The intestinal mucosa represents a primary site of HIV replication during the peak of infection (3
). Vaccination through the GI mucosa could potentially induce mucosal immunity to prevent virus entry and/or limit the spread of infection. Oral immunization is the most convenient way to deliver mucosal vaccines. Although several studies have reported that the use of this route of immunization of Ad vectors encoding various virus antigens generated antibody responses in the serum and mucosal secretions (33
), the administration of Ads encoding Gag or Env from HIV-1 or SIV elicited weak or undetectable intestinal mucosa responses (24
). The major obstacles to successful induction of mucosal immunity and/or systemic immunity via GI delivery of vaccine have not been defined previously. In this study utilizing murine intestinal explant cultures and in vivo gene expression assessments, we demonstrate that the combination of a low-pH environment and the presence of proteases as well as the mucus/glycocalyx in the GI tract prevents rAd5 from effectively transducing the intestinal cells when delivered p.o. Therefore, p.o. immunization of vaccine vectors must overcome or bypass these barriers in the GI tract in order to be effective. We also show that the ileum is the most receptive site for rAd transduction and that intraileal injection of rAd5 results in a significantly higher transgene expression in the ileum than does p.o. delivery. In addition, intraileal immunization with rAd5 did not induce significant systemic antivector neutralizing activity, since the rAd5 i.m. administration was effective in the homologous rAd5 prime-rAd5 boost regimen. This is in agreement with our previous work demonstrating the absence of circulating neutralizing antibodies following p.o. administration of rAd5 and rAd41 (17
). It has also been shown that preexisting immunity to the vaccine carrier did not impair p.o. vaccination with Ad vectors in mice (45
). Thus, the ileum represents a valid target for delivery of rAd vectors.
Utilization of a route of administration that bypassed the barriers to ileal transduction revealed the potency of rAd5 as a mucosal immunogen. A single immunization of mice with rAd5 encoding HIV-1 gp140B via intraileal injection generated significantly higher numbers of H-2Dd
T cells in both mucosal and systemic compartments and antigen-specific IFN-γ-, TNF-α-, and IL-2-producing CD4+
T cells in the spleen than did p.o. administration (Fig. ). In fact, p.o. immunization did not generate any detectable immune response after a single dose in our study. These results indicate that the higher transduction from intraileal injections than from p.o. delivery (Fig. ) is associated with the stronger immune responses to HIV-1 gp140B. Systemic boosting with rAd5-gp140B dramatically increased CD8+
T-cell responses in the mice primed via the intraileal route in both systemic and mucosal compartments compared to responses in the group primed p.o. (Fig. , right) as well as splenic CD4+
T-cell responses compared to those in the mice primed p.o. or via ileal injection (Fig. , left). Whether the activation of CD4+
T cells would provide more target cells for HIV acquisition will be addressed in nonhuman primate (NHP) studies, though a previous study in which such mucosal responses were detected showed that these responses led to protection (21
). Oral dosing with replication-competent Ad vectors generates systemic immune responses in nonhuman primates, but multiple primes and protein boosting are required for protective efficacy against SIVmac251
). Oral vaccination with replication-competent Ad4 and Ad7 has been shown to generate protective Ad neutralizing antibodies in humans (11
), indicating that replication-competent vectors elicit antivector neutralizing antibodies, which may compete with transgene-specific responses or may prevent multiple p.o. administrations of the same vector and affect efficacy. In addition, the magnitude and contribution of the transgene-specific response in mediating protection by replication-competent rAd remain unknown in this model because the gut mucosal immune responses were not measured and compared to systemic responses. Thus, there are potential efficacy and regulatory concerns with replication-competent rAds, and further studies are needed to assess the potential efficacy of gut mucosal immunization against lentivirus infection in NHP. Direct mucosal introduction of replication-defective vectors offers an alternative approach to address questions of immunogenicity and efficacy in this model. Although cellular responses were robust after intraileal injection, we did not detect a significant level of IgG or IgA antibody from fecal extracts (data not shown). Some animals showed IgA responses from the vaginal washes, but the responses were low and inconsistent. Though collection methods for mucosal secretions can be optimized (19
), it is likely that these responses are low in magnitude in the GI tract.
While the results reported here describe the immunogenicity of rAd5- and rAd35-based vectors, we have previously analyzed rAd41, an enteric tropic Ad, for its ability to generate immune responses through direct ileum lumenal administration (17
). When rAd41 was administered directly to the lumen of the ileum, it efficiently primed antigen-specific cellular immunity. Ad41, a member of the Ad species F, has been reported to bind/uptake more efficiently than Ad5 in the isolated intestinal loops of rats. Furthermore, gene delivery with Ad5 to differentiated enterocytes in vitro and to the rat jejunum in vivo is extremely inefficient (7
). However, the immune responses induced by Ad41 and Ad5 through intraileal injection priming and i.m. boosting were not significantly different, which highlighted that vectors derived from mucosal pathogens, both enteric and respiratory, can be used in these regimens. However, due to the prevalence of neutralizing antibodies to Ad5 and Ad41 in humans, the development and use of alternative serotypes of Ad vectors for virus vaccines remain a priority. Ad35 is one relatively well-characterized rare serotype vector and has been associated with infections of the kidney and urinary tract. Therefore, the fiber swap study was done for two reasons: (i) to define the contribution of fiber versus hexon to transduction of mucosal epithelium and (ii) to generate vectors resistant to anti-Ad41 hexon neutralizing antibodies, should they be mediated by fiber. Here, we found that rAd35 did not transduce the organ cultures of intestinal segments as efficiently as rAd5 and that rAd35 was less immunogenic in the ileal priming regimen, thus allowing us to address these questions. Unexpectedly, grafting the long fiber or the short fiber of Ad41 to the rAd35 vector did not improve the transduction efficiency of intestinal explants or immunogenicity, suggesting that the Ad41 fiber is not the only determinant of enteric tropism. However, these results suggest that the immunogenicity of rAd vectors in vivo correlates with the efficiency of transduction of intestinal explants ex vivo. We conclude that the lower small intestine represents a target for antigen delivery with certain rAd vectors. Further development of special formulations to deliver rAd to the ileum should be emphasized for practical vaccination.
Our results suggest that it is important to pursue a mucosal route of administration of rAd vaccine vectors for HIV. The strong cellular immune responses induced by intraileal injection, further strengthened by i.m. boost, suggest a strategy for protection of gut-associated lymphoid tissue lymphocytes from HIV-1 infection. Possible mechanisms of immune stimulation include persistent antigen expression from lamina propria cells transduced by the rAd vector (7
). Further characterization of the immune responses induced by the vaccination regimens employed in this work, such as the balance of CD8+
T-cell responses, the polyfunctionality of the T cells, and the duration of the immune response, could provide a better prediction as to which is the most important type of immune response for protection against HIV-1 (22
). Regarding the applicability of these data to other species, as well as the uncertain applicability to humans, this study represents one of a stepwise series of studies to determine whether mucosal immunity can be enhanced by different rAd vectors with alternative routes of administration. Having been demonstrated with rodents, this will next be evaluated with nonhuman primate challenge models to determine its contribution to immune protection. Should these results support the concept of a mucosal vaccine, an effort will be made to formulate an Ad vector for delivery to the ileum in humans, since the site-specific delivery technology to deliver drugs and vaccines to the ileum has been developed with partial success so far (14
The use of rAd5 vaccines in populations with moderate to high neutralizing antibodies to Ad5 based on the STEP trial (3a
) results is a concern. It will be difficult to define mechanisms responsible for this effect or even to document it definitively. At the same time, rAd5 vectors are well characterized and induce strong immune responses. There are numerous differences in vector design, insert combination, and immunization regimen that may result in different performances in future clinical studies. Regardless of its future clinical utility, rAd5 remains a useful model vector for inducing mucosal immunity and for determining how mucosal immune responses contribute to protection. Once these key questions are addressed, rAd5 vectors can be used if justified by efficacy/safety considerations, or, if concerns remain, other vectors can be substituted.