Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp. having more than 300 serovars. These serovars can infect a variety of hosts, some being asymptomatic carriers and others showing varied symptoms of mild to severe infection. Since lipopolysaccharide (LPS) is the major antigen which defines serovar specificity, this different course of infection may be attributed to a differential innate response against this antigen. Previous studies have shown that Leptospira LPS is less endotoxic. However, it is unclear whether there is a difference in the ability of LPS isolated from different serovars to modulate the innate response. In this study, we purified LPS from three widely prevalent pathogenic serovars, i.e. Icterohaemorrhagiae strain RGA, Pomona, Hardjo, and from non-pathogenic L. biflexa serovar semeranga strain Potac 1 collectively termed as L-LPS and tested their ability to modulate innate response in macrophages from both resistant (mice) and susceptible (human and bovine) hosts. L-LPS induced differential response being more proinflammatory in mouse and less proinflammatory in human and bovine macrophages but overall less immunostimulatory than E. coli LPS (E-LPS). Irrespective of serovar, this response was TLR2-dependent in humans, whereas TLR4-dependent/CD14-independent in mouse using MyD88 adapter and signalling through P38 and ERK-dependent MAP kinase pathway. L-LPS-activated macrophages were able to phagocytose Leptospira and this effect was significantly higher or more pronounced when the macrophages were stimulated with L-LPS from the corresponding serovar. L-LPS activated both canonical and non-canonical inflammasome, producing IL-1β without inducing pyroptosis. Further, L-LPS induced both TNF-mediated early and NO-mediated late apoptosis. Altogether, these results indicate that L-LPS induces a differential innate response that is quite distinct from that induced by E-LPS and may be attributed to the structural differences and its atypical nature.

Monophosphoryl lipid A (MPLA), a TLR4 agonist, is a clinically approved vaccine adjuvant, but its complex structure and occasional toxicity limit broader use. Synthetic small-molecule TLR4 agonists offer advantages such as ease of synthesis, lower cost, and reduced toxicity. In this study, we conducted structure-based virtual screening of the ZINC database to identify novel TLR4-targeting small molecules across human, murine, and bovine species. Three lead compounds—NSF-418, NSF-501, and NSF-951—were selected based on favorable binding interactions and subjected to in vitro and in vivo evaluation. NSF-951 emerged as a potent TLR4 agonist, inducing strong proinflammatory cytokine responses (IL-6, TNF-α), upregulating CD80 and CD86 expression, and promoting macrophage maturation. Conversely, NSF-418 and NSF-501 acted as antagonists by suppressing MPLA-induced responses. In murine immunization studies, NSF-951, alone or with Alum (AF007), significantly enhanced OVA-specific antibody and T-cell responses without observable toxicity. These findings suggest that NSF-951 is a promising, cost-effective TLR4 agonist with strong immunostimulatory and adjuvant potential. Further studies are warranted to assess its performance with other antigens and adjuvant combinations, supporting its development as a next-generation adjuvant for veterinary and human vaccines.

Leptospirosis is a globally significant zoonotic disease. The current inactivated vaccine offers protection against specific serovars but does not provide complete immunity. Various surface antigens, such as Leptospira immunoglobulin-like proteins (LigA and LigB), have been identified as potential subunit vaccine candidates. However, these antigens require potent adjuvants for effectiveness. Bacterial lipopolysaccharides (LPSs), including lipid A, are a well-known immunostimulant, and clinical adjuvants often contain monophosphoryl lipid A (MPLA). Being less endotoxic, we investigated the adjuvant properties of lipid A isolated from L. interrogans serovar Pomona (PLA) in activating innate immunity and enhancing antigen-specific adaptive immune responses. PLA activated macrophages to a similar degree as MPLA, albeit at a higher dose, suggesting that it is less potent in stimulation than MPLA. Mice immunized with a variable portion of LigA (LAV) combined with alum and PLA (LAV-alum-PLA) exhibited significantly higher levels of LAV-specific humoral and cellular immune responses compared to alum alone but similar to those induced by alum-MPLA. The adjuvant activity of PLA resembles that of MPLA and is primarily achieved through the increased recruitment, activation, and uptake of antigens by innate immune cells. Furthermore, like MPLA, PLA formulation establishes a long-lasting memory response. Notably, PLA demonstrated superior potency than MPLA formulation and provided sterilizing immunity against the leptospirosis in a hamster model. Overall, our study sheds light on the adjuvant properties of Leptospira lipid A and offers promising avenues for developing LPS-based vaccines against this devastating zoonotic disease.

Leptospirosis is a zoonotic disease of global importance. The current killed vaccine provides serovar-specific protection without sterilizing immunity. Several surface antigens, including Leptospira immunoglobulin-like proteins (LigA and LigB), have been identified as subunit vaccine candidates; however, they require potent adjuvants. Bacterial Lipopolysaccharide (LPS), including lipid A, is a well-known immunostimulatory agent, and the formulation of Monophosphoryl Lipid A (MPLA) in Alum is a clinical adjuvant. Being less endotoxic, we tested the adjuvant activity of lipid A purified from L. interrogans serovar Pomona (PLA) for its ability to activate innate and enhance antigen-specific adaptive immune response. PLA-induced activation of macrophages similar to levels induced by MPLA, albeit at a much higher dose, indicating that it is less stimulatory than MPLA. Mice immunized with a Variable portion of LigA (LAV) formulated in Alum and PLA (LAV-Alum-PLA) induced significantly higher levels of LAV-specific humoral and cellular immune response than Alum but similar to levels induced by Alum-MPLA. The adjuvant activity of PLA seems to be quite similar to MPLA and primarily mediated through enhanced recruitment, activation, and uptake of antigens by innate immune cells. Moreover, like MPLA, the PLA formulation was able to generate a long-term memory response. Most importantly, PLA demonstrated better potency than MPLA formulation and generated sterilizing immunity against challenge in a hamster model of leptospirosis. Altogether, our study has provided important insight into the adjuvant activity of Leptospira lipid A and has opened avenues for the development of LPS-based vaccines against this dreadful zoonosis.