Nancy M. Cladel

Immunological cross-reactivity to laboratory-produced HPV-11 virions of polysera raised against bacterially derived fusion proteins and synthetic peptides of HPV-6b and HPV-16 capsid proteins

Polysera raised in rabbits to bacterially derived fusion proteins and synthetic peptides of the L1 and L2 ORFs of HPV-6b and -16 were tested for cross-reactivity to laboratory-produced infectious HPV-11 virions. The polysera were analyzed in a series of five different immunological assays including immunoperoxidase staining of the koilocytotic nuclei in sections of formalin-fixed, paraffin-embedded as well as fresh frozen sections of HPV-11 experimental condylomas generated in the athymic nude mouse xenograft system, ELISA, Western blots, and neutralization of infectious HPV-11 virions. ELISA and Western blot assays were used to determine whether the polysera identified external or internal epitopes on HPV-11 virions, and whether there was cross-reactivity to BPV-1 or laboratory-produced CRPV virions. Seven of a total of 12 sera were positive for reactivity to HPV-11 in one or more assays, but none of the reactivity was directed to external epitopes on the intact virions as determined by ELISA. None of the L1 products generated group-specific antigen (GSA) antisera including a synthetic peptide spanning the GSA site. The combination of assays clearly demonstrated that apparent false positive and false negative reactivities of different antisera were obtained for each assay system tested. Thus, no single assay could be used reliably to determine the true antiviral reactivity of a given polysera.

An HLA-A2.1-Transgenic Rabbit Model to Study Immunity to Papillomavirus Infection

We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8+ T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82–90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition, vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82–90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy.

High efficiency induction of papillomas in vivo using recombinant cottontail rabbit papillomavirus DNA

Plasmids containing cottontail rabbit papillomavirus (CRPV) DNA can induce papillomas in vivo, but efficiency has been low. The aim of the present investigation was to explore some of the technical variables involved in inoculation of rabbits with recombinant CRPV DNA in attempts to improve both yield and consistency of papilloma induction. It was found that induction of epidermal hyperplasia, with either a mixture of turpentine and acetone or phorbol esters, produced a marked increase in papilloma yield. An additional powerful factor was the use of very vigorous, cutaneous scarification, sufficient to penetrate the papillary dermis and produce bleeding. When used in combination, papilloma yields were consistent and often reached 90-100% of inoculated sites. A number of other variables which did not consistently affect papilloma yield were tested. These included bleb and puncture injections, plasmid dose, vector type, occlusive dressings, lipofection reagent, carrier DNA, and different methods for plasmid DNA extraction and purification. It is concluded that the most important variables in improving papilloma yields were prior induction of epidermal hyperplasia and vigorous cutaneous scarification.

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.