Binding and neutralization efficiencies of monoclonal antibodies, Fab fragments, and scFv specific for L1 epitopes on the capsid of infectious HPV particles.

We compared the neutralization abilities of individual monoclonal antibodies (MAb) of two large panels reactive with L1 epitopes of HPV-11 or HPV-16. Binding titers were compared using both L1-only VLPs and L1/L2 pseudovirions. While the VLPs were antigenically similar to the pseudovirions, clear differences in the surface exposure of some epitopes were evident with the HPV-16 particles. To determine whether all antibody binding events are equivalent in their neutralizing effect on infectious HPV virions or pseudovirions, the binding and neutralization titers for individual MAbs were used to calculate the relative neutralization efficiency for each antibody. HPV neutralization was achieved by all MAbs capable of strong binding to either linear or conformation-sensitive epitopes on pseudovirus particles. Our data suggest, however, that some L1 epitopes may be more neutralization-sensitive than other surface epitopes, in that successful infection can be blocked by varying degrees of epitope saturation. Additionally, the effective neutralization of virions by several monovalent Fab fragments and single-chain variable fragments (scFv) demonstrates that viral neutralization does not require HPV particle aggregation or L1 crosslinking. Identification of capsid protein structures rich in neutralization-sensitive epitopes may aid in the development of improved recombinant vaccines capable of eliciting effective and long-term antibody-mediated protection against multiple HPV types.

Protective cell-mediated immunity by DNA vaccination against Papillomavirus L1 capsid protein in the Cottontail Rabbit Papillomavirus model.

Papillomavirus major capsid protein L1 has successfully stimulated protective immunity against virus infection by induction of neutralizing antibodies in animal models and in clinical trials. However, the potential impact of L1-induced protective cell-mediated immune (CMI) responses is difficult to measure in vivo because of the coincidence of anti-L1 antibody. In this study, we tested the hypothesis that L1 could activate CMI, using the Cottontail Rabbit Papillomavirus (CRPV)-rabbit model. A unique property of this model is that infections can be initiated with viral DNA, thus bypassing all contributions to protection via neutralizing anti-L1 antibody. DNA vaccines containing either CRPV L1, or subfragments of L1 (amino-terminal two-thirds of L1 [L1N] and the carboxylterminal two-thirds of L1 [L1C]), were delivered intracutaneously into rabbits, using a gene gun. After three booster immunizations, the rabbits were challenged with several viral DNA constructs: wild-type CRPV, CRPV L1ATGko (an L1 ATG knockout mutation), and CRPV-ROPV hybrid (CRPV with a replacement L1 from Rabbit Oral Papillomavirus). Challenge of L1 DNA-vaccinated rabbits with wild-type CRPV resulted in significantly fewer papillomas when compared with challenge with CRPV L1ATGko DNA. Significantly smaller papillomas were found in CRPV L1-, L1N-, and L1C-vaccinated rabbits. In addition, rabbits vaccinated with either L1 or L1N grew significantly fewer and smaller papillomas when challenged with CRPV-ROPV hybrid DNA. Therefore, CRPV L1 DNA vaccination induced CMI responses to CRPV DNA infections that can contribute to protective immunity. Cross-protective immunity against CRPV L1 and ROPV L1 was elicited in these CRPV L1- and subfragment-vaccinated rabbits.

Differential antibody responses to a distinct region of human papillomavirus minor capsid proteins.

A peptide derived from the human papillomavirus type 16 (HPV-16) minor capsid protein, L2, has previously been reported to induce cross-neutralizing antibodies in mice. In this report, four HPV L2 peptides, including the HPV-16 peptide and its HPV type 6 and 11 homologues, along with extended peptides containing a conserved set of amino acids, were used to immunize rabbits and mice. Antibody responses were evaluated for specificity and ability to neutralize viral infection in vitro with a quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) assay. All peptide immunizations resulted in cognate and cross-peptide reactivity, but this did not translate equally into recognition of full-length protein, VLP, or neutralization of virus in vitro. This report provides the first evidence of cross-neutralization of authentic HPV by antiserum to L2 peptides. Comparison of the anti-peptide serum reactivity, especially with regard to neutralization of virus, indicates that the extended peptides may offer more potential to induce adequate responses for cross-protective immunity.

Mucosally-derived HPV-40 can infect both human genital foreskin and cutaneous hand skin tissues grafted into athymic mice.

HPV-40 is a rare HPV type that has been detected only in genital mucosal tissues. This HPV type is very closely related to HPV-7, which has a predominantly cutaneous tissue tropism. We have shown, previously, that an isolate of HPV-40 (described here as HPV-40(Hershey) or HPV-40(H)) productively infected genital tissues. In this study, HPV-40(H) was tested for productive infection of cutaneous tissue. Fetal hand skin fragments were incubated with infectious HPV-40(H) and implanted subrenally into athymic mice. After 120 days, xenografts showed morphological changes consistent with HPV-40(H) infection and were HPV-40 DNA in situ positive and capsid antigen positive. The results demonstrated that hand skin can support HPV-40(H) infection thereby indicating that this viral type has the capacity to infect both genital mucosal and cutaneous tissues.

Gene gun-mediated intracutaneous vaccination with papillomavirus E7 gene delays cancer development of papillomavirus-induced skin papillomas on rabbits.

High-risk human papillomavirus (HPV) E6 and E7 viral oncogenes are expressed in HPV-associated cancers, and thus represent tumor-specific antigens. We used the cottontail rabbit papillomavirus (CRPV) rabbit model to test whether vaccination with either the E6 or E7 genes alone could prevent or delay carcinoma development. CRPV-induced papillomas on 24 rabbits were allowed to grow for 3 months without any treatment intervention. An immunization protocol using gene gun-mediated intracutaneous administration of DNA plasmids encoding the E6 or the E7 gene or vector only, respectively was initiated at this time point. Carcinoma development was followed up to 24 months after virus infection. Within this period, five rabbits died due to other causes but without carcinoma; one from the vector control group, and two each from the E6- and E7-vaccinated groups. The remaining seven rabbits from the vector control group developed carcinoma within 7-17 months. The remaining six E6-vaccinated rabbits developed cancer within 8-15 months. There was no delay in cancer development for the E6-vaccinated rabbits compared to the vector-injected rabbits. Some delay in cancer development in the remaining E7-vaccinated rabbits was observed; one developed cancer at month 23 and a second was without cancer at month 24. In addition, some E7-vaccinated rabbits with primary skin carcinomas had fewer lung metastases (<2) compared to vector-vaccinated controls (20+). These results suggested that gene gun-mediated intracutaneous immunization with papillomavirus early gene E7 but not E6 delayed carcinoma development of papillomavirus-induced lesions.