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 an emerging zoonotic and neglected disease across the world causing huge loss of life and economy. In this study, we did whole genome sequencing of one Leptospira isolate and a comparative genomic analysis with 69 other species of Leptospira available in RefSeq database provided insight into taxonomic and evolutionary relationship between species. AAI and whole genome based phylogenomic analysis established 3 clusters of Leptospira i.e. pathogenic, intermediate and saprophytic correlating with level of virulence of species. Leptospira has large closed core genome of 1038 genes and an open pan genome with 20,822 genes. The mobilome related genes were found mainly in pan-genome of pathogenic clade. A total of 498 genes have been identified as virulomes, with 329 virulent genes exhibiting presence/absence in various Leptospira species contributing to each species specific virulence profile. The hierarchical clustering of the congregated pathogenic genes yielded five groups, each with a distinct pattern of predominant genes that were either unique or common among clades, indicating lineage uniqueness. Most of the virulent gene pool identified were significantly enriched in COG functional categories of Nucleotide transport and metabolism, Intracellular trafficking, secretion and vesicular transport, cell motility and amino acid transport & metabolism. Pathogenic leptospires exhibit fewer clade-specific genes than non-pathogenic and intermediate leptospires, indicating gene loss and gain events in the evolution of pathogenic leptospires from non-pathogenic. The study’s clade-specific and virulent genes can be utilised as markers for defining clade and associated virulence levels in any new Leptospira isolates. Wet-lab validation of virulent genes will help in accurately targeting pathogenic pathways of Leptospira and controlling leptospirosis.

The effect of breed on milk components—fat, protein, lactose, and water—has been observed to be significant. As fat is one of the major price-determining factors for milk, exploring the variations in fat QTLs across breeds would shed light on the variable fat content in their milk. Here, on whole-genome sequencing, 25 differentially expressed hub or bottleneck fat QTLs were explored for variations across indigenous breeds. Out of these, 20 genes were identified as having nonsynonymous substitutions. A fixed SNP pattern in high-milk-yielding breeds in comparison to low-milk-yielding breeds was identified in the genes GHR, TLR4, LPIN1, CACNA1C, ZBTB16, ITGA1, ANK1, and NTG5E and, vice versa, in the genes MFGE8, FGF2, TLR4, LPIN1, NUP98, PTK2, ZTB16, DDIT3, and NT5E. The identified SNPs were ratified by pyrosequencing to prove that key differences exist in fat QTLs between the high- and low-milk-yielding breeds.

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.

India, home to the world’s largest cattle population, hosts native dairy breeds essential to its agricultural economy because of their adaptability and resilience. This study characterizes the genomes of five prominent breeds—Gir, Kankrej, Red Sindhi, Sahiwal, and Tharparkar—highlighting their unique genomic characteristics. The de novo assemblies ranged from 2.70 to 2.78 Gb in size, with 90% of the genomes assembled in just 56 to 1663 scaffolds. The use of reference-guided scaffolding further enhanced these genomes, resulting in 93.3%–96.7% pseudomolecule coverage with strong BUSCO scores (94.1%–95.5%). Comparative analyses revealed 87%–95% synteny with the Brahman genome and identified 19.84–153.16 Mb of structural rearrangements per genome, including inversions, translocations, and duplications. Synteny diversity analysis uncovered 10 643 perfectly collinear regions spanning 87.3 Mb and 6622 hotspots of rearrangement (HOT regions) covering 55.18 Mb. These HOT regions, characterized by high synteny diversity, were significantly enriched with immune-related genes. Moreover, immune-related gene clusters, including major histocompatibility complex, natural killer complex, and leukocyte receptor complex, were identified within HOT regions in the desi reference genome. Our findings provide valuable insights into the genetic diversity of desi cattle breeds. The high-quality genome assemblies generated in this study will serve as valuable resources for future research in genetic improvement, disease resistance, and environmental adaptation.