Delivery of Human Immunodeficiency Virus Vaccine Vectors to the Intestine Induces Enhanced Mucosal Cellular Immunity

Effective vaccines for human immunodeficiency virus type 1 (HIV-1) will likely need to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4⁺ T-cell depletion. While replication-competent recombinant adenovirus (rAd) vectors can stimulate adenovirus-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific immunogens has proven challenging. In this study, we have systematically identified barriers to effective gut delivery of rAd vectors and identified sites and strategies to induce potent cellular and humoral immunity. Vector-mediated gene transfer by rAd5 was susceptible to low-pH buffer, gastric and pancreatic proteases, and extracellular mucins. Using ex vivo organ explants, we found that transduction with rAd5 was highest in the ileum and colon among all intestinal segments. Transgene expression was 100-fold higher after direct surgical introduction into the ileum than after oral gavage, with rAd5 showing greater potency than the rAd35 or the rAd41 vector. A single immunization of rAd5 encoding HIV-1 gp140B to the ileum stimulated potent CD8⁺ T-cell responses in the intestinal and systemic compartments, and these responses were further enhanced by intramuscular rAd5 boosting. These studies suggest that induction of primary immune responses by rAd5 gut immunization and subsequent systemic boosting elicits potent antigen-specific gut mucosal responses.Human immunodeficiency virus type 1 (HIV-1) infection is characterized by uncontrolled virus replication and cytopathicity in the intestinal mucosa, the site of major T-cell depletion after primary infection. The gastrointestinal (GI) tract is the predominant site of a pronounced CD4⁺ T-cell loss in the early stages of HIV infection and simian immunodeficiency virus (SIV) infection in the nonhuman primate model (3, 23, 26, 43). It has been suggested that a mucosal vaccine which generates HIV-specific CD8⁺ T cells in the gut could prevent the loss of CD4⁺ cells in gut-associated lymphoid tissue, establishment of infection, or spread of virus (13, 34). Therefore, targeted delivery of vaccines to the GI tract to stimulate mucosal responses has the potential to improve the efficacy of immune protection against HIV-1; however, the site of gene-based transduction and the barriers to vaccine delivery have not been well defined.Adenoviruses (Ads) have been used extensively as vectors for both gene transfer and vaccine development. They offer several advantages as tools for vaccine delivery, such as the ability to transduce both dividing and nondividing cells, relative safety and stability in vivo, ease of production in high titers, and lack of integration (2, 35). These vectors are promising because parenteral administration in both animals and humans has been shown to generate strong and long-lasting humoral and cellular immune responses. The immune responses surpass those achieved with other types of gene vectors and genetic vaccines (5, 38, 46). As a result, recombinant Ad (rAd) vectors have been developed and tested as vaccine vehicles to immunize against a number of pathogens (4, 10, 15, 18, 41).Orally (p.o.) delivered vaccines are attractive in theory because of their ease of administration and potential to deliver antigen to gut-associated lymphoid tissue, permitting induction of immune responses in both mucosal and systemic compartments. At the same time, p.o. delivery of replication-defective rAd vectors has posed a challenge and has met with variable levels of success. Immunization with rAd5 encoding rabies virus antigens, influenza virus antigens, or other antigens has generated some protection against infection in animal models (9, 27, 31, 39, 41), but p.o. immunization has elicited much lower CD8⁺ T-cell responses than systemic delivery (33), and a much higher dose is required to induce immune responses (37). We have recently shown in an HIV vaccine model that rAd41, a human enteric Ad-based vector, induced potent CD8⁺ T-cell responses in both systemic and mucosal compartments when primed p.o. or in the ileum (17). The previous study showed that rAd41 vectors delivered through direct ileal injection elicited mucosal cell immunity, but whether other rAd vectors could stimulate these responses and which factors affected delivery and immunogenicity were unknown. In this report, we have investigated the mechanisms associated with the low immunogenicity of rAd5 dosed through the p.o. route in mice. The purpose was to identify barriers to effective delivery of rAd vectors to gut tissues and to ascertain sites and strategies for induction of potent cellular and humoral immunity. To investigate the mechanism of the low immunogenicity of rAd vectors through the p.o. route and develop effective delivery of rAd5 and rare serotype rAd35 vectors as gut mucosal HIV vaccines, we have analyzed the obstacles to p.o. immunization, characterized vector transgene expression, and systematically compared immune responses induced by p.o. and local immunization strategies. These studies demonstrated that the higher immune responses were strongly associated with higher gene expression in the intestine and support further study of gut mucosal immunization in SIV challenge models as a potential HIV vaccine strategy.