This study provides evidence that the female cervicovaginal mucosa can be an effective site for induction and boosting of effector memory CD8+
T cell responses using a non-replicating viral vector. Previous studies involving live viruses or attenuated replicating vectors left open the possibility that the presence of these cells in the cervicovaginal mucosa might have resulted from their immune induction at remote sites. Trafficking of the virus/vector and expression of the antigen in other tissues could lead to subsequent recruitment of immunocytes to the cervicovaginal mucosa. We have focused our studies on the induction of effector T cell responses, rather than antibodies, because the efficient induction of cervicovaginal mucosa T cells by a non-replicating vaccine had not previously been demonstrated. In contrast, it is now recognized, thanks to the remarkable efficacy of the commercial HPV prophylactic vaccines, that systemic immunization with a protein antigen, in this case a virus-like particle, can induce protective levels of antibodies in the female cervicovaginal mucosa (48
Antigen expression by the HPV PsV, which is transient because the infected keratinocytes are sloughed during the normal squamous differentiation process, is limited to “wounded” cervicovaginal keratinocytes because infection occurs in conjunction with epithelial disruption, which allows binding of the virus particle to the basement membrane before they infect the keratinocytes (34
). Therefore, the genetically delivered antigen is expressed in the proinflammatory and potentially immunogenic setting of disrupted epithelium rather than in an undisturbed tissue that is more likely to be tolerogenic (3
). Other characteristics of HPV PsV, such as the ability of the capsid to trigger an innate immune response, may also contribute to making them exceptionally potent inducers of genital CD8+
T cell responses (52
This is the first study to our knowledge to demonstrate the preferential induction of large numbers of intraepithelial CD103+CD8+ T cells (IELs) in the cervicovaginal mucosa after Ivag immunization with a non-replicating vector. Unfortunately, because of technical limitations inherent in the available KdM282-tetramers, we were unable to monitor in situ antigen-specific CD8+ T cells by M2-tetramer staining on tissue sections of the cervicovaginal mucosa. However, the paucity of preexisting CD8+ T cells in the cervicovaginal epithelium of young mice that have not been bred, and the fact that the exceptional increase in genital CD8+ T cells was attributable entirely to the expansion of KdM282-tetramer–positive CD8+ T cells as shown by flow cytometry, strongly imply that the intraepithelial CD8+ T cells induced after HPV PsV Ivag immunization are specific for the immunodominant epitope M282–90. The low number of cervicovaginal T cells prior to vaccination led us to focus on absolute numbers of T cells induced rather than on their relative percentage in the tissue, since the latter calculation can produce an overestimate of the response in tissues with low preexisting numbers of T cells.
Based on the large number of CD3+
T cells detected histologically in most tissue sections from vaccinated mice, it is our impression that the flow cytometry analysis substantially underestimates the true number of T cells induced in the cervicovaginal mucosa, which might result from the incomplete liberation of T cells from the tissue by our extraction procedures. In future studies, it will be important to determine whether higher numbers of preexisting IELs in the cervicovaginal epithelium inhibit induction of antigen-specific CD8+
T cells after Ivag HPV PsV vaccination and whether the pool of memory IEL CD8+
T cells can expand further, as previously reported for the pool of memory CD8+
T cells in PBMCs (54
Some vaccination strategies have sought to increase the strength and durability of effector memory CD8+
T cell responses by inducing persistent antigen expression with live virus vectors (12
). Our results demonstrate that even transient antigen expression can induce strong, durable responses. It is noteworthy that we did not observe an acute contraction phase in genital CD8+
T cells after Ivag vaccination. Our findings support accumulating evidence that intraepithelial T cells, especially effector memory cells, may in general be longer lived than systemic effector T cells, as befits their putative role as guardians of epithelial surfaces. It is unclear whether the marked acquisition of an effector memory phenotype observed in the genital CD8+
T cells over time is due to the evolution of the phenotype of individual cells or gradual replacement of effector with effector memory cells, for instance, by preferential survival or homeostatic proliferation.
The ability to preferentially induce large numbers of antigen-specific IELs by vaccination should facilitate future studies of this unique class of lymphocytes. Our characterization indicates that primary CD8+
T cells depend on CD4+
T cell help, which is consistent with their priming occurring in lymph nodes. However, the role of CD4+
T cells during at the booster immunization step remains to be assessed. The recruitment of circulating T cells to the genital mucosa has been previously described and requires CD4+
T cells as well as expression of CXCR3 by circulating memory CD8+
T cells (55
). Our FTY720 and BrdU experiments strongly implicate local mucosal proliferation during the secondary response to keratinocyte-expressed antigen, but it does not address the role of CD4+
T cells or whether inflammation-driven recruitment from the circulation was required. While this is, to our knowledge, the first evidence of this phenomenon in a mucosal tissue after vaccination, several recent studies have presented evidence that peripheral proliferation of primed CD8+
T cells in response to antigen can occur in other settings, for instance, in tumors or neuronal tissues (26
). In future studies, it will be important to determine whether the “wounded” keratinocytes, which can express costimulatory and MHC I molecules, can directly present antigen for in situ proliferation of CD8+
T cells or whether cross-presentation by professional APCs or membrane transfer from keratinocytes to DCs, referred to as cross-dressing, are involved in the recall of memory T cells in the cervicovaginal mucosa (57
). In addition, the trafficking patterns of IELs within the epithelium and between the epithelium and other tissues are poorly understood, and this system might allow for their study.
There are numerous reports of the induction of CD8+
T cell responses in the cervicovaginal mucosa after systemic vaccination with non-replicating vectors (7
), and we confirmed these observations using our Ad5-MM2 vector in an intramuscular prime/boost regimen. However, this regimen was qualitatively and quantitatively inferior to the Ivag prime/boost immunization regimen. In spite of the relatively high numbers of cervicovaginal Kd
T cells measured by flow cytometry, there was no increase in IEL number after systemic prime/boost immunization, which raises the possibility that the T cells extracted from the cervicovaginal tissues after the systemic immunization procedures may have largely been retained within the mucosal vasculature or were loosely associated with the submucosa. The unique ability of HPV PsV Ivag vaccination, compared with Ad5 i.m. vaccination, to confer enhanced protection against a local viral challenge is associated with the unique induction of CD8 IELs and their upregulation of CD103. Intraepithelial memory CD8+
T cells would be able to respond to infection more promptly than their circulating counterparts, but one could also speculate that the expression of CD103 at the surface of cervicovaginal IEL CD8+
T cells would contribute to enhancing protection, as this integrin has been shown to participate in the cytotoxic activity of human tumor-infiltrating CD8+
T cells (58
). Also, the fact that CD103 expression was restricted to Kd
T cells from the cervicovaginal mucosa and expressed up to 6 months after boost (our unpublished observations) suggests a role for the cervicovaginal microenvironment in CD103 induction. This conjecture is consistent with recent reports indicating that persistent tissue-resident memory T cells upregulate CD103 in peripheral tissues but not in other lymphoid organs (27
). Furthermore, the limited CD103 expression in the cervicovaginal CD8+
T cells induced after i.m. immunization may explain the absence of intraepithelial CD8+
T cells in i.m. immunized mice and suggests that de novo antigen expression in wounded keratinocytes is driving the upregulation of CD103.
The phenotypic and functional characterization of the genital CD8+ T cells induced after genital HPV PsV vaccination is consistent with their being functional effector memory cells with potential antiviral activity. First, most cells were polyfunctional, as they simultaneously secreted IFN-γ and TNF-α. Second, they exhibited in vivo cytotoxic activity against autologous cells pulsed with the immunodominant M282–90 peptide. Third, they were associated with a 1,000-fold reduction in the mean cervicovaginal mucosa viral load of recombinant VV expressing the M282–90 peptide after vaginal inoculation. It is noteworthy that this viral challenge model is rather stringent, since protection is entirely mediated by CD8+ T cells.
The ability to preferentially induce long-lived antigen-specific intraepithelial CD8+
T cells may be an attractive feature for a vaccine designed to prevent a viral infection that is initiated in the cervicovaginal mucosa such as HIV or HSV. Having sentinel T cells at the initial site of infection has been shown to lead to a more rapid response in the skin during HSV-1 infection and thereby control infection before it can spread systemically (57
). Indeed, recruitment of systemic T cells may take too long to effectively control the initial phase of infection, which is an important determinant of clinical outcome. Having local T cells can increase the local ratio of virus-specific CD8+
T cells to virus-infected cells and provide enhanced protection (29
). For application to HIV vaccines, it is noteworthy that our Ivag vaccination protocol did not increase the number of cervicovaginal mucosa CD4+
T helper cells, so it would not be expected to increase the pool of infection targets, which could thereby potentiate infection.
Transudated systemic antibodies and effector IELs ideally could act synergistically to prevent or control infection of the genital mucosa, and vaccines inducing both major limbs of the adaptive immune response should be considered. If the absence of genital IELs after systemic immunization of mice is predictive for humans, then the role of IELs should be critically evaluated in future HIV or HSV clinical trials of candidate vaccines (61
). Using HPV-encapsidated DNA or other viral vectors to locally induce effector CD8+
IELs should be a high-priority approach to evaluate difficult vaccine targets, such as HSV or HIV.
Vaccines that induce cervicovaginal mucosa IELs might also be considered for therapeutic vaccines, particularly against HSV herpetic lesions and HPV-induced intraepithelial neoplasia. Abatement of recurrent genital herpes lesions was recently correlated with the appearance of CD8+
T cells located between the nerve endings and the adjacent epithelial cells at the site of previous herpetic lesions (63
). Generation of HSV-specific effector memory T cells at these sites by vaccination might have a similar effect. A marked recruitment of CD8+
T cells into the epithelium of HPV-induced cervical intraepithelial neoplasia was observed in lesions that subsequently regressed (30
). In contrast, a T cell infiltrate in the submucosa, along with its exclusion from the overlying dysplastic epithelium, was observed in lesions that persisted. Induction of HPV-specific IELs by vaccination might overcome this apparent barrier to clearance.
Finally, the feasibility of translating our vaccination strategy to women should be considered. We have recently shown that our HPV PsV–based vaccination strategy is not restricted to mice, with studies in non-human primates demonstrating efficient gene delivery and immunogenicity against SIV antigens in Depo-Provera–treated monkeys (35
). The mice in this study were similarly progesterone treated to ensure that they had uniformly thin cervicovaginal epithelium and so relatively consistent vector infection. However, this treatment might be more critical in mouse studies than in human applications for two reasons. First, the thickness of the cervicovaginal epithelium appears to be more hormonally dependent in mice, with estrogen-dominated mice exhibiting highly keratinized squamous epithelium. The thickness of the squamous epithelium varies less across the menstrual cycle in women. Second, the simple columnar epithelium of the endocervix begins rather high up the endocervical channel in mice and is not readily exposed to N-9 and PsV unless the cervix is cannulated (34
). In contrast, the endocervical epithelium is exposed in women, particularly in young women. It might be predicted that the simple columnar epithelium of the endocervix would be exceptionally susceptible to N-9 disruption and PsV infection, and this susceptibility to infection would not be expected to vary substantially across the menstrual cycle. It will be important to further determine in non-human primate models whether IELs can similarly be induced in the simple columnar epithelium of the endocervix throughout the menstrual cycle, particularly since the endocervix and/or the transformation zone between endo- and ectocervix may be especially susceptible to viral transmission.
The requirement for epithelial permeabilization or disruption in conjunction with vaccination might also raise safety concerns. For this reason, we purposely chose to use a disrupting agent that is an over-the-counter product containing N-9 (Conceptrol) that is approved for unregulated Ivag application in women. While N-9–containing spermicides used repeatedly may increase susceptibility to some sexually transmitted infections, these agents could be acceptable for a limited number of applications as part of a vaccination protocol. Although the use of N-9–containing products was associated with an increased risk of HIV transmission (64
), the epithelial disruption they induce is transient, and refraining from unprotected sexual activity for several days after their use as part of a vaccination regimen would likely mitigate this risk. Finally, the experience from clinical trials of topical microbicides against STDs provides a rational basis for developing a relatively simple gel-based vaccine for topical application.