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The route of vaccination is important in influencing immune responses at the initial site of pathogen invasion where protection is most effective. Immune responses required for mucosal protection can differ vastly depending on the individual pathogen. For some mucosal pathogens, including acute self-limiting infections, high-titer neutralizing Abs that enter tissue parenchyma or transude into the mucosal lumen are sufficient for clearing cell-free virus. However, for pathogens causing chronic infections such as HIV, hepatitis C virus, herpes viruses, mycobacteria, and fungal and parasitic infections, a single arm of the immune response generated by systemic vaccination may be insufficient for protection. Induction of the mucosal innate and adaptive immune systems, including CD4+ T help, Th17, high avidity CD8+ CTL, and secretory IgA and IgG1 neutralizing Abs, at the site of pathogen entry may be required for effective protection against highly invasive pathogens that lead to chronic infection and may be generated predominantly by mucosal vaccination.

Effective frontline defenses against HIV-1 will require targeting vaccines to mucosal tissue in order to induce alphabeta CD8(+) lymphocytes in mucosal effector sites (lamina propria and intraepithelial compartment) as well as antibody secreting plasma cells that can neutralize and limit free virus. A concerted second wave of assault against the virus will require the activation and recruitment of antigen specific memory CD4(+) and CD8(+) T cells in mesenteric lymph nodes and distal secondary lymphoid organs. New delivery strategies targeting the "right" DC subsets in combination with delivery of mucosal adjuvants and innate signals for activating DC will be essential for mucosal vaccines in order to circumvent the naturally tolerogenic environment and the induction of Tregs. Mucosal delivery of antigen in combination with inflammatory signals has been shown to empower systemic immunization by directing responses to mucosal sites for imprinting optimum mucosal memory. Here, we discuss novel vaccine strategies and adjuvants for optimizing mucosal delivery of HIV vaccines.

The gastrointestinal and vaginal mucosa are the major routes of natural HIV transmission, with the former being the primary reservoir for virus replication. The crucial need for a global HIV vaccine underscores the effort to develop vaccines capable of eliciting mucosal immune responses. Vaccines that induce high levels of effective mucosal immunity should impact viral replication rate and prevent dissemination of virus from the mucosa into the systemic circulation. In our opinion, the major effort for effective HIV vaccine should be concentrated on generating protective immunity at the site of viral entry (i.e. the gastrointestinal and vaginal mucosae). Here we examine the current information regarding the role of mucosal immunity in prevention of HIV transmission and discuss strategies for mucosal AIDS vaccine development.

To improve the safety of recombinant vaccinia virus vaccines, modified vaccinia virus Ankara (MVA) has been employed, because it has a replication defect in most mammalian cells. Here we apply MVA to human immunodeficiency virus type 1 (HIV-1) vaccine development by incorporating the envelope protein gp160 of HIV-1 primary isolate strain 89.6 (MVA 89.6) and use it to induce mucosal cytotoxic-T-lymphocyte (CTL) immunity. In initial studies to define a dominant CTL epitope for HIV-1 89.6 gp160, we mapped the epitope to a sequence, IGPGRAFYAR (from the V3 loop), homologous to that recognized by HIV MN loop-specific CTL and showed that HIV-1 MN-specific CTLs cross-reactively recognize the corresponding epitope from strain 89.6 presented by H-2Dd. Having defined the CTL specificity, we immunized BALB/c mice intrarectally with recombinant MVA 89.6. A single mucosal immunization with MVA 89.6 was able to elicit long-lasting antigen-specific mucosal (Peyer's patch and lamina propria) and systemic (spleen) CTL responses as effective as or more effective than those of a replication-competent vaccinia virus expressing 89.6 gp160. Immunization with MVA 89.6 led to (i) the loading of antigen-presenting cells in vivo, as measured by the ex vivo active presentation of the P18-89.6 peptide to an antigen-specific CTL line, and (ii) the significant production of the proinflammatory cytokines (interleukin-6 and tumor necrosis factor alpha) in the mucosal sites. These results indicate that nonreplicating recombinant MVA may be at least as effective for mucosal immunization as replicating recombinant vaccinia virus.

Mucosal tissues are major sites of HIV entry and initial infection. Thus, the induction of a mucosal cytotoxic T lymphocyte (CTL) response is an important feature for an effective HIV vaccine. However, little is known about approaches to induce such a protective CTL response in the mucosa. Here for the first time we show that intrarectal immunization with a synthetic, multideterminant HIV peptide plus cholera toxin adjuvant induced long-lasting, antigen-specific CTL memory in both the inductive (Peyer's patch) and effector (lamina propria) mucosal sites, as well as in systemic sites (spleen), whereas systemic immunization induced specific CTL only in the spleen. Cholera toxin adjuvant, while enhancing the response, was not essential. The CTL recognized target cells either pulsed with HIV peptide or expressing endogenous whole envelope glycoprotein of Mr 160,000 (gp160). Exploring the requirements for CTL induction, we show that mucosal CTL responses are both interleukin 12 and interferon-gamma dependent by using antibody-treated and knock-out mice. Finally, to determine whether a mucosal response is actually protective against local mucosal challenge with virus, we show that intrarectal immunization with the synthetic HIV peptide vaccine protected mice against infection via mucosal challenge with a recombinant vaccinia virus expressing HIV-1IIIB gp160. These studies provide an approach to development of an HIV vaccine that induces CTL immunity in the mucosal and systemic immune systems and protects against mucosal infection with a virus expressing HIV-1 gp160.