IMMUNIZATION VIA THE COLONIC MUCOSA USING ADENOVIRAL VECTORS

Sexually-transmitted diseases (STDs) are among the most common causes of illness in the world. The annual incidence of STDs is rising, from 250 million in 1990 to 340 million in 2002. Viral infection is a frequent cause of STDs and the mucosal surfaces are the natural sites of transmission of viruses. Both genital and rectal tracts are involved in transmission of viral SIDs. To infect a host, a virus has to penetrate mucosal immunologic barriers, including lumenal immunoglobulin A (lgA) intervention, epithelial defenses, and lamina propria (LP) lymphocyte-mediated protection. The breach of the mucosal immune system can lead to virus spreading to the rest of the body, thereby causing life-threatening disease. Induction of effective mucosal immunity is essential for the host to control local viral infection and prevent SID development. Immune responses are initiated when pathogen-derived antigen (e.g., viral antigen) is encountered and taken by antigen-presenting cells (APes), especially dendritic cells (Des). After antigen processing, Des present immunogenic determinants to and activate naIve lymphocytes in mucosal lymphoid tissues. Activated lymphocytes leave the lymphoid tissue and, via the bloodstream, migrate to the LP. These effector cells exert a series of immune functions, such as cytokine production, cytotoxicity and antibody secretion. Accordingly, the mucosal immune system is divided into the inductive and effector site. Gut-associated lymphoid tissues (GALT) represent the inductive site of the gastrointestinal (GI) system. The GALT within the rectal mucosa iii mainly consists of iliac lymph nodes (lLN) , Peyer's patch-like aggregated lymphoid follicles (ALF) and isolated lymphoid follicles (ILF). The ILN have been identified as the principal inductive site, whereas the role for mucosally located lymphoid follicles remains poorly understood. To elicit specific mucosal immune responses against virus infection, a viral antigen must be introduced into the mucosal immune system, importantly the inductive site. Efficient antigen delivery ensures the success of inducing effective mucosal immunity. Both the mucus layer and the integral epithelial monolayers form barriers against the passage of proteins and particulate matter across the epithelium. Previous studies demonstrated that these barriers could be overcome by mucus removal with ethanol treatment followed by utilizing viral vectors such as replication-deficient adenoviral vectors (Adv) engineered to encode heterologous antigen genes. Adv can infect a broad range of mammalian cells, including human and ~ouse epithelial cells, and has proved to be efficient in transferring genes to the colonic mucosa. It has been discovered that mucosal immunity can be induced at multiple mucosal sites, but rarely via a systemic route. This phenomenon is largely due to the mechanism of the common mucosal immunologic system (CMIS), within which activated mucosal lymphocytes migrate from one mucosal site (e.g., the upper respiratory tract mucosa) to another (e.g. the genital tract). Thus, the concept of CMIS is used as a guiding principle for mucosal vaccine design. However, emerging results have suggested that distant iv mucosal immunization is less effective than local immunization and that CMIS might comprise several anatomical-based grouped networks having different lymphocyte homing mechanisms. In this context, the vaginal (local mucosal) immunization regimen might improve local protection against genital STDs. Following the similar logic, rectally-induced immune responses might have potential to combat rectal STD infections. As both genital and rectal mucosae are drained by the ILNs, the putative genito-rectal associated lymphoid tissue, rectal immunization might be an alternative solution to genital immunization, especially to deal with the problem of immunization in the male genitourinary tract. The major goal of this study was to evaluate the effects of Adv-based local mucosal immunization via the rectum of mice in the induction of mucosal immunity against virus infection at the rectal as well as genital mucosa. A non-Invasive intrarectal (IR) delivery method (pipetting and Dermabond® following a ethanol enema) for Adv was developed in the present study to provide better gene transfer for induction of mucosal immune responses. The first approach was to reinvestigate gene transfer to the mouse colon after intrarectal (lR) administration. The transgene was found highly expressed at days 1-3 and mainly confined to the colon. Gene expression was not only identified within the epithelium but also immediately beneath the epithelium, probably due to the penetration of Adv through epithelial cells. Mucosal immune responses were examined in an antigen model using ovalbumin (OV A). After IR immunization with Adv encoding OVA (AdOV A), the frequency of LP interferon (lFN)y secreting cells was detected as early as day 4, and continually progressed upward. The v appearance of ILN IFN-y-secreting cells was transient, and this was mirrored by the ILN cytolytic activities. CDS+ T cells were stimulated to produce IFN-y, and cytolytic activities depended largely on CDS. Also, IR immunization with Adv induced Thl T-cell responses and local production of specific IgA. When challenged with recombinant vaccinia virus expressing OV A, immunized mice completely controlled local viral infection and prevented virus dissemination from the rectal tissue. Thus, an infectious mouse model of herpes simplex virus type 2 (HSV -2) given via the rectum was developed in the study and used to further validate the IR immunization regimen. Mice immunized with Adv encoding glycoprotein B (AdgB) were protected from rectal challenge of HSV -2 at absolute lethal doses. Clinical pathology and virus replication were remarkably reduced and virus-shedding period was significantly lessened. CDS+ T cells, IFN-y and interleukin (lL)-12 appeared to play an essential role in such protective immunity. Protection of mice against HSV-2 challenge in the vaginal tract was also achieved, thus indicating that rectal immunization could also confer protective genital immunity. In comparison with the intranasal (distant mucosal) and subcutaneous (systemic non-mucosal) immunization, rectal immunization proved to be more effective in the induction of rectal immune responses including the frequency of IFN-y secreting cells and IgA production, and to provide better protection against rectal or vaginal HSV-2 challenge. All these results underscored the importance of applying local mucosal immunization to induce mucosal immunity at both rectal and genital tracts. In conclusion, IR administration with Adv by the new delivery method efficiently