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Papillomavirus infection is a major antecedent of anogenital malignancy. We have previously established that the L1 and L2 capsid genes of papillomavirus have suboptimal codon usage for expression in mammalian cells. We now show that the lack of immunogenicity of polynucleotide vaccines based on the L1 gene can be overcome with codon modified L1, which induces strong immune responses, including conformational virus neutralising antibody and delayed type hypersensitivity. Conjugation of a ubiquitin gene to a hybrid gene incorporating L1 and the E7 non-structural papillomavirus protein improved E7 specific CTL responses, and induced protection against an E7 expressing tumour, but induced little neutralising antibody. However, a mixture of ubiquitin conjugated and non-ubiquitin conjugated polynucleotides induced virus neutralising antibody and E7 specific CD8 T cells. An optimal combined prophylactic/therapeutic viral vaccine might therefore comprise ubiquitin conjugated and non-ubiquitinated genes, to induce prophylactic neutralising antibody and therapeutic cell mediated immune responses.

Mice transgenic for E6/E7 oncogenes of Human Papillomavirus type 16 display life-long expression of E6 in lens and skin epithelium, and develop inflammatory skin disease late in life, which progresses to papillomata and squamous carcinoma in some mice. We asked whether endogenous expression of E6 induced a specific immunological outcome, i.e. immunity or tolerance, or whether the mice remained immunologically naïve to E6. We show that prior to the onset of skin disease, E6 transgenic mice did not develop a spontaneous E6-directed antibody response, nor did they display T-cell proliferative responses to dominant T-helper epitope peptides within E6. In contrast, old mice in which skin disease had arisen, developed antibodies to E6. We also show that following immunisation with E6, specific antibody responses did not differ significantly among groups of E6-transgenic mice of different ages (and therefore of different durations and amounts of exposure to endogenous E6), and non-transgenic controls. Additionally, E6 immunisation-induced T-cell proliferative responses were similar in E6-transgenic and non-transgenic mice. These data are consistent with the interpretation that unimmunised E6-transgenic mice that have not developed inflammatory skin disease remain immunologically naïve to E6 at the B- and Th levels. There are implications for E6-mediated tumorigenesis in humans, and for the development of putative E6 therapeutic vaccines.

Recombinant bacille Calmette-Guerin (BCG) based vaccine delivery systems could potentially share the safety and effectiveness of BCG. We therefore prepared recombinant BCG vaccines which expressed the L1 late protein of the human papillomavirus (HPV) 6b or the E7 early protein of the HPV 16. The two recombinants were evaluated as immunogens in C57BL/6J and BALB/c mice, and compared with a conventional protein/adjuvant system using E7 or L1 mixed with Quil-A adjuvant. rBCG6bL1 and rBCG16E7 primed specific immune responses, represented by DTH, T-proliferation and antibody, and rBCG16E7 induced cytotoxic immune response to E7 protein. The magnitude of the observed responses were less than those elicited by protein/adjuvant vaccine. As recombinant BCG vaccines expressing HPV6bL1 or HPV16E7 persist at low levels in the immunised host, they may be beneficial to prime or retain memory responses to antigens, but are unlikely to be useful as a single component vaccine strategy.

Subjects with genital warts were immunized three times or more with HPV6b VLPs without adjuvant. All immunized subjects had DTH to HPV6b L1 protein. Of 32 subjects, nine had HPV6b specific antibody prior to immunization and 22 acquired antibody with immunization. VLP specific antibody increased following a single immunization in 6 of 8 subjects with low level antibody at recruitment. Complete regression of genital warts was observed in 25 of 33 evaluable subjects over the 20-week observation period. We conclude that immunization with HPV6b L1 VLPs without adjuvant induces immunity to the L1 protein epitopes recognised during natural infection, and may accelerate regression of warts.

We have determined the post-translational modifications of the major capsid protein, L1 of human papillomavirus (HPV) type 6b. Since this virus cannot be cultured in the laboratory to obtain sufficient material for a study, a recombinant L1 protein produced in a vaccinia virus expression system was used in this investigation. Our results show that this protein is phosphorylated at serine residues and is also glycosylated. No myristoylation or palmitoylation was detected. The fraction of L1 protein incorporated into virus-like particles was not glycosylated. Since recombinant L1 protein is a potential human vaccine candidate, knowledge of the post-translation modifications of this protein may prove useful for the design of anti-HPV vaccines.

The presence of T-helper epitopes within the E7 transforming protein of human papillomavirus type 18 (HPV18) was sought using a series of overlapping synthetic 15-20 mer peptides spanning the entire 105 amino acid sequence of this protein. Two H-2k restricted T-helper epitopes were defined, comprising 44VNHQHLPARRA55 and 81DDLRAFQQLF90 as the minimal T proliferative epitopes. Peptides containing these epitopes were able to provide cognate help to B epitopes from HPV18E7 protein for production of antibody to this protein in vivo in CBA/CaH mice. No H-2b or H-2d restricted epitopes were demonstrable, and in H-2d mice this was associated with poor antibody response to the E7 protein. There is no "promiscuous" T-helper epitope in HPV18 E7 comparable to the 49DRAHYNI55 sequence in HPV16 E7, and restricted T-helper epitope availability may be a determinant of poor immune responses to this protein after natural infection.

Synthetic peptides can be tailor-made to include any B or T epitopes desired from a single or multiple antigens or organisms. However, peptides in general are not very immunogenic and have not proven easy to incorporate into immunogenic vaccines. ISCOMs is an adjuvant system that has the capability not only to enhance the humoral immunogenicity of a protein but has also been shown to induce cell-mediated immune responses in animals. Synthetic peptide ISCOM vaccines are few because of the difficulty in incorporation of these peptides into ISCOMs. We have shown in this study that non-immunogenic peptides could be made immunogenic by polymerisation, and these polymers could be incorporated into ISCOMs to give highly immunogenic vaccines. Synthetic 20mer peptides containing known B and T-helper epitopes from the E7 protein of the cervical cancer associated human papillomavirus type 16 (HPV16 E7) have been used here as model immunogens. We have compared the humoral immunity induced by these peptides as polymers or as copolymers with a lipid binding 20mer peptide (LAP 20), with or without incorporation into ISCOMs. Unpolymerised peptide elicited no measurable antibody. When polymerised peptide was administered with CFA, or in phosphate-buffered saline (PBS) without adjuvant, or incorporated into ISCOMs, antibodies recognising both the immunising peptide and HPV16 E7 protein were produced. For equal quantities of administered peptide (5 micrograms), ISCOMs gave higher titres of antibody than CFA or PBS. Polymerised peptides induced high antigen-specific IgG2a:IgG1 ratios, which increased with multiple immunisations. These data indicate that polymerised peptides could be incorporated into ISCOMs to form efficient immunogens which may elicit a Th1 type response.