Women are most commonly infected by HIV-1 through heterosexual contact and immune mechanisms at or within the female GT would be expected to provide a crucial first barrier to transmission. As nAb that neutralize the countless HIV-1 variants remain elusive, many of the vaccines currently in clinical trials focus on the induction of HIV-1-specific CD8+ T-cells. Such response cannot prevent the initial infection, but if present at the port of entry, might rapidly eliminate infected cells and thus thwart or potentially prevent spread of the virus.
We showed in mice that a homologous prime-boost regimen using AdC vectors expressing gag induces transgene product-specific CD8+
T-cells that could be isolated from the GT [13
]. This previous paper used intracellular cytokine staining assays, which may not be optimal for the study of the GT-derived lymphocytes. Here we extended these studies testing different routes of immunization, more efficacious heterologous prime-boost regimens, and assessed migratory patterns of such cells.
It is known that nasal immunization is able to induce immune responses not only in the respiratory tract but also at the GT [23
]. Results reported here show that CD8+
T-cells, which home to the female GT, can be induced by i.n. immunization but this response is not sustained. In addition, vaginal booster immunization, as would be experienced in human vaccine recipients against HIV-1, causes only a slight local increase in i.n.-induced antigen-specific CD8+
T- cells and fails to increase responses systemically. Last but not least, i.n. immunization may be problematic for some vectors as this route allows access of the vaccine into the central nervous system. I.vag immunization, as reported by others [24
], only induces very low levels of antigen-specific CD8+
T-cells, which combined with logistic problems in humans should discourage further pursuit of this route of immunization for Ad vectors.
Results are more promising after i.m. immunization, which not only elicits antigen-specific CD8+
T-cells in systemic tissues but also high and sustained responses within the GT, as also reported recently by another group [25
]. A second immunization given i.m. causes a robust booster effect within the GT of i.m.-primed mice, and gag-specific CD8+
T- cells remain detectable for at least one year. I.m. immunization is thus overall superior at inducing genital CD8+
T- cell responses by AdC vectors compared to i.n. immunization, and offers the added benefit of also eliciting potent systemic CD8+
T-cell responses, which may serve as a second layer of defense in case the virus breaks through the mucosal barrier. These findings are in agreement with a study in mice showing that intraperitoneal infection with lymphocytic choriomeningitis virus is superior to i.n. infection for the induction of CD8+
T-cell responses in the vaginal mucosa [26
]. Other reports reached similar conclusions upon vaccination with protein vaccines [27
], DNA vaccines [28
], or DNA vaccines combined with Modified Vaccinia Ankara and Semliki Forest vectors [29
]. Taken together, these results indicate that induction of CD8+
T-cell responses at mucosal sites upon i.m. immunization is independent of a given vaccine platform.
Antigen-experienced CD8+ T-cells may traffic to the GT with the help of specific sensors that remain to be identified, or alternatively this process may be random. To gain further insight into the vaccine-induced CD8+ T-cells that homed to the GT, we conducted a detailed phenotypical analysis of gag-specific CD8+ T-cells induced by the different immunization protocols, comparing cells isolated from spleen, blood, ILN and the GT at different times after immunization. In some assays we also tested cells isolated from NALT; the latter were tested for comparison as a population of cells homing to a distinct mucosal site. Phenotypes of gag-specific CD8+ T-cells isolated from systemic sites and the GT were phenotypically distinct, and this was especially pronounced at 1 year after the i.m./i.m. prime-boost vaccine regimen. The phenotypes suggest that most tet+CD8+ T-cells present in the GT remain fully activated and would be expected to start target cell lysis immediately upon encounter of infected cells.
We evaluated markers that are known to be up-regulated on cells derived from the intestinal mucosa. Studies have demonstrated high levels of CD69 expression on intestinal CD8+
], but expression of CD69 was not increased in the GT at any of the time points analyzed. Although α4
has been linked to the genital migration of subsets of CD4+
], and is a well-known marker for homing of T-cells to the intestinal mucosa, our results do not suggest that α4
affects homing of CD8+
T-cells to the GT. CD103 was slightly increased in tet+
T-cells from the GT at early time points, and by 1 year after immunization became strongly up-regulated. In the adoptive transfer experiment, CD103 was low on the gag-specific CD8+
T-cells isolated from the vaccinated donors and upon transfer remained low on cells isolated from all compartments but the GT, where an increase was observed. Again, these data argues against the notion that CD103 supports mucosal homing but rather suggest that CD103 may contribute to the retention of CD8+
T-cells within the GT. The adoptive transfer experiment also showed that gag-specific CD8+
T-cells from the spleen could readily migrate to the GT to a similar extend as observed in vaccinated mice. This argues against the need for a distinct differentiation pathway during activation to allow for migration of CD8+
T-cells to the mucosa, as had been described for T-cell homing to GALT [32
] or for CD4+
T-cells of the female GT [33
]. On the other hand, the observation that at 2 weeks upon i.m. immunization frequencies of Gag-specific CD8+ T-cells were ~10 fold higher in blood but only ~ 2 fold higher in the GT than upon i.n. immunization argues for a deliberate homing process that is dictated by the conditions under which the CD8+
T-cells are initially stimulated. We would therefore assume that migration of activated CD8+
T-cells to the GT is in part random and affected by their overall frequencies in blood, and in part driven by the expression of yet to be identified homing markers. In either case, we would assume that activated CD8+
T-cells receive signals from the microenvironment that favor their retention once they reach the GT leading to an enrichment of this cells at the mucosal surface, which is the port of entry for many pathogens.
The functionality of genital CD8+
T-cells remains to be investigated in more depth. Our data thus far show that T-cells from the GT produce IFN-γ but not IL-2 as has also been reported for genital T-cells in SIV-infected NHP [34
]. In our study, gag-specific CD8+
T-cells from the GT expressed high levels of granzyme B, perforin and Ki-67, which suggests that they are highly activated cells able to immediately commence target cell lysis and proliferation. Other authors have demonstrated atypical T-cells within mucosal surfaces [22
] and we speculate that the high levels of lytic enzymes seen in memory-type CD8+
T-cells from the GT could be a result of a specific microenvironment.
In summary, data presented here show that i.m. immunization with a replication defective AdC vector in mice induces a robust transgene product-specific CD8+ T-cell response within the GT that can be enhanced by a booster immunization given i.m. The response is sustained and can still be detected 1 year after immunization. Vaccine-induced genital CD8+ T-cells are functional, they carry lytic enzymes and release cytokines upon antigenic stimulation. Taken together, results shown should allow for guarded optimism that potent vaccines administered i.m. may induce a genital barrier to HIV-1 infection in women. In fact, systemic regimens would be preferable over mucosal ones in humans due to logistical factors and the lack of interference by flora or menstrual cycle, which may profoundly affect mucosal vaccine efficacy.