The long-term immune response after HPV16 peptide vaccination in women with low-grade pre-malignant disorders of the uterine cervix: a placebo-controlled phase II study.

The capacity of a low-dose HPV16 synthetic long-peptide vaccine (HPV16-SLP) to induce an HPV16-specific T-cell response as well as to establish long-term immunologic memory in patients with low-grade abnormalities of the cervix was determined in a placebo-controlled, double-blinded phase II study. In addition, the effect of a booster vaccination after 1 year was evaluated. Patients received either the HPV16-SLP or a placebo at the start of the study. After 1 year, the vaccinated patients were again randomized to receive the HPV16-SLP or a placebo. Patients were followed for 2 years. HPV16-specific T-cell responses were determined in pre- and post-vaccination blood samples by ELISPOT, proliferation assay and cytokine assays. We show that the HPV16-specific T-cell responses detected after vaccination are clearly due to vaccination and that reactivity was maintained for at least 2 years. Interestingly, a booster vaccination after 1 year especially augmented the HPV16-specific Th2 response. Furthermore, pre-existing immunity to HPV16 was associated with a stronger response to vaccination and with more side effects, reflected by flu-like symptoms. We conclude that two low-dose injections of HPV16-SLP can induce a strong and stable HPV16-specific T-cell response that lasts for at least 1 year. If booster vaccination is required, then polarizing adjuvant should be added to maintain the Th1 focus of the vaccine-induced T-cell response.

Addition of interferon-α to the p53-SLP® vaccine results in increased production of interferon-γ in vaccinated colorectal cancer patients: a phase I/II clinical trial.

We previously established safety and immunogenicity of a p53 synthetic long peptides (p53-SLP®) vaccine. In the current trial, we investigated whether combination of interferon-alpha (IFN-α) with p53-SLP® is both safe and able to improve the induced p53-specific IFN-γ response. Eleven colorectal cancer patients successfully treated for metastatic disease were enrolled in this study. Of these, nine patients completed follow-up after two injections with p53-SLP® together with IFN-α. Safety and p53-specific immune responses were determined before and after vaccination. Furthermore, cryopreserved PBMCs were compared head-to-head to cryopreserved PBMCs obtained in our previous trial with p53-SLP® only. Toxicity of p53-SLP® vaccination in combination with IFN-α was limited to Grade 1 or 2, with predominantly small ongoing swellings at the vaccination site. All patients harbored p53-specific T cells after vaccination and most patients showed p53-specific antibodies. Compared to the previous trial, addition of IFN-α significantly improved the frequency of p53-specific T cells in IFN-γ ELISPOT. Moreover, in this trial, p53-specific T cells were detectable in blood samples of all patients in a direct ex vivo multiparameter flowcytometric assay, opposed to only 2 of 10 patients vaccinated with p53-SLP® only. Finally, patients in this trial displayed a broader p53-specific immunoglobulin-G response, indicating an overall better p53-specific T-helper response. Our study shows that p53-SLP® vaccination combined with IFN-α injection is safe and capable of inducing p53-specific immunity. When compared to a similar trial with p53-SLP® vaccination alone the combination was found to induce significantly more IFN-γ producing p53-specific T cells.

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.

Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses.

One half of a group of 20 patients with human papillomavirus type 16 (HPV16)-induced vulvar intraepithelial neoplasia grade 3 displayed a complete regression (CR) after therapeutic vaccination with HPV16 E6/E7 synthetic long peptides. Patients with relatively larger lesions generally did not display a CR. To investigate immune correlates of treatment failure, patients were grouped according to median lesion size at study entry, and HPV16-specific immunity was analyzed at different time points by complementary immunological assays. The group of patients with smaller lesions displayed stronger and broader vaccine-prompted HPV16-specific proliferative responses with higher IFNgamma (P = 0.0003) and IL-5 (P < 0.0001) levels than patients with large lesions. Characteristically, this response was accompanied by a distinct peak in cytokine levels after the first vaccination. In contrast, the patient group with larger lesions mounted higher frequencies of HPV16-specific CD4(+)CD25(+)Foxp3(+) T cells (P = 0.005) and displayed a lower HPV16-specific IFNgamma/IL-10 ratio after vaccination (P < 0.01). No disparity in T memory immunity to control antigens was found, indicating that the differences in HPV-specific immunity did not reflect general immune failure. We observed a strong correlation between a defined set of vaccine-prompted specific immune responses and the clinical efficacy of therapeutic vaccination. Notably, a high ratio of HPV16-specific vaccine-prompted effector T cells to HPV16-specific CD4(+)CD25(+)Foxp3(+) T cells was predictive of clinical success. Foxp3(+) T cells have been associated previously with impaired immunity in malignancies. Here we demonstrate that the vaccine-prompted level of this population is associated with early treatment failure.

Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia.

BACKGROUND: Vulvar intraepithelial neoplasia is a chronic disorder caused by high-risk types of human papillomavirus (HPV), most commonly HPV type 16 (HPV-16). Spontaneous regression occurs in less than 1.5% of patients, and the rate of recurrence after treatment is high. METHODS: We investigated the immunogenicity and efficacy of a synthetic long-peptide vaccine in women with HPV-16-positive, high-grade vulvar intraepithelial neoplasia. Twenty women with HPV-16-positive, grade 3 vulvar intraepithelial neoplasia were vaccinated three or four times with a mix of long peptides from the HPV-16 viral oncoproteins E6 and E7 in incomplete Freund's adjuvant. The end points were clinical and HPV-16-specific T-cell responses. RESULTS: The most common adverse events were local swelling in 100% of the patients and fever in 64% of the patients; none of these events exceeded grade 2 of the Common Terminology Criteria for Adverse Events of the National Cancer Institute. At 3 months after the last vaccination, 12 of 20 patients (60%; 95% confidence interval [CI], 36 to 81) had clinical responses and reported relief of symptoms. Five women had complete regression of the lesions, and HPV-16 was no longer detectable in four of them. At 12 months of follow-up, 15 of 19 patients had clinical responses (79%; 95% CI, 54 to 94), with a complete response in 9 of 19 patients (47%; 95% CI, 24 to 71). The complete-response rate was maintained at 24 months of follow-up. All patients had vaccine-induced T-cell responses, and post hoc analyses suggested that patients with a complete response at 3 months had a significantly stronger interferon-gamma-associated proliferative CD4+ T-cell response and a broad response of CD8+ interferon-gamma T cells than did patients without a complete response. CONCLUSIONS: Clinical responses in women with HPV-16-positive, grade 3 vulvar intraepithelial neoplasia can be achieved by vaccination with a synthetic long-peptide vaccine against the HPV-16 oncoproteins E6 and E7. Complete responses appear to be correlated with induction of HPV-16-specific immunity.

A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions.

The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping long-peptide vaccine to stimulate the HPV16-specific T-cell response, to enhance the infiltration of HPV16-specific type 1 T cells into the lesions of patients with HPV16+ high-grade cervical squamous intraepithelial lesion (HSIL) and HPV clearance. This was a placebo-controlled randomized phase II study in patients with HPV16-positive HSIL. HPV16-specific T-cell responses were determined pre- and post-vaccination by ELISPOT, proliferation assay and cytokine assays in PBMC and HSIL-infiltrating lymphocytes, and delayed-type hypersensitivity skin tests. Motivational problems of this patient group to postpone treatment of their premalignant lesions affected the inclusion rates and caused the study to stop prematurely. Of the accrued patients, 4 received a placebo and 5 received 1-2 vaccinations. Side effects mainly were flu-like symptoms and injection site reactions. A strong HPV-specific IFNγ-associated T-cell response was detected by ELISPOT in all vaccinated patients. The outcome of the skin tests correlated well with the ELISPOT analysis. The cytokine profile associated with HPV16-specific proliferation varied from robust type 1 to dominant type 2 responses. No conclusions could be drawn on vaccine-enhanced T-cell infiltration of the lesion, and there was no HPV clearance at the time of LEEP excision. Thus, vaccination of HSIL patients results in increased HPV16-specific T-cell immunity. Further development of this type of treatment relies on the ability to motivate patients and in the reduction in the side effects.

Induction of p53-specific immunity by a p53 synthetic long peptide vaccine in patients treated for metastatic colorectal cancer.

PURPOSE: The tumor-associated self-antigen p53 is commonly overexpressed in cancer, including colorectal cancer, and can serve as a target for immunotherapy. The safety and immunogenicity of a p53 synthetic long peptide (p53-SLP) vaccine were investigated in patients treated for metastatic colorectal cancer. EXPERIMENTAL DESIGN: Ten patients were vaccinated twice with a set of 10 overlapping p53-SLP in a phase I/II trial. Both the safety and the breadth, magnitude, and polarization of vaccine-induced p53-specific T cells was evaluated in blood samples drawn before and after vaccination by IFN-gamma enzyme-linked immunospot, proliferation, cytokine secretion, and multiparameter flow cytometry. The migratory capacity of p53-specific T cells was evaluated by assessing their presence in a biopsy of the second vaccination site. RESULTS: Toxicity was limited to grade 1/2, mostly at the vaccination site. p53-specific T-cell responses were induced in 9 of 10 colorectal cancer patients as measured by IFN-gamma enzyme-linked immunospot, proliferation, and cytokine bead array. In 6 of 9 tested patients, p53-specific T-cell reactivity persisted at least 6 months. Furthermore, p53-specific T cells isolated from the vaccination site were characterized as CD4+ T cells producing both T-helper types 1 and 2 cytokines on stimulation with p53 peptide and p53 protein. Multiparameter flow cytometry revealed that only a minor population of the p53-specific CD4+ T cells was optimally polarized. CONCLUSIONS: The p53-SLP vaccine is safe and capable to induce p53-specific T-cell responses in patients treated for colorectal cancer. New trials should focus on improving the polarization of the p53-SLP vaccine-induced T-cell response.

Vaccination against Oncoproteins of HPV16 for Noninvasive Vulvar/Vaginal Lesions: Lesion Clearance Is Related to the Strength of the T-Cell Response.

PURPOSE: Therapeutic vaccination with human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides (SLP) is effective against HPV16-induced high-grade vulvar and vaginal intraepithelial neoplasia (VIN/VaIN). However, clinical nonresponders displayed weak CD8(+) T-cell reactivity. Here, we studied if imiquimod applied at the vaccine site could improve CD8(+) T-cell reactivity, clinical efficacy, and safety of HPV16-SLP (ISA101). EXPERIMENTAL DESIGN: A multicenter open-label, randomized controlled trial was conducted in patients with HPV16(+) high-grade VIN/VaIN. Patients received ISA101 vaccination with or without application of 5% imiquimod at the vaccine site. The primary objective was the induction of a directly ex vivo detectable HPV16-specific CD8(+) T-cell response. The secondary objectives were clinical responses (lesion size, histology, and virology) and their relation with the strength of vaccination-induced immune responses. RESULTS: Forty-three patients were assigned to either ISA101 with imiquimod (n = 21) or ISA101 only (n = 22). Imiquimod did not improve the outcomes of vaccination. However, vaccine-induced clinical responses were observed in 18 of 34 (53%; 95% CI, 35.1-70.2) patients at 3 months and in 15 of 29 (52%; 95% CI, 32.5-70.6) patients, 8 of whom displayed a complete histologic response, at 12 months after the last vaccination. All patients displayed vaccine-induced T-cell responses, which were significantly stronger in patients with complete responses. Importantly, viral clearance occurred in all but one of the patients with complete histologic clearance. CONCLUSIONS: This new study confirms that clinical efficacy of ISA101 vaccination is related to the strength of vaccine-induced HPV16-specific T-cell immunity and is an effective therapy for HPV16-induced high-grade VIN/VaIN. Clin Cancer Res; 22(10); 2342-50. ©2016 AACRSee related commentary by Karaki et al., p. 2317.