FLAGELLIN AS AN ADJUVANT FOR AN ANTIGEN DELIVERY SYSTEM BASED ON CELL WALL DERIVED PARTICLES FROM LACTOCOCCUS LACTIS

SILVESTRE D.; MORENO G.; ARGÜELLES M.H.; TOMÁS FARIÑA J.; PERI IBAÑEZ E.S.; MANDILE M.G.; GLIKMANN G.; CASTELLO A.A.; RUMBO M.; TEMPRANA C.F.

Modalidad virtual

Congreso; LVII Reunión Anual de SAIB - XVI Congreso Anual de SAMIGE; 2021

Mucous surfaces represent an entry site into the human body for numerous pathogens and, therefore, developing vaccines that generate a protective immune response at these sites is of great interest. However, immunization via the mucosa entails some obstacles, such as the enzymatic degradation of antigens or the induction of immunotolerance, among others. In this context, lactic acid bacteria have emerged as a potential antigen delivery system for mucous surfaces. In particular, our group is focused on developing a platform based on Lactococcus lactis cell wall derived particles (CWDP). Previously, we managed to obtain L. lactis that express rotavirus VP6 protein anchored on the outside of their cell wall, although mucosal immunization with said recombinant L. lactis did not induce a specific humoral response. On the contrary, when the CWDP of the same L. lactis containing VP6 were administered intranasally, a specific anti-rotavirus immune response was achieved. However, CWDP-VP6 only conferred protection against infection when co-administered with an adjuvant. Given that flagellin has been proposed as a mucosal adjuvant due to its ability to activate receptors of the immune system such as TLR5, our new focus is to increase the immunogenicity of CWDP using FliC131. We use this mutant of Salmonella flagellin, with a deletion in its antigenic domains, because anti-flagellin antibodies generated after repeated administrations could impair its adjuvanticity.Recently, we generated L. lactis that express FliC131 or a chimeric protein FliC131-VP6, by fusing their coding sequences using SOE-PCR. The expression of both recombinant proteins was evaluated by SDS-PAGE and their identity confirmed with western blots using anti-rotavirus or anti-flagellin antibodies. Subsequently, the corresponding CWDP-FliC131 and CWDP-FliC131-VP6 were obtained and the presence of the recombinant proteins re-confirmed with flow cytometry. Additionally, the concentration of both FliC131 and FliC131-VP6 was determined by SDS-PAGE and bands densitometry. Lastly, their ability to activate the TLR5 receptor was evaluated in vitro using the Caco-CCL20-Luc reporter cell line. The results show that the concentrations of FliC131 and FliC131-VP6 in the CWDP obtained are 2,1 μg/μl and 0,2 μg/μl, respectively. Finally, under the conditions tested in vitro, only CWDP-FliC131 managed to activate the TLR5 receptor. In this work, we obtained in a simple and inexpensive way CWDP from L. lactis containing FliC131 or FliC131-VP6, with the former retaining its ability to activate TLR5 in vitro. The next step in our research is to evaluate, through in vivo assays in a murine model, the adjuvanticity of CWDP-FliC131 and, eventually, its capacity to induce a protective immune response against rotavirus infection when co-administered with CWDP-VP6 or when using only CWDP-FliC131-VP6.