimulation, mimicking the B7-1 costimulatorymolecule to induce the Th1-protective response [119].

Chromatographic fractionation of the saponin R adjuvant ofthe Leishmune vaccine revealed the presence of the QS21saponin and the deacylsaponins (saponins that lack the hydro-phobic moiety acylated in C28) as the most active adjuvantcomponents [57]. However, slightly different from what was

Figure 3. Lutzomyia longipalpis midguts experimentally infected through an in vivo membrane assay with hamster spleen amastigotes of Leishmania chagasi, blood and preimmune dog serum or dog Leishmune®-vaccinated serum. Agglutinates of promastigotes of Leishmania are shown inside the white circles in the midgut of an insect fed with preimmune serum. Two individual promastigotes are pointed out by the black arrows. The empty midgut is shown inside the black circle of the midgut of the sand fly fed on a pool of sera of Leishmune-vaccinated dogs. Fresh preparation, phase contrast, 400× magnification.

Predog sera Leishmune®

dog seraPreimmunedog sera



Table 3. Infection index of sand flies fed on preimmune Leishmune® or visceral leishmaniasis dog sera.

Dog treatment(pool of sera)

Insects Infected sand fly Promastigote/insect Infection index Infection (%) Parasite load reduction (%)

Preimmune 12345678910

-+-++++-+-

022,000054,00010,00034,00010,80006,0000

Total 10 6/10 = 0.6 22,800 ± 18,382 13,680 100.0 0.0

Leishmune® 12345678910

----+---++

000018,4000008,8008,000

Total 10 3/10 = 0.3 11,733 ± 5,787 3,520 25.7 74.3

Preimmune 123456789101112131415

++-++--++--++++

5,60018,80004,40017,6000001,600008,80040012,8003,280

Total 15 10/15 = 0.66 8,142 ± 6,819 5,374 100.0 0.0

Infected 1234567891011121314151617

+-+++-+++++-+--+-

5,20003,200160,00024,40007,0002,00025,6004001,20003,80000160,0000

Total 17 11/17 = 0.65 35,709 ± 62,073 23,211 431.9 -331.9

Promastigote/insect rates are expressed as individual values and as mean averages ± standard deviation.Infection index: Ratio of infected phlebotomines x average number of promastigotes in insect midguts.



described previously [119], not only the intact QS21 but the dea-cylated saponins also induced a Th1 response, evidenced by thehighest and nonsignificantly different increases in DTH, CD4+

T lymphocytes in spleen, IFN-γ secretion, bodyweight gain anda similar and pronounced reduction of parasite burden in liver(95% for QS21 and 86% for deacylsaponins; p > 0.05) ofL. chagasi-challenged mice [58]. While QS21 showed mild toxic-ity, significant adjuvant effect on the anti-FML humoralresponse before and after infection and a decrease in liver relativeweight, the deacylsaponins showed no toxicity, less hemolysis,an increase in antibody and DTH responses after infection anda stronger Leishmania-specific in vitro splenocyte proliferation[58], indicating that the hydrophobic moiety of the saponins wasnot essential for its complete adjuvanticity. Recently publishedresults confirm the ability of the Leishmune vaccine to inducechanges in T lymphocytes, particularly in CD8+ T cells, andactivate phagocytes in vaccinated dogs [120].

The CP05 saponin from Calliandra pulcherrima Benth showsremarkable similarities to the QS21 saponin of Q. saponariaMolina and similar outstanding adjuvant potential [121]. Bothshare a monoterpene hydrophobic moiety, a glycidic chainattached to the triterpene C28 and three sugars attached to C3.Different from QS21, the CP05 does not show the aldehydegroup in C4-triterpene [121]. We immunized mice with FML for-mulated either with intact saponin (CP05), the monoterpene-deprived saponin (BS), the C28 carbohydrate-deprived saponin(HS) or with the sapogenin fraction, and challenged withL. chagasi [122]. While the CP05 induced 90% survival and92.1% parasite reduction, a 100% survival and 94.1% protec-tion were detected after the BS vaccine treatment, indicating thatthe monoterpene acylated moiety, absent in the BS vaccine, isnot necessary for the induction of a protective global Th1response. Only the DTH response of BS vaccines was mildlylower. Maximal anti-FML antibody, CD4+ and CD8+ Leishma-nia-specific lymphocytes, IFN-γ splenocyte secretion, reductionin parasite load and survival was also detected for the BS vaccine.The HS-FML vaccine showed diminished responses in all testedvariables, except for IFN-γ secretion, indicating that the integrityof the carbohydrate moiety attached to C28 is mandatory forthese functions. No protection was induced by the sapogenin-FML, indicating that the CP05 triterpene lacking the C4 alde-hyde group is not an immunostimulating compound. No contri-bution to protection was detected by the CP05 saponin treat-ment alone, supporting the specificity of the FML antigen [122].

Before the launch of the Leishmune vaccine in Brazil, a safetyanalysis was required to establish the possible degree of toxicityof the formulation [98] that contains 90 µg of QS21 and 97 µgof the deacylated saponins per dose [58]. A group of 600 healthyand asymptomatic dogs from Brazilian canine VL-endemicareas was vaccinated with three doses of Leishmune [97]. Safetyevaluation was performed for 14 days after each vaccine injec-tion and disclosed transient reactions of local pain (40.87%),anorexia (20.48%), apathy (24.17%), local swelling reactions(15.90%), vomit (2.4%) and diarrhea (1.5%) [98]. All effects

showed significantly correlating declines from the first to thethird dose. Most of the noticed reactions of pain (73%), ano-rexia (79%) and local swelling (84.7%) were mild. No signifi-cant differences between puppies and adults dogs were found inthe number of adverse reactions. Adult dogs, however, devel-oped 94.5% of the small swelling reactions (<3 cm), indicatingthat they are more resistant to the inflammatory response pro-moted by the saponins. There were no deaths due to anaphy-laxis and only two dogs (0.1%) showed allergic reactions (facialedema and itching) after the third dose. Transient alopecia oninjection site occurred in only five poodles (0.28%) with totalrecovery and no need for treatment. All mild adverse events inresponse to Leishmune injection were transient and disappearedbefore the following vaccine dose, confirming the tolerability ofthe vaccine. The Leishmune preparation was less hemolytic(HD50 = 180 µg/ml) than expected for a QS21 saponin-con-taining vaccine, indicating that its formulation with the FMLantigen diminished the potential in vitro toxicity [98].

Preliminary recent results suggest that protection induced bythe FML–QS21 vaccine is also related to the activation of abradykinin-mediated inflammatory response at the site of injec-tion, which stimulates immature dendritic cells through theirB1R and B2R surface receptors, thereby triggering a Th1response against L. chagasi [123]. Therefore, part of the adverseinflammatory response induced by the saponin [98] is responsiblefor the protective response of the vaccine [123].

GP36 & NH of the FML antigenFML electrophoretic analysis revealed the presence of several pro-teic bands. Two of them, 36 and 55 kDa, were also stained forcarbohydrates [20]. In western blots, rabbit anti-FML hyper-immune serum reacted with the 36-kDa band [20]. A total of23 hybridomas secreting IgG and seven hybridomas secretingIgM were cloned. A total of 22 IgG clones recognized the36-kDa band of FML, and a single clone recognized the 55-kDaband. All the IgM clones recognized only the 55-kDa band. Noclone recognized both bands or other subfractions of the FML.The integrity of the GP36 glycidic moiety was necessary for itsantigenic function [20]. No crossreactivity between these twoFML fractions was detected. No antigenic homology could bedetected among the 36- and 55-kDa bands of FML and theGP63 major surface leishmanial antigen [20]. The 36-kDa glyco-protein was identified as the major FML antigenic fraction, a sur-face glycoprotein antigen of L. donovani and was designatedGP36 [20]. The GP36 antigen was recognized by sera fromhuman kala-azar patients [25]. Since no crossreaction was detectedwith sera from patients with tegumentary (dermal) leishmaniasis,Chagas’ disease or with sera from normal healthy subjects, GP36was considered to be a marker for human kala-azar [25].

As described previously for the FML antigen [16] and inglycoproteic fractions formerly isolated from L. donovani byOlafson et al. [18], a majority of acid and nonpolar residueswere detected among GP36 components: 13.1% aspartic



acid, 11.0% glutamic acid, 7.6% glycine, 10.2% alanine,7.4% valine and 10.1% leucine. On the other hand, fucose andmannose that were previously characterized in the FML gly-cidic moiety [16] were found in the GP36 glycidic moiety [120].The analysis of acetylated residues performed by gas chromatog-raphy revealed the presence of two different types of fucose resi-dues (2,3-Me2-fucose and 2,4-Me2-fucose) and a majority of2,3,6-Me3-mannose units and tri-Me3-galactose residues, corre-sponding to short linear chains of 4-O-substituted mannopyran-ose alternating with 3-O- and 4-O-substituted fucopyranose res-idues. GP36, then, is a L. donovani-specific antigen thatcontains the sugar ligands necessary for macrophage–parasiterecognition and penetration [124].

The GP36 was isolated by chemical elution plus sonication andused for Balb/c mouse vaccination in combination with saponin,by the subcutaneous route, inducing a strong and specific immu-nogenic and protective effect against experimental VL shown bythe increase of specific IgG (mainly IgG2a) antibodies (82.6%), theDTH to promastigote lysate (37.8%), the ganglia lymphocytes invitro cellular proliferative response to GP36 (53.5%) and thedecrease of liver parasite burden (68.1%) [124]. Saponin-treatedcontrols reacted significantly differently from GP36-vaccinatedanimals for all the assayed variables (p < 0.05). GP36 induced sig-nificant protection against murine VL at concentrationscommonly used for vaccination with recombinant antigens [124].

The gene that encodes an antigenic protein component ofGP36 was cloned on the basis of a partial peptide sequence [91].Based on the predicted open-reading frame, the single-copy genewas identified as a NH (NH36) with significant similarity tofamily members identified from other kinetoplastids. In contrastto the FML antigen, which recognizes both subclinical asympto-matic and clinical overt cases of kala-azar, recombinant NH36reacted mainly with sera of symptomatic dogs in an ELISA assay[91]. Most dogs (18 out of 25) showed a higher IgG1 responseagainst NH36, which was significantly correlated to the increasein the number of symptoms (p = 0.0031) correspondent to theadvancement of the disease. Thus, NH36 is a potential marker ofdisease in zoonotic VL [91].

NH36 has a predicted molecular mass of 34.236 kDa and iscomposed of 314 amino acids [202]. Its sequence alignmentshowed that the predicted protein possessed 95.2% identityand 96.2% similarity to the L. major NH (LmNH) [125], 80.2%identity and 85.7% similarity to the inosine uridine NH ofCrithidia fasciculata (CfIUNH) [126] and 26.8% identity and36.4% similarity to the inosine adenosine guanosine NH ofTrypanosoma brucei brucei (TbIAGNH) [127].

The NH36 is a vital enzyme for parasite cell division [91]. Ithydrolyzes foreign DNA nucleosides, releasing purines or pyri-midines to be used in the synthesis of the parasite’s own DNA.Since these enzymes have not been identified in mammal cells,the NH becomes a potential target for chemotherapy. Itsrecently found genetic polymorphism justified its use in molec-ular taxonomy and in multilocus enzyme electrophoresis-basedphylogenetic analysis of the L. donovani complex [128,129].

NH36 was tested recently as a bivalent DNA vaccine(VR1012–NH36), in comparison with the FML and the recom-binant NH36 antigens formulated with saponin, and was immu-noprotective against visceral (L. chagasi) and tegumentary(L. mexicana) murine leishmaniasis [56]. Significant reduction ofparasitic load compared with controls was achieved after FMLand NH36 vaccination (79%) in L. chagasi-infected mice and byFML vaccination (27%) in L. mexicana experiments. The high-est protection was developed by mice immunized with theVR1012-NH36 DNA vaccine (88% against L. chagasi and 47%against L. mexicana). Protection was related to IFN-γ -producingCD4+ T cells, characteristic of the induction of a Th1-typeimmune response [56]. Our results showed a strong and specificimmunoprotection induced by the NH36 DNA vaccine againstVL and a milder but also protective effect against the tegumentardisease, pointing out its potential use in a bivalent immunopro-phylactic vaccine tool for the control of both endemics [56]. Therecent achievement of mice protection from challenge withL. major, using the recombinant NH [130], and from challengewith L. amazonensis, using the NH DNA vaccine [57], stronglysupports our results. An important immunogenic potential wasdemonstrated for the NH, since strong protection was achievedwith no adjuvant addition to the DNA vaccine [57] and eitherwith or without IL-12 addition to the recombinant protein [130].

Immunotherapy against L. chagasi infection was also assayedusing two doses of 100- or 20-µg NH36 DNA vaccine and, as apossible immunomodulator, aqueous garlic extract [68], which waspreviously effective in immunotherapy of cutaneous murine leish-maniasis [131]. Liver parasitic load was reduced by 100 µg (91%)and 20 µg (77%) of the DNA vaccine, and by 20 µg of the DNAvaccine plus garlic (76%). Survival was 33% for saline controls,100% for the 100-µg vaccine and 83 and 67% for the 20-µg vac-cine with and without garlic addition, respectively. Garlic treat-ment alone did not reduce parasite load but increased survival(100%). The NH36 DNA vaccine has potential in the therapyand control of VL, while the mild protective effect of garlic mightbe related to unspecific enhanced IFN-γ splenocyte secretion [68].

A Phase I–IIa study on the immunoprophylactic effect of theNH36 DNA vaccine is under progress, with protection so far seenin four out of six vaccinated dogs [132]. The prophylactic effect ofthe NH36 DNA vaccine is particularly outstanding since, differentfrom what was reported for other Leishmania DNA vaccines [72],this assay used no adjuvant addition and had a high experimentalvirulent challenge (7 × 108 amastigotes of L. chagasi) [132].

The protective effect for mice and dog vaccinations and thestrong antigenic potential for human and canine serodiagnosisraise the interest in identification of the NH36 epitopes for anti-body interactions and for MHC class I and class II receptors. Forthis purpose, we obtained three recombinant fragments of NH36of molecular weight 10,845.5, 10,327.9 and 13,101.1 Da, respec-tively, by molecular cloning in the pET28 system. Study of theinteraction of these peptides with splenocytes or ganglia cells ofimmunized animals will disclose valuable information leading tofurther development of a potential synthetic vaccine against VL.



Expert commentaryMany Leishmania genes have been identified as vaccine candi-dates. DNA vaccines are more stable, do not require cold chainnor addition of bovine calf sera in culture media used for large-scale preparations of parasites. However, the slow knowledgetransfer from the laboratory to industry, the good manufacturingpractice regulations that dampen down the industrial interest, thepoorly developed biotechnological industry and the lack of scien-tists in regulatory agencies of underdeveloped countries, the ethi-cal constraints on research in animals and the increasing dogchemotherapy in Europe, where human leishmaniasis is less fre-quent, contribute to the continuous use of first-generation andlive vaccines and to the delay in the arrival of combined DNAvaccines to public health. Our prediction is that vaccines for leish-maniasis in the forthcoming years will be of the second-generationtype, composed of complex native antigens and well-developedadjuvants. Different from other important re-emergent zoonoseswith wild reservoir animals, and being a canid zoonoses, VL hasthe advantage of being relatively easier to control in the domesticdog population, both in America and in the Mediterranean. Inthis scenario, the broader use of the FML–saponin Leishmunevaccine, the only canine Leishmania vaccine licensed for prophy-lactic use to date, will have a positive impact on the control of VL,reducing the canine and human incidence of the disease and theneed of culling seropositive infected dog reservoire.

Five-year viewThe perspective for the forthcoming years is that the increase inthe use of Leishmune will reduce the canine and human inci-dence of VL in Brazil. The development and license of new diag-nostic kits that allow the differentiation of the seropositive Leish-mania-infected from the seropositive Leishmune-vaccinated dogswill allow the broader use of Leishmune in Brazil, were the epide-miological enquire is based on serology. When the seropositivity

of vaccinated dogs will no longer be considered as a problem forcontrol campaign, the increase of the saponin adjuvant concen-tration in the formulation will be feasible, since its safety hasalready been shown. In that way, the vaccine will not only be pro-phylactic but also therapeutic. In Europe, where neither serologi-cal control nor culling is performed, and where the human inci-dence is lower, the vaccine could be launched directly using thehigher saponin concentration. In the next few years, we intend toidentify the principal epitopes of the NH36 antigen, in order tobetter understand the genesis of the immunogenic response ofthe Leishmune and the NH36 DNA vaccine, and to developnew synthetic diagnostic methods and/or new synthetic vaccines.

Acknowledgements

We are grateful to I Amorim, E Freitas, MN Melo, MSM Michalick,ES Dias and A Costa-Val for the great effort made to obtain thexenodiagnostic results and to I Silva-Antunes and A Aguiar Morgado fromthe Center for Zoonoses Control, Araçatuba, São Paulo, Brazil, for theofficial epidemiological data. The authors are gratefull to David Strakerfor English review.

Financial & competing interests disclosure

This work was financially supported by Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq) (Edital Milênio420067/2005, Edital Universal 473830/2007-8 and productitivityfellowship 301215/2007-3), Fundação de Amparo à Pesquisa do Estadodo Rio de Janeiro (FAPERJ-PRONEX, CNE fellowshipE-26/152824/2006, Edital Pensa Rio E-26/110305/2007 and EditalFAPERJ-INFRA E-26/110132) and Fundação de Amparo à Pesquisa doEstado de São Paulo (FAPESP 2006/02832-0). The authors have noother relevant affiliations or financial involvement with any organizationor entity with a financial interest in or financial conflict with the subjectmatter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Key issues

• A licensed second-generation, a recombinant and a DNA vaccine against canine visceral leishmaniasis were developed based on the fucose–mannose glycoproteic ligand (FML) complex of Leishmania donovani and its nucleoside hydrolase NH36 main antigen.

• The search for a relevant L. donovani glycoconjugate candidate disclosed the FML as the most immunogenic.

• The FML ELISA was useful in human and dog diagnosis and prognosis and in human-cure monitoring and blood-bank control.

• The FML ELISA is also a useful tool in epidemiological control of the disease.

• The FML second-generation vaccine development involved selection of routes and adjuvants, Phase I–III assays in mice, hamsters and dogs, and the structure–function study of the saponin adjuvants.

• The canine prophylactic vaccine is licensed in Brazil under the name of Leishmune®. It contains the QS21 and deacylated saponins of Quillaja saponaria Molina as adjuvants and renders the vaccinated dogs noninfectious.

• Leishmune with increased adjuvant concentration is also immunotherapeutic.

• Antibodies generated in dogs vaccinated with Leishmune impede the development of Leishmania in the sand fly vector, blocking transmission of the disease.

• The nucleoside hydrolase NH36 of the FML antigen is its main antigenic compound and specific marker of dog and human visceral leishmaniasis and a NH36 DNA vaccine showed to be prophylactic in mice and dogs and immunotherapeutic in mice.



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Patents

201 Universidade Federal do Rio de Janeiro, Clarisa B. Palatnik de Sousa and Radovan Borojevic, Brazilian patent INPI: (pipeline) PI1100173-9.

202 The European Molecular Biology Laboratory, Genbank™ and DDJB data bases, access number: AY007193.

Websites

301 World Health Organization-TDR. The TDR fifteenth programm report. Research progress 1999–2000. New and improved tools. 2003www.who.int/tdr/research/progress 9900/tools/vdr.htm(Accessed 5 February 2008).

302 Bernardo RR, Palatnik de Sousa CB, Parente JP. N-linked oligosaccharide structures of the FML antigen of Leishmania (L.) donovani. Mem. Inst. Oswaldo Cruz MEMIOC. 93, 179 (1998)www.scielo.br/pdf/mioc/v93s2/bc.pdf

303 US Food and Drug Administrationwww.fda.gov/bbs/topics/ANSWERS/ANS00360.html

304 Dumonteil E, McMahon-Pratt D, Price VL. Report of the fourth TDR/IDRI meeting on second generation Vaccines against leishmaniaisis. UNDP/World Bank/WHO Special Programme for Research & Training in Tropical Diseases (TDR). TDR/WHO (Ed.) Geneva SW,10, (2001)www.who.int/leishmaniasis/resources/documents/en/TDR_PRD_LEISH_VAC_01.1.pdf.

Affiliations

• Clarisa B Palatnik-de-SousaInstituto de Microbiologia, CCS, UFRJ, Avda Carlos Chagas 373, Caixa Postal 68040, 21941-590 Cidade Universitária, Ilha do Fundão, Rio de Janeiro, BrazilTel.: +55 21 2562 6742 ext. 145Fax: +55 21 2560 [email protected]

• André de Figueiredo BarbosaInstituto Oswaldo Cruz, CEP 21045-900 Rio de Janeiro, [email protected]

• Sandra Maria OliveiraInstituto Oswaldo Cruz, CEP 21045-900 Rio de Janeiro, [email protected]

• Dirlei NicoInstituto de Microbiologia Prof. Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Avda Carlos Chagas 373, Cid. Universitária, Ilha do Fundão, Caixa Postal 68040 CEP 21941-590 Rio de Janeiro, BrazilTel.: +55 21 2560 6742Fax: +55 21 2560 [email protected]

• Robson Ronney BernardoUniversidade Estácio de Sá, Fac. Farmácia. Avda Ducídio Cardoso 2900, CEP 22631-052, Barra da Tijuca, Rio de Janeiro, BrazilTel.: +55 21 2432 [email protected]

• Wania R SantosBiomanguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil

• Mauricio M RodriguesCINTERGEN, UNIFESP-Escola Paulista de Medicina, Rua Mirasol 207, Vila Clementino, CEP 04044-010, São Paulo, [email protected]

• Irene SoaresDepartamento de Análises Clínicas e Toxicológicas, Universidade de São Paulo, Avenue prof. Lineu prestes 580, BL17, CEP05508-900, São Paulo, [email protected]

• Gulnara P Borja-CabreraInstituto de Microbiologia Prof. Paulo de Góes, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Avda Carlos Chagas 373, Cid. Universitária, Ilha do Fundão, Caixa Postal 68040 CEP 21941-590 Rio de Janeiro, BrazilTel.: +55 21 2560 6742Fax: +55 21 2560 [email protected]

FML vaccine against canine visceral leishmaniasis: from second-generation to synthetic vaccine

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Transcript of FML vaccine against canine visceral leishmaniasis: from second-generation to synthetic vaccine