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immunity and nonhumoral immunity are important in the control of established HPV infection and HPV-related lesions.

In order to prevent the development of lesions, eliminate existing lesions, or even eliminate malignancies, a therapeutic vaccine should target HPV antigens that are continuously expressed in the infected cells and cancer cells. HPV encodes eight papillo-mavirus genes (see Table 1) that are potential "nonself" targets for a therapeutic vaccine. Infected basal epithelial cells usually do not express E4, L1, or L2 proteins at detectable levels. Additionally, the E1, L1, and L2 genes and especially the E2 gene are frequently lost in HPV-associated malignancies during integration. Indeed these genes are dispensable for transformation, and loss of E2 is in fact believed to enhance this process. E5 is not considered an optimal target because it is not required for transformation. It is also poorly immunogenic, probably reflecting its location predominantly within membranes. In contrast, the remaining viral oncoproteins are potential target antigens as they are expressed throughout the viral life cycle and help to regulate progression of the disease. E6 and E7 are critical for HPV replication and cervical epithelial transformation, and are expressed throughout the viral life cycle. As described earlier, because E2, a negative regulator of E6 and E7, is often deleted during the HPV transformation process, E6 and E7 genes are further upregulated in cervical cancer cells. Thus, E6 and E7 are important targets for HPV therapeutic vaccines because they are not expressed in normal cells, but are expressed in all HPV infected cells and they cannot be lost by the tumor as this results in apoptosis. And, whereas E2 is a poor target for immunotherapy of cervical cancer, E1 and E2 represent potential targets for induction of papilloma and CIN regression.

Live-vector vaccines, peptide or protein vaccines, nucleic acid vaccines, cell-based vaccines, and combined approach vaccines have all been tested in the development of HPV therapeutic vaccines both preclinically and now, more frequently in patients. However, despite many successes in curing mice of transplantable tumor models, human trials of many therapeutic vaccines have provided little or no benefit to patients with HPV malignancies. Thus the focus is on the findings in patients to form future improvements in vaccine strategies, combination of treatments, better animal models, and designing immunological assays that better correlate with clinical outcome. Table 5 is a summary of the advantages and disadvantages of the HPV therapeutic strategies and the progress these approaches have made in clinical trials.

The most extensive studies have been performed with peptide-based, viral-vector and naked DNA vaccines. Peptide-based therapeutic vaccines are stable, easily produced, and safe. Unfortunately, they are also immunogenically weak and therefore require adjuvants; and with respect to cell-mediated immunity, these vaccines are also MHC-specific. Synthetic peptides representing two HPV16 E7-encoded, HLA-A0201-restricted cytotoxic T lymphocyte epitopes have been studied in patients with recurrent or residual cervical carcinoma refractory to conventional treatment (87-89). Additionally, a vaccine consisting of a 9-amino acid peptide from HPV-16 E7 amino acids 12-20 was tested in women with high-grade cervical or vulvar intraepithelial neoplasia who were positive for HPV-16 and were HLA-A2 (90). These HPV peptide vaccines have been shown to be both safe and well tolerated in humans. However, HPV-specific immune responses generated by these vaccines have been weak and correlated poorly with the few clinical responses. A subgroup of patients vaccinated with these peptide epitopes do show measured regression of lesions and even clearance of dysplasia. However, neither peptide-specific proliferative cytotoxic T-lymphocyte (CTL) responses nor lesion regression are consistently detected.

The live vector vaccines, especially those that are capable of replication in the host, are generally highly immunogenic and can induce strong immune responses. However, there are significant safety concerns, particularly in cancer patients with weakened immune systems. The prevalence of pre-existing vector immunity that may decrease the effectiveness of the vaccine and inhibit repeated vaccination is another issue with this vaccination strategy that must be considered. Additionally, vector antigens may become immunodominant on the HPV antigen carried on the vector, thus interfering with the formation of an efficacious anti-HPV immune response. Some live vector vaccines have passed preclinical evaluations and proceeded into clinical trials. For example, a recombinant vaccinia virus encoding HPV-16 and HPV-18 E6/E7 (called TA-HPV) was tested in clinical trials. Importantly, TA-HPV was well tolerated and T-cell immune responses were observed after vaccination in some patients with highgrade CIN, early invasive cervical cancer, and even advanced cervical cancer (91-93). The vaccine was also given to patients with HPV-associated vulvar or vaginal intra-epithelial neoplasia with specific immune responses observed (94,95). In a study conducted by Baldwin and colleagues, five out of 12 patients had at least a 50% reduction in lesion diameter after 24 weeks, and one patient showed complete regression of lesion after vaccination (94). TA-HPV has also been used in conjunction with the fusion protein TA-CIN using a prime-boost strategy (see Table 5). Another recombinant vaccinia virus encoding E2 (called MVA E2) has been tested in patients with CIN. Although, utilized as a genetic therapy, the vaccine might potentially generate HPV-reactive E2-specific immune responses (96).

DNA vaccines, most commonly in the form of naked DNA expression plasmids, have several beneficial features for HPV therapeutic vaccine development. DNA vectors are easily produced and can be engineered to express tumor antigenic peptides or proteins. The stability and purity of DNA vaccines are even higher than peptide or protein vaccines. DNA vaccines also have the ability to produce antigenic proteins and peptides in antigen-presenting cells during an extended period. Thus, the amount of antigen delivered to the immune system is potentially higher than for peptide and protein vaccines. This is also an issue when expressing viral oncogenes, and therefore E6 and E7 containing inactivating mutations are frequently used. Using DNA vaccines to express proteins, thus allowing the cell to generate its own peptide-MHC complex, bypasses the MHC restriction whereas maintaining higher CTL responses than current protein vaccines in preclinical studies. DNA vaccines can also be repeatedly applied to the same patient safely and effectively, unlike vaccines, which utilize a live vector. But like peptide vaccines, DNA vaccines are limited by their low immunogenicity. This limited immunogenicity may be enhanced by expression of only the relevant epitopes to facilitate antigen processing and presentation.

Some DNA-based vaccines have passed preclinical evaluations and proceeded into clinical trials. A clinical trial in patients with high-grade anal intraepithelial lesions (97) and another in CIN-2/3 patients (98), tested a plasmid DNA vaccine, ZYC101 (ZYCOS Inc), which encodes multiple HLA-A2-restricted epitopes derived from the HPV-16 E7 protein. Subjects were shown to tolerate the vaccine well. Similar to many

Table 5

Characteristics of Therapeutic HPV Vaccine Approaches

Approach

Advantages

Disadvantages

Clinical trials

Peptide-based

Protein-based

Stable

Easy to produce Safe

Can incorporate multiple epitopes Can enhance peptides for MHC binding

Easy to produce Multiple known adjuvants No HLA restriction Can produce fusion proteins for enhanced immunogenicity

Weakly immunogenic Must determine epitopes HLA restriction

Weakly immunogenic Usually better induction of antibody response than CTL response

Study of toxicity and anti-tumor immune responses in patients with recurrent or residual cervical carcinoma refractory to conventional treatment, receiving vaccinations with synthetic peptides representing two HPV16 E7-encoded, HLA-A*0201-restricted cytotoxic T lymphocyte epitopes and a pan-HLA-DR-binding T-helper epitope, PADRE, in adjuvant (87,88) Study of a vaccine consisting of a 9-amino acid peptide from amino acids 12-20 encoded by the E7 gene emulsified with incomplete Freund's adjuvant in women with high-grade cervical or vulvar intraepithelial neoplasia who were positive for HPV-16 and were HLA-A2 positive (90) Study of HLA-A*0201-restricted, HPV-16 E7 lipopeptide vaccine in eliciting cellular immune responses in women with refractory cervical cancer (89) Study of immunogenicity of PD-E7/AS02, an HPV-16 E7 protein-based vaccine linked to the first 108 amino acids of Haemophilus influenzae protein D, formulated in the GlaxoSmithKline Biologicals adjuvant AS02B, in women with oncogenic HPV-positive CIN (128)

Vector-based: Viral

Vector-based: Bacterial

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