Cross-protective immunity against leptospirosis elicited by a live, attenuated lipopolysaccharide mutant

Abstract

Background: Leptospira species cause leptospirosis, a zoonotic disease found worldwide. Current vaccines against leptospirosis provide protection only against closely related serovars.

Methods: We evaluated an attenuated transposon mutant of Leptospira interrogans serovar Manilae (M1352, defective in lipopolysaccharide biosynthesis) as a live vaccine against leptospirosis. Hamsters received a single dose of vaccine and were challenged with the homologous serovar (Manilae) and a serologically unrelated heterologous serovar (Pomona). Comparisons were made with killed vaccines. Potential cross-protective antigens against leptospirosis were investigated.

Results: Live M1352 vaccine induced superior protection in hamsters against homologous challenge. The live vaccine also stimulated cross-protection against heterologous challenge, with 100% survival (live M1352) versus 40% survival (killed vaccine). Hamsters receiving either vaccine responded to the dominant membrane proteins LipL32 and LipL41. Hamsters receiving the live vaccine additionally recognized LA3961/OmpL36 (unknown function), Loa22 (OmpA family protein, recognized virulence factor), LA2372 (general secretory protein G), and LA1939 (hypothetical protein). Manilae LigA was recognized by M1352 vaccinates, whereas LipL36 was detected in Pomona.

Conclusion: This study demonstrated that a live, attenuated vaccine can stimulate cross-protective immunity to L. interrogans and has identified antigens that potentially confer cross-protection against leptospirosis.

Figure 1.

Protection against challenge with Leptospira interrogans serovar Manilae. Hamsters (groups of 10) were vaccinated with a single intraperitoneal injection, with the dose indicated, of live M1352 vaccine, heat-killed (HK) wild-type (WT) Manilae vaccine, or EMJH medium. After 14 d, hamsters were challenged with 10³ WT Manilae leptospires and monitored for 21 d. Vaccine efficacy was assessed by hamster survival (A), culture of leptospires from kidney tissue (B), and evidence of lung hemorrhage in each animal (C). **P < .01 (Fisher exact test).

Figure 2.

Protection against 3 different challenge doses with Leptospira interrogans serovar Manilae. Hamsters (groups of 10) were vaccinated with a single intraperitoneal injection of 10⁷ live M1352 leptospires, 10⁷ heat-killed (HK) Manilae leptospires, or EMJH medium. After 14 d, hamsters were challenged with the dose indicated of wild-type Manilae and monitored for 21 d. Vaccine efficacy was assessed by hamster survival (A), culture of leptospires from kidney tissue (B), and evidence of lung hemorrhage in each animal (C). * P < .05 (Fisher exact test).

Figure 3.

Vaccine protection against heterologous leptospiral challenge. Hamsters (groups of 10) were vaccinated with a single intraperitoneal injection of 10⁷ live M1352, heat-killed (HK) Manilae, formalin-killed (FK) M1352, or HK M1352 leptospires. EMJH medium was used as a negative control. After 14 d, hamsters were challenged with a dose of 10³ Leptospira interrogans serovar Manilae (serogroup Pyrogenes) leptospires (A, D, and G), 10⁴ serovar Pomona (serogroup Pomona) leptospires (B, E, and H), or 10⁶ Leptospira borgpetersenii serovar Hardjobovis (serogroup Sejroe) leptospires (C, F, and I) and monitored for 21 d. The experiment was conducted up to 3 times and data were pooled (the total number of hamsters tested in each condition is indicated on the x-axis in parentheses). * P < .05; ** P < .01 (Fisher exact test).

Figure 4.

Western blot analysis of the immune response to leptospiral lipopolysaccharide. Proteinase K-digested whole-cell lysates of Leptospira interrogans serovar Manilae (M) or Pomona (P) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a membrane, and probed with serum samples from individual vaccinated hamsters.

Figure 5.

Humoral response to Leptospira interrogans after vaccination with the live or heat-killed (HK) vaccine. Membrane preparations from L. interrogans serovar Manilae (A-C) or Pomona (D-F) were separated by 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gels were stained with colloidal Coomassie blue (A and D) or transferred and probed with pooled serum samples from 5 hamsters vaccinated with either HK serovar Manilae (B and E) or live M1352 (C and F).