Immunogenicity and Protective Efficacy of T-Cell Epitopes Derived From Potential Th1 Stimulatory Proteins of Leishmania (Leishmania) donovani

Abstract

Development of a suitable vaccine against visceral leishmaniasis (VL), a fatal parasitic disease, is considered to be vital for maintaining the success of kala-azar control programs. The fact that Leishmania-infected individuals generate life-long immunity offers a viable proposition in this direction. Our prior studies demonstrated that T-helper1 (Th1) type of cellular response was generated by six potential recombinant proteins viz. elongation factor-2 (elF-2), enolase, aldolase, triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and p45, derived from a soluble antigenic fraction (89.9-97.1 kDa) of Leishmania (Leishmania) donovani promastigote, in treated Leishmania patients and golden hamsters and showed significant prophylactic potential against experimental VL. Moreover, since, it is well-known that our immune system, in general, triggers production of specific protective immunity in response to a small number of amino acids (peptide), this led to the identification of antigenic epitopes of the above-stated proteins utilizing immunoinformatics. Out of thirty-six, three peptides-P-10 (enolase), P-14, and P-15 (TPI) elicited common significant lymphoproliferative as well as Th1-biased cytokine responses both in golden hamsters and human subjects. Further, immunization with these peptides plus BCG offered 75% prophylactic efficacy with boosted cellular immune response in golden hamsters against Leishmania challenge which is indicative of their candidature as potential vaccine candidates.

Keywords: T-cell epitopes; Th1 stimulatory proteins; hamsters; human PBMCs; immunoinformatics; peptides; protective response; visceral leishmaniasis.

Figure 1

(A) Lymphoproliferative response in lymphocytes derived from the mesenteric lymph node of uninfected, infected as well as treated Leishmania-infected hamsters against 36 Th1 stimulatory peptides in comparison to their parent recombinant proteins. The concentration of peptides, as well as recombinant proteins, was adjusted to 1 ng/ml of cells. Each bar represents the pooled data of hamsters (Mean ± SE). Statistical significant differences were assessed using a paired t-test amid mean values of treated groups stimulated with synthetic peptides and their respective recombinant proteins. (B) Lymphoproliferative response in PBMCs of healthy contacts, active VL patients as well as treated VL patients against 36 Th1 stimulatory peptides and their parent recombinant proteins. The concentration of peptides, as well as recombinant proteins, was adjusted to 1 ng/ml of cells. Each bar represents the pooled data (Mean ± SE) and the levels of statistical significance were assessed between the mean values of treated groups, stimulated with synthetic peptides and their respective recombinant proteins by paired t-test. *p < 0.05; **p < 0.01; and ***p < 0.001.

Figure 2

Cytokines level in the peptide-stimulated culture supernatant of lymphocyte derived from uninfected, infected, and treated Leishmania-infected hamsters (A) as well as in PBMCs derived from healthy contacts, active VL as well as treated VL patients (B). The levels of significance were calculated using unpaired t-test between infected and treated groups (*p < 0.05 and **p < 0.01).

Figure 3

Parasite load in splenic dab smear of vaccinated hamsters (A), DTH response to SLD as footpad swelling (B) and lymphoproliferative response in vaccinated hamsters against SLD (C) in comparison to the unimmunized infected hamsters on days 60 and 90 p.c. One-way ANOVA was used to calculate the statistical significance between the vaccinated and infected group (*p < 0.05; **p < 0.01; ***p < 0.001, and ****p < 0.0001).

Figure 4

Cytokines level in serum samples of vaccinated hamsters at day 60 (A) and day 90 (B) p.c. Significance values indicate the difference in cytokine concentration between infected and treated hamsters (*p < 0.05; **p < 0.01; and ***p < 0.001) and was calculated using One-way ANOVA.

Figure 5

Cytokine mRNA expression profile of spleen of normal, infected, and vaccinated hamsters on days 60 and 90 p.c. by quantitative real-time-PCR. The levels of significance were calculated using unpaired t-test between infected and treated groups (*p < 0.05; **p < 0.01; and ***p < 0.001).