Fusion of a tumour-associated antigen to HIV-1 Tat improves protein-based immunotherapy of cancer.

BACKGROUND: The ultimate success of cancer vaccination is primarily dependent upon the generation of tumour-specific CTLs. Protein-based vaccination, while safe, poorly elicits such CTL responses. As fusion of an antigen to the HIV-1 Tat transduction domain was reported to increase MHC class I presentation and CTL responses in vitro, we tested the potency of this approach to augment tumour-directed responses. MATERIALS AND METHODS: Human papillomavirus type 16 (HPV16) E7 proteins, fused (E7-Tat) or not (E7) to Tat carboxy-terminal region, were produced and studied in vitro and in vivo. RESULTS: E7-Tat, not E7, penetrated the cell membrane and was transcriptionally active. In vitro, E7-Tat induced higher IFN-gamma production from E7-specific T-cells than E7. In C57BL/6 mice, E7-Tat mixed with Quil A generated enhanced prophylactic and therapeutic suppression of HPV16-positive C3 tumour outgrowth. Similar, but greatly enhanced E7-specific effector and helper T-cell responses were elicited following E7-Tat/Quil A rather than E7/Quil A vaccination. CONCLUSION: This study offers a new strategy for improving subunit cancer vaccines.

Pre-clinical immunogenicity and anti-tumour efficacy of a deleted recombinant human papillomavirus type 16 E7 protein.

BACKGROUND: Current vaccination strategies against Human papillomavirus (HPV)-induced ano-genital cancers mostly target E7 from HPV16. However, the oncogenic nature of E7 raises potential human safety issues. Although the modifications abrogating the E7 transforming potential have been well characterized, their effect on E7 immunogenicity has been poorly studied. In this study, we evaluated the vaccine potential of an HPV16 E7 protein deleted from the entire pRb-binding motif. MATERIALS AND METHODS: Purified recombinant deleted (E7delta21-26) and wild-type (His6-E7 and E7WT) E7 proteins were studied in pre-clinical mice models. RESULTS: In C57BL/6 mice, E7delta21-26 formulated with the Quil A adjuvant generated systemic E7-specific cytotoxic T-cell and antibody responses similar to those induced following His6-E7/Quil A and E7WT/Quil A vaccinations. E7delta21-26/Quil A injections efficiently protected animals from challenge with the HPV16-expressing tumours, C3 and TC-1. Moreover, therapeutic vaccination with adjuvant-modified E7 suppressed or significantly decreased C3 tumour outgrowth. CONCLUSION: E7delta21-26 could represent a safe and efficient vaccine candidate against E7-containing tumour cells.

DNA vaccines against HPV-16 E7-expressing tumour cells.

BACKGROUND: Genetic immunisation induces the endogenous production of the encoded antigens, which favours their presentation by MHC class I molecules. The E7 protein from "high risk" Human Papillomavirus (HPV) is constitutively expressed in cervical cancer and represents a target for immunotherapy. MATERIALS AND METHODS: Several E7-encoding DNA vaccines were constructed including unmodified E7 and E7 fused to ubiquitin or to the Invariant chain in order to increase the presentation of E7-derived peptides by MHC class I or II molecules, respectively. These vaccines were administered i.m. to C57BL/6 mice that were subsequently challenged with E7-positive tumour cell lines expressing different levels of MHC class I molecules. RESULTS: The E7-Ii fusion sequence protected a number of animals from tumour challenging. No differences were associated with the MHC class I status of the challenging cell lines. CONCLUSION: Engineering the intracellular pathway for antigen presentation is able to produce a valid therapeutic response even against tumours with down-regulated MHC class I.

DNA vaccine encoding endosome-targeted human papillomavirus type 16 E7 protein generates CD4+ T cell-dependent protection.

Human papillomavirus type 16 is commonly implicated in cervical cancers. The viral genome encodes potential targets like the oncoprotein E7, expressed in transformed cells but thought to represent a poorly immunogenic antigen. We describe in this work a DNA-based vaccination protocol aimed at inducing an efficient anti-E7 immune response in vivo. Plasmids allowing the expression of the E7 protein in distinct cellular compartments were generated and assayed in an in vivo model of tumor growth. Our data demonstrate that mice vaccinated with a plasmid encoding for an E7 protein fused to a domain of the MHC class II-associated invariant chain (IiE7) were protected against tumor challenge. Mice immunized against an ubiquitinated form of E7 (Ub(Ala)E7) failed to control tumor growth. Protection induced by IiE7 was correlated with the development of CD8+ CTL and required the presence of CD4+ cells. In vitro studies confirmed that the IiE7 fusion protein was expressed at high levels in the endosomal compartment of transfected cells, while the natural and the ubiquitin-modified form of E7 were mainly nuclear. The present study suggests that an efficient anti-tumor response can be induced in vivo by DNA constructs encoding for E7 protein forms localizing at the endosomal compartment.

Phase I/II trial of immunogenicity of a human papillomavirus (HPV) type 16 E7 protein-based vaccine in women with oncogenic HPV-positive cervical intraepithelial neoplasia.

PURPOSE: Infection with oncogenic human papillomavirus (HPV) and HPV-16 in particular is a leading cause of anogenital neoplasia. High-grade intraepithelial lesions require treatment because of their potential to progress to invasive cancer. Numerous preclinical studies have demonstrated the therapeutic potential of E7-directed vaccination strategies in mice tumour models. In the present study, we tested the immunogenicity of a fusion protein (PD-E7) comprising a mutated HPV-16 E7 linked to the first 108 amino acids of Haemophilus influenzae protein D, formulated in the GlaxoSmithKline Biologicals adjuvant AS02B, in patients bearing oncogenic HPV-positive cervical intraepithelial neoplasia (CIN). METHODS: Seven patients, five with a CIN3 and two with a CIN1, received three intramuscular injections of adjuvanted PD-E7 at 2-week intervals. Three additional CIN1 patients received a placebo. CIN3 patients underwent conization 8 weeks postvaccination. Cytokine flow cytometry and ELISA were used to monitor antigen-specific cellular and antibody responses from blood taken before and after vaccine or placebo injection. RESULTS: Some patients had preexisting systemic IFN-gamma CD4+ (1/10) and CD8+ (5/10) responses to PD-E7. Vaccination, not placebo injection, elicited systemic specific immune responses in the majority of the patients. Five vaccinated patients (71%) showed significantly increased IFN-gamma CD8+ cell responses upon PD-E7 stimulation. Two responding patients generated long-term T-cell immunity toward the vaccine antigen and E7 as well as a weak H. influenzae protein D (PD)-directed CD4+ response. All the vaccinated patients, but not the placebo, made significant E7- and PD-specific IgG. CONCLUSIONS: The encouraging results obtained from this study performed on a limited number of subjects justify further analysis of the efficacy of the PD-E7/AS02B vaccine in CIN patients.