Sublethal infection of C3H/HeNJ against Leptospira interrogans serovar Pomona.

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira spp. Leptospires can infect a variety of mammalian species. Golden Syrian hamsters are mostly used to study acute leptospirosis. However, the immunopathogenic mechanism is poorly understood because immunological reagents for hamsters are limited. This study aimed to establish C3H/HeNJ mice as an animal model for leptospirosis. Five-week-old C3H/HeNJ mice were infected with either low (103 cells) or high (106 cells) inoculum dose of Leptospira interrogans serovar Pomona. All mice were investigated for survival rate, leptospiral load and histopathology of target organs, antibody levels, and cytokine production (IFN-γ, IL-6 and IL-10) at day 28 post-infection. All infected mice survived and did not develop acute lethal infection. However, C3H/HeNJ mice infected with 106 cells of leptospires showed kidney colonization of leptospires and pathological changes in the lung and kidney including renal fibrosis. The glomerular size in PAS-D stained kidney tissues of C3H/HeNJ mice infected with 106 cells of leptospires was significantly reduced compared to that of mice infected with 103 cells of leptospires and non-infected mice. High-dose leptospires induced significantly greater levels of IFN-gamma and IL-6 than low-dose leptospires, but IL-10 level was not significantly different. Moreover, 106 leptospiral cells induced predominant IgG2a isotype suggesting Th1-like response. These results suggest that C3H/HeNJ mice may be used as a sublethal model of leptospirosis.

Designing Adjuvant Formulations to Promote Immunogenicity and Protective Efficacy of Leptospira Immunoglobulin-Like Protein A Subunit Vaccine.

The leptospirosis burden on humans, especially in high-risk occupational groups and livestock, leads to public health and economic problems. Leptospirosis subunit vaccines have been under development and require further improvement to provide complete protection. Adjuvants can be used to enhance the amplitude, quality, and durability of immune responses. Previously, we demonstrated that LMQ adjuvant (neutral liposomes containing monophosphoryl lipid A (MPL) and Quillaja saponaria derived QS21 saponin) promoted protective efficacy of LigAc vaccine against Leptospira challenge. To promote immunogenicity and protective efficacy of the subunit vaccines, three alternative adjuvants based on neutral liposomes or squalene-in-water emulsion were evaluated in this study. LQ and LQuil adjuvants combined the neutral liposomes with the QS21 saponin or Quillaja saponaria derived QuilA® saponin, respectively. SQuil adjuvant combined a squalene-in-water emulsion with the QuilA® saponin. The immunogenicity and protective efficacy of LigAc (20 µg) formulated with the candidate adjuvants were conducted in golden Syrian hamsters. Hamsters were vaccinated three times at a 2-week interval, followed by a homologous challenge of L. interrogans serovar Pomona. The results showed that LigAc combined with LQ, LQuil, or SQuil adjuvants conferred substantial antibody responses and protective efficacy (survival rate, pathological change, and Leptospira renal colonization) comparable to LMQ adjuvant. The LigAc+LQ formulation conferred 62.5% survival but was not significantly different from LigAc+LMQ, LigAc+LQuil, and LigAc+SQuil formulations (50% survival). This study highlights the potential of saponin-containing adjuvants LMQ, LQ, LQuil, and SQuil for both human and animal leptospirosis vaccines.

Identification of in vivo expressed proteins in live attenuated lipopolysaccharide mutant that mediates heterologous protection against Leptospira spp.

Leptospirosis vaccines that elicit broad protection against a range of pathogenic Leptospira spp. would overcome a major drawback of currently licensed bacterin vaccines. Live attenuated vaccine produced from a lipopolysaccharide (LPS) mutant strain of L. interrogans serovar Manilae M1352 (Live M1352) stimulated better protective efficacy than heat killed M1352 (HK M1352) against a heterologous challenge with L. interrogans serovar Pomona. To identify antigens of Live M1352 potentially responsible for cross protection, in vivo-induced antigen technology (IVIAT), a powerful tool to identify in vivo-induced (ivi) genes expressed during infection, was employed in this study. Pooled sera from hamsters immunized with Live M1352 were sequentially adsorbed with various preparations of in vitro grown M1352. The pre-adsorbed sera were used to screen a genomic expression library of M1352. Nineteen strongly reactive clones were selected for DNA sequencing. These ivi genes are conserved in most Leptospira strains. Four selected genes including LIMLP_04965 (tolB), LIMLP_01535, LIMLP_06785 (fliI), and LIMLP_14930 were confirmed for their upregulated expression in kidneys of infected hamsters by RT-qPCR, suggesting their role in leptospiral infection. These ivi proteins represent potential targets for vaccine candidates that warrant further investigation for their protective efficacy.

A Comparison of Intramuscular and Subcutaneous Administration of LigA Subunit Vaccine Adjuvanted with Neutral Liposomal Formulation Containing Monophosphoryl Lipid A and QS21.

Leptospirosis vaccines with higher potency and reduced adverse effects are needed for human use. The carboxyl terminal domain of leptospiral immunoglobulin like protein A (LigAc) is currently the most promising candidate antigen for leptospirosis subunit vaccine. However, LigAc-based vaccines were unable to confer sterilizing immunity against Leptospira infection in animal models. Several factors including antigen properties, adjuvant, delivery system, and administration route need optimization to maximize vaccine efficacy. Our previous report demonstrated protective effects of the recombinant LigAc (rLigAc) formulated with liposome-based adjuvant, called LMQ (neutral liposome combined with monophosphoryl lipid A and Quillaja saponaria fraction 21) in hamsters. This study aimed to evaluate the impact of two commonly used administration routes, intramuscular (IM) and subcutaneous (SC), on immunogenicity and protective efficacy of rLigAc-LMQ administrated three times at 2-week interval. Two IM vaccinations triggered significantly higher levels of total anti-rLigAc IgG than two SC injections. However, comparable IgG titers and IgG2/IgG1 ratio was observed for both routes after the third immunization. The route of vaccine administration did not influence the survival rate (60%) and renal colonization against lethal Leptospira challenge. Importantly, the kidneys of IM group showed no pathological lesions while the SC group showed mild damage. In conclusion, IM vaccination with rLigAc-LMQ not only elicited faster antibody production but also protected from kidney damage following leptospiral infection better than SC immunization. However, both tested routes did not influence protective efficacy in terms of survival rate and the level of renal colonization.

Reduced Renal Colonization and Enhanced Protection by Leptospiral Factor H Binding Proteins as a Multisubunit Vaccine Against Leptospirosis in Hamsters.

Subunit vaccines conferring complete protection against leptospirosis are not currently available. The interactions of factor H binding proteins (FHBPs) on pathogenic leptospires and host factor H are crucial for immune evasion by inhibition of complement-mediated killing. The inhibition of these interactions may be a potential strategy to clear leptospires in the host. This study aimed to evaluate a multisubunit vaccine composed of four known leptospiral FHBPs: LigA domain 7-13 (LigAc), LenA, LcpA, and Lsa23, for its protective efficacy in hamsters. The mono and multisubunit vaccines formulated with LMQ adjuvant, a combination of neutral liposome, monophosphoryl lipid A, and Quillaja saponaria fraction 21, induced high and comparable specific antibody (IgG) production against individual antigens. Hamsters immunized with the multisubunit vaccine showed 60% survival following the challenge by 20 LD50 of Leptospira interrogans serovar Pomona. No significant difference in survival rate and pathological findings of target organs was observed after vaccinations with multisubunit or mono-LigAc vaccines. However, the multisubunit vaccine significantly reduced leptospiral burden in surviving hamsters in comparison with the monosubunit vaccines. Therefore, the multisubunit vaccine conferred partial protection and reduced renal colonization against virulence Leptospira infection in hamsters. Our multisubunit formulation could represent a promising vaccine against leptospirosis.