A self-assembled Pirococcus abyssi exosome complex displaying outer membrane proteins of Leptospira interrogans elicits protective immunity against leptospirosis.

Leptospirosis is a widespread zoonosis caused by Leptospira spp. New vaccine strategies are being explored to enhance both the magnitude and duration of protection. In this study, we developed a novel recombinant vaccine formulation, using the Pyrococcus abyssi exosome complex to present immunogenic regions of key Leptospira antigens (LigANI, LigBrep, and LipL32). A co-expression vector containing the P. abyssi exosome proteins fused to the three Leptospira antigens was constructed, generating the EXOLEP (EXOsome + LEPtospira) multivalent complex, and the polyproteins were expressed in Escherichia coli. The stability of EXOLEP was confirmed through in vitro and in vivo assays, demonstrating remarkable resistance, remaining stable at temperatures up to 100 °C and extended shelf life. In hamster model, a hybrid EXOLEP formulation (EXOLEP Copenhageni + Leptospira bacterin from serovar Canicola) provided 80-100 % protection (P < 0.05) against Leptospira serovars Copenhageni and Canicola. Additionally, the hybrid formulation induced a balanced Th1/Th2 immune response, evidenced by upregulation of IFN-γ, TNF-α, IL-10, and TGF-β, along with significant levels (P < 0.05) of IgG1 and IgG2/3 isotypes against all Leptospira antigens. The formulation maintained its efficacy after one year of storage at 4 °C. These results suggest that the EXOLEP hybrid is a promising, cost-effective, and scalable candidate for veterinary leptospirosis vaccines.