LACTOBACILLUS CASEI EXPRESSING THE MAJOR CAPSID PROTEIN L1 OF HPV16 INDUCES SYSTEMIC AND VAGINAL MUCOSAL IMMUNE RESPONSE IN MICE AFTER INTRAVAGINAL IMMUNIZATION

Karina Araujo Aires (Instituto Butantan); Sílvia Boschi Bazan (Instituto Butantan); Ivana Barros Campos (Instituto Butantan); Maria Leonor Sarno de Oliveira (Instituto Butantan); Aurora Marques Cianciarullo (Instituto Butantan); Paulo Lee Ho (Instituto Butantan)

Introduction: Infections with HPV16 are closely associated with the development of human cervical carcinoma. Nowadays, the most promising vaccines against HPV16 infection is based on L1 major capsid protein, which self-assembles in structures similar to HPV, named Virus-like Particles (VLPs). Since HPV16 have the genital mucosa as a host infection site, a prophylactic safe and low cost mucosal vaccine would constitute an interesting alternative to parenteral vaccines. In this work, we used the constitutive P1 promoter from Lactococcus lactis for the expression of HPV16 L1 in Lactobacillus casei. Objectives: The objective of this work was evaluate the constitutive expression of L1 in L. casei, the production of VLPs, and the potential of the intravaginal immunization as a effective mucosal route for systemic and vaginal induction of HPV16 VLPs-specific IgA and IgG antibodies in mice immunized with HPV16 L1-expressing L. casei (L. casei/L1C). Methods: The expression of L1 was evaluated in L. casei/L1C extracts carrying pT1NX/L1C expression plasmid by SDS-PAGE and confirmed by western-blot using the HPV16 L1-specific antibody Camvir. The production of VLPs was analyzed by electron microscopy. Six to eight-week-old female C57BL/6 mice were used for intravaginal immunization experiments. Mice was treated with medroxyprogesterone acetate, five days before each vaccine dose for estral cycle synchronization. L. casei expressing constitutively L1 were grown until the OD₆₀₀ was 1 and then the concentration was adjusted to 10¹⁰ CFU per 10 µL (1 dose). Groups of six anesthetized mice were innoculated intravaginally with 10¹⁰ CFU of L. casei or L. casei/L1C on three consecutive days. Three administrations, one priming and two boosts, were performed at 2-weeks intervals, for a total of nine doses. Ten days after the last dose, individual vaginal secretions and serum samples were collected for analysis of HPV-16 VLP-specific IgA and IgG antibodies by ELISA. Results and Discussion: The expression of the 56 kDa L1 protein by L. casei/L1C was confirmed by western-blot with Camvir antibody, and ultrastructural analysis showed the production of VLPs. Intravaginal immunization with L. casei/L1C elicited serum VLPs-specific IgG antibodies in mice (U-Test, p=0,005; p<0,05). However, vaginal VLP-specific IgA was only induced after an intranasal booster immunization with a yeast-produced HPV-16 VLPs sub-optimal dose (U-Test, p=0,04; p<0,05). Mice immunized with L. casei (L1-non-expressing) did not present IgA after VLP-intranasal booster. The results indicate that T and B lymphocytes of the vaginal mucosa were previously stimulated by L. casei/L1C after vaginal immunization. The production of HPV16-VLP by Lactobacillus and the induction of VLP-specific systemic and mucosal antibodies open the possibility for the development of new live mucosal prophylactic vaccines.