Chimeric epitope vaccine against Leptospira interrogans infection and induced specific immunity in guinea pigs

Abstract

Background: Leptospirosis is an important reemerging zoonosis, with more than half a million cases reported annually, and is caused by pathogenic Leptospira species. Development of a universal vaccine is one of the major strategic goals to overcome the disease burden of leptospirosis. In this study, a chimeric multi-epitope protein-based vaccine was designed and tested for its potency to induce a specific immune response and provide protection against L. interrogans infection.

Results: The protein, containing four repeats of six T- and B-cell combined epitopes from the leptospiral outer membrane proteins, OmpL1, LipL32 and LipL21, was expressed and purified. Western blot analysis showed that the recombinant protein (named r4R) mainly expressed in a soluble pattern, and reacted with antibodies raised in rabbit against heat-killed Leptospira and in guinea pigs against the r4R vaccine. Microscopic agglutination tests showed that r4R antisera was immunological cross-reactive with a range of Chinese standard reference strains of Leptospira belonging to different serogroups. In guinea pigs, the r4R vaccine induced a Th1-biased immune response, as reflected by the IgG2a/IgG1 ratio and cytokine production of stimulated splenocytes derived from immunized animals. Finally, r4R-immunized guinea pigs showed increased survival of lethal Leptospira challenges compared with PBS-immunized animals and tissue damage and leptospiral colonization of the kidney were reduced.

Conclusions: The multi-epitope chimeric r4R protein is a promising antigen for the development of a universal cross-reactive vaccine against leptospirosis.

Keywords: Cross protection; Leptospira; Multi-epitope; Outer membrane protein; Vaccine.

Fig. 1

Characterization of the expressed and purified chimeric r4R protein. a SDS-PAGE analysis of the expression and purification steps of the r4R protein. Pellet (lane 1 and 3) and supernatant (lane 2 and 4) fractions of lysates from E. coli BL21 (DE3) cells containing the empty vector pET28a (lane 1 and 2) or pET28a-4R (lane 3 and 4) were electrophoresed in a 10 % SDS-PAGE gel. M indicates the protein ladder. Lane 5 contains the purified recombinant 4R protein. b Anti-Leptospira Western blot analysis. The purified chimeric r4R protein was run on a SDS-PAGE gel and transferred to a PVDF membrane. Serum from heat-killed L. interrogans strain Lai immunized rabbits was used as a primary antibody to detect r4R. Serum from PBS injected rabbits was used as control. c Anti-r4R Western analysis. The purified chimeric r4R protein was run on SDS-PAGE gel and transferred to a PVDF membrane. Serum from guinea pigsimmunized with PBS (negative control) or chimeric protein, were used as primary antibody

Fig. 2

Response of IgG subclasses on the chimeric protein. IgG subclasses induced by the chimeric protein in guinea pigs were detected by ELISA

Fig. 3

Proliferative and immune responses of splenocytes immunized with chimeric protein. Guinea pigs were immunized with the chimeric r4R protein. Ten days after the last immunization, the splenocytes were isolated, stimulated with the r4R protein and cultured for 72 h. a Splenocyte proliferation analysis of PBS, ConA and r4R stimulated splenocytes. b ELISA measurements of IL-4 and INF-γ secreted by pooled control, ConA and r4R stimulated splenocytes and lymphocytes in the culture supernatants

Fig. 4

Histopathological effects of Leptospira challenges on immunized guinea pig tissues. Representative images of liver, kidney and lung tissues of healthy guinea pigs (a) or guinea pigs immunized with the chimeric r4R protein (b) or PBS (c) and challenged with L. interrogans strain Lai. Tissues were stained with hematoxylin and eosin

Fig. 5

Colonization of L. interrogans in chimeric r4R immunized guinea pigs. Representative images of kidney lysates (a) or urine samples (b) that were collected from healthy control guinea pigs (CK) of guinea pigs immunized with r4R or PBS and subsequently challenged with L. interrogans strain Lai. All samples were strained with the Fontana silver staining method and observed using an optical microscope