Timothy D. Culp

A Protective and Broadly Cross-Neutralizing Epitope of Human Papillomavirus L2

ABSTRACT We generated a monoclonal antibody, RG-1, that binds to highly conserved L2 residues 17 to 36 and neutralizes human papillomavirus 16 (HPV16) and HPV18. Passive immunotherapy with RG-1 was protective in mice. Antiserum to the HPV16 L2 peptide comprising residues 17 to 36 (peptide 17-36) neutralized pseudoviruses HPV5, HPV6, HPV16, HPV 18, HPV31, HPV 45, HPV 52, HPV 58, bovine papillomavirus 1, and HPV11 native virions. Depletion of HPV16 L2 peptide 17-36-reactive antibodies from cross-neutralizing rabbit and human L2-specific sera abolished cross-neutralization and drastically reduced neutralization of the cognate type. This cross-neutralization of diverse HPVs associated with cervical cancer, genital warts, and epidermodysplasia verruciformis suggests the possibility of a broadly protective, peptide-based vaccine.

Keratinocyte-Secreted Laminin 5 Can Function as a Transient Receptor for Human Papillomaviruses by Binding Virions and Transferring Them to Adjacent Cells

ABSTRACT Human papillomaviruses (HPVs) replicate only in the terminally differentiating epithelium of the skin and mucosa. While infection of basal keratinocytes is considered a requirement for permissive infection, it remains unclear whether virions can specifically target basal cells for adsorption and uptake following epithelial wounding. We present evidence that HPV binds specifically to laminin 5 (LN5), a component of the extracellular matrix (ECM) secreted by migrating and basal keratinocytes. HPV type 11 capsids colocalized with LN5 in the ECM secreted by vaginal keratinocytes. Binding of both virions and virus-like particles to purified LN5 and to the LN5-rich ECM secreted by cultured keratinocytes was effectively blocked by pretreatment with anti-LN5 antibodies. HPV capsid binding to human cervical mucosa sections included the basement membrane which contains LN5. Cultured keratinocytes expressing α6 integrin, a transmembrane protein known to bind LN5, were readily infected by virions preadsorbed to LN5-containing substrates, whereas mutant keratinocytes lacking α6 integrin were relatively resistant to infection via this route. These findings suggest a model of natural HPV infection in which proliferating keratinocytes expressing α6 integrin at the site of epithelial wounding might be targeted by virions adsorbed transiently to LN5 secreted by migrating keratinocytes.

Protection of Rabbits against Challenge with Rabbit Papillomaviruses by Immunization with the N Terminus of Human Papillomavirus Type 16 Minor Capsid Antigen L2

ABSTRACT Current L1 virus-like particle (VLP) vaccines provide type-restricted protection against a small subset of the human papillomavirus (HPV) genotypes associated with cervical cancer, necessitating continued cytologic screening of vaccinees. Cervical cancer is most problematic in countries that lack the resources for screening or highly multivalent HPV VLP vaccines, suggesting the need for a low-cost, broadly protective vaccinogen. Here, N-terminal L2 polypeptides comprising residues 1 to 88 or 11 to 200 derived from HPV16, bovine papillomavirus type 1 (BPV1), or cottontail rabbit papillomavirus (CRPV) were produced in bacteria. Rabbits were immunized with these N-terminal L2 polypeptides and concurrently challenged with CRPV and rabbit oral papillomavirus (ROPV). Vaccination with either N-terminal L2 polypeptides of CRPV effectively protected rabbits from CRPV challenge but not from papillomas induced by cutaneous challenge with CRPV genomic DNA. Furthermore, papillomas induced by CRPV genomic DNA deficient for L2 expression grew at the same rate as those induced by wild-type CRPV genomic DNA, further suggesting that the L2 polypeptide vaccines lack therapeutic activity. Neutralizing serum antibody titers of >15 correlated with protection (P < 0.001), a finding consistent with neutralizing antibody-mediated protection. Surprisingly, a remarkable degree of protection against heterologous papillomavirus types was observed after vaccination with N-terminal L2 polypeptides. Notably, vaccination with HPV16 L2 11-200 protected against cutaneous and mucosal challenge with CRPV and ROPV, respectively, papillomaviruses that are evolutionarily divergent from HPV16. Further, vaccination with HPV16 L2 11-200 generates broadly cross-neutralizing serum antibody, suggesting the potential of L2 as a second-generation preventive HPV vaccine antigen.

Varicella-zoster virus infection induces autophagy in both cultured cells and human skin vesicles.

ABSTRACT When grown in cultured cells, varicella-zoster virus (VZV) forms many aberrant light particles and produces low titers. Various studies have explored the reasons for such a phenotype and have pointed to impaired expression of specific late genes and at lysosomal targeting of egressing virions as possible causes. In the studies presented here, we report that the autophagic degradation pathway was induced at late time points after VZV infection of cultured cells, as documented by immunoblot analysis of the cellular proteins LC3B and p62/SQSTM1, along with electron microscopy analysis, which demonstrated the presence of both early autophagosomes and late autophagic compartments. Autophagy was induced in infected cells even in the presence of phosphonoacetic acid, an inhibitor of viral late gene expression, thus suggesting that accumulation of immediate-early and early viral gene products might be the major stimulus for its induction. We also showed that the autophagic response was not dependent on a specific cell substrate, virus strain, or type of inoculum. Finally, using immunofluorescence imaging, we demonstrated autophagosome-specific staining in human zoster vesicles but not in normal skin. Thus, our results document that this innate immune response pathway is a component of the VZV infectious cycle in both cultured cells and the human skin vesicle, the final site of virion formation in the infected human host.

Preclinical model to test human papillomavirus virus (HPV) capsid vaccines in vivo using infectious HPV/cottontail rabbit papillomavirus chimeric papillomavirus particles.

ABSTRACT A human papillomavirus (HPV) vaccine consisting of virus-like particles (VLPs) was recently approved for human use. It is generally assumed that VLP vaccines protect by inducing type-specific neutralizing antibodies. Preclinical animal models cannot be used to test for protection against HPV infections due to species restriction. We developed a model using chimeric HPV capsid/cottontail rabbit papillomavirus (CRPV) genome particles to permit the direct testing of HPV VLP vaccines in rabbits. Animals vaccinated with CRPV, HPV type 16 (HPV-16), or HPV-11 VLPs were challenged with both homologous (CRPV capsid) and chimeric (HPV-16 capsid) particles. Strong type-specific protection was observed, demonstrating the potential application of this approach.

Quantitative RT-PCR assay for HPV infection in cultured cells

Early events in the life cycle of the human papillomaviruses (HPV) have been difficult to investigate due to both the scarcity of authentic HPV virions and limitations in assays to detect and quantify nonpermissive infections in monolayer cell culture. We have developed a quantitative reverse transcription-PCR (QRT-PCR) assay for the E1( wedge )E4 transcript of HPV-11. This assay is both sensitive, and capable of differentiating between infections caused by a wide range of virus input. The QRT-PCR assay measured accurately the relative amount of viral transcripts present in samples during validation experiments using RNAs from three cell lines. Infections in all three cell lines, using titrations of HPV-11 virions ranging from 20 to 600 particles per cell, produced linear expression profiles suggesting that these multiplicities of infection are below the saturation level for viral uptake and transcription. Comparison of the QRT-PCR assay with the commonly used nested RT-PCR assay revealed that although the nested RT-PCR assay was more sensitive, it did not differentiate between infections caused by >1000-fold difference in viral inputs. Potential applications of the QRT-PCR assay are demonstrated in experiments measuring the ability of a capsid-specific monoclonal antibody and a nonspecific microbicide to block HPV-11 infection.

Papillomavirus Particles Assembled in 293TT Cells Are Infectious In Vivo

ABSTRACT Papillomaviruses (PVs) demonstrate both tissue and species tropisms. Because PVs replicate only in terminally differentiating epithelium, the recent production of infectious PV particles in 293 cells marks an important breakthrough. In this article, we demonstrate that infectious PV particles produced in 293TT cells can cause papillomatous growths in the natural host animal. Moreover, we show that species-matched PV genomes can be successfully delivered in vivo by a heterologous, species-mismatched PV capsid. Additionally, our results indicate that the addition of the simian virus 40 origin of replication to the papillomavirus genome increases the production of infectious papillomavirus particles by increasing genome amplification in the transfected 293TT cells.

Binding and neutralization characteristics of a panel of monoclonal antibodies to human papillomavirus 58.

Human papillomavirus (HPV) 58 is a high-risk HPV type associated with progression to invasive genital carcinomas. We developed six monoclonal antibodies (mAbs) against HPV58 L1 virus-like particles that bind conformational epitopes on HPV58. The hybridoma cell lines were adapted to serum- and animal component-free conditions and the mAb supernatants were affinity-purified. The six mAbs neutralized HPV58 pseudoviruses (PsVs) and ‘quasivirions’ with different capacities. The mAbs differed in their ability to prevent PsV58 attachment to HaCaT cells, to the extracellular matrix (ECM) deposited by HaCaT cells, to heparin and to purified human laminin 5, a protein in the ECM. These mAbs provide a unique set of tools to study the binding properties of a previously untested, high-risk HPV type and the opportunity to compare these characteristics with the binding of other HPV types.