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Vaccination with the minor capsid protein L2, notably the 17-36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17-36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum±MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17-36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant., (Copyright © 2015. Published by Elsevier Ltd.)

The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well known and computer-predicted A2-restricted epitopes. In the current study we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope that has been relocated to positions within the CRPV E7 gene and the CRPV L2 gene. Epitope expression from both the E7 protein and the L2 protein resulted in increased protection against viral DNA challenge of the HLA-A2.1 transgenic rabbits as compared to control-vaccinated rabbit groups. These data indicate that proteins expressed at both early and late time points during a natural papillomavirus infection can be targeted by epitope-specific immunity and indicate this immunity is increased to early rather than late expressed proteins of papillomaviruses. This study also highlights the broad utility of the HLAA2.1 transgenic rabbit model for testing numerous immunological factors involved in vaccine generated protective immunity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

DNA vaccines delivered subcutaneously by gene-gun have generated strong protective and therapeutic immunity in rabbits. Recent studies have shown that peptides delivered by the mucosal routes also stimulate local and systemic immune responses. Since mucosal delivery is easier to administer and more cost-effective when compared to gene-gun delivery, we were interested to learn whether mucosally delivered peptides would prime protective immunity comparable to that of gene-gun-delivered DNA in rabbits. Our newly developed HLA-A2.1 transgenic rabbit model was used to test the hypothesis. We chose an HLA-A2.1 restricted cottontail rabbit papillomavirus (CRPV) E1 epitope (E1/303-311, MLQEKPFQL) for the peptide immunization studies because it provided complete protection when used as a DNA vaccine. Adjuvant has been widely used to boost immunity for vaccines. In this study, three adjuvants reported to be effective for rabbits (TT helper motif, PADRE and CpG2007) were tested with the peptide vaccine. Peptide alone or fused to TT helper or PADRE to create chimeric peptides was delivered by two mucosal routes (ocular and intranasal) together. Partial protection was found in HLA-A2.1 transgenic rabbits when peptide was delivered mucosally in the presence of adjuvant. When a subsequent booster of a half-dose of the corresponding DNA vaccine was delivered, complete protections were achieved. We conclude that mucosal peptide immunization can be combined with a single DNA vaccination to provide strong protective immunity in rabbits.

Recent phylogenic studies indicate that DNA recombination could have occurred in ancient papillomavirus types. However, no experimental data are available to demonstrate this event because of the lack of human papillomavirus infection models. We have used the cottontail rabbit papillomavirus (CRPV)/rabbit model to study pathogenesis and immunogenicity of different mutant genomes in vivo. Although the domestic rabbit is not a natural host for CRPV infection, it is possible to initiate infection with naked CRPV DNA cloned into a plasmid and monitor papilloma outgrowth on these animals. Taking advantage of a large panel of mutants based on a CRPV strain (Hershey CRPV), we tested the hypothesis that two non-viable mutant genomes could induce papillomas by either recombination or complementation. We found that co-infection with a dysfunctional mutant with an E2 transactivation domain mutation and another mutant with an E7 ATG knock out generated papillomas in rabbits. DNA extracted from these papillomas contained genotypes from both parental genomes. Three additional pairs of dysfunctional mutants also showed similar results. Individual wild type genes were also shown to rescue the function of corresponding dysfunctional mutants. Therefore, we suggest that complementation occurred between these two non-viable mutant PV genomes in vivo.

Cottontail rabbit papillomavirus (CRPV)/rabbit model is widely used to study pathogenesis of papillomavirus infections and malignant tumor progression. Recently, we established HLA-A2.1 transgenic rabbit lines and demonstrated efficacy for the testing of immunogenicity of a well-known A2-resticted epitope (HPV16E7/82-90) [Hu J, Peng X, Schell TD, Budgeon LR, Cladel NM, Christensen ND. An HLA-A2.1-transgenic rabbit model to study immunity to papillomavirus infection. J Immunol 2006;177(11):8037-45]. In the present study, we screened five HLA-A2.1 restricted epitopes from CRPVE1 (selected using online MHCI epitope prediction software) and constructed a multivalent epitope DNA vaccine (CRPVE1ep1-5). CRPVE1ep1-5 and a control DNA vaccine (Ub3) were then delivered intracutaneously onto normal and HLA-A2.1 transgenic rabbits, respectively, by a helium-driven gene-gun delivery system. One, two or three immunizations were given to different groups of animals from both New Zealand White outbred and EIII/JC inbred genetic background. Two and three immunizations with CRPVE1ep1-5 DNA vaccine provided complete protection against viral DNA infection of HLA-A2.1 transgenic rabbits from both genetic backgrounds but not in the control-vaccinated groups. One immunization, however, failed to protect HLA-A2.1 transgenic rabbits against viral DNA infection. This study further demonstrated that the HLA-A2.1 transgenic rabbits can be used to test the immunogenicity of HLA-A2.1 restricted epitopes identified by MHCI epitope predication software.