Heterologous DNA prime-protein boost immunization with RecA and FliD offers cross-clade protection against leptospiral infection

Abstract

The emergence of >300 serovars of Leptospira confounded the use of generalized bacterin, the whole cell lysate, as vaccines to control leptospirosis. Because of substantial genetic and geographic heterogeneity among circulating serovars, one vaccine strain per serovar cannot be efficacious against all the serovars. We have performed heterologous DNA prime-protein boost vaccination challenge studies in hamsters using in vivo expressed, leptospiral recombinase A (RecA) and flagellar hook associated protein (FliD). We prepared the monovalent recombinant protein, plasmid DNA, and DNA prime protein boost adjuvant vaccines. The whole cell bacterin served as a control. Our data show that (i) RecA and FliD have multiple immunogenic B and T-cell epitopes with highly conserved domains among most prevalent pathogenic Leptospira spp., (ii) humoral and cell mediated immune responses were induced remarkably, (iii) provides significant protection against homologous (Autumnalis strain N2) and cross-clade heterologous (Canicola strain PAI-1) challenge infection for the heterologous prime-protein boost (∼91-100%) and, the DNA vaccine (∼75-83%). Recombinant protein vaccine shows only partial protection (∼58-66%), (iv) RecA prime-protein boost vaccine shows sterilizing immunity, with heterologous protection. This RecA/FliD prime-protein boost strategy holds potential for vaccination against animal leptospirosis and for a better control of zoonotic transmission.

Figure 1

Conservation of the predicted epitopes in RecA and FliD. (A-B) The predicted B-cell epitopes were highlighted in sequence alignment as well as in 3D structure of RecA and FliD. RecA contains highly conserved Walker A motif and Walker B motif and FliD contains N terminus region, middle region and (C) terminus region. (C) Heat map showing predicted B-cell epitopes based on BCPred, conservancy and VaxiJen scores in RecA and FliD. A–BCPred score (Control score -0.8), B–VaxiJEN score (Control score -0.4). Blank or excluded were shown as X through the cells. (D) Epitope conservancy: A–L. interrogans serovar Autumnalis (AGW25358, EMN53408), B–L. interrogans serovar Australis (EMY22543, OOB99359), C–L. interrogans serovar Canicola (EKO69724, EKO71148), D–L. interrogans serovar Copenhageni (AAS70334, AAS69344), E–L. interrogans serovar Icterohaemorrhagiae (EKP23302, EMO07105), F–L. interrogans serovar Pomona (EMF34262, EMJ59305), G–L. kirschneri serovar Grippotyphosa (EJO70816, EJO70003), H–L. noguchii (WP_004424072, WP_004450466). (E–H) Heat map showing number of predicted T- cell strong binder major histocompatibility complex (MHC-I and II) epitopes in RecA and FliD. (E) List of predicted immunogenic T-cell epitopes for HLA-A allelic variants. (F) List of predicted immunogenic T-cell epitopes for HLA-B allelic variants. (G) List of predicted immunogenic T-cell epitopes for DQA1/DQB1 and DPA1/DPB1 locus. (H) List of predicted immunogenic T-cell epitopes for DRB1/3/4/5 locus.

Figure 2

Evaluation of humoral immune response in control and immunized hamster groups by IgG ELISA. (A) rRecA antigen used, (B) rFliD antigen used. Graphs represent the Mean ± SD of the optical density of the sera obtained on day 0 (pre-vaccination), on 14^th, 21^st (before booster) and 42^nd days (before challenge). ns-no significance, *P < 0.05, **P < 0.01, ***P < 0.001. P values were obtained through comparison with the negative control (PBS) using a Tukey’s multiple comparisons test by 2way ANOVA.

Figure 3

Evaluation of cell mediated immune response in control and immunized hamster groups by qRT-PCR. (A) TNFα, (B) IL-10, (C) IL-4, D) IL-12p40, (E) IFN-γ. The relative CT (ΔΔ CT) method was used to quantify cytokine gene expression: CTs were normalized against the GAPDH gene CT (Δ CT) and then compared to the same normalized gene in the PBS or pEGFPN3 immunized hamster group (calibrator). The control groups were set to 1. ns-no significance, *P < 0.05, **P < 0.01, ***P < 0.001. P values were obtained through comparison with the negative control (PBS or pEGFPN3) using a Tukey’s multiple comparisons test by 2way ANOVA.

Figure 4

Immunoprotective potential of immunized hamster groups after challenge. (A) Survival graph of animals immunized with different vaccines after N2 (homologous) (B) PAI-1 (heterologous) lethal challenge and the survival curves were compared using log-rank analysis. Values are in Mean ± SD of different (0–28) days after challenge. Two-tailed P value was determined by Fisher exact test in comparison to the result of different groups observed as follows: rRecA vs recA-pEFGPN3 + rRecA (<0.01); recA-pEGFPN3 vs recA-pEFGPN3 + rRecA (ns); rFliD vs fliD-pEGFPN3 + rFliD (<0.10); fliD-pEGFPN3 vs fliD-pEGFPN3 + rFliD (ns); rRecA vs recA-pEGFPN3 (<0.10); rFliD vs fliD-pEGFPN3 (<0.10) for homologous challenge with Autumnalis N2 (A) and rRecA vs recA-pEFGPN3 + rRecA (<0.05); recA-pEGFPN3 vs recA-pEFGPN3 + rRecA (ns); rFliD vs fliD-pEGFPN3 + rFliD (<0.05); fliD-pEGFPN3 vs fliD-pEGFPN3 + rFliD (ns); rRecA vs recA-pEGFPN3 (ns); rFliD vs fliD-pEGFPN3 (ns) for heterologous challenge with Canicola PAI-1 (B). ns- Not significant (P > 0.01).