In the United States, CIN2/3 is common, with an estimated age-adjusted incidence ranging from 30-60 per 100,000 women.(6
) If left undetected and/or untreated, a subset of CIN2/3 lesions will progress to invasive squamous cell carcinomas. Conversely, complete histologic regression does occur spontaneously in up to 25% of HPV16-associated high grade dysplasia.(1
) Since conventional histopathological assessment of biopsy tissue at the time of diagnosis does not predict regression, all CIN2/3 lesions are treated by either surgical resection or ablation. Surgical resection of cervical dysplasias has been associated with subsequent risk of preterm delivery in several large studies.(8
) The development of immunotherapeutic strategies would prevent morbidity even in high-resource settings. Moreover, because a subset of CIN2/3 lesions do indeed regress, this patient population is a potentially informative cohort in which to identify immune correlates of lesion regression versus persistence.
This single-plasmid HPV16 DNA vaccine construct was well tolerated in healthy patients with biopsy-confirmed CIN2/3 associated with HPV16. In addition, three of nine patients in the highest dose cohort had complete histologic regression of established CIN2/3 in the study window. Using an overnight Elispot assay, no patients had significant changes in recognition of E7 in the initial 19 weeks. However, we did detect new responses in 5/9(55.6%) of subjects in the highest dose cohort. Using an assay which included a cycle of in vitro stimulation, T cell responses to E7 were increased subsequent to vaccination in 8/15 patients. However, overall, HPV-specific T-cell responses were not of greater magnitude than those we have identified in unvaccinated subjects with HPV16+ CIN2/3. Vaccination was not associated with changes in serum IgG antibody titers to the vaccine antigen, within the study window.
The long interval before detection of responses was unexpected. However, maturing clinical data from other investigators have also identified new responses to DNA vaccine antigens after a protracted interval.(11
) Other investigators assessing IFNγ responses to heterologous DNA prime-viral vector-based boost vaccination regimens have also uncovered different kinetics of response to vaccine antigens in direct ex vivo
assays compared to cultured assays of patient-matched specimens.(12
) Responses identified using cultured assays correlated with long-lasting T cell responses. The identification of the kinetics of response to vaccination will have obvious implication in the design of boosting strategies in humans.
Many investigators have demonstrated the safety, tolerability, and feasibility of DNA vaccination, in cohorts encompassing a spectrum of health, from healthy naïve cohorts to patients with end-stage disease. While DNA vaccine constructs are relatively simple to engineer and to produce, in humans, the potency of DNA vaccination alone is limited. In fact, the immunogenicity of the DNA vaccines in humans becomes obvious only after heterologous boosting. Clinical experience with DNA vaccination using other antigenic targets, notably malaria and HIV, have uncovered greater efficacy of priming when DNA vaccination is used as a priming vaccination in a heterologous prime-boost regimen, compared to homologous vaccination strategies. (13
) In our preclinical model, heterologous E7-targeted vaccination using DNA priming and vaccinia-based boosting is considerably more immunogenic than homologous vaccination with either DNA DNA or vaccinia-vaccinia, or vaccinia-DNA regimens.(18
) Because our target patient population is essentially healthy, our first trial needed to demonstrate safety of the vaccine construct used alone. It does appear possible to elicit measureable changes in HPV-specific T cell responses in patients with established preinvasive HPV disease.
Pre-existing adaptive immune responses to E6, presumably reflecting previous exposure and immune recognition, did not appear to be associated with ability to respond to vaccination with this E7-targeted construct. In this cohort, we identified endogenous responses to E6 more frequently than responses to E7, and were overall, of greater magnitude. Several lines of evidence suggest that the E6 oncoprotein may well be more immunogenic than E7. While functional T cell recognition of both antigens have been measured in peripheral blood in unvaccinated clinical cohorts, memory CD4+ T cell responses against HPV16 E6 are found frequently in healthy subjects without disease,(19
) and conversely, the absence of CD4 recognition of E6 correlates with higher incidence of disease. (21
) In a recently published phase I trial assessing vaccination with E6 and E7, long peptides in subjects with late-stage cervical cancer, subjects who underwent vaccination with both E6 and E7 in the same limb displayed dominant responses to E6; vaccination with E6 peptides and E7 peptides targeting separate sets of draining lymph nodes, in contrast, increased the magnitude of response to E7, and did not affect the magnitude of responses to E6.(22
) Therefore, in our next clinical trial, we will assess the effect of heterologous, viral-based boost vaccination on immunogenicity, and will also include E6 as an immunotherapeutic target.
Finally, another, plausible reason that HPV-specific immune responses measured in the peripheral blood do not correlate completely with regression of CIN2/3 is that relevant immune variables are likely to be compartmentalized at the site of the lesion. Since high grade dysplastic lesions develop only in the clinical setting of a chronic, mucosally-sequestered viral infection in the context of many co-existing commensal organisms, the immunologic ‘default’ set-point is likely one of localized immune ‘privilege’, i.e., one in which immunogenicity is suppressed.(23
) Although localized immune suppression is widely observed in the clinical setting of many frankly invasive diseases, it is likely to play a role well before transition to malignancy in the case of HPV-associated dysplasia. Indeed, Frazer et al indicate a critical role for the generation of local lesional inflammation for effective tumor immunotherapy.(24
) In an animal model that used HPV16 E6 and E7 as nominal antigens, even in the face of circulating antigen-specific effector T cells, local acute inflammation was necessary to allow antigen-specific effector T cells to eliminate epithelium expressing these antigens, even in primed hosts. Early clinical data suggests an increased likelihood of response to local immune modulation in the presence of pre-existing measureable HPV-specific T cell responses(25
) Based on these data, our next clinical trial of sequential heterologous vaccination will also assess the effect of topical imiquimod on access to lesions.
Statement of Clinical Relevance
Human papillomavirus (HPV) causes 10% of malignancies in women. Despite the availability of prophylactic vaccines, because barriers to obtaining prophylaxis are significant, the need to pursue therapeutic strategies remains real. Because the viral oncoproteins (E6 and E7) are functionally required for disease initiation and persistence, they present compelling immunotherapeutic targets.
In the US, high grade cervical dysplasia, the immediate precursor to cervical cancer is common. We developed an HPV16 E7 targeted vaccine which had considerable therapeutic effect in the preclinical TC-1 model. Here we report clinical, safety, and immunologic outcomes of a single-institution, investigator-initiated phase I dose-escalation trial in women with HPV16+ high grade dysplasia.