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. 2015 Apr 10;4(4):e35.
doi: 10.1038/cti.2015.6. eCollection 2015 Apr.

From mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE

Affiliations

From mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE

Rhea N Coler et al. Clin Transl Immunology. .

Abstract

Key antigens of Leishmania species identified in the context of host responses in Leishmania-exposed individuals from disease-endemic areas were prioritized for the development of a subunit vaccine against visceral leishmaniasis (VL), the most deadly form of leishmaniasis. Two Leishmania proteins-nucleoside hydrolase and a sterol 24-c-methyltransferase, each of which are protective in animal models of VL when properly adjuvanted- were produced as a single recombinant fusion protein NS (LEISH-F3) for ease of antigen production and broad coverage of a heterogeneous major histocompatibility complex population. When formulated with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE), a Toll-like receptor 4 TH1 (T helper 1) promoting nanoemulsion adjuvant, the LEISH-F3 polyprotein induced potent protection against both L. donovani and L. infantum in mice, measured as significant reductions in liver parasite burdens. A robust immune response to each component of the vaccine with polyfunctional CD4 TH1 cell responses characterized by production of antigen-specific interferon-γ, tumor necrosis factor and interleukin-2 (IL-2), and low levels of IL-5 and IL-10 was induced in immunized mice. We also demonstrate that CD4 T cells, but not CD8 T cells, are sufficient for protection against L. donovani infection in immunized mice. Based on the sum of preclinical data, we prepared GMP materials and performed a phase 1 clinical study with LEISH-F3+GLA-SE in healthy, uninfected adults in the United States. The vaccine candidate was shown to be safe and induced a strong antigen-specific immune response, as evidenced by cytokine and immunoglobulin subclass data. These data provide a strong rationale for additional trials in Leishmania-endemic countries in populations vulnerable to VL.

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Figures

Figure 1
Figure 1
Recognition of NH and SMT antigens in patients from a L. donovani endemic area in Bangladesh. (a) Total IgG against L. donovani SLA, NH and SMT are shown. Antibodies against the antigens were measured by ELISA in sera (used at 1:400 dilution) in VL individuals (n=25), asymptomatic individuals (n=32) and non-endemic controls (n=24). (b) PMBCs were recalled with 10 μg ml−1 L. donovani SLA, NH or SMT and analyzed for CD4 T cells production of indicated cytokines by flow cytometry. (a and b) Black bars indicate median OD for each group. Statistical significance indicated is vs the non-endemic normal group; statistic was calculated by Kolmogorov–Smirnov t-test. ****P<0.0001.
Figure 2
Figure 2
LEISH-F3 construct and characterization. (a) Schematic of LEISH-F3 (NS) fusion protein. (be) SDS-PAGE and immunoblot of LEISH-F3 (NS). (b) LEISH-F3 was run in reducing and nonreducing conditions on a 4–20% Tris-glycine gel. (c) Immunoblot of LEISH-F3 with rabbit polyclonal antibody to LEISH-F3; 100 ng each of LEISH-F3, NH and SMT; 27 μg of L. donovani whole-cell lysate (WCL). (d) Immunoblot of 2 μg of LEISH-F3 and HMS174 E. coli lysate with antibody to HMS174 E. coli. (e, f) Immunoblot of LEISH-F3 and its component antigens, NH and SMT, with mouse monoclonal antibody (mAb) against (e) NH or (f) SMT; 100 ng each of LEISH-F3, NH and SMT were loaded.
Figure 3
Figure 3
LEISH-F3+GLA-SE is immunogenic and generates an antigen-specific TH1 memory response. BALB/c mice were immunized with the indicated formulations three times, at 3-week intervals, for the analysis of LEISH-F3-specific immune responses. (a) Serum was collected 4 weeks after the final immunization and analyzed for LEISH-F3-specific antibodies by ELISA end-point titration (n=5). Statistics within isotypes (vs saline) and between isotypes by Sidack's multiple comparison test. *P<0.05 and ****P<0.0001. (be) LEISH-F3-specific immune responses were analyzed by recall of splenocytes with 10 μg ml−1 LEISH-F3 from mice 4 weeks after the final immunization. (b) ELISPOT assay of IFNγ and IL-5 (n=4); statistics by Dunnett's multiple comparison test within each cytokine vs saline. ****P<0.0001. (c) Cytokine bead array of cytokines in the supernatant of splenocytes recalled for 72 h (n=3). Statistics by Dunnett's multiple comparison test vs saline. *P<0.05. (d) Percentage of CD4 T cells expressing CD154 (n=5); statistics by Dunnett's multiple comparison test. *P<0.05 and ****P<0.0001. (e and f) Percentages of polyfunctional CD4 T cells expressing combinations of IFNγ, TNF and IL-2 (n=5). (f) Statistics by Dunnett's multiple comparison test within polyfunctional cytokine groups vs saline. *P<0.05, **P<0.01 and ****P<0.0001. (g) Major histocompatibility complex (MHC-II) epitope mapping of the LEISH-F3 fusion protein in LEISH-F3+GLA-SE-immunized mice. Statistics by Student's t-test vs media. *P<0.05 and **P<0.01.
Figure 4
Figure 4
CD4 memory T cells generated by vaccination with LEISH-F3+GLA-SE protect against experimental VL infection. (a and c) BALB/c and (b and d) C57BL/6 mice were immunized with the indicated vaccines three times, at 3-week intervals. (ac) Four weeks after the final immunization, mice were infected with (a and b) 1 × 106L. donovani or (c) 5 × 106 L. infantum parasites. (d) Three weeks after the final immunization, mice received three intraperitoneal injections, on 3 consecutive days, of 0.5 mg of the indicated depleting antibody; 3 weeks later, mice were infected with 1 × 106 L. donovani parasites intravenously. (ad) Livers were harvested 3 weeks after infection and analyzed by reverse transcription-PCR (RT-PCR) to determine the total parasite burden. (a and b) Statistics by Tukey's multiple comparison test. *P<0.05 and ***P<0.005. (c and d) Statistics by unpaired t-test between indicated groups. *P<0.05, **P<0.01 and ****P<0.001.
Figure 5
Figure 5
Clinical trial CONSORT diagram. Flow chart shows the number of subjects entering the study from enrollment, allocation and follow-up (FU). Subjects missing the second and third injection dose either declined to continue or were lost to follow-up. Subjects were in follow-up for 1 year after the third injection. The safety population is defined as all subjects who received at least one study injection; the per-protocol population comprises subjects who received all three study injections and completed day 84.
Figure 6
Figure 6
LEISH-F3 formulated with GLA-SE is immunogenic in humans. Thirty-six healthy adult subjects were immunized on days 0, 28 and 56 with 20 μg LEISH-F3+2 μg GLA-SE (n=12), 20 μg LEISH-F3+5 μg GLA-SE (n=12) and 20μg LEISH-F3 protein alone (n=12). The immunogenicity of the vaccine was evaluated by assessing antibody and T-cell responses at days 0, 35, 63, 84 and 168 and days 0, 7, 35, 63, 84 and 168, respectively. (a and b) ELISAs for titers of LEISH-F3-specific antibodies (total and IgG subclasses and total IgE) in volunteer serum were conducted for the indicated time points. (c) Quantitative T-cell responses to LEISH-F3 was measured by IL-2, IL-5, IL-10, IFN-γ and TNF cytokine production in whole blood Luminex assay (WBA). Nine subjects were excluded from the per-protocol population immunology summaries resulting in evaluable groups such as: 2 μg GLA-SE (n=8), 5 μg GLA-SE (n=10) and LEISH-F3 alone (n=9). P-value for comparison was performed for various treatment groups: between 2 and 5 μg GLA-SE vaccine groups and between vaccine (2 and 5 μg GLA-SE vaccine groups combined) and 20 μg LEISH-F3 alone. P-values were considered significant at the 0.05 significance level. *P-values significant at 0.05 significance level when compared between vaccine (2 and 5 μg GLA-SE vaccine groups combined) and 20 μg LEISH-F3 alone. **P-values significant at 0.05 significance level when compared between vaccine (2 and 5 μg GLA-SE vaccine groups separately).

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