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.

Purpose: The study aims to evaluate the effect of a human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides vaccine on the antigen-specificT-cell response in cervical cancer patients. Experimental Design: Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51. HPV16-specificT-cell immune responses were analyzed by evaluating the magnitude, breadth, type, and polarization by proliferation assays, IFNg-ELISPOT, and cytokine production and phenotyped by theT-cell markers CD4, CD8, CD25, and Foxp3. Results: Vaccine-induced T-cell responses against HPV16 E6 and E7 were detected in six of six and five of six patients, respectively.These responses were broad, involved both CD4 + and CD8 Foxp3+ phenotype. Conclusions: The HPV16 E6 and E7 synthetic long peptides vaccine is highly immunogenic, in that it increases the number and activity of HPV16-specific CD4 + and CD8 + Tcells to a broad array of epitopes in all patients. The expansion of CD4 + and CD8 + tumor-specific T cells, both considered to be important in the antitumor response, indicates the immunotherapeutic potential of this vaccine. Notably, part of the vaccine-inducedTcells display a CD4 + CD25 + Foxp3+ phenotype that is frequently associated with regulatory T-cell function, suggesting that strategies to disarm this subset of Tcells should be considered as components of immunotherapeutic modalities against HPV-induced cancers.Cervical cancer is the second most common cancer in women worldwide (1), and it is the first cancer recognized by the WHO to be 100% attributable to the infection with a high-risk type of human papillomavirus (HPV; ref. 2). HPV type 16 (HPV16) is the most common carcinogenic type and is found in around 50% of invasive cervical tumors worldwide (3, 4).

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 IFNγ (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 IFNγ/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.human papilloma virus | immunomonitoring | therapeutic vaccine | regulatory T cells

Purpose: To determine the toxicity, safety, and immunogenicity of a human papillomavirus 16 (HPV16) E6 and E7 long peptide vaccine administered to end-stage cervical cancer patients. Experimental Design:Three groups of end-stage cervical cancer patients (in total n = 35) were s.c. vaccinated with HPV16 E6 combined with or separated from HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant, four times at 3-week intervals. Group 1 received 300 Ag/ peptide at a single site and group 2 received 100 Ag/peptide of the E6 peptides in one limb and 300 Ag/peptide of the E7 peptides in a second limb. Group 3 received separate injections of E6 and E7 peptides, each at a dose of 50 Ag/peptide. The primary end point was to determine safety and toxicity of the HPV16 long peptides vaccine. In addition, the vaccine-induced T-cell response was assessed by IFNg enzyme-linked immunospot. Results: No toxicity beyond grade 2 was observed during and after four vaccinations. In a few patients, transient flu-like symptoms were observed. Enzyme-linked immunospot analysis of the vaccine-induced immune response revealed that coinjection of the E6 and E7 peptides resulted in a strong and broad T-cell response dominated by immunity against E6. Injection of the E6 and E7 peptides at two different sites increased the E7 response but did not affect the magnitude of the E6-induced immune response. Conclusions: The HPV16 E6 and E7 long peptide-based vaccine is well tolerated and capable of inducing a broad IFNg-associated T-cell response even in end-stage cervical cancer patients.Close to 100% cervical cancers are caused by persistent infection with high-risk human papillomaviruses (HPV; ref. 1). Of the different high-risk HPV types that can cause cervical cancer (2), HPV16 alone is responsible worldwide for more than half of all cases of cervical cancer (1, 2). Genital infection with high-risk HPV is very common and normally cleared within 1 year. However, in a minority (f1%) of the infected individuals, the HPV persists, ultimately resulting in genital neoplastic lesions.Currently, a preventive HPV vaccine is on the market, which effectively prevents this persistent infection and associated disease by the induction of neutralizing antibodies against the envelope proteins of HPV16 and HPV18, the HPV type second on the list of most frequent HPV types associated with cancer. Although these vaccines are very efficient in the prevention of persistent infection by HPV16 and HPV18 (3 -8) and have potential for prevention of cervical intraepithelial neoplasia (9 -11), there is no evidence for efficacy against established .It is generally accepted that virus-infected cells can only be effectively dealt with by cell-mediated adaptive T-cell immunity. Consequently, immunotherapy capable of inducing robust HPV16-specific T-cell immunity is highly desirable for eradication of established HPV16 infection and diseases caused thereby, such as cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, cervical cancer, other anoge...

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 vaccineinduced p53-specificTcells was evaluated in blood samples drawn before and after vaccination by IFN-g enzyme-linked immunospot, proliferation, cytokine secretion, and multiparameter flow cytometry. The migratory capacity of p53-specificT 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-g enzymelinked 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 bothT-helper types 1and 2 cytokines on stimulation with p53 peptide and p53 protein. Multiparameter flow cytometry revealed that only a minor population of the p53-specific CD4 + Tcells was optimally polarized. Conclusions: The p53-SLP vaccine is safe and capable to induce p53-specificT-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.

BackgroundHuman 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.MethodsPatients 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.ResultsNo 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.ConclusionsThe 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.

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 þ T-cell reactivity. Here, we studied if imiquimod applied at the vaccine site could improve Results: Forty-three patients were assigned to either ISA101 with imiquimod (n ¼ 21) or ISA101 only (n ¼ 22). Imiquimod did not improve the outcomes of vaccination. However, vaccineinduced 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.

A series of glycolipids have been prepared which contain a cluster galactoside moiety with high affinity for the hepatic asialoglycoprotein receptor and a bile acid ester moiety which mediates stable incorporation into liposomes. Loading of liposomes with these glycolipids at a ratio of 5% (w/w) resulted in efficient recognition and uptake of the liposomes by the liver. Preinjection with asialofetuin almost completely inhibited the uptake, establishing that the liposomes were selectively recognized and processed by the asialoglycoprotein receptor on liver parenchymal cells. In contrast, a glycolipid content of 50% (w/w) led to a liver uptake that could not be inhibited by preinjection with asialofetuin, indicating that the liposomes were now processed by the Gal/Fuc-recognizing receptor on liver macrophages. The results presented in this study are important for future targeting of water-soluble and amphiphilic drugs, enveloped in these glycolipid-laden liposomes, to parenchymal liver cells.