Leptospira Lipid A Is a Potent Adjuvant That Induces Sterilizing Immunity against Leptospirosis.

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.

Differential modulation of innate immune response by lipopolysaccharide of Leptospira.

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-1ß 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.

LigA formulated in AS04 or Montanide ISA720VG induced superior immune response compared to alum, which correlated to protective efficacy in a hamster model of leptospirosis.

Leptospirosis is a zoonotic disease of global importance. The current vaccine provides serovar-specific and short-term immunity and does not prevent bacterial shedding in infected animals. Subunit vaccines based on surface proteins have shown to induce protection in an animal model. However, these proteins were tested with non-clinical adjuvants and induced low to moderate protective efficacy. We formulated a variable region of Leptospira immunoglobulin-like protein A (LAV) in clinical adjuvants, AS04 and Montanide ISA720VG, and then evaluated the immune response in mice and protective efficacy in a hamster model. Our results show that animals immunized with LAV-AS04 and LAV-Montanide ISA720VG (LAV-M) induced significantly higher levels of LAV-specific antibodies than LAV-Alum. While LAV-Alum induced Th2 response with the induction of IgG1 and IL-4, AS04 and LAV-M induced a mixed Th1/Th2 response with significant levels of both IgG1/IL-4 and IgG2c/IFN-γ. Both LAV-AS04 and LAV-M induced the generation of a significantly higher number of cytotoxic T cells (CTLs). The immune response in LAV-AS04- and LAV-M-immunized animals was maintained for a long period (>180 days) with the generation of a significant level of B- and T-cell memory. The strong immune response by both vaccines correlated to enhanced recruitment and activation of innate immune cells particularly DCs at draining lymph nodes and the formation of germinal centers (GCs). Furthermore, the immune response generated in mice correlated to protective efficacy in the hamster model of leptospirosis. These results indicate that LAV-AS04 and LAV-M are promising vaccines and can be further evaluated in clinical trials.

Screening of Surface-Exposed Lipoproteins of Leptospira Involved in Modulation of Host Innate Immune Response.

Leptospira, a zoonotic pathogen, is capable of causing both chronic and acute infection in a susceptible host. Surface-exposed lipoproteins play a major role in modulating the host immune response by activating the innate cells like macrophages and dendritic cells or evading complement attack and killing by phagocytes like neutrophils to favor pathogenesis and establish infection. In this study, we screened some surface-exposed lipoproteins known to be involved in pathogenesis to assess their possible role in immune modulation (innate immune activation or evasion). Surface proteins of the Len family (LenB, LenD, and LenE), Lsa30, Loa22, and Lipl21 were purified in recombinant form and then tested for their ability to activate macrophages of the different host (mouse, human, and bovine). These proteins were tested for binding with complement regulators like Factor H (FH), C4 Binding Protein (C4BP), and host protease Plasminogen (PLG) and also as nucleases to access their possible role in innate immune evasion. Our results show that, of various proteins tested, Loa22 induced strong innate activation and Lsa30 was least stimulatory, as evident from the production of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-α) and expression of surface markers [CD80, CD86, and major histocompatibility complex class II (MHCII)]. All the tested proteins were able to bind to FH, C4BP, and PLG; however, Loa22 showed strong binding to PLG correlating to plasmin activity. All the proteins except Loa22 showed nuclease activity, albeit with a requirement of different metal ions. The nuclease activity of these proteins correlated to in vitro degradation of neutrophil extracellular trap (NET). In conclusion, our results indicate that these surface proteins are involved in innate immune modulation and may play a critical role in assisting the bacteria in invading and colonizing the host tissue for persistent infection.

Deciphering the Role of Leptospira Surface Protein LigA in Modulating the Host Innate Immune Response.

Leptospira, a zoonotic pathogen, is known to infect various hosts and can establish persistent infection. This remarkable ability of bacteria is attributed to its potential to modulate (activate or evade) the host immune response by exploiting its surface proteins. We have identified and characterized the domain of the variable region of Leptospira immunoglobulin-like protein A (LAV) involved in immune modulation. The 11th domain (A11) of the variable region of LigA (LAV) induces a strong TLR4 dependent innate response leading to subsequent induction of humoral and cellular immune responses in mice. A11 is also involved in acquiring complement regulator FH and binds to host protease Plasminogen (PLG), there by mediating functional activity to escape from complement-mediated killing. The deletion of A11 domain significantly impaired TLR4 signaling and subsequent reduction in the innate and adaptive immune response. It also inhibited the binding of FH and PLG thereby mediating killing of bacteria. Our study discovered an unprecedented role of LAV as a nuclease capable of degrading Neutrophil Extracellular Traps (NETs). This nuclease activity was primarily mediated by A11. These results highlighted the moonlighting function of LigA and demonstrated that a single domain of a surface protein is involved in modulating the host innate immune defenses, which might allow the persistence of Leptospira in different hosts for a long term without clearance.

Leptospira surface adhesin (Lsa21) induces Toll like receptor 2 and 4 mediated inflammatory responses in macrophages.

Leptospirosis is zoonotic and emerging infectious disease of global importance. Little is understood about Leptospira pathogenesis and host immune response. In the present work we have investigated how Leptospira modulates the host innate immune response mediated by Toll-like receptors (TLRs) via surface exposed proteins. We screened Leptospira outer membrane/surface proteins for their ability to activate/inhibit TLR2/4 signaling in HEK293 cell lines. Of these the 21 kDa Leptospira surface adhesin, Lsa21 had strong TLR2 and TLR4 activity leading to production of proinflammatory cytokines and expression of costimulatory molecules in mouse macrophages. This activity of Lsa21 on innate response was dependent on activation of mitogen activated protein kinases (MAPKs) via stimulating the rapid phosphorylation of p38, JNK and activation of transcription factor NF-κB. Additionally, neutralizing antibodies against TLR2 and TLR4 significantly inhibited cytokine secretion and attenuated Lsa21 induced phosphorylation of p38 and JNK. Furthermore, Lsa21 induced cytokine levels were significantly lower in TLR2-/- and TLR4-/- than in wild type mouse macrophage cell lines. Confocal microscopy and molecular docking confirmed that Lsa21 interacted with both TLR2 and TLR4. These results indicate that Lsa21 is a potent TLR2 and TLR4 agonist that induces strong innate response and may play important role in Leptospira pathogenesis.