ÿþ<HTML><HEAD><TITLE>25º Congresso Brasileiro de Microbiologia </TITLE><link rel=STYLESHEET type=text/css href=css.css></HEAD><BODY aLink=#ff0000 bgColor=#FFFFFF leftMargin=0 link=#000000 text=#000000 topMargin=0 vLink=#000000 marginheight=0 marginwidth=0><table align=center width=700 cellpadding=0 cellspacing=0><tr><td align=left bgcolor=#cccccc valign=top width=550><font face=arial size=2><strong><font face=Verdana, Arial, Helvetica, sans-serif size=3><font size=1>25º Congresso Brasileiro de Microbiologia </font></font></strong><font face=Verdana size=1><b><br></b></font><font face=Verdana, Arial,Helvetica, sans-serif size=1><strong> </strong></font></font></td><td align=right bgcolor=#cccccc valign=top width=150><font face=arial size=2><strong><font face=Verdana, Arial, Helvetica, sans-serif size=1><font size=1>ResumoID:1889-1</font></em></font></strong></font></td></tr><tr><td colspan=2><br><br><table align=center width=700><tr><td>Área: <b>Fermentação e Biotecnologia ( Divisão J )</b><p align=justify><strong><P><FONT SIZE=3><SPAN LANG=EN-US><B><EM>LACTOBACILLUS CASEI </EM>EXPRESSING THE MAJOR CAPSID PROTEIN L1 OF HPV16 INDUCES SYSTEMIC AND VAGINAL MUCOSAL IMMUNE RESPONSE IN MICE AFTER INTRAVAGINAL IMMUNIZATION</B></SPAN></FONT></P></strong></p><p align=justify><b><u>Karina Araujo Aires </u></b> (<i>Instituto Butantan</i>); <b>Sílvia Boschi Bazan </b> (<i>Instituto Butantan</i>); <b>Ivana Barros Campos </b> (<i>Instituto Butantan</i>); <b>Maria Leonor Sarno de Oliveira </b> (<i>Instituto Butantan</i>); <b>Aurora Marques Cianciarullo </b> (<i>Instituto Butantan</i>); <b>Paulo Lee Ho </b> (<i>Instituto Butantan</i>)<br><br></p><b><font size=2>Resumo</font></b><p align=justify class=tres><font size=2>&nbsp; <P class=western style="MARGIN-BOTTOM: 0cm" align=justify><FONT size=3><SPAN lang=en-US><STRONG>Introduction: </STRONG></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">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 </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"><EM>Lactococcus lactis</EM></SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"> for the expression of HPV16 L1 in </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"><EM>Lactobacillus casei</EM></SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">.</SPAN></SPAN></FONT><FONT size=3><SPAN style="FONT-WEIGHT: normal"> </SPAN></FONT><FONT size=3><STRONG>Objectives: </STRONG></FONT><FONT size=3></FONT><FONT size=3><SPAN style="FONT-WEIGHT: normal">The objective of </SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">this</SPAN></SPAN></FONT><FONT size=3><SPAN style="FONT-WEIGHT: normal"> </SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">work</SPAN></SPAN></FONT><FONT size=3><SPAN style="FONT-WEIGHT: normal"> was evaluate the constitutive expression of L1 in </SPAN></FONT><FONT size=3><I><SPAN style="FONT-WEIGHT: normal">L. casei</SPAN></I></FONT><FONT size=3><SPAN style="FONT-WEIGHT: normal">, 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 </SPAN></FONT><FONT size=3><I><SPAN style="FONT-WEIGHT: normal">L. casei </SPAN></I></FONT><FONT size=3><SPAN style="FONT-STYLE: normal"><SPAN style="FONT-WEIGHT: normal">(</SPAN></SPAN></FONT><FONT size=3><I><SPAN style="FONT-WEIGHT: normal">L. casei</SPAN></I></FONT><FONT size=3><SPAN style="FONT-STYLE: normal"><SPAN style="FONT-WEIGHT: normal">/L1C)</SPAN></SPAN></FONT><FONT size=3><SPAN style="FONT-WEIGHT: normal">. </SPAN></FONT><FONT size=3><STRONG>Methods:</STRONG></FONT><FONT size=3><SPAN style="FONT-WEIGHT: normal"> The expression of L1 was evaluated in </SPAN></FONT><FONT size=3><I><SPAN style="FONT-WEIGHT: normal">L. casei</SPAN></I></FONT><FONT size=3><SPAN style="FONT-STYLE: normal"><SPAN style="FONT-WEIGHT: normal">/L1C</SPAN></SPAN></FONT><FONT size=3><SPAN style="FONT-WEIGHT: normal"> 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. </SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">Mice was treated with medroxyprogesterone acetate, five days before each vaccine dose for estral cycle synchronization. </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">L. casei</SPAN></I></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"> expressing constitutively L1 were grown until the OD</SPAN></SPAN></FONT><SUB><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">600</SPAN></SPAN></FONT></SUB><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"> was 1 and then the concentration was adjusted to 10</SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"><SUP>10</SUP></SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"><SUP> </SUP>CFU per 10 </SPAN></SPAN></FONT><FONT face="Times New Roman, serif"><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">µ</SPAN></SPAN></FONT></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">L (1 dose). Groups of six anesthetized mice were innoculated intravaginally with 10</SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"><SUP>10</SUP></SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"> CFU of </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">L. casei</SPAN></I></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"> or </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">L. casei/</SPAN></I></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-STYLE: normal"><SPAN style="FONT-WEIGHT: normal">L1C</SPAN></SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"> 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. </SPAN></SPAN></FONT><FONT size=3><STRONG>Results and Discussion:</STRONG></FONT><FONT size=3><SPAN style="FONT-WEIGHT: normal"> The expression of the 56 kDa L1 protein by </SPAN></FONT><FONT size=3><I><SPAN style="FONT-WEIGHT: normal">L. casei</SPAN></I></FONT><FONT size=3><SPAN style="FONT-WEIGHT: normal">/L1C was confirmed by western-blot with Camvir antibody, and ultrastructural analysis showed the production of VLPs. </SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">Intravaginal immunization with </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">L. </SPAN></I></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">casei/L1C elicited serum VLPs-specific IgG antibodies in mice (U-Test, </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">p</SPAN></I></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">=0,005; </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">p</SPAN></I></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-STYLE: normal"><SPAN style="FONT-WEIGHT: normal">&lt;0,05</SPAN></SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">). 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, </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">p</SPAN></I></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-STYLE: normal"><SPAN style="FONT-WEIGHT: normal">=0,04; </SPAN></SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">p</SPAN></I></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-STYLE: normal"><SPAN style="FONT-WEIGHT: normal">&lt;0,05)</SPAN></SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">. Mice immunized with </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">L. casei</SPAN></I></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"> (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 </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">L. casei</SPAN></I></SPAN></FONT><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal">/L1C after vaginal immunization. The production of HPV16-VLP by </SPAN></SPAN></FONT><FONT size=3><SPAN lang=en-US><I><SPAN style="FONT-WEIGHT: normal">Lactobacillus</SPAN></I></SPAN></FONT><FONT color=#000000><FONT size=3><SPAN lang=en-US><SPAN style="FONT-WEIGHT: normal"> and the induction of VLP-specific systemic and mucosal antibodies open the possibility for the development of new live mucosal prophylactic vaccines.</SPAN></SPAN></FONT></FONT></P> <P class=western style="MARGIN-BOTTOM: 0cm" align=justify><FONT face="Times New Roman, serif"><SPAN lang=en-US><B>Supported by</B></SPAN></FONT><FONT face="Times New Roman, serif"><B>: FAPESP, CNPq, Fundação Butantan.</B></FONT></P></font></p><br><b>Palavras-chave: </b>&nbsp;Lactobacillus, HPV16, Virus-like Particles, intravaginal immunization, mucosal vaccine</td></tr></table></tr></td></table></body></html>