HPV16 synthetic long peptide (HPV16-SLP) vaccination therapy of patients with advanced or recurrent HPV16-induced gynecological carcinoma, a phase II trial.

BACKGROUND: Human papilloma virus type 16 (HPV16)-induced gynecological cancers, in particular cervical cancers, are found in many women worldwide. The HPV16 encoded oncoproteins E6 and E7 are tumor-specific targets for the adaptive immune system permitting the development of an HPV16-synthetic long peptide (SLP) vaccine with an excellent treatment profile in animal models. Here, we determined the toxicity, safety, immunogenicity and efficacy of the HPV16 SLP vaccine in patients with advanced or recurrent HPV16-induced gynecological carcinoma. METHODS: Patients with HPV16-positive advanced or recurrent gynecological carcinoma (n = 20) were subcutaneously vaccinated with an HPV16-SLP vaccine consisting of a mix of 13 HPV16 E6 and HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant. The primary endpoints were safety, toxicity and tumor regression as determined by RECIST. In addition, the vaccine-induced T-cell response was assessed by proliferation and associated cytokine production as well as IFNγ-ELISPOT. RESULTS: No systemic toxicity beyond CTCAE grade II was observed. In a few patients transient flu-like symptoms were observed. In 9 out of 16 tested patients vaccine-induced HPV16-specific proliferative responses were detected which were associated with the production of IFNγ, TNFα, IL-5 and/or IL-10. ELISPOT analysis revealed a vaccine-induced immune response in 11 of the 13 tested patients. The capacity to respond to the vaccine was positively correlated to the patient's immune status as reflected by their response to common recall antigens at the start of the trial. Median survival was 12.6 ± 9.1 months. No regression of tumors was observed among the 12 evaluable patients. Nineteen patients died of progressive disease. CONCLUSIONS: The HPV16-SLP vaccine was well tolerated and induced a broad IFNγ-associated T-cell response in patients with advanced or recurrent HPV16-induced gynecological carcinoma but neither induced tumor regression nor prevented progressive disease. We, therefore, plan to use this vaccine in combination with chemotherapy and immunomodulation.

Generating HPV specific T helper cells for the treatment of HPV induced malignancies using TCR gene transfer.

BACKGROUND: Infection with high risk Human Papilloma Virus (HPV) is associated with cancer of the cervix, vagina, penis, vulva, anus and some cases of head and neck carcinomas. The HPV derived oncoproteins E6 and E7 are constitutively expressed in tumor cells and therefore potential targets for T cell mediated adoptive immunotherapy. Effective immunotherapy is dependent on the presence of both CD4+ and CD8+ T cells. However, low precursor frequencies of HPV16 specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer purposes. An alternative to generate HPV specific CD4+ and CD8+ T cells is TCR gene transfer. METHODS: HPV specific CD4+ T cells were generated using either a MHC class I or MHC class II restricted TCR (from clones A9 and 24.101 respectively) directed against HPV16 antigens. Functional analysis was performed by interferon-γ secretion, proliferation and cytokine production assays. RESULTS: Introduction of HPV16 specific TCRs into blood derived CD4+ recipient T cells resulted in recognition of the relevant HPV16 epitope as determined by IFN-γ secretion. Importantly, we also show recognition of the endogenously processed and HLA-DP1 presented HPV16E6 epitope by 24.101 TCR transgenic CD4+ T cells and recognition of the HLA-A2 presented HPV16E7 epitope by A9 TCR transgenic CD4+ T cells. CONCLUSION: Our data indicate that TCR transfer is feasible as an alternative strategy to generate human HPV16 specific CD4+ T helper cells for the treatment of patients suffering from cervical cancer and other HPV16 induced malignancies.

Evaluation of immunological cross-reactivity between clade A9 high-risk human papillomavirus types on the basis of E6-Specific CD4+ memory T cell responses.

CD4(+) T cell responses against the E6 oncoprotein of human papillomavirus (HPV) type 16 and 5 closely related members of clade A9 (HPV31, 33, 35, 52, and 58) were charted in peripheral blood mononuclear cell cultures from healthy subjects and patients who underwent HPV16 E6/E7-specific vaccination. Initial analyses with overlapping peptide arrays showed that approximately one-half of the responding subjects displayed reactivity against corresponding E6 peptides from >or=2 HPV types. This suggested immunological cross-reactivity and complicated retrospective evaluation of the infection history of the healthy subjects. Importantly, further dissection of the response by means of enriched and clonal T cell cultures (with protein antigen instead of peptides) revealed that CD4(+) T cells that are capable of efficiently reacting against E6 antigen from multiple HPV types are rare and only occur when epitope sequences are highly conserved. Our data indicate that natural and vaccine-induced HPV16 E6-specific CD4(+) T cell responses are unlikely to mediate efficient cross-protection against other clade A9 members.

A prospective study on the natural course of low-grade squamous intraepithelial lesions and the presence of HPV16 E2-, E6- and E7-specific T-cell responses.

This study investigates the clinical course of low grade squamous intraepithelial lesions (LSIL), HPV status and HPV16-specific immune response in a large prospective study of 125 women with LSIL followed cytologically, virologically and histologically. Women with low-grade abnormal smears were recruited and followed-up for one year. Colposcopy, cervical biopsy for histology and brushings for HPV typing was performed at recruitment, 6 months (no biopsy) and upon completion of the study at one year. HPV16-specific T-cell responses were analysed by interferon-gamma ELISPOT at entry, 6 and 12 months. Infection with multiple HPV types was detected in 70% of all patients, HPV16 was found in 42% of the patients. LSIL lesions progressed to HSIL in 24%, persisted in 60% and regressed to normal in 16% of the patients. No difference was observed in the clearance rate of infections with single or multiple HPV types among the groups with a different histological outcome. HPV16-specific type 1 T-cell responses were detected in only half of the patients with an HPV16+ LSIL, and predominantly reactive to HPV16 E2 and E6. Interestingly, the presence of HPV16 E2-specific T-cell responses correlated with absence of progression of HPV16+ lesions (p = 0.005) while the detection of HPV16 E6 specific reactivity was associated with persistence (p = 0.05). This large prospective study showed that the majority of LSIL persisted or progressed within the first year.This was paralleled by immune failure as most of the patients with an HPV16+ LSIL failed to react to peptides of HPV16 E2, E6 or E7.

Skin reactions to human papillomavirus (HPV) 16 specific antigens intradermally injected in healthy subjects and patients with cervical neoplasia.

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n = 11) and healthy individuals (n = 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV16+ patients (<8 days) than in healthy subjects (8-25 days). The development of late skin reactions in healthy subjects was associated with the appearance of circulating HPV16-specific T cells and the infiltration of both HPV16-specific CD4+ Th1/Th2 and CD8+ T cells into the skin. These data show that the intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of circulating HPV16-specific T cells. The use of this test to measure HPV16-specific immunity is currently tested in a low resource setting for the measurement of spontaneously induced T-cell responses as well as in our HPV16 vaccination trials for the detection of vaccine-induced immunity.

Association of cervical cancer with the presence of CD4+ regulatory T cells specific for human papillomavirus antigens.

Because of their important role in the maintenance of self-tolerance, CD4(+) regulatory T cells prevent autoimmune diseases but also curtail the efficacy of T cell immune responses against cancers. We now show that this suppressive action of CD4(+) regulatory T cells is not limited to cancers displaying tumor-associated self antigens, such as melanomas, but also extends to human papillomavirus (HPV)-positive cervical cancers that express foreign tumor antigens. HPV-specific CD4(+) T cells isolated from lymph node biopsies of cervical cancer patients were found to suppress proliferation and cytokine (IFN-gamma, IL-2) production by responder T cells. The capacity of HPV-specific CD4(+) T cells to exert this suppressive effect depended on their activation by cognate HPV antigen and on close-range interactions with responder T cells. HPV-specific CD4(+) regulatory T cells were also retrieved from cervical cancer biopsies, suggesting that they interfere with the anti-tumor immune response at both the induction and effector levels. Our findings offer a plausible explanation for the observed failure of the tumor-specific immune response in patients with cervical carcinoma.