Coated Bacterial Vaccines: A new approach for non-heterologous antigen surface display on B. subtilis and its use in recombinant tetanus vaccine development

HARGUINDEGUY INES; HIRIART YANINA; GORGOJO JUAN P.; ALARCÓN, VANINA; CAVALITTO, SEBASTIÁN F; ORTIZ, GASTÓN E

Rosario

Congreso; LIX Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2023

Tetanus is an acute and frequently fatal infectious disease, caused by the tetanus toxin produced by Clostridium tetani. The current tetanus vaccine is effective but its production presents several drawbacks, including the need for manipulating toxigenic strains of C tetani and the handling of toxin. In this context, it has been demonstrated the effectiveness of the non-toxic C-terminal fragment of tetanus toxin (TTFC) to induce immunity against this toxin. Thus, this fragment has been used for development of vectorized vaccine based on surface antigen presentation, such as Live Bacterial Vaccines (LBVs) and Gram-positive Potentiating Matrix Particles (BLPs). In this way, our group has developed an antigen presentation system called Coated Bacterial Vaccines (CBVs). This system involves the exogenous production of the target vaccine antigen fused to the C-terminal end of S-layer proteins of Lactobacillus spp (FasTAG®). This domain exhibits a strong affinity for Gram-positive bacterial membranes, such as Bacillus subtilis. Consequently, the chimeric antigen can be produced in heterologous expression systems such as Escherichia coli. Subsequently, the intrinsic affinity of this can be used to coat the surface of chemically inactivated Gram-positive bacteria, which act as antigen-presenting carriers. Based on the above, the purpose of this work was to evaluate the functionality of the CBVs system to developed a recombinant tetanus vaccine based on model antigen TTFC. For this purpose, the recombinant antigen TTFC-FasTAG® was produced in E. coli and displayed on the surface of chemical inactivated B. subtilis to formulate the CBVs. Next, so as to analyze the vaccination capacity of CBVs, BALB/c mice were immunized intraperitoneally with three doses of 5 ug/dose TTFC. In order to evaluated the response, the anti-TTFC serum antibody levels were determined by ELISA. The results show that vaccinated mice increased the levels of specific anti-TTFC IgG antibody titer in sera to log=4, similar to reported for commercial vaccine immunization protocols. Moreover, to characterize the Th cell response induce by CBVs, we analyzed the distribution of TTFC-specific IgG isotypes present in sera of immunized mice. Immunization with CBVs induced higher IgG1 antibody titers compared to IgG2a, resulting in a Th2-polarized response, thus inducing a strongly humoral immune response. Furthermore, so as to determine whether the presence of specific antibodies correlated with protection, animals were challenged with 100xLD50 of purified toxin and demonstrated to raise complete protection against this toxin. Finally, the set of experiments carried out here validate the potential and functionality of this system and showed that it was able to confer active immunity to a murine model. Our results provide a theoretical basis for the development of a safe and effective recombinant tetanus vaccine as a potential substitute for the current toxoid vaccine.