SummaryThe induction and maintenance of long-term CTL memory at mucosal surfaces may be a critical component of protection against mucosal pathogens and is one goal towards development of effective mucosal vaccines. In these studies we have functionally evaluated short and longterm CTL memory in systemic and respiratory or genital-associated lymphoid tissues following mucosal or systemic routes of immunization. Our results indicate that shortly after immunizing mice with a recombinant adenovirus vector expressing glycoprotein ~ (gB) of herpes simplex virus (AdgB8), gB-specific CTL memory responses were observed in systemic and mucosal immune compartments regardless of the route of inoculation. In contrast, several months after immunization, anamnestic CTL responses compartmentalized exclusively to mucosal or systemic lymphoid tissues after mucosal or systemic immunization, respectively. Furthermore, the compartmentalized CTL memory responses in mucosal tissues were functionally observed for longer than 1.5 yr after intranasal immunization, and CTL precursor frequencies one year after immunization were comparable to those seen shortly after immunization. Therefore, to our knowledge, this is the first functional demonstration that the maintenance of anti-viral memory CTL in mucosal tissues is dependent on the route of immunization and the time of assessment. These results have important implications for our understanding of the development, maintenance, and compartmentalization of functional T cell memory and the development and evaluation of vaccines for mucosal pathogens, such as HSV and HIV.

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.

Autoimmune destruction of islets in the pancreas leads to the development of insulin-dependent diabetes mellitus (IDDM). Replacement of insulin-producing tissue by transplantation of islets provides a cure to disease but requires immunosuppression or a means of controlling anti-graft immune responses. To promote islet survival we have utilized a local approach by expressing immunoregulatory molecules in islet grafts. The results presented here show that the human immunodeficiency virus (HIV)-based lentiviral vector is capable of stably transducing whole islets. Foreign reporter gene expression was observed both in vitro and in vivo 30 days after transplantation. Grafts containing insulin-positive beta-islet cells expressing foreign protein indicate that transduction does not interfere with glucose regulation. The absence of inflammatory infiltrates in grafts suggests that transduction does not activate the immune system. When islets transduced with an HIV vector expressing IL-4 were transplanted into diabetes-prone mice, animals were protected from autoimmune insulitis and islet destruction. As demonstrated by proliferative and cytokine analysis, protection was consistent with a switching of islet-antigen-specific T cell responses toward a Th2 phenotype. These results suggest that HIV-based lentivirus vectors can efficiently transduce islet cells with genes encoding potentially therapeutic molecules, for possibly managing diabetes.

This study demonstrates that the levels of gB-specific IgG and IgA in vaginal washes of mice immunized intranasally (i.n.) with a recombinant adenovirus vector expressing herpes simplex virus (HSV) glycoprotein B (AdgB8) vary inversely with each other and are dependent on the stage of the estrous cycle. Anti-gB IgA titers in vaginal washes were significantly higher during estrus than diestrus or proestrus, whereas specific IgG titers were significantly higher during diestrus than estrus. This was further demonstrated in hormone-treated mice, where progesterone administration induced a diestrus-like state that resulted in elevated specific IgG-to-IgA ratios. Interestingly, unimmunized mice were only susceptible to intravaginal (ivag) infection with HSV-2 during diestrus. Mice immunized i.n. with AdgB8 and given progesterone were protected from a lethal intravaginal HSV-2 challenge, despite the fact that virus replication was present for 4 days postchallenge. Further, high numbers of gB-specific IgA and IgG antibody-secreting cells were present in both the genital tracts and the draining iliac lymph nodes of i.n.-immunized, but not unimmunized, mice 6 days following ivag HSV-2 challenge. These results demonstrate that the levels of specific antibodies in the female genital tract are dependent on the stage of the estrous cycle. Furthermore, i.n. AdgB8 immunization provided a significant level of protection and specific IgA and IgG antibody-secreting cells in the genital tissues during resolution of an ivag infection with HSV-2.