AbstractVisceral leishmaniasis is a chronicand lethal parasite disease against which no human vaccine is available.Hepato- splenomegaly and a progressive suppression of the cellular immune response are among its most important clinical signs. The characteristic cellular immunosupression was described as being mediated in part, through the spatial segregation of dendritic cells (DCs) and T cell lymphocytes due to altered frequencies and migration capabilities of DCs. In this investigation, we measured the spleen/body relative weight, the spleen parasite load and the total counts of spleen DCs of C57BL6 mice infected with Leishmania chagasi. All the variables achieved their maximum at 30 days after infection. We detected in infected animals a 5.08 fold increase of spleen relative weight, a 19.6 fold increase of parasite load and a 4.55 increase of total DCs counts, when compared to naïve controls. We further analysed the efficacy of the NH36 and F3 vaccines formulated in saponin in prevention of visceral leishmaniasis. When compared to the infected controls, both vaccines determined strong protection. The F3 vaccine induced the highest efficacy showing 95% and 49% reduction the parasite load and splenomegaly, respectively. The NH36 vaccine, on the other hand, developed a slightly lower but still significant protection reducing by 87% the parasite load and by 39% the spleen relative weight. Both vaccines also prevented the increase in total counts of DCs with no significant difference between them (36% by the NH36 and 26% by the F3 vaccine). Our results suggest that vaccination against murine visceral leishmaniasis with the NH36 vaccine can prevent the development of the disease by preventing the DCs dysfunction-related immunosupression. Additionally, they disclose the potential use of the NH36 C-terminal moiety, the F3 peptide for optimization of the vaccine efficacy

Morrot, Alexandre

Nico, Dirlei

Palatnik-de-Sousa, Clarisa Beatriz

Elsevier - Publisher Connector 

Vaccination with Nucleoside Hydrolase (NH36) of L.(L.) Donovani or its C-terminal Portion (F3) in Formulation with Saponin Prevents the Increase of the Proportions of Spleen Dendritic Cells in Murine Experimental Visceral Leishmaniasis 

1877-282X © 2014 Elsevier B.V. This is an open access article under the CC BY-NC-ND license 
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Selection and peer-review under responsibility of the 7th Vaccine Conference Organizing Committee.
doi: 10.1016/j.provac.2014.07.010 
Available online at www.sciencedirect.com
ScienceDirect
7th Vaccine & ISV Congress, Spain, 2013 
Vaccination with Nucleoside hydrolase (NH36) of L.(L.) donovani or its C-terminal 
portion (F3) in formulation with saponin prevents the increase of the proportions of 
spleen dendritic cells in murine experimental visceral leishmaniasis  
Dirlei Nico, Alexandre Morrot, Clarisa Beatriz Palatnik-de-Sousa* 
Insituto de Microbiologia Paulo de Góes, Universidade Federal de Rio de Janeiro, Avda Carlos Chagas 373. Cidade Universitaria, Ilha do 
Fundão,Rio de Janeiro, CEP 21941-902, Brazil. 
Abstract 
Visceral leishmaniasis is a chronicand lethal parasite disease against which no human vaccine is available.Hepato-
splenomegaly and a progressive suppression of the cellular immune response are among its most important clinical 
signs. The characteristic cellular immunosupression was described as being mediated in part, through the spatial 
segregation of dendritic cells (DCs) and T cell lymphocytes due to altered frequencies and migration capabilities of 
DCs. In this investigation, we measured the spleen/body relative weight, the spleen parasite load and the total counts 
of spleen DCs of C57BL6 mice infected with Leishmania chagasi. All the variables achieved their maximum at 30 
days after infection. We detected in infected animals a 5.08 fold increase of spleen relative weight, a 19.6 fold 
increase of parasite load and a 4.55 increase of total DCs counts, when compared to naïve controls. We further 
analysed the efficacy of the NH36 and F3 vaccines formulated in saponin in prevention of visceral leishmaniasis. 
When compared to the infected controls, both vaccines determined strong protection. The F3 vaccine induced the 
highest efficacy showing 95% and 49% reduction the parasite load and splenomegaly, respectively. The NH36 
vaccine, on the other hand, developed a slightly lower but still significant protection reducing by 87% the parasite 
load and by 39% the spleen relative weight.  Both vaccines also prevented the increase in total counts of DCs with 
no significant difference between them (36% by the NH36 and 26% by the F3 vaccine). Our results suggest that 
vaccination against murine visceral leishmaniasis with the NH36 vaccine can prevent the development of the disease 
by preventing the DCs dysfunction-related immunosupression. Additionally, they disclose the potential use of the 
NH36 C-terminal moiety, the F3 peptide for optimization of the vaccine efficacy. 
Keywords: Visceral leishmaniasis; Leishmania donovani; Leishmania chagasi; C57BL6 mice; spleen/body relative 
weight; splenomegaly; dendritic cells  
© 2014 The Authors. Published by Elsevier B.V. 
Selection and peer-review under responsibility of the 7th Vaccine Conference Organizing Committee. 
*Corresponding author. Tel.: +55-21-25626742; fax:+55-21-25608344 
E-mail address: immgcpa@micro.ufrj.br 
  Els vier B.V. This is an open acc ss article under the CC BY-NC-ND license 
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Selection and peer-review under responsibility of the 7th Vaccine Conference Organizing Committee.
 Procedia in Vaccinology  8 ( 2014 )  58 – 61 
59 Dirlei Nico et al. /  Procedia in Vaccinology  8 ( 2014 )  58 – 61 
 
Visceral leishmaniasis is a chronic parasite disease which occurs as a canine zoonosis caused by 
Leishmania infantum/ L. chagasi in the Americas, the Middle East, Central Asia, China and the Mediterranean and 
as an anthroponosisprovoked by L. donovani in India and Central Africa. No human vaccine against VL is available 
[1]. Annually, 0.2 to 0.4 million new human cases of VL are registered [2]. More than 90% of them occur in India, 
Bangladesh, Sudan, South-Sudan, Ethiopia andBrazil [2].The human disease is lethal if not treated early after the 
onset of clinicopathological abnormalities that include: malaise, anaemia, cachexia, hypergammaglobulinaemia, 
hepato-splenomegaly and progressive suppression of the cellular immune response [1].  
The geographical encounter between DCs and T cells in the T cell areas of secondary lymphoid organs 
determines the success of cellular immunity after infection. In spleen, DCs should migrate from the marginal zone 
(MZ) to the periarteriolar sheath (PALS), to meet T cells in response to constitutively express chemokine gradients 
of CCL21 and CCL19. In contrast to that, a defective DC migration plays a major role in the pathogenesis of VL. 
Indeed, the characteristic cellular immunosupression of VL is mediated in part, through the spatial segregation of 
DCs and T cells [3]. 
In the BALB/c and C57BL/6 mice models of VL, an increase in the absolute numbers of spleen CD11c+ 
DCs during the progression of L. donovaniinfection was described [3]. During this period, the parasite number 
increased and severe splenomegaly became evident [3]. Furthermore, DCs of L. donovani infected mice showed a 
defective migration to the periarteriolar lymphoid sheath (PALS) which prevented the antigen presentation to T cells 
and the subsequent T cell activation and optimal induction of immune response against the parasite [3]. 
We developed the first vaccine licensed against canine VL (Leishmune®) which is composed of the FML 
(Fucose mannose ligand) antigen of L. donovani and QS21 and deacylated saponins of Quillaja saponaria. The 
vaccine induces 98% protection to canine VL in healthy dogs [4], reduces the human and canine incidence of 
disease [5], the number of exposed parasites to insect vectors [6] and is a transmission blocking vaccine [7, 8]. The 
main antigen of FML is the Nucleoside hydrolase of Leishmania donovani (NH36) which protects mice and dogs 
from VL [revised in 8]. Vaccination with the NH36 C-terminal moiety, the F3 peptide, formulated with saponin, 
induced the strongest protection (88-97%) against VLthat was mediated by a CD4+-T cell driven immune response 
[9]. 
As a first step in the evaluation of the impact of vaccination with NH36 or F3, in prevention of the 
defective distribution and function of spleen´s DCs, we here assessed the variation of splenomegaly, parasite load 
and absolute numbers of CD11c+ DCsin spleens of C57BL6 vaccinated mice that were further infected with L. 
chagasi. 
Eight week old C57BL6 females were vaccinated with three doses of 100 μg of NH36 or the F3 peptide obtained as 
described before [9] and formulated in saponin, through the sc route, with weekly interval. On week four,the 
intradermal response against L. donovani promastigotes was evaluated and a challenge with 3 x 107 amastigotes of 
L. chagasi obtained from infected hamsters was performed by intravenous injection via the tail vein. Mice were 
sacrificed at day 15, 30 and 45 after infection, for evaluation of splenomegaly (% of spleen weight/ body weight), 
parasite load determination in Giemsa stained spleen smears (Leishman Donovan units-LDU= number of 
amastigotes/ 500 spleen cells x spleen weight in mg) and total counts of spleen DCs. To isolate the DCs spleen were 
harvested aseptically and disrupted with DNAse and collagenase. The cell suspensions were counted in 
haemocytometer and labelled with anti-CD11c (DCs surface marker)-magnetic beads. DCs were further separated 
by chromatography in magnetic columns, recovered by mechanical force, centrifuged, incubated with CD11-c-FITC 
antibody and counted.All mouse studies followed the guidelines set by the National Institute of Health, USA, the EU 
Directive 2010/63/EU and the Institutional Animal Care and Use Committee approved the animal protocols 
(Biophysics Institute-UFRJ, Brazil, protocol IMPPG-007).We use the Kruskall Wallis and Mann Whitney non-
parametrical tests for comparison of means. 
 The respective evolution of the spleen/body relative weight, spleen parasite load and total counts of spleen 
DCs along the time are summarized, respectively in Figure 1 A, B and C. When compared to normal uninfected 
mice  (Figure 1A-C), the splenomegaly, the number of parasites (LDU units) and the number of DCs of infected 
mice were significantly increased and developed exponential slopes from day 15 to day 30 followed by a plateau 
detected from day 30 to day 45 after infection. Our data slightly differ from the results obtained by Ato et al. [3]who 
used the same mice model but L. donovani infection. Ato et al. [3] expressed the relative splenomegaly, as the  
 
 
60   Dirlei Nico et al. /  Procedia in Vaccinology  8 ( 2014 )  58 – 61 
ratio of the spleen weight to body weight of infected mice divided by that of naïve mice. Despite they described 
ratios close to 1on day 14, the peak achieved on day 28 was lower (1.7) than the one we detected in L. chagasi 
infected mice (5.08 = 3.41 in infected/0.67in naïve mice) suggesting a more advanced pathology caused by infection 
with L. chagasi. Ato et al. (2002) did not follow up the infection up to 45 days [3].In agreement with the higher 
levels of spleen relative weight, the numbers of spleen LDU achieved after L. chagasi infection (39.2 at day 15 and 
86.2 at day 30) (Figure 1 B) were much higher than those reported after L. donovani infection (2 at day 15 and 10 at 
day 30) [3]. Indeed, the L. chagasi parasite loadshowed a 19.6 and 8.62 respective fold increases, for day 15 and day 
30, respectively,above those achieved in the L. donovani model [3].Additionally, the total number of DCs was 
higher in our L. chagasi model (45.55 x 106 DCs at day 30)(Figure 1 C) than in the L. donovani model (10 x 106) 
[3].Ato et al., used an infective challenge only 1.5 fold lower (2 x 107 amastigotes of L. donovani) [3] than the one 
we used in this L. chagasi model of infection (2 x 107 amastigotes). Therefore, the 5.08 fold increase in spleen 
relative weight, 19.6 fold increase in parasite load and 4.55 increase in total DCs counts could not attributed to the 
higher inoculum but instead, to moreaggressive aspects of the infection by L. chagasi. 
 
 
 
Fig. 1. Variation of the (A) spleen/body relative weight, (B) spleen parasite load and (C) total counts of DCs in spleen, along the time (days).  
Effect of the preventive vaccination with NH36sap and F3sap on the (D) spleen/body relative weight, (E) spleen parasite load and (F). Results of 
two identical experiments with n=7 mice per group in each experiment. 
 
 
We further analysed the efficacy of the NH36 and F3 vaccines formulated in saponin (NH36sap and F3sap) 
in prevention of the increase in spleen relative weight, spleen parasite load and increase of DCs total provoked by 
the challenge with L. chagasi(Figure 1 D-F). The comparison was performed at day 30 after infection, when these 
deleterious effects showed their highest incidence (Figure 1 A-C). As expected, the infection enhanced the levels of 
spleen/body relative weight (Fig. 1D), and DCs counts (Fig. 1 F) above those exhibited by naïve controls. 
Additionally, when compared to the infected controls, both vaccines determined strong protection.A significantly 
higher efficacy was developed by the F3 vaccinewhich reduced by 95% the parasite load (Fig. 1 E) and by 49% the 
splenomegaly (Fig. 1 D). The NH36 vaccine, on the other hand, developed a slightly lower but still significant 
protection reducing by 87% the parasite load and by 39% the spleen relative weight. Both vaccines also prevented 
61 Dirlei Nico et al. /  Procedia in Vaccinology  8 ( 2014 )  58 – 61 
the increase in total counts of DCs with no significant difference between them (36% by the NH36 and 26% by the 
F3 vaccine) (Figure 1 D-F). 
Our results of the L. chagasi model support the previous report of Ato et al., [3] who described the increase 
of the frequency of total DCs in spleens of L. donovani infected mice during the period of increase of parasite load 
and pathology. Furthermore, we showed that the severity of parasitic and clinical signs is higher during the infection 
caused by L. chagasi. The NH36 and its C-terminal moiety, the F3 peptide, showed high efficacy in vaccination 
against this very severe infection. The F3 peptide was identified before as the domain of the NH36 of L. donovani 
responsible for its immunogenicity and protective efficacy against murine VL [9]. Immunization with F3 exceeds in 
36.73 ± 12. 33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and 
IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T 
cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated 
mice [9]. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong 
correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted 
CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p=0.011) that was long-lasting 
[9]. In this investigation we further demonstrate that, in the more susceptible C57BL6 mice model, protection 
induced by the NH36 vaccine is also directed to its F3 peptide. In addition, we showed that the modulation of the 
immunoprotective response against VL induced by F3 is related also to the prevention of the increase of the total 
numbers of DCs in spleens. The analysis of the migration capabilities of the spleen DCs is under progress. 
 
Acknowledgements 
 This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ, 
Fellowships 301215-2007-3, 302039/2010-4, 559756/2010-0 and grant 404400/ 2012-4) and by Fundação de 
Amparo à Pesquisa do Estado de Rio de Janeiro (FAPERJ, grants 102733/2008 and 102957/2011 and Fellowships 
E-26/102415/2010 and E-26/110535/2010).  
 
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Dirlei Nico

Alexandre Morrot

Clarisa Beatriz Palatnik-de-Sousa

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Procedia in Vaccinology

Vaccination with Nucleoside Hydrolase (NH36) of L.(L.) Donovani or its C-terminal Portion (F3) in Formulation with Saponin Prevents the Increase of the Proportions of Spleen Dendritic Cells in Murine Experimental Visceral Leishmaniasis

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Vaccination with Nucleoside Hydrolase (NH36) of L.(L.) Donovani or its C-terminal Portion (F3) in Formulation with Saponin Prevents the Increase of the Proportions of Spleen Dendritic Cells in Murine Experimental Visceral Leishmaniasis

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