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Background: Human papillomavirus (HPV) etiology has become evident in head and neck cancers (HNCs) and HPV positivity showed a strong association with its malignant progression. Since aberrant DNA methylation is known to drive carcinogenesis and progression in HNCs, we investigated to determine target gene(s) associated with this modification., Methods: We characterized epigenetic changes in tumor-related genes (TRGs) that are known to be associated with HNC development and its progression., Results: The expression levels of 42 candidate HNC-associated genes were analyzed. Of these, 7 TGRs (CHFR, RARβ, GRB7, EREG, RUNX2, RUNX3, and SMG-1) showed decreased expressions in HPV-positive ( + ) HNC cells compared with HPV-negative ( - ) HNC cells. When gene expression levels were compared corresponding to the DNA methylation conditions, GRB7 and EREG showed significant differential expression between HPV + and HPV - cells, which suggested these genes as primary targets of epigenetic regulation in HPV-induced carcinogenesis. Furthermore, treatment with a demethylation agent, 5-aza-2'-deoxycytidine (5-aza-dc), caused restoration of EREG expression and was associated with hypomethylation of its promoter in HPV + cells, while no changes was noted in HPV - cells. EREG promoter hypermethylation in HPV + cells was confirmed using methylation-specific PCR (MS-PCR)., Conclusion: We conclude that EREG is the target of epigenetic regulation in HPV + HNCs and its suppressed expression through promoter hypermethylation is associated with the development of HPV-associated HNCs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This study evaluated the integration and methlyation of human papillomavirus type 16 (HPV16) in head and neck squamous cell carcinoma (HNSCC) and its oral precursor, high-grade oral epithelial dysplasia (hgOED). Archival samples of HPV16-positive hgOED ( N = 19) and HNSCC ( N = 15) were evaluated, along with three HNSCC (UMSCC-1, -47 and -104) and two cervical cancer (SiHa and CaSki) cell lines. HgOED cases were stratified into three groups with increasing degrees of cytologic changes (mitosis, karyorrhexis and apoptosis). The viral load was higher and the E2/E6 ratio lower (indicating a greater tendency toward viral integration) in group 3 than in groups 1 or 2 ( p = 0.002, 0.03). Methylation was not observed in hgOED cases and occurred variably in only three HNSCC cases (26.67%, 60.0% and 93.3%). In HNSCC cell lines, lower E7 expression correlated with higher levels of methylation. HgOED with increased cytologic change, now termed HPV-associated oral epithelial dysplasia (HPV-OED), exhibited an increased viral load and a tendency toward DNA integration, suggesting a potentially increased risk for malignant transformation. More detailed characterization and clinical follow-up of HPV-OED patients is needed to determine whether HPV-OED is a true precursor to HPV-associated HNSCC and to clarify the involvement of HPV in HNSCC carcinogenesis., Competing Interests: CONFLICTS OF INTEREST All authors have no conflicts of interest.

The Florida manatee (Trichechus manatus latirotris) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.

Previous studies of naturally occurring mouse papillomavirus (PV) MmuPV1-induced tumors in B6.Cg-Foxn1 nu/nu mice suggest that T cell deficiency is necessary and sufficient for the development of such tumors. To confirm this, MmuPV1-induced tumors were transplanted from T cell-deficient mice into immunocompetent congenic mice. Consequently, the tumors regressed and eventually disappeared. The elimination of MmuPV1-infected skin/tumors in immunocompetent mice was consistent with the induction of antitumor T cell immunity. This was confirmed by adoptive cell experiments using hyperimmune splenocytes collected from graft-recipient mice. In the present study, such splenocytes were injected into T cell-deficient mice infected with MmuPV1, and they eliminated both early-stage and fully formed tumors. We clearly show that anti-tumor T cell immunity activated during tumor regression in immunocompetent mice effectively eliminates tumors developing in T cell-deficient congenic mice. The results corroborate the notion that PV-induced tumors are strongly linked to the immune status of the host, and that PV antigens are major anti-tumor antigens. Successful anti-PV T cell responses should, therefore, lead to effective anti-tumor immune therapy in human PV-infected patients., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Reported cytologic alterations associated with high-risk human papillomavirus (HR-HPV) in oral epithelial dysplasia (HPV-OED) need further characterization., Study Design: Archival cases of high-grade oral epithelial dysplasia (hgOED) (N = 38) were assigned a cytologic score (CS) based on the average number of mitotic, karyorrhectic, and apoptotic cells per high-power field. Three groups were then generated on the basis of increasing CS: Focal (group 1, N = 14), Intermediate (group 2, N = 12), and Diffuse (group 3, N = 12). Polymerase chain reaction-based HPV genotyping and p16 immunohistochemistry were performed., Results: HR-HPV was found significantly more in group 3 (83.3%) compared with groups 1 and 2 (group 1&2; 42.9% and 41.7%, respectively; P = .047). HPV16 predominated in HR-HPV-positive cases (90.5%). By location, the tongue or the floor of mouth was associated with all groups (P = .04). Increasing CS was associated with a slightly younger age (P = .04) and increased expression of p16 (P = .005). CS and p16 expression were not sensitive but were highly specific predictors for HR-HPV presence. Based on limited follow-up information, HPV-OED does not differ in clinical aggressiveness compared with conventional OED., Conclusions: Increased CS in hgOED is strongly associated with HR-HPV (mostly HPV16) and p16 expression. CS and p16 expression are specific predictors of HR-HPV presence. Further molecular study and long-term follow-up of HPV-OED are needed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an alternative to the currently available virus-like particle (VLP) vaccines, and would be much less expensive. We optimized methods to express HPV16 L1 protein and purify VLPs from tobacco (Nicotiana benthamiana) leaves transfected with the magnICON deconstructed viral vector expression system. L1 proteins were extracted from agro-infiltrated leaves using a series of pH and salt mediated buffers. Expression levels of L1 proteins and VLPs were verified by immunoblot and ELISA, which confirmed the presence of sequential and conformational epitopes, respectively. Among three constructs tested (16L1d22, TPL1d22, and TPL1F), TPL1F, containing a full-length L1 and chloroplast transit peptide, was best. Extraction of HPV16 L1 from leaf tissue was most efficient (> 2.5% of total soluble protein) with a low-salt phosphate buffer. VLPs were purified using both cesium chloride (CsCl) density gradient and size exclusion chromatography. Electron microscopy studies confirmed the presence of assembled forms of HPV16 L1 VLPs. Collectively; our results indicated that chloroplast-targeted transient expression in tobacco plants is promising for the production of a cheap, efficacious HPV16 L1 VLP vaccine. Studies are underway to develop plant VLPs for the production of a cervical cancer vaccine.

Few papers have had a greater impact on the health of the human species than the simple, yet elegant, observations and clinical trials of Edward Jenner with what was at the time called the Cow Pox. In fact, this was a naturally attenuated rodent (probably rat) pox that could infect horses and, through farriers and farm hands, dairy cattle. While commonly called the Cow Pox at the time, Jenner's transmission studies between humans used infectious materials from horses. His methods provided protection from the serious effects of smallpox infections. In 1977, smallpox was considered to be eradicated, although people continue to be infected by pox viruses from other mammalian species. We consider this to be our 'favorite historical paper' because it emphasizes careful clinical observation followed by relatively simple clinical testing can have a profound influence on human health, even when almost nothing is known about the underlying molecular mechanisms. Continued follow-up with strict attention to detail resulted in a crude but effective way to deal with an epidemic, methods still used today for containing infectious diseases., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Infection by mouse papillomavirus (PV), MmuPV1, of T cell-deficient, B6.Cg-Foxn1(nu)/J nude mice revealed that four, distinct squamous papilloma phenotypes developed simultaneously after infection of experimental mice. Papillomas appeared on the muzzle, vagina, and tail at or about day 42days post-inoculation. The dorsal skin developed papillomas and hair follicle tumors (trichoblastomas) as early as 26days after infection. Passive transfer of hyperimmune sera from normal congenic mice immunized with MmuPV1 virus-like particles (VLPs) to T cell-deficient strains of mice prevented infection by virions of experimental mice. This study provides further evidence that T cell deficiency is critical for tumor formation by MmuPV1 infection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Because a combination of retinoic acid, interferon-alpha, and radiation therapy demonstrated synergistic action and effectiveness to treat advanced cervical cancers in earlier studies, we designed this randomized phase 2 open-label trial to assess efficacy and safety of interferon alpha-2b (IFN) and 13-cis-retinoic acid (RA) administered concomitantly with radiation therapy (IFN-RA-radiation) to treat stage III cervical cancer., Methods and Materials: Stage III cervical cancer patients were randomized to study and control groups in a 1:1 ratio. All patients were treated with radiation therapy; study arm patients received IFN (3 × 10(6) IU subcutaneously) 3 times a week for 4 weeks and daily RA (40 mg orally) for 30 days starting on day 1 of radiation, whereas control arm patients received weekly cisplatinum (40 mg/m(2)) for 5 weeks during radiation. Patients were followed for 3 years. The primary endpoint was overall survival at 3 years., Results: Patients in the study (n=104) and control (n=105) groups were comparable for clinicopathological characteristics, radiation therapy-related variables and treatment response. Proportions of disease-free patients in the study and control groups were 38.5% and 44.8%, respectively, after median follow-up of 29.2 months. Hazard ratios were 0.67 (95% confidence interval [CI]: 0.44-1.01) and 0.69 (95% CI: 0.44-1.06) for overall and disease-fee survival, respectively, comparing the study group to control, and demonstrated an inferior outcome with RA-IFN-radiation, although differences were statistically nonsignificant. Kaplan-Meier curves of disease-free and overall survival probabilities also showed inferior survival in the study group compared to those in the control. Acute toxicities of chemoradiation were significantly higher with 2 acute toxicity-related deaths., Conclusions: Treatment with RA-IFN-radiation did not demonstrate survival advantage over chemoradiation despite being less toxic. The trends predicted an inferior outcome with the RA-IFN combination., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Human papillomavirus (HPV) typing of oral lesions microscopically consistent with multifocal epithelial hyperplasia (MEH) was performed to identify potential novel clinical presentations., Study Design: MEH (N = 22 lesions, 17 patients) and squamous papilloma control samples (N = 9 lesions, 9 patients) were compared by using polymerase chain reaction-based HPV genotyping. Student's t tests were used to compare continuous characteristics., Results: Of the study cases, 86.4% of MEH and only 11% of controls were positive for HPV (P = .0002). In MEH lesions, 45.5% contained HPV32, 36.4% HPV6, and 4.5% HPV40. MEH lesions were mostly multifocal (50%) and occurred in HIV-negative patients (81.3%). They predominated on the labial/buccal mucosa (63.3%), and there were significant differences between groups by anatomic site (P < .0001). HPV32, but not HPV6, was detected in known HIV-positive patients., Conclusions: A novel clinical presentation of MEH associated with HPV32 in HIV-negative, middle-aged to older adults is reported here. One case with HPV40 is the first to be reported. Future detection protocols should include HPV32, as it may be currently overlooked., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We isolated two new manatee papillomavirus (PV) types, TmPV3 and TmPV4, from a Florida manatee (Trichechus manatus latirostris). Two PV types were previously isolated from this species. TmPV1 is widely dispersed amongst manatees and a close-to-root PV; not much is known about TmPV2. The genomes of TmPV3 and TmPV4 were 7622 and 7771 bp in size, respectively. Both PVs had a genomic organization characteristic of all PVs, with one non-coding region and seven ORFs, including the E7 ORF that is absent in other cetacean PVs. Although these PVs were isolated from separate genital lesions of the same manatee, an enlarged E2/E4 ORF was found only in the TmPV4 genome. The full genome and L1 sequence similarities between TmPV3 and TmPV4 were 63.2 and 70.3 %, respectively. These genomes shared only 49.1 and 50.2 % similarity with TmPV1. The pairwise alignment of L1 nucleotide sequences indicated that the two new PVs nested in a monophyletic group of the genus Rhopapillomavirus, together with the cutaneotropic TmPV1 and TmPV2.

Background: Human papillomavirus (HPV)-positive oropharyngeal cancer is associated with improved survival and treatment response as compared to HPV-negative cancers. P16 overexpression is widely accepted as a surrogate marker for HPV positivity., Methods: A total of 92 serum samples from 75 head and neck squamous cell carcinoma (HNSCC) patients were examined for HPV16 and 18 E7 antibodies by ELISA. Available tissue was tested for HPV-DNA by PCR, and p16 immunohistochemistry was obtained from a deidentified database., Results: Of 75 HNSCC patients, 25 were HPV E7 seropositive. Seropositivity was strongly associated with cancers of the oropharynx, and correlated with positive p16 immunohistochemistry (IHC) and HPV-DNA. Post-treatment serum was available in a limited subset of patients, revealing a decrease in antibody titers following response to treatment., Conclusions: HPV E7 seropositivity correlated with positive tumor HPV-DNA and p16 expression, and was strongly associated with cancers of the oropharynx. E7 serology warrants further study as a potential biomarker in HPV-positive HNSCC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Current vaccines against Human Papillomavirus (HPV) are highly effective and based on recombinant virus-like particles (VLPs) of the major capsid protein L1. Since these vaccines are HPV type-specific and expensive for global implementation, an alternative, broader-spectrum immunogen would be the N-terminus of the minor capsid protein L2 that induces low titered broadly cross-neutralizing antibodies. Here we analyzed the reactivity of different synthetic L2 peptides containing N-terminus amino acids 17-36 in order to test their antigenicity., Methods: Different synthetic peptides were designed to target the 17-36 amino acid sequences, present in highly antigenic amino-terminus of L2 protein. Six different peptides including Cys22-Cys28 disulfide bonded cyclized L2 peptide were examined for their antigenicity against mouse monoclonal antibody RG-1 and rabbit polyclonal antisera to HPV L2 by enzyme-linked immunosorbent assay (ELISA)., Results: Here we report that the cyclized form of synthetic L2 peptide, which is formed through Cys22-Cys28 disulfide bridges, has the highest reactivity to antibodies than other synthetic L2 peptides., Conclusion: A cyclized L2 peptide has potential to be an excellent candidate to formulate a low-cost, broadly protective pan-oncogenic HPV vaccine., (Copyright © 2015. Published by Elsevier Inc.)

Papillomaviruses (PVs) induce papillomas, premalignant lesions, and carcinomas in a wide variety of species. PVs are classified first based on their host and tissue tropism and then their genomic diversities. A laboratory mouse papillomavirus, MmuPV1 (formerly MusPV), was horizontally transmitted within an inbred colony of NMRI-Foxn1(nu)/Foxn1nu (nude; T cell deficient) mice of an unknown period of time. A ground-up, filtered papilloma inoculum was not capable of infecting C57BL/6J wild-type mice; however, immunocompetent, alopecic, S/RV/Cri-ba/ba (bare) mice developed small papillomas at injection sites that regressed. NMRI-Foxn1(nu) and B6.Cg-Foxn1(nu), but not NU/J-Foxn1(nu), mice were susceptible to MmuPV1 infection. B6 congenic strains, but not other congenic strains carrying the same allelic mutations, lacking B- and T-cells, but not B-cells alone, were susceptible to infection, indicating that mouse strain and T-cell deficiency are critical to tumor formation. Lesions initially observed were exophytic papillomas around the muzzle, exophytic papillomas on the tail, and condylomas of the vaginal lining which could be induced by separate scarification or simultaneous scarification of MmuPV1 at all four sites. On the dorsal skin, locally invasive, poorly differentiated tumors developed with features similar to human trichoblastomas. Transcriptome analysis revealed significant differences between the normal skin in these anatomic sites and in papillomas versus trichoblastomas. The primarily dysregulated genes involved molecular pathways associated with cancer, cellular development, cellular growth and proliferation, cell morphology, and connective tissue development and function. Although trichoepitheliomas are benign, aggressive tumors, few of the genes commonly associated with basal cell carcinoma or squamous cells carcinoma were highly dysregulated.

Correctly folded virus-like particles (VLPs) of papillomavirus (PV) display conformationally dependent epitopes that are type specific, maintained on authentic virions, and induce neutralizing antibodies. Alignment of the L1 amino acid (aa) sequences of 84 PVs revealed that the lengths of their N-termini are diverse and that multiple, possible initiation methionine (met) codons exist. The L1 gene of MusPV (MmuPV1), that naturally infects immunodeficient laboratory mouse strain (NMRI-Foxn1(nu)/Foxn1(nu)), has four met codons at the 1st, 2nd, 28th, and 30th aas from its N-terminus. Of these, the 3rd and 4th mets, that are at the 28th and 30th aa position from the N-termius, respectively, are located at the position where most PVs have their first met. These two mets, located at the 9th and 11th from the YLPP conserved aas of most PVs, should be considered as consensus initiation codons of PV L1s. Three L1 proteins of MusPV, starting from the 2nd, 3rd, and 4th mets, were expressed using a baculovirus expression system and characterized for their ability to self-assemble into VLPs. While MusPV L1 proteins starting from the 2nd met expressed an L1 protein that did not fold into VLPs, the L1s starting from the 3rd and 4th mets generated correct VLPs in abundant quantities. We now conclude that the highest quantity and best quality VLPs are made from the consensus L1 met of MusPV., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mouse parvoviruses (MPVs) are small, single-stranded, 5 kb DNA viruses that are subclinical and endemic in many laboratory mouse colonies. MPVs cause more distinctive deleterious effects in immune-compromised or genetically-engineered mice than immuno-competent mice. At the University of Louisville (U of L), there was an unexpected increase of MPV sero-positivity for MPV infections in mouse colonies between January 2006 and February 2007, resulting in strategic husbandry changes aimed at controlling MPV spread throughout the animal facility. To investigate these MPVs, VP2 genes of seven MPVs were cloned and sequenced from eight documented incidences by PCR technology. The mutations in these VP2 genes were compared to those found at the Genbank database (NCBI; http://www.ncbi.nlm.nih.gov) and an intra-institutional phylogenetic tree for MPV infections at U of L was constructed. We discovered that the seven MPV isolates were different from those in Genbank and were not identical to each other. These MPVs were designated MPV-UL1 to 7; none of them were minute virus of mice (MVMs). Four isolates could be classified as MPV1, one was classified as MPV2, and two were defined as novel types with less than 96% and 94% homology with existing MPV types. Considering that all seven isolates had mutations in their VP2 genes and no mutations were observed in VP2 genes of MPV during a four-month time period of incubation, we concluded that all seven MPVs isolated at U of L between 2006 and 2007 probably originated from different sources. Serological survey for MPV infections verified that each MPV outbreak was controlled without further contamination within the institution., (Copyright © 2013. Published by Elsevier Inc.)

Background. A significant number of non-small-cell lung cancers (NSCLC) have human papillomavirus (HPV) DNA integrated in their genome. This study sought to further establish HPV's possible etiologic link to NSCLC by evaluating an immune response to HPV's oncogene, E7, in patients with NSCLC. Patients and Methods. Antibodies (IgG) in serum against E7 for HPV 16 and 18 in 100 patients with NSCLC were examined by enzyme-linked immunosorbent assay (ELISA). Results. Sixteen NSCLC patients were found to have a high titration of IgG for HPV oncogenic E7 protein. 23.5% of adenocarcinomas (AC,) and 15.4% of squamous cell carcinomas (SCC) were positive for IgG against HPV E7. HPV-18 (11%) had a slightly higher frequency than HPV-16 (6%). Of the six positive cases for HPV-16, 3 were AC, 2 SCC, and 1 NOS (not otherwise specified). For the 11 HPV-18 positives, 7 were AC, and 4 SCC. The one case with IgG against HPV 16 and 18 was AC. One case had high cross-reactive levels against E7 of HPV 16 and 18. Two (28%) of 7 patients who reported never smoking were positive for HPV, and 12 (13.6%) of 88 smokers were HPV positive. Conclusions. The study detected high levels of IgG against E7 in 16% of NSCLC patients. This adds evidence to a potential role of HPV in the pathogenesis of NSCLC.

The number of studies addressing neoplasia in marine mammals has recently increased, giving rise to concern whether such lesions could be reflective of an emerging infectious disease. Eight species-specific viruses, seven papillomaviruses (PVs) and two herpesviruses (HVs) have separately been shown to be associated with genital tumors in Atlantic bottlenose dolphins (Tursiops truncatus, Tt): TtPV1-6, as well as HVs provisionally assigned the names DeHV4 and -5 (Delphinid HVs). A definite causal role of these viruses in cell transformation remains to be demonstrated. Concurrent PV- and HV-infection has never been reported in marine mammals. DNA extractions from biopsies of genital tumors derived from 15 free-ranging Atlantic bottlenose dolphins were selected for molecular examination. Polymerase chain reaction (PCR) analyses revealed the presence of DeHV4, while a serological screening using an antibody-based TtPV enzyme-linked immunosorbent assay (ELISA) demonstrated previous and/or current infection of the HV-positive dolphins with at least one TtPV type. Therefore, care must be taken when drawing conclusions about viral causalities in tumor development, since the "hit and run" and other mechanisms have been described for types of both viral families. This study presents the first evidence of marine mammals having a history of PV- as well as HV-infection and discusses the disputed effects of viral co-infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

MusPV, a novel papillomavirus (PV) that naturally infects laboratory mice, was isolated and characterized from a colony of NMRI-Foxn1(nu)/Foxn1(nu) (nude) mice in India. Because MusPV may have been missed during routine pathogen screening of mice in colonies worldwide, a variety of detection methods are described to detect MusPV. The clinical and histologic lesions of productive MusPV infections fit PV-associated features, including papillomas, koilocytes within the stratum granulosum of the hyperplastic/acanthotic papillomatous epithelium, and the presence of intranuclear virus particles in koilocytotic cells visualized by electron microscopy. Antiserum against disrupted PV virions, isolated from another species (canine), identified conserved viral antigens in productively infected cells by immunohistochemistry. A rolling circle technique was used to amplify viral circular DNAs followed by endonuclease restriction enzyme digestion to determine the correct size of PV DNA. Consensus PV degenerative primers, My09/11, commonly used to detect many different types of PVs by polymerase chain reaction (PCR), particularly mucosotropic HPVs, also identified MusPV and all rodent PVs tested. Since there was one nucleotide mismatch between the My09/11 primer set and the MusPV template, a new primer set, MusPV-My09/11, was designed to specifically detect MusPV in latent infections and spontaneous MusPV-induced papillomas. Southern blot analysis verified the presence of full size PV DNA in infected tissues. Virus-like particles (VLPs), generated from MusPV L1 genes, provided a substrate for serological testing of naturally and experimentally infected mice. In summary, a series of diagnostic assays were developed and validated to detect MusPV infection in skin tumors and serological response in laboratory mice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In 1997, cutaneous papillomatosis caused by Florida manatee (Trichechus manatus latirostris [Tm]) papillomavirus 1 (TmPV1) was detected in seven captive manatees at the Homosassa Springs Wildlife State Park, Florida, USA, and, subsequently, in two wild manatees from the adjacent Homosassa River. Since then, papillomatosis has been reported in captive manatees housed in other locations, but not in wild animals. To determine TmPV1 antibody prevalence in captive and wild manatees sampled at various locations throughout Florida coastal regions, virus-like particles, composed of the L1 capsid protein of TmPV1, were generated with a baculovirus expression system and used to measure anti-TmPV1 antibodies in an enzyme-linked immunosorbent assay. Serologic analysis of 156 manatees revealed a TmPV1 antibody prevalence of 26.3%, with no significant difference between captive (n=39) and wild (n=117) manatees (28.2% and 25.6%, respectively). No antibody-positive wild animal showed PV-induced cutaneous lesions, whereas papillomatosis was observed in 72.7% of antibody-positive captive manatees. Our data indicate that Florida manatees living in the wild are naturally infected by TmPV1 but rarely show TmPV1-induced papillomatosis. Hence, it appears that the wild population would not be harmed in a case of contact with captive animals without visible lesions and productive infections, which could be thus released into the wild.

A papillomavirus (PV) that naturally infects laboratory mice will provide an extremely valuable tool for PV research. We describe here the isolation, cloning and molecular analysis of the first novel laboratory-mouse PV, designated MusPV. This agent, recently identified in the tissues from florid and asymmetrical papillomas on the face of nude mice (NMRI-Foxn1(nu)/Foxn1(nu)), was demonstrated to be transmissible to immunocompetent mice (Ingle et al., 2010). The MusPV genome is 7510 bp in length, is organized similarly to those of other PVs and has at least seven ORFs (E1, E2, E4, E6, E7, L1 and L2). Phylogenetic analysis indicates that MusPV belongs to the π genus together with four other rodent PVs (McPV2, MaPV1, MmiPV and RnPV1). Of the rodent PVs, MusPV appears most closely related to Mastomys coucha PV (McPV2), with 65 % genomic homogeneity and 80 % L1 amino acid similarity. Rodent PVs, except for MnPV1, do not contain any identifiable retinoblastoma protein (RB) binding sites. MusPV has one putative RB-binding site on the E6 protein but not on the E7 protein. Non-coding regions (NCRs) of PVs maintain multiple binding sites for transcription factors (TFs). The NCR of MusPV has numerous sites for TF binding, of which at least 13 TFs are common to all PVs in the π genus. MusPV provides a potentially valuable, novel mouse model to study mechanisms of infection, oncology and novel preventive and therapeutic approaches in mice that can be translated to diseases caused by human PVs.

Certain types of human papillomavirus (HPV) induce cancers, especially cervical cancers in women. A meta-analysis of the literature suggests that HPV is also associated with 20%-25% of non small cell lung carcinoma (NSCLC). Merkel cell Polyomavirus (MCPyV) causes most Merkel cell carcinomas in immunocompromised hosts, and is associated with some squamous carcinomas of skin in immunocompetent individuals. Since both oncogenic viruses appear to involve the tonsils and, therefore, have clear access to the lungs, we examined that the possible association of HPV and MCPyV infections with lung cancers, especially, NSCLC. DNAs were extracted from 51 frozen tissues from 30 lung cancer patients, and examined for the presence of HPV and MCPyV by PCR and DNA sequencing analysis. Clinical data was correlated with the viral status. HPVs were only detected in 5 adenocarcinomas (16.7% of all lung cancers examined). Three were positive for HPV-16, 1 for HPV-11 and 1 had an unknown HPV type DNA. None was identified in benign tissue. MCPyV DNA was detected in 5 NSCLCs (16.7%). Three of the 5 were identified in squamous carcinomas, 1 in adenocarcinoma, and 1 in an unspecified NSCLC. Two additional samples were positive for MCPyV DNA within benign adjacent lung tissue only. In one adenocarcinoma, HPV-11 was identified in an adenocarcinoma, and MCPyV DNA was detected in the adjacent "benign" tissue. HPV and MCPyV were directly associated with 33.3% of NSCLC. Further studies are necessary to determine if polyomavirus and papillomavirus are necessary risk factors for some cases of NSCLC., (Copyright © 2010. Published by Elsevier Inc.)

Genital epithelial tumors of Atlantic bottlenose dolphins (Tursiops truncatus [Tt]) and Burmeister's porpoises (Phocoena spinipinnis) were formerly shown to be associated with papillomavirus (PV) infection. Papillomaviruses are highly prevalent viruses involved in the development of various tumor types in a wide range of animals, and so-called high-risk PVs contribute to malignant progression. In marine mammals, the incidence and prevalence of PV infection, transmission pathways, and persistence of infection are largely unknown. Using virus-like particles of bottlenose dolphin PV type 1 (TtPV1) as the antigen, enzyme-linked immunosorbent assay (ELISA) studies were conducted to evaluate PV antibody prevalence in bottlenose dolphins. In total, sera obtained from 115 dolphins were examined. Fifty-one percent of captive dolphins (n=18 of 35) and 90% of free-ranging dolphins (n=72 of 80) were antibody positive. Higher ELISA reactivity was observed among males compared with females. Sexually immature dolphins appeared more likely to seroconvert with age. Besides determining their PV antibody prevalence, each animal was also assessed for the presence of orogenital tumors. Interestingly, the mean age of free-ranging dolphins with tumors (n=21) was 11.2 yr compared with 29.9 yr in captive dolphins with tumors (n=9). Results from the current study suggest PV infection in bottlenose dolphins is common, that the main route of PV transmission among them may be horizontal, and that orogenital neoplasia may develop in early life stages of certain free-ranging bottlenose dolphins.

Goal: Human Papillomavirus (HPV) is found to be increasingly implicated in head and neck cancers. The objective of this study was to determine the primary site of origin of HPV positive squamous carcinomas metastatic to lymph nodes of the neck., Methods: Surgical pathology records from January 1, 2000 to July 31, 2007 were used to identify surgically removed neck lymph nodes with the diagnosis of metastatic squamous carcinoma. Specimens in formalin-fixed, paraffin-embedded blocks were examined for HPV (+) by analyzing sequencing data generated by PCR and immunostaining for the expression of the p16INK biomarker, which is overexpressed if Rb is not present. H & E stained slides were also reviewed for histological classification. The available retrospective demographics were extracted from the charts to determine trends of confounding factors., Results: Of the 43 patient samples, 41 contained adequate DNA to test for HPV. The mean age of the 41 patients was 62 years. All of the patients smoked and 39/41 patients consumed alcohol. The overall HPV (+) incident rate was 27% (11/41) by PCR with strongly diffuse or strong focal p16 staining. 9 of the 34 males and 2 of the 7 females had HPV (+) carcinomas. The average age of the 2 HPV (+) females was 44, compared to the HPV (-) females who averaged 70. The average age of the HPV (+) males was 56 compared with the average age 55 of the HPV (-) males. HPV (+) carcinomas appeared to arise from multiple sites in the oropharynx, particularly the tonsils and tongues, including unknown primaries. By histological exam, most metastatic HPV(+) squamous carcinomas were poorly differentiated (basaloid) microscopically and grossly cystic., Conclusion: The 27% HPV (+) squamous cancers metastatic to neck lymph node originated from multiple sites in the oropharynx. The HPV (+) female population, although a total of only 2, tended to be much younger than the HPV (-) ones, whereas the HPV (+) male population was similar in age to the HPV (-) male population.

Papillomaviruses are small nonenveloped DNA viruses that infect squamous epithelial cells. These viruses have been found in many organisms. Human papillomaviruses (HPVs) give rise to a large spectrum of epithelial lesions, mainly benign hyperplasia (eg, warts and papillomas) with low malignant potential. There is a subgroup of HPV, the "high-risk" HPV, which is associated with precancerous and cancerous lesions. A small fraction of people infected with high-risk HPV will develop cancers that usually arise many years after the initial infection (Psyrri and Dimaio, Nat Clin Pract Oncol. 2008;5:24-31). Nonsmall cell lung cancer is a heterogeneous disease. The most common histologic subtypes include squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Despite different histologies, nonsmall cell lung cancers are often classified together because of similarities in approach and management of the disease. In this article, we reviewed the current literature on lung cancer and HPV. On the basis of this data, we suggested a possible mechanism of carcinogenesis induced by HPV.

Carcinogenic human papillomaviruses (HPVs) that cause cervical cancer preferentially infect basal, metaplastic squamous cells of the transformation zone. If infection persists, and a vegetative infection ensues, a premalignant lesion may develop with the potential to progress into an invasive squamous cell carcinoma. Papillomavirus prophylactic vaccines target the systemic immune system for induction of neutralizing antibodies that protect the basal cells against infection. Because the carcinogenic HPVs are susceptible to neutralization by antibodies for 9-48 h after reaching the basal cells, both low and high titered HPV type-specific antibodies induced by HPV L1 and L2-based vaccines are highly efficacious. The greatest burden of HPV-associated cancers occurs in poor areas of the world where women do not have access to routine gynecological care. The burden of HIV/AIDS in these same regions of the world has added to the burden of HPV-associated disease. There is an urgent need for a cost-effective, broad-spectrum HPV prophylactic vaccine in developing countries, which necessitates substantial cost subsidization of the virus-like particle (VLP) based vaccines licensed in industrialized countries or an alternative approach with second-generation vaccines that are specifically designed for delivery to women in resource-poor communities.

Papillomaviruses (PVs) are a group of small, non-enveloped DNA viruses that cause mucosal or cutaneous neoplasia in a variety of animals. Whilst most papillomas will regress spontaneously, some may persist or undergo malignant transformation. In this study, aggressive, persistent and extensive warts were observed on the hands and feet of a cynomolgus macaque (Macaca fascicularis). The presence of PV in the wart biopsies was identified by immunohistochemistry and PCR amplification of PV DNA. The genomic DNA of this PV was cloned and sequenced, and the PV was designated M. fascicularis papillomavirus type 1 (MfPV-1). Its genome was 7588 bp in length and the organization of its putative open reading frames (E1, E2, E6, E7, L1, L2 and E4) was similar to that of other PVs. MfPV-1 had a short non-coding region (NCR) of 412 bp. Molecular analysis of MfPV-1 genomic DNA classified it into the genus Betapapillomavirus, to which all epidermodysplasia verruciformis (EV)-type PVs belong. Diseases caused by PVs of the genus Betapapillomavirus are usually associated with natural or iatrogenic immunosuppression. The genomic characterization performed in this study showed that MfPV-1 clustered within the genus Betapapillomavirus and also contained EV-type-specific motifs in its NCR. Further characterization of this virus and its host interactions may allow us to develop a non-human primate model for human betapapillomaviruses, a genus populated by human PV types causing EV.

Papillomaviruses (PVs) have been shown as being the etiologic agents of various benign and malignant tumours in many vertebrate species. In dolphins and porpoises, a high prevalence of orogenital tumours has recently been documented with at least four distinct novel species-specific PV types detected in such lesions. Therefore, we generated the immunological reagents to establish a serological screening test to determine the prevalence of PV infection in Atlantic bottlenose dolphins [(Tursiops truncatus (Tt)]. Using the baculovirus expression system, virus-like particles (VLPs) derived from the L1 proteins of two TtPV types, TtPV1 and TtPV2, were generated. Polyclonal antibodies against TtPV VLPs were produced in rabbits and their specificity for the VLPs was confirmed. Electron microscopy and enzyme-linked immunosorbent assay (ELISA) studies revealed that the generated VLPs self-assembled into particles presenting conformational immunodominant epitopes. As such, these particles are potential antigen candidates for a TtPV vaccine. Subsequently, the VLPs served as antigens in initial ELISA tests using sera from six bottlenose dolphins to investigate PV antibody presence. Three of these sera were derived from dolphins with genital tumour history and showed positive PV ELISA reactivity, while the remaining sera from lesion-free dolphins were PV antibody-negative. The results suggest that the developed screening test may serve as a potential tool for determining PV prevalence and thus for observing transmission rates in dolphin populations as the significance of PV infection in cetaceans starts to unfold.

Human papilloma virus (HPV) is the major cause of invasive cervical cancer (ICC). The study aim was to determine the prevalence of HPV genotypes and to correlate HPV types with response to radiotherapy. A total of 43 cervical biopsies collected from sequentially enrolled patients were analyzed by DNA amplification with MY09/MY11 primers and sequenced to determine the HPV genotype. Samples with multiple infections were resolved by multiplex PCR, combined with array primer extension (APEX). HPV DNA was detected in 40 of 43 (93%) samples. Nine different HPVs, including the most common types -16 (53%) and -18 (13%) were detected. Other types were HPV 31, 33, 45, 52, 58, 66 and 68. Single HPV types were found in 33 of 40 samples (82%) and multiple types in 7 of 40 samples (18%). The following significant predictors were identified: a) HPV 58 was most significant (p=0.02), followed by HPV 18 (p=0.04) associated with lack of treatment response; b) tumor size (p=0.042) and treatment response (p=0.025) elicited association with HPV infection type; c) treatment failure were found to be nearly 5-fold higher in case of multiple infections than of single infection (57% versus 12%) (odds ratio = 9.66; 95% CI 1.6-6.00). d) Multiple HPV infections correlated most prominently with lack of treatment compared with single type infection (p=0.005). Hence, patients with multiple infections, large tumor size, and HPV 58 and/or 18, are at risk of treatment failure and need to be followed for response and suitable inter-ventions done for a favorable outcome.

Phylogenetic analysis of novel dolphin (Tursiops truncatus) papillomavirus sequences, TtPV1, -2, and -3, indicates that the early and late protein coding regions of their genomes differ in evolutionary history. Sliding window bootscan analysis showed a significant a change in phylogenetic clustering, in which the grouped sequences of TtPV1 and -3 move from a cluster with the Phocoena spinipinnis PsPV1 in the early region to a cluster with TtPV2 in the late region. This provides indications for a possible recombination event near the end of E2/beginning of L2. A second possible recombination site could be located near the end of L1, in the upstream regulatory region. Selection analysis by using maximum likelihood models of codon substitutions ruled out the possibility of intense selective pressure, acting asymmetrically on the viral genomes, as an alternative explanation for the observed difference in evolutionary history between the early and late genomic regions of these cetacean papillomaviruses.

The papillomaviruses form a large group of species-specific pathogens that cause epithelial proliferations in a wide spectrum of animal hosts. Previous reports demonstrated a relatively high frequency of a variety of skin lesions in captive European harvest mice. The Micromys minutus papillomavirus (MmPV) was isolated from one of these lesions found on a captive European harvest mouse in a regional zoo in Chicago. In this study we present the entire genomic sequence of MmPV. The MmPV genome is organized into the seven classical papillomaviral open reading frames. Phylogenetic analysis places MmPV together with a papillomavirus (PV) isolated from a Syrian golden Hamster (HaOPV) in the genus Pipapillomavirus. The similar clustering pattern of the MmPV-HaOPV pair and their rodent hosts support the hypothesis of papillomaviral and host co-phylogenetic descent. The availability of the complete genomic sequence of a mouse PV should allow researchers to use MmPV as a model for PV carcinogenesis.

Background: Estimating evolutionary rates for slowly evolving viruses such as papillomaviruses (PVs) is not possible using fossil calibrations directly or sequences sampled over a time-scale of decades. An ability to correlate their divergence with a host species, however, can provide a means to estimate evolutionary rates for these viruses accurately. To determine whether such an approach is feasible, we sequenced complete feline PV genomes, previously available only for the domestic cat (Felis domesticus, FdPV1), from four additional, globally distributed feline species: Lynx rufus PV type 1, Puma concolor PV type 1, Panthera leo persica PV type 1, and Uncia uncia PV type 1., Results: The feline PVs all belong to the Lambdapapillomavirus genus, and contain an unusual second noncoding region between the early and late protein region, which is only present in members of this genus. Our maximum likelihood and Bayesian phylogenetic analyses demonstrate that the evolutionary relationships between feline PVs perfectly mirror those of their feline hosts, despite a complex and dynamic phylogeographic history. By applying host species divergence times, we provide the first precise estimates for the rate of evolution for each PV gene, with an overall evolutionary rate of 1.95 x 10(-8) (95% confidence interval 1.32 x 10(-8) to 2.47 x 10(-8)) nucleotide substitutions per site per year for the viral coding genome., Conclusion: Our work provides evidence for long-term virus-host co-speciation of feline PVs, indicating that viral diversity in slowly evolving viruses can be used to investigate host species evolution. These findings, however, should not be extrapolated to other viral lineages without prior confirmation of virus-host co-divergence.

A novel papillomavirus (PV) was isolated from a genital condyloma of a free-ranging bottlenose dolphin inhabiting the coastal waters of Charleston Harbor, SC, USA: Tursiops truncatus papillomavirus type 2 (TtPV2). This novel virus represents the first isolated North American cetacean PV and the first American bottlenose dolphin PV. After the viral genome was cloned, sequenced and characterized genetically, phylogenetic analyses revealed that TtPV2 is most similar to the only published cetacean PV isolated and characterized thus far, Phocoena spinipinnis PV type 1 (PsPV1). A striking feature of the genome of TtPV2, as well as that of PsPV1, is the lack of an E7 open reading frame, which typically encodes one of the oncogenic proteins believed to be responsible for malignant transformation in the high-risk mucosotropic human papillomaviruses (HPVs). TtPV2 E6 contains a PDZ-binding motif that has been shown to be involved in transformation in the case of high-risk genital HPVs.

Equus caballus papillomavirus type 1 (EcPV-1) was isolated from a cutaneous papilloma, the most common neoplasm in horses. The complete EcPV-1 nucleotide sequence and genomic organization were determined. Phylogenetic analysis showed that EcPV-1 is a close-to-root papillomavirus, with only distant relationships to the fibropapillomaviruses and the benign cutaneous papillomaviruses. To produce EcPV-1 virus-like particles (VLPs), the EcPV-1 L1 major capsid protein was expressed in insect cells using a recombinant baculovirus vector. The self-assembled EcPV-1 VLPs were morphologically indistinguishable from wild type papillomavirus virions. Monoclonal antibodies were developed against intact and denatured EcPV-1 VLPs. When tested by ELISA, all monoclonal antibodies produced against intact (#18) and some against denatured EcPV-1 VLPs (#16) reacted with intact EcPV-1 VLPs only, demonstrating that the VLPs carry type-specific conformational as well as linear epitopes on their surface. Recombinant EcPV-1 VLPs offer the potential of a noninfectious vaccine to prevent and eradicate equine cutaneous papillomatosis.

By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (Trichechus manatus latirostris), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the Trichechus manatus latirostris papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of Sirenia. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus.

The Florida manatee (Trichechus manatus latirostris) is one of the most endangered marine mammals in American coastal waters. Naturally resistant to infectious disease, the manatee immune system appears highly developed to protect it against the harsh marine environment and the effects of human-related injury. In 1997, seven captive Florida manatees developed multiple, cutaneous, pedunculated papillomas over a period of 6 months. Approximately 3 years later, four of the seven manatees developed multiple, cutaneous, sessile papillomas topically and clinically distinct from the initial lesions, some of which are still present. Histologic, ultrastructural, and immunohistochemical features indicated that the two distinct phenotypic lesions were caused by papillomaviruses (PVs). Preliminary immunologic data correlated with daily clinical observations suggested that the manatees were immunologically suppressed and that the papillomas were caused by activation of latent PV infections and reinoculation from active infections. The emergence of PV-induced papillomas in captive manatees, the possibility of activation of latent infection or transmission of active infection to free-ranging manatees, and the underlying cause of immune suppression predisposing manatees to develop viral papillomatosis are serious concerns for the future management of this highly endangered species., (Copyright 2002 Elsevier Science.)

Cervical cancer is a sexually transmitted disease caused by the human papillomavirus (HPV), especially HPV-16 and -18. Of the half million new cases of cervical cancer reported yearly, 20% occur in India. Mass cancer screening programs to detect and treat cervical cancer and its precursor lesions are not available in India and most other developing countries because of the lack of resources. Curative and palliative treatments are not the same for all patients with cervical cancer because the result depends on the immunological response of the patient. This article describes the natural history of cervical carcinogenesis and the rational behind various modalities of prevention and treatment for the practising gynecological oncologist. Prophylactic vaccines against HPV-16 and -18 and therapeutic vaccines against cervical cancers should be able to overcome the logistical problems that now exist to screen, diagnose and treat cervical cancer and its precursor lesions.

The pathogenesis of carcinogenic human papillomavirus (HPV) infections of the cervix includes early induction of peripheral tolerance of tissue-infiltrating lymphocytes and an imbalanced Th2 response to HPV early virus proteins. As lesions become progressively dysplastic, major histocompatibility complex (MHC)-1 molecules are down-regulated on the surface of abnormal keratinocytes. When the target of MHC-1 class-restricted cytotoxic lymphocytes disappears, immune deviation to a Th2 response becomes more dominant. After severely dysplastic lesions become invasive, cervical cancer cells die and release HPV E6 and E7 oncoproteins that react with anti-E6 and anti-E7 antibodies to form insoluble immune complexes in antibody excess under the continuing influence of immune deviation. On the basis of this knowledge of the pathogenesis of advanced cervical cancer, we believe that successful immunotherapeutic treatments of these patients will use a vaccine formulation that will break peripheral tolerance in association with biological response modifiers that will enable the patient's immune system to switch classes from Th2 to Th1 while up-regulating MHC-1 molecules on cancer cells. Like prophylactic vaccines against HPV, successful therapeutic vaccine against cervical cancer may have to be universal rather than individualized to be efficacious., (Copyright 2001 Elsevier Science.)

Persistent infection with human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Neutralizing epitopes present on the major coat protein, L1, have not been well characterized, although three neutralizing monoclonal antibodies (MAbs) had been identified by using HPV-16 pseudovirions (R. B. Roden et al., J. Virol. 71:6247-6252, 1997). Here, two of these MAbs (H16.V5 and H16.E70) were demonstrated to neutralize authentic HPV-16 in vitro, while the third (H16.U4) did not. Binding studies were conducted with the three MAbs and virus-like particles (VLPs) composed of the reference L1 sequence (114K) and three variant L1 sequences: Rochester-1k (derived from viral stock DNA), GU-1 (derived from cervical biopsy DNA), and GU-2 (derived from biopsy DNA, but containing some sequence changes likely to be artifactual). While all three MAbs bound to 114K and Rochester-1k VLPs, GU-1 VLPs were not recognized by H16.E70, and both H16.E70 and H16.V5 failed to bind to GU-2 VLPs. Site-directed mutagenesis was used to replace disparate amino acids in the GU-2 L1 with those found in the 114K L1. Alteration of the amino acid at position 50, from L to F, completely restored H16.V5 binding and partially restored H16.E70 binding, while complete restoration of H16.E70 binding occurred with GU-2 VLPs containing both L50F and T266A alterations. Immunization of mice with L1 variant VLPs revealed that GU-2 VLPs were poorly immunogenic. The L50F mutant of GU-2 L1, in which the H16.V5 epitope was restored, elicited HPV-16 antibody responses comparable to those obtained with 114K VLPs. These results demonstrate the importance of the H16.V5 epitope in the generation of potent HPV-16 neutralizing antibody responses.

There is increasing evidence for widespread occurrences of infection with Epidermodysplasia verruciformis-related human papillomaviruses, both in the general population and in immunosuppressed patients. In order to test for the prevalence of antibodies directed against the native L1 epitopes exposed on the surface of the virions, we have established an IgG-specific enzyme-linked immunosorbent assay with L1 virus-like particles of the Epidermodysplasia verruciformis-specific human papillomavirus 8 as antigen to screen 567 representative serum samples from the general population and immunosuppressed/dermatologic patients. Among healthy European donors (n = 210), 7.6% were found to be seropositive. In a group of renal transplant recipients (n = 185) the antibody prevalence was elevated to 21.1%, irrespective of the presence or absence of skin cancer. High positivity rates could be detected among (i) immunocompetent patients with nonmelanoma skin tumors (45.6%, n = 79) and (ii) Psoralene/UVA treated psoriasis patients (42.9%, n = 42). In contrast, anti-human papillomavirus 8-virus-like particle antibodies were found in only 6.8% of Hodgkin lymphoma patients (n = 44).

Recently, the L1 capsid protein of canine oral papillomavirus (COPV) has been used as an effective systemic vaccine that prevents viral infections of the oral mucosa. The efficacy of this vaccine is critically dependent upon native L1 conformation and, when purified from Sf9 insect cells, the L1 protein not only displays type-specific, conformation-dependent epitopes but it also assembles spontaneously into virus-like particles (VLPs). To determine whether VLP formation was coupled to the expression of conformation-dependent epitopes, we generated a series of N- and C-terminal L1 deletion mutants and evaluated their ability to form VLPs (by electron microscopy) and to react with conformation-dependent antibodies (by immunofluorescence microscopy). We found that (a) deletion of the 26 C-terminal residues generated a mutant protein which formed VLPs efficiently and folded correctly both in the cytoplasm and in the nucleus; (b) further truncation of the L1 C terminus (67 amino acids) resulted in a capsid protein which formed VLPs but which failed to express conformational epitopes; (c) deletion of the first 25 N-terminal amino acids also abolished expression of conformational epitopes (without altering VLP formation) but the native conformation of this deletion mutant could be restored by the addition of the human papillomavirus type 11 N terminus. These results demonstrate that VLP formation and conformational epitope expression can be dissociated and that the L1 N terminus has a critical role in protein folding. In addition, it appears that correct L1 protein folding is not dependent upon the nucleoplasmic environment.

This review concentrates on recent advances in human papillomavirus vaccine development. Strategies for prophylactic HPV subunit vaccines utilizing recombinantly synthesized, immunogenic virus-like particles are discussed. Therapeutic strategies focusing on the induction of cell-mediated immunity and gene manipulation for the treatment of established HPV-associated disease are also reviewed.

During the last 10 yr, we have derived monoclonal antibodies from animals immunized with denatured bovine papillomaviruses type 1 major capsid (L1) protein, mapped their corresponding immunodominant epitopes to within a single amino acid (aa), and compared the reactivity of authentic L1 proteins to the predicted response by collinear analysis of the aa sequences of the same and other papillomaviruses (PVs). The data obtained from this approach has provided us with new insights into the sensitivity and specificity of the antibody response to viral proteins. We have included here some observations and conclusions that appear to be generic for the immune response, some of which might have applications for working with linear epitopes in other experimental systems.

Virus-like-particles (VLPs) of various papillomavirus (PV) types have been produced by expressing recombinant L1 proteins in eukaryotic cells. Although VLPs have the same ultrastructural appearance as native virions and their immunogenicity appears to be similar, their antigenicity has not been carefully evaluated. For this reason, the antigenicity of intact bovine PV type 1 (BPV-1) virions was compared with that of BPV-1 recombinant L1 VLPs by ELISA using a well-characterized panel of polyclonal and monoclonal antibodies generated against intact and denatured BPV-1 particles. The structural integrity of the authentic virions and recombinant VLPs was verified by electron microscopy. The specificity of antibodies raised against intact BPV-1 virions and their reactivity with VLPs revealed that the immunodominant, type-specific, conformational epitopes of intact virions were reproduced on VLPs. However, many monoclonal antibodies that define cross-reactive, non-conformational (linear) epitopes cryptic to the authentic BPV-1 virion tested positively when reacted with intact VLPs. One monoclonal antibody, which recognizes a BPV-1 and deer PV surface conformational epitope, did not react with VLPs. Therefore, although VLPs can be used to immunize animals against infection, the external exposure of broadly cross-reactive epitopes of intact L1 VLPs suggests that the use of L1 VLPs in antigenicity studies such as serological screening should be done with caution.

Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.

Antibodies reactive to HPV types 6 and 11 were tested in ELISA and HPV-11 neutralization assays to determine whether these closely related types shared cross-reactive neutralizing epitopes. A series of HPV-11 neutralizing monoclonal antibodies (N-MAbs) that targeted conformational epitopes on infectious HPV-11 and HPV-11 L1 virus-like particles (VLPs) were tested for type-specificity of reactivity using intact HPV-6 L1 VLPs. Polyclonal antisera generated against intact HPV-6 L1 VLPs were also tested for HPV-11 neutralizing capacity using the athymic mouse xenograft system. The results demonstrated that conformationally dependent neutralizing epitopes on HPV-11 were very type-specific. Three of the four HPV-11 N-MAbs were negative for binding to HPV-6 L1 VLP, and the fourth demonstrated binding to HPV-6 L1 VLPs that was several orders of magnitude weaker than its binding to HPV-11 L1 VLP. The polyclonal anti-HPV-6 L1 VLP antiserum was only partially protective against HPV-11 infectivity even at a low dilution of 1:100. In contrast, polyclonal anti-HPV-11 L1 VLP antiserum was completely protective at dilutions greater than 1:10,000.

Human papillomaviruses (HPVs) cause a variety of cutaneous warts, mucosal condylomata, and dysplasias and are etiologic in cervical cancer. Papillomavirus (PV) conformational epitopes on the surface of virions are type-specific and are the target of neutralizing antibodies. In this study, we describe two methods of in vitro expression of HPV major capsid (L1) proteins which mimicked conformational epitopes and demonstrate their type specificity and ability to react with neutralizing and/or conformation-dependent antibodies. The L1 open reading frames (ORFs) for HPV-1, 6, 11, and 16 were molecularly cloned into a SV 40 expression vector and the encoded gene products were expressed in mammalian (cos) cells. Similarly, the L1 ORFs for HPV-6, 11, 16, and 18 were molecularly cloned into recombinant baculovirus and the encoded gene products were expressed in insect (SF9) cells. The expressed L1 proteins reacted by immunofluorescence and immunoprecipitation with polyclonal and monoclonal antibodies generated against their corresponding native virions and by Western blotting with antibodies that recognized nonconformational epitopes of denatured virions. The recombinant L1 proteins expressed conformational epitopes in both cos and Sf9 cells that were type-specific and displayed neutralizing epitopes. The ability to express, purify, and qualitate the reactivity of recombinant L1 proteins will now permit the serologic analysis of host response to HPV infection and the development of prophylactic PV subunit vaccines.

Previous studies demonstrated that the human papillomavirus (HPV) type 1 L1 protein, expressed in cos cells by an SV40-based vector, displays conformational epitopes characteristic of native virions. In this study, we analyzed the expression of HPV-1, HPV-6, and HPV-11 L1 proteins in order to determine the forms of conformational epitopes expressed by recombinant L1 proteins. Using both immunofluorescence and immunoprecipitation techniques, polyclonal and monoclonal antibodies (MAbs) generated against native HPV-11 virions reacted with expressed L1 proteins of HPV-6 and/or HPV-11, but not HPV-1. Similarly, polyclonal antibodies and MAbs generated against HPV-1 virions reacted with the expressed L1 protein of HPV-1, but not HPV-6 or HPV-11. Of two MAbs that neutralized HPV-11 infection of murine fetal foreskin xenografts, one reacted with the expressed L1 protein of both HPV-6 and HPV-11, and the other reacted with HPV-11 only. A nonneutralizing conformationally dependent MAb reacted with the expressed L1 protein of both HPV-6 and HPV-11. These results demonstrate that expressed HPV L1 proteins retain type-specific, neutralizing, and nonneutralizing conformational epitopes and that cos cells may be utilized to evaluate host immune responses to such epitopes.