Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis

Abstract

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen^-/-) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen^-/- parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen^-/- immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen^-/- infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen^-/- immunized hamsters. This study demonstrates that the LmCen^-/- parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial.

Fig. 1. Live attenuated LmCen^−/− parasites do not cause any pathology in hamster model and do not cause any infection in sand flies.

a Lesion size was monitored every week in hamsters injected with 10⁶-total stationary phase either LmWT or LmCen^−/− parasites by intradermal (ID) injection. Ear lesion diameters were measured at indicated days post inoculation. Results (SD) are representative cumulative effect of two (3 d, 15 d, and 28 d) to three (49 d) independent experiments, (p values were determined by Mann–Whitney two-tailed test). b Photographs of representative ears of LmWT and LmCen^−/− immunized hamsters at 49 days post inoculation. Red arrow indicates the lesion development. c, d Parasite load in the ear (c) and draining lymph node (dLN) (d) of LmWT and LmCen^−/− immunized hamsters were determined by serial dilution assay at 3, 15, and 28 days (n = 6/group of hamsters) and 49 days (n = 15/group of hamsters) post inoculation. Results (Mean ± SD) represent cumulative effect of two (15 d) to three (49 d) independent experiments (p values were determined by Mann–Whitney two-tailed test). e Schematic representation of xenodiagnoses to determine infectiousness of immunized hamsters for sand flies. f Photographs of representative ears of LmWT and LmCen^−/− infected hamsters for xenodiagnoses at 2- and 8- weeks post inoculation. Red arrows indicate the lesion development. g After exposed with infected hamsters (n = 6/group), blood-fed flies were isolated, and parasite positive flies were identified after dissection of flies isolated from both LmWT and LmCen^−/− infected groups at 4 days post-blood feeding and 8 days post-blood feeding. Results (Mean ± SD) are representative one experiment. h Schematic representation of immune-suppression by DXM treatment of LmCen^−/− immunized hamsters. i Photographs of representative ears of LmWT, LmCen^−/−, and LmCen^−/− + DXM treated hamsters. Red arrow indicates the lesion development. j Ear lesion diameters were measured after 4 weeks of DXM treatment (total 15 weeks post parasite infection) in LmWT (n = 6) and LmCen^−/− (n = 12) and LmCen^−/− + DXM (n = 12) treated hamsters. Results (Mean ± SD) represent cumulative effect of two independent experiments, 1 ear, total 6–12 hamsters per group (p values were determined by Mann–Whitney two-tailed test). k, l Parasite load in the inoculated ear (k) and dLN (l) of each group of hamsters (LmWT, n = 6; LmCen^−/−, n = 12; and LmCen^−/− + DXM, n = 12) were determined by limiting dilution assay. Results (Mean ± SD) represent cumulative effect of two independent experiments (p values were determined by Mann–Whitney two-tailed test). BF Blood Fed, DXM Dexamethasone.

Fig. 2. Immunogenicity of LmCen^-−/− parasites in hamsters.

a Heat map showing the differential gene expression in the spleen (with or without 24 h of L. major freeze–thaw antigen restimulation; ±FTAg) of LmWT infected and LmCen^-/- immunized hamsters at 7 weeks post inoculation. Downregulation and upregulation of the transcripts are shown in blue, yellow and pink respectively. Transcripts are annotated in the left side according to their general functions. For heat map, results were shown as log₁₀ fold change over naive hamster. b Expression profile of IFN-γ, TNF-α, IL-4, IL-21, IL-10, and IFN-γ/IL-10 Ratio in the spleen which was evaluated by RT-PCR. The expression levels of genes of interest were determined by the 2^−ΔΔCt method; samples were normalized to either γ-actin expression or 18S RNA and determined relative to expression values from naive hamsters. The results were pooled of two independent experiments. Results (Mean ± SD) represent cumulative effect of two independent experiments (n = 4 for Naive, n = 9 for LmWT, and n = 8 for LmCen^−/−) (p values were determined by Mann–Whitney two-tailed test). c Ingenuity pathway analysis comparing the expression of the immune markers in LmWT and LmCen^−/− groups showing upstream regulators as predicted by the IPA is shown. Most upstream regulators are shared between the LmWT and LmCen^−/− infections as shown in the area of intersection. The statistical analysis is provided in Supplementary Information.

Fig. 3. LmCen^−/− immunized hamster protects and induces pro-inflammatory type of immune response upon challenge with L. donovani.

a Schematic representation of experimental plan to determine the efficacy and immune response of LmCen^−/− parasites hamsters against L. donovani via needle challenge. b, c Parasite load were determined by limiting dilution after various periods post challenge and expressed as number of parasites per Spleen (b) and per gram of Liver (c). LmCen^−/− immunized (Imm Chal) (n = 6 for 1.5 and 3 months and n = 10 for 9 and n = 11 for 12 months) and age-matched nonimmunized (Non-Imm Chal) hamsters (n = 6 for 1.5 and 3 months and n = 10 for 9 and 12 months) after various periods of post-needle challenge (1.5, 3, 9, and 12 month) with L. donovani. Results (Mean ± SD) are representative of cumulative effect of two independent experiments (p values were determined by Mann–Whitney two-tailed test). d Heat map showing the differential gene expression in the splenocytes (with or without 24 h of L. donovani freeze–thaw antigen restimulation; ±FTAg) of age-matched nonimmunized (Non-Imm Chal) and LmCen^−/− immunized (Imm Chal) hamsters after 1.5 months of L. donovani challenge by needle injection (n = 6/group). According to the general function, transcripts are annotated in the left side. Downregulation and upregulation of the transcripts are shown in pink, yellow and blue, respectively. For heat map, results were shown as log₁₀ fold change over naive hamster. e Expression profile of IFN-γ, TNF-α, IL-4, IL-21, IL-10, and IFN-γ/IL-10 Ratio in the spleen, which was evaluated by RT-PCR. The data were normalized to γ-Actin expression and shown as the fold-change relative to age-matched naive hamster. Results (Mean ± SD) represent cumulative effect of two independent experiments (p values were determined by Mann–Whitney two-tailed test).

Fig. 4. LmCen^−/− immunization confers protection against sand fly bite transmitted fatal L. donovani infection in hamsters.

a Schematic representation of the experimental plan. b Kaplan–Meier survival curves of LmCen^−/−-immunized hamsters (Imm Chal; blue lines, n = 8) following challenge with L. donovani-infected sand flies and compared with age-matched nonimmunized-challenged group (Non Imm Chal; red lines, n = 8). c Picture of hamsters from each group after 9 months of sand fly-transmitted L. donovani challenge. d Photographs (left panel) of representative two spleen samples of both LmCen^−/−-immunized and nonimmunized hamsters following 9 months post challenge as well as one age-matched naive hamster is shown. Right panel showing stamp smear of respective spleens stained with H&E, black arrows indicates intracellular parasites (bar-20 μm). e, f Parasite load in the spleen (e) and liver (f) (n = 8, n = 10, and n = 5 for 3.5M, 9M, and 12M post challenge, respectively, of nonimmunized and immunized-challenged groups). Results represent (the geometric means with 95% Cl) cumulative effect of two (3.5MPC and 9MPC) and one (12MPC) independent experiment respectively (p values were determined by Mann–Whitney two-tailed test). g Liver sections are from animals at 3.5 months after challenge and stained with H&E (left panel ×20 and right panel ×100 magnification). h–j IFN-γ (h) and IL-10 (i) expression in the spleen of LmCen^−/− immunized and age-matched nonimmunized hamsters (n = 8/per group) was evaluated by qPCR following 3.5 months post-L. donovani-infected sand fly challenge. The ratio of IFN-γ/IL-10 expression in the spleen was also determined (j). Results (the geometric means with 95% Cl) represent the cumulative effect of two independent experiments (p values were determined by Mann–Whitney two-tailed test). WPI weeks postimmunization, MPC months post challenge.

Fig. 5. GLP-grade LmCen^−/− parasites immunization confers protection in hamsters.

a Production of GLP-grade LmCen^−/− parasite. b Growth behavior of parasite during bioreactor cultivation. c Morphology (magnification ×400) of the parasite during 4 days of bioreactor cultivation. d Photographs (left panel) of representative spleen samples of both LmCen^−/−-immunized and nonimmunized hamsters following 10 months post challenge as well as one age-matched naive hamster is shown. e, f Parasite load in the spleen (e) and liver (f) of hamsters either immunized with GLP-grade LmCen^−/− (Imm Chal, n = 6) parasites or age-matched nonimmunized control (Non-Imm Chal, n = 5) were determined following 10 months of post challenge with L. donovani-infected sand flies. Results plotted as geometric means with 95% Cl from one experiment (p values were determined by Mann–Whitney two-tailed test). g Kaplan–Meier survival curves of GLP-grade LmCen^−/−-immunized hamsters (Imm Chal; blue lines, n = 6) following challenge with L. donovani-infected sand flies and compared with age-matched nonimmunized-challenged group (Non-Imm Chal; red lines, n = 5).

Fig. 6. GLP-grade LmCen^−/− immunization confers long-term protection against fatal L. donovani infection in hamsters.

a Schematic representation of experimental plan to determine the long-term protective efficacy of LmCen^−/− parasites against L. donovani infection through needle injection. b, c Spleen (b) and liver (c) parasite burden of GLP-grade LmCen^−/− immunized (Imm Chal, n = 6) and age-matched nonimmunized (Non-Imm Chal, n = 5) hamsters were determined at 8 months post-needle challenge. Results (means ± SD) are from one experiment (p values were determined by Mann–Whitney two-tailed test). MPI Months post immunization, MPC months post challenge.

Fig. 7. GLP-grade LmCen^−/− induce host pro-inflammatory immune response in human PBMCs.

a Schematic representation of experiment plan to determine immune response of LmCen^−/− in human PBMCs. b–d Scatter dot plots showing the levels (pg/ml) of pro-inflammatory (IFN-γ) (b) and anti-inflammatory (IL-10) (c) cytokine as well as IFN-γ/IL-10 ratio (d) in culture supernatant of PBMCs from Non-endemic region after 48 h of infection with LmCen^−/− parasites by ELISA. Control group was left uninfected. Results (Mean ± SD) are representative of one experiment (p values were determined by Mann–Whitney two-tailed t test).