A defined subunit vaccine that protects against vector-borne visceral leishmaniasis

Abstract

Vaccine development for vector-borne pathogens may be accelerated through the use of relevant challenge models, as has been the case for malaria. Because of the demonstrated biological importance of vector-derived molecules in establishing natural infections, incorporating natural challenge models into vaccine development strategies may increase the accuracy of predicting efficacy under field conditions. Until recently, however, there was no natural challenge model available for the evaluation of vaccine candidates against visceral leishmaniasis. We previously demonstrated that a candidate vaccine against visceral leishmaniasis containing the antigen LEISH-F3 could provide protection in preclinical models and induce potent T-cell responses in human volunteers. In the present study, we describe a next generation candidate, LEISH-F3+, generated by adding a third antigen to the LEISH-F3 di-fusion protein. The rationale for adding a third component, derived from cysteine protease (CPB), was based on previously demonstrated protection achieved with this antigen, as well as on recognition by human T cells from individuals with latent infection. Prophylactic immunization with LEISH-F3+formulated with glucopyranosyl lipid A adjuvant in stable emulsion significantly reduced both Leishmania infantum and L. donovani burdens in needle challenge mouse models of infection. Importantly, the data obtained in these infection models were validated by the ability of LEISH-F3+/glucopyranosyl lipid A adjuvant in stable emulsion to induce significant protection in hamsters, a model of both infection and disease, following challenge by L. donovani-infected Lutzomyia longipalpis sand flies, a natural vector. This is an important demonstration of vaccine protection against visceral leishmaniasis using a natural challenge model.

Fig. 1

Recognition of truncated cysteine protease B protein (ΔCPB) by L. donovani-affected individuals. In a, serum antibodies against ΔCPB were measured by ELISA in samples from VL patients (VL; n = 23) and non-endemic control individuals (NEC; n = 24). Each point represents the response of each individual sample with black bars indicating the mean OD for each group. In b, peripheral blood mononuclear cells from subjects living in either a highly endemic (n = 20) or non-endemic (n = 10) region were incubated with L. donovani SLA or ΔCPB prior to analyses by flow cytometry to identify CD4 T cells producing IFN-γ, TNF or IL-2, respectively. Each point represents the response of each individual sample with black bars indicating the mean for each group. Positive responses were determined as having optical densities (OD) or cell populations greater than the mean plus 3 s.e.m of that observed with NEC. Statistical significance was calculated by Kolmogorov–Smirnov test, *p < 0.05, ***p < 0.001, and ****p < 0.0001

Fig. 2

Construction and characterization of the LEISH-F3+ fusion protein. In a, a cartoon depiction of the LEISH-F3+ fusion protein is shown. In b, LEISH-F3+ fusion protein was characterized by immunoblot. Recombinant LEISH-F3+ (lane 1), Nucleoside hydrolase (NH; lane 2), Sterol-24-c-methyl-transferase (SMT; lane 3) or truncated cysteine protease B protein (ΔCPB; lane 4); were loaded at 100 ng each lane into gels. Blots were developed with mouse polyclonal or monoclonal antibodies as indicated, derived from the same original gel. The cartoon depiction of the fusion protein is the authors own rendering

Fig. 3

Immune recognition of each component within LEISH-F3+. C57BL/6 mice were injected a total of three times with 1 μg LEISH-F3+ protein, or a molar equivalence of the individual NH, SMT and ΔCPB proteins alone, formulated with GLA-SE. One month after the final immunization spleens were removed to prepare single cell suspensions (n = 3). In a, cells were incubated with antigen indicated in the box for 4 days then cytokine content in the culture supernatant determined by ELISA. In b, cells were subjected to flow cytometry to identify antigen-experienced CD4 T cells and the cytokine protection profile (various combinations of IFN-γ, IL-2, or TNF). Data are shown as mean and s.e.m, three mice per group. Data are representative of results obtained in two similar experiments

Fig. 4

Immunization with LEISH-F3+ reduces Leishmania infection. Mice were subcutaneously injected a total of three times with 5 μg protein formulated with GLA-SE, then 1 month after the final immunization were infected by intravenous injection of Leishmania spp. promastigotes. Livers were removed 1 month after inoculation and parasite burdens were determined by qPCR. In a and b, C57BL/6 and BALB/c mice, respectively, were infected with L. donovani. In c and d, C57BL/6 and BALB/c mice, respectively, were infected with L. infantum. Each point represents the burden of each individual mouse, with the bars indicating the mean and s.e.m for each group with 7–10 mice per group. Data are representative of results obtained with each protein in 2–3 independent experiments

Fig. 5

Immunization with LEISH-F3+/GLA-SE reduces parasite burden in hamsters infected with L. donovani during sand fly blood meals. In a, hamsters were injected a total of three times with 5 ug protein formulated with GLA-SE (step 1), then 1 month after the final immunization were infected by bites of L. donovani-infected sand flies (step 2), then monitored (step 3). In b, hamster weights were checked just prior to exposure to sand flies then monitored throughout the infection phase of the experiment. Each point depicts the group mean at the given time. In c, spleens were removed 9 months after parasite inoculation and burdens were determined by qPCR. Each point represents the burden of each individual animal, with the bars indicating the median of 11–12 hamsters per group. Statistical significance was calculated by Wilcoxon-signed rank sum analysis. Data are representative of results obtained in two similar experiments. The images are the authors own renderings