EVALUATION OF CELL WALL-DERIVED PARTICLES FROM LACTOCOCCUS LACTIS WITH FLAGELLIN AS AN ADJUVANT FOR MUCOSAL IMMUNIZATION

DALILA SILVESTRE; GRISELDA MORENO; MARCELO H. ARGÜELLES; MARINA E. BIEDMA; JULIETA TOMÁS FARIÑA; ESTEFANÍA S. PERI IBAÑEZ; MARCELO G. MANDILE; GRACIELA GLIKMANN; ALEJANDRO A. CASTELLO; MARTÍN RUMBO; C. FACUNDO TEMPRANA

Mar del Plata

Congreso; REUNIÓN ANUAL DE SOCIEDADES DE BIOCIENCIAS; 2022

Sociedad Argentina de Investigación Clínica

Lactic acid bacteria have emerged as a potential antigen delivery system for mucous surfaces. In particular, our aim is to develop a platform based on Lactococcus lactis cell wall-derived particles (CWDP). We previously generated CWDP that contained rotavirus VP6 protein, which conferred protection against infection when co-administered intranasally with dmLT in a murine model. Because flagellin has been proposed as a mucosal adjuvant, our goal is to increase the immunogenicity of CWDP using FliC131, a mutant of Salmonella flagellin. We generated L. lactis that express FliC131 on their cell wall. The CWDP-FliC131 obtained were evaluated by SDS-PAGE, Western blot, and flow cytometry. Moreover, the concentration of FliC131 was determined by SDS-PAGE and bands densitometry. The ability of CWDP-FliC131 to activate the TLR5 receptor was evaluated in vitro with the HEK-hTLR5 reporter cell line. The results confirmed the identity of FliC131 and showed a concentration of 2,1 mg/ml. Additionally, CWDP-FliC131 stimulated TLR5 under the tested conditions. To evaluate the adjuvanticity of CWDP-FliC131 in vivo, groups of five mice were immunized intranasally with PBS, OVA, CWDP-FliC131, OVA plus CWDP-FliC131 or OVA plus CWDP-NZ9000 (derived from non-recombinant L. lactis NZ9000). After three doses, samples of serum, bronchoalveolar lavage (BAL), and intestinal lavage were collected. Both CWDPFliC131 plus OVA (CFO) and CWDP-NZ9000 plus OVA (NZO) induced anti-OVA IgG and IgA detectable by ELISA in serum and BAL. Even though there were no statistically significant differences, four mice in the CFO group tested positive for serum IgA versus only one in the NZO group. In conclusion, CWDP were immunogenic when administered intranasally, although the adjuvant effect of FliC131needs to be further tested using different doses of CWDP-FliC131 and increasing the number of mice to reduce the variability associated with the intranasal immunization and the biological response of each mouse.