Recombinant Adeno-Associated Virus Expressing Human Papillomavirus Type 16 E7 Peptide DNA Fused with Heat Shock Protein DNA as a Potential Vaccine for Cervical Cancer

Abstract

In this study, we explore a potential vaccine for human papillomavirus (HPV)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus ( Cervical carcinoma remains one of the most common malignancies worldwide, with 500,000 new cases diagnosed each year (5) and 200,000 cervical cancer deaths annually (21). Human papillomavirus type 16 (HPV-16) is the predominant etiologic agent of cervical cancer and carries three transforming oncogenes, E5, E6, and E7 (10,20,45). Their products are thus unique tumor antigens and can be ideally used as tumor vaccines (7). Because E6 and E7 oncoproteins are consistently retained and expressed, the E6 and E7 oncogenes become more attractive targets for T-cell-based immunotherapy of cervical cancer. Evidence for the value of HPV antigen-directed immunotherapy against cervical cancer comes from the experimental and natural papillomavirus-associated tumors that can be controlled by immunization with E7 antigen. Previous studies have used different modes of immunization, such as (i) recombinant E7 vaccinia viruses (2,3,16,24,28,42), (ii) syngeneic cells transfected with E7 (8, 9), (iii) E7 proteinpulsed dendritic cells (12, 37), (iv) peptides corresponding to a cytotoxic T-lymphocyte (CTL) epitope in E7 with incomplete Freund's adjuvant (13), (v) E7 vaccine based on papillomavirus-like particles (17,25,31), and (vi) Salmonella enterica serovar Typhimurium expressing E7 epitope inserted into hepatitis B virus core (26). These studies demonstrate that CTLs are likely to be the most effective immunological effector mechanisms.Adeno-associated virus (AAV), a single-stranded virus, has been studied as a vector for gene therapy (29, 41). Classified as a defective human parvovirus, AAV has many natural features that are attractive for a human gene therapy vector, such as nonpathogenicity, targeted integrating capability, and a broad host range (human, simian, murine, canine, and avian). In sharp contrast to other viral vectors that have been used in vaccination, such as vaccinia virus or adenovirus, AAV vectors do not express any viral genes. The only viral DNA that must be included in an AAV vector is the 145-bp inverted terminal repeat. Since naked DNA is used for immunization, the only gene expressed by AAV vectors is that for the antigen itself.Since HPV-16 E7 is a transforming oncoprotein (20), dangerous side effects such as transformation are not anticipated with a protein vaccine. Peptide vaccination with a CTL epitope to prevent the outgrowth of a tumor is a safe and effective immunotherapeutic method (13). However, a peptide vaccine combined with a toxic adjuvant such as incomplete Freund's adjuvant sometimes can lead to rapid tumor growth through specific T-cell tolerance induction (36). Recently, Mycobacterium tuberculosis heat shock protein 70 (hsp70) has been used as an adjuvant-free carrier to stimulate the humoral and cellular response to a full-length human immunodeficiency virus p24 (33) that is covalently linked to hsp...

Immunity to adeno-associated virus serotype 2 delivered transgenes imparted by genetic predisposition to autoimmunity

“…However, in a majority of such literature, AAV2-delivered transgenes that have been reported tobe capable of inducing immune responses that are either pathogen-specific antigens when AAV2 vectors were specifically designed as vaccine, [38][39][40] or highly pathogenic in nature (eg ovalbumin, b-Gal). In contrast, the transgenes used in our study (eg GFP) have not been shown to elicit antibody responses when delivered by AAV into immunocompetent animals.…”

Secretory heat-shock protein as a dendritic cell-targeting molecule: a new strategy to enhance the potency of genetic vaccines

“…HSPs have been used to enhance the potency of DNA vaccines by others, 46,47,49,50 but there are several important differences between their strategy and ours. The E7/ HSP70 fusion protein in the present report is designed to be secreted by transfected cells and targeted to DCs, as compared to cytoplasmic expression of transfected cells in previous systems.…”

Immunization strategy against cervical cancer involving an alphavirus vector expressing high levels of a stable fusion protein of human papillomavirus 16 E6 and E7

“…Several approaches have been described for the induction of HPV-specific CTL responses such as immunization with recombinant viruses, HPV-specific proteins or peptides, E7 linked to heat shock proteins or intracellular sorting molecules, peptides/proteins loaded on dendritic cells, DNA-or RNA-transfected dendritic cells (DCs) or cell lines. [8][9][10][11][12][13] We are exploiting the Semliki Forest virus (SFV) expression system. 5,[14][15][16][17] This vector represents an ideal system for the induction of cellular immune responses against the oncoproteins HPV E6 and E7 since, apart from its high efficiency and biosafety, SFV RNA does not integrate and SFV infection is cytolytic by apoptosis.…”