A T-Cell Epitope-Based Multi-Epitope Vaccine Designed Using Human HLA Specific T Cell Epitopes Induces a Near-Sterile Immunity against Experimental Visceral Leishmaniasis in Hamsters

Abstract

Visceral leishmaniasis is a neglected tropical disease affecting 12 million people annually. Even in the second decade of the 21st century, it has remained without an effective vaccine for human use. In the current study, we designed three multiepitope vaccine candidates by the selection of multiple IFN-γ inducing MHC-I and MHC-II binder T-cell specific epitopes from three previously identified antigen genes of Leishmania donovani from our lab by an immuno-informatic approach using IFNepitope, the Immune Epitope Database (IEDB) T cell epitope identification tools, NET-MHC-1, and NET MHC-2 webservers. We tested the protective potential of these three multiepitope proteins as a vaccine in a hamster model of visceral leishmaniasis. The immunization data revealed that the vaccine candidates induced a very high level of Th1 biased protective immune response in-vivo in a hamster model of experimental visceral leishmaniasis, with one of the candidates inducing a near-sterile immunity. The vaccinated animals displayed highly activated monocyte macrophages with the capability of clearing intracellular parasites due to increased respiratory burst. Additionally, these proteins induced activation of polyfunctional T cells secreting INF-γ, TNF-α, and IL-2 in an ex-vivo stimulation of human peripheral blood mononuclear cells, further supporting the protective nature of the designed candidates.

Keywords: IFN-γ epitope; Leishmania; T cell; epitopes; hamster; sterile immunity; vaccine.

Figure 1

General scheme for epitope selection and designing of multiepitope construct using peptide regions identified as T cell epitopes by IEDB for MHC-I and MHC-II, NETMHC-1, and NETMHC-2. Representative image for epitope selection from F2/1 (AY-180912) for the 64 kDa construct. Sequence alignment by Clustal Omega (EMBL-EBI). Details are available in the Supplementary Materials.

Figure 2

Mean parasite load and splenic index from the spleen of different animal groups. LDU = Leishman Donovan unit. (number of amastigotes/number of nucleated cells × weight of the organ(mg)). Splenic index, the ratio of (spleen weight/whole body weight) × 100. n = 8. Mean ± SEM. Statistical comparison between Infected and test group. Student’s t-test. Level of significance, ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

Mean promastigote growth in different groups at day 0, day 5, day 7, and day 14. n = 8. Box and whiskers for median value, lower, and upper quartile. * Difference statistically significant as determined by ANOVA. Level of significance, ns p > 0.05, ** p ≤ 0.01, **** p≤ 0.00001.

Figure 4

(A) Parasite clearance observed at different time intervals. Statistical comparison between healthy and test groups. Average MFI (mean fluorescent intensity) at different time points from adherent peritoneal cells of different animal groups. (B) Variation in ROS production levels over 24 h. ROS is expressed relative to healthy control, ROS production index (RPI) = MFI test group/MFI healthy group. The dashed line indicates the expression levels of healthy cells. CFA: Freund’s adjuvant. n = 8, mean values ± SEM. (A) Statistical comparison of Student’s t-test between the test group and healthy group. Level of significance, ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01.

Figure 5

Relative fold change in gene expression levels of (A) IFN-γ, (B) IL-12, (C) TNF-α, (D) IL-21, (E) TBX21, (F) L selectin, (G) IL-10, and (H) TGF-β from immunized and unimmunized animal’s spleen. n = 8 for 64 kDa and 36 kDa, n = 7 for 29 kDa and n = 5 for the infected. Mean values ± SEM. Statistical comparison between infected and test group, Student’s t-test. Level of significance, ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.00001.

Figure 6

Bar graph comparing the frequency of CD3+ T cells secreting IFN-γ, TNF-α, IL-2, and IL-10. (A) Frequency of polyfunctional CD3+ T cells expressing IFN-γ, TNF-α, and IL-2 simultaneously. (B) Frequency of dual positive CD3+ T cells expressing IFN-γ and TNF-α simultaneously. (C) Frequency of CD3+ T cells expressing IFN-γ. (D) Frequency of CD3+ T cells expressing TNF-α. (E) Frequency of CD3+ T cells expressing IL-2. (F) Frequency of cells expressing IL-10. n = 4. Symbols for individual values, • LPSA, ■ 64 kDa, ▲ 36 kDa, ▼ 29 kDa, ♦ PMA & ● PHA. Mean values and SD. Statistical comparison between LPSA and test group. Student’s t-test. Level of significance, ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01.