Immune Responses in Oral Papillomavirus Clearance in the MmuPV1 Mouse Model.

Human papillomavirus (HPV)-induced oropharyngeal cancer now exceeds HPV-induced cervical cancer, with a noticeable sex bias. Although it is well established that women have a more proficient immune system, it remains unclear whether immune control of oral papillomavirus infections differs between sexes. In the current study, we use genetically modified mice to target CCR2 and Stat1 pathways, with the aim of investigating the role of both innate and adaptive immune responses in clearing oral papillomavirus, using our established papillomavirus (MmuPV1) infection model. Persistent oral MmuPV1 infection was detected in Rag1ko mice with T and B cell deficiencies. Meanwhile, other tested mice were susceptible to MmuPV1 infections but were able to clear the virus. We found sex differences in key myeloid cells, including macrophages, neutrophils, and dendritic cells in the infected tongues of wild type and Stat1ko mice but these differences were not observed in CCR2ko mice. Intriguingly, we also observed a sex difference in anti-MmuPV1 E4 antibody levels, especially for two IgG isotypes: IgG2b and IgG3. However, we found comparable numbers of interferon-gamma-producing CD8 T cells stimulated by E6 and E7 in both sexes. These findings suggest that males and females may use different components of innate and adaptive immune responses to control papillomavirus infections in the MmuPV1 mouse model. The observed sex difference in immune responses, especially in myeloid cells including dendritic cell (DC) subsets, may have potential diagnostic and prognostic values for HPV-associated oropharyngeal cancer.

Passive Immunization with a Single Monoclonal Neutralizing Antibody Protects against Cutaneous and Mucosal Mouse Papillomavirus Infections.

We have established a mouse papillomavirus (MmuPV1) model that induces both cutaneous and mucosal infections and cancers. In the current study, we use this model to test our hypothesis that passive immunization using a single neutralizing monoclonal antibody can protect both cutaneous and mucosal sites at different time points after viral inoculation. We conducted a series of experiments involving the administration of either a neutralizing monoclonal antibody, MPV.A4, or control monoclonal antibodies to both outbred and inbred athymic mice. Three clinically relevant mucosal sites (lower genital tract for females and anus and tongue for both males and females) and two cutaneous sites (muzzle and tail) were tested. At the termination of the experiments, all tested tissues were harvested for virological analyses. Significantly lower levels of viral signals were detected in the MPV.A4-treated female mice up to 6 h post-viral inoculation compared to those in the isotype control. Interestingly, males displayed partial protection when they received MPV.A4 at the time of viral inoculation, even though they were completely protected when receiving MPV.A4 at 24 h before viral inoculation. We detected MPV.A4 in the blood starting at 1 h and up to 8 weeks postadministration in some mice. Parallel to these in vivo studies, we conducted in vitro neutralization using a mouse keratinocyte cell line and observed complete neutralization up to 8 h post-viral inoculation. Thus, passive immunization with a monoclonal neutralizing antibody can protect against papillomavirus infection at both cutaneous and mucosal sites and is time dependent. IMPORTANCE This is the first study testing a single monoclonal neutralizing antibody (MPV.A4) by passive immunization against papillomavirus infections at both cutaneous and mucosal sites in the same host in the mouse papillomavirus model. We demonstrated that MPV.A4 administered before viral inoculation can protect both male and female athymic mice against MmuPV1 infections at cutaneous and mucosal sites. MPV.A4 also offers partial protection at 6 h post-viral inoculation in female mice. MPV.A4 can be detected in the blood from 1 h to 8 weeks after intraperitoneal (i.p.) injection. Interestingly, males were only partially protected when they received MPV.A4 at the time of viral inoculation. The failed protection in males was due to the absence of neutralizing MPV.A4 at the infected sites. Our findings suggest passive immunization with a single monoclonal neutralizing antibody can protect against diverse papillomavirus infections in a time-dependent manner in mice.

Depo Medroxyprogesterone (DMPA) Promotes Papillomavirus Infections but Does Not Accelerate Disease Progression in the Anogenital Tract of a Mouse Model.

Contraceptives such as Depo-medroxyprogesterone (DMPA) are used by an estimated 34 million women worldwide. DMPA has been associated with increased risk of several viral infections including Herpes simplex virus-2 (HSV-2) and Human immunodeficiency virus (HIV). In the current study, we used the mouse papillomavirus (MmuPV1) anogenital infection model to test two hypotheses: (1) contraceptives such as DMPA increase the susceptibility of the anogenital tract to viral infection and (2) long-term contraceptive administration induces more advanced disease at the anogenital tract. DMPA treatments of both athymic nude mice and heterozygous NU/J (Foxn1nu/+) but ovariectomized mice led to a significantly increased viral load at the anogenital tract, suggesting that endogenous sex hormones were involved in increased viral susceptibility by DMPA treatment. Consistent with previous reports, DMPA treatment suppressed host anti-viral activities at the lower genital tract. To test the impact of long-term contraceptive treatment on the MmuPV1-infected lower genital tract, we included two other treatments in addition to DMPA: 17ß-estradiol and a non-hormone based contraceptive Cilostazol (CLZ, Pletal). Viral infections were monitored monthly up to nine months post infection by qPCR. The infected vaginal and anal tissues were harvested and further examined by histological, virological, and immunological analyses. Surprisingly, we did not detect a significantly higher grade of histology in animals in the long-term DMPA and 17ß-estradiol treated groups when compared to the control groups in the athymic mice we tested. Therefore, although DMPA promotes initial papillomavirus infections in the lower genital tract, the chronic administration of DMPA does not promote cancer development in the infected tissues in our mouse model.

The environmental pollutant and tobacco smoke constituent dibenzo[def,p]chrysene is a co-factor for malignant progression of mouse oral papillomavirus infections.

HPV infections in the oral cavity that progress to cancer are on the increase in the USA. Model systems to study co-factors for progression of these infections are lacking as HPVs are species-restricted and cannot grow in preclinical animal models. We have recently developed a mouse papillomavirus (MmuPV1) oral mucosal infection model that provides opportunities to test, for the first time, the hypothesis that tobacco carcinogens are co-factors that can impact the progression of oral papillomas to squamous cell carcinoma (SCC). Four cohorts of mice per sex were included: (1) infected with MmuPV1 and treated orally with DMSO-saline; (2) infected with MmuPV1 and treated orally with the tobacco carcinogen, dibenzo[def,p]chrysene (DBP); (3) uninfected and treated orally with DMSO-saline, and (4) uninfected and treated orally with DBP. Oral swabs were collected monthly for subsequent assessment of viral load. Oral tissues were collected for in situ viral DNA/RNA detection, viral protein staining, and pathological assessment for hyperplasia, papillomas and SCC at study termination. We observed increased rates of SCC in oral tissue infected with MmuPV1 and treated with DBP when compared to mice treated with DBP or virus individually, each of which showed minimal disease. Virally-infected epithelium showed strong levels of viral DNA/RNA and viral protein E4/L1 staining. In contrast, areas of SCC showed reduced viral DNA staining indicative of lower viral copy per nucleus but strong RNA signals. Several host markers (p120 ctn, p53, S100A9) were also examined in the mouse oral tissues; of particular significance, p120 ctn discriminated normal un-infected epithelium from SCC or papilloma epithelium. In summary, we have confirmed that our infection model is an excellent platform to assess the impact of co-factors including tobacco carcinogens for oral PV cancerous progression. Our findings can assist in the design of novel prevention/treatment strategies for HPV positive vs. HPV negative disease.

Antibody-Mediated Immune Subset Depletion Modulates the Immune Response in a Rabbit (Oryctolagus cuniculus) Model of Epstein-Barr Virus Infection.

Epstein-Barr Virus (EBV) is a γ-herpesvirus which infects over 90% of the adult human population. Most notably, this virus causes infectious mononucleosis but it is also associated with cancers such as Hodgkin and Burkitt lymphoma. EBV is a species-specific virus and has been studied in many animal models, including nonhuman primates, guinea pigs, humanized mice, and tree shrews. However, none of these animal models are considered the "gold standard" for EBV research. Recently, rabbits have emerged as a viable alternative model, as they are susceptible to EBV infection. In addition, the EBV infection progresses after immune suppression with cyclosporine A (CsA), modeling the reactivation of EBV after latency. We sought to refine this model for acute or active EBV infections by performing antibody-mediated depletion of certain immune subsets in rabbits. Fourteen 16 to 20-wk old, NZW rabbits were intravenously inoculated with EBV and concurrently treated with either anti-CD4 T-cell antibody, anti-pan-T-cell antibody (anti CD45), CSA, or, as a control, anti-HPV antibody. Rabbits that received the depleting antibodies were treated with CsA 3 times at a dose of 15 mg/kg SC once per day for 4 d starting at the time of EBV inoculation then the dose was increased to 20 mg/kg SC twice weekly for 2 wk. Weights, temperatures, and clinical signs were monitored, and rabbits were anesthetized once weekly for blood collection. When compared with the control group, anti-CD4-treated rabbits had fewer clinical signs and displayed higher levels of viral DNA via qPCR in splenocytes; however, flow cytometry results showed only a partial depletion of CD4 T-cells. Treatment with anti-pan-T-cell antibody did not result in noticeable T-cell depletion. These data suggest the EBV-infected rabbit is a promising model for testing antiviral medications and prophylactic vaccines for EBV.

Papillomavirus can be transmitted through the blood and produce infections in blood recipients: Evidence from two animal models.

Human papillomaviruses (HPV) contribute to most cervical cancers and are considered to be sexually transmitted. However, papillomaviruses are often found in cancers of internal organs, including the stomach, raising the question as to how the viruses gain access to these sites. A possible connection between blood transfusion and HPV-associated disease has not received much attention. Here we show, in rabbit and mouse models, that blood infected with papillomavirus yields infections at permissive sites with detectable viral DNA, RNA transcripts, and protein products. The rabbit skin tumours induced via blood infection displayed decreased expression of SLN, TAC1, MYH8, PGAM2, and APOBEC2 and increased expression of SDRC7, KRT16, S100A9, IL36G, and FABP9, as seen in tumours induced by local infections. Furthermore, we demonstrate that blood from infected mice can transmit the infection to uninfected animals. Finally, we demonstrate the presence of papillomavirus infections and virus-induced hyperplasia in the stomach tissues of animals infected via the blood. These results indicate that blood transmission could be another route for papillomavirus infection, implying that the human blood supply, which is not screened for papillomaviruses, could be a potential source of HPV infection as well as subsequent cancers in tissues not normally associated with the viruses.

BET bromodomain inhibitors show anti-papillomavirus activity in vitro and block CRPV wart growth in vivo.

The DNA papillomaviruses infect squamous epithelium and can cause persistent, benign and sometimes malignant hyperproliferative lesions. Effective antiviral drugs to treat human papillomavirus (HPV) infection are lacking and here we investigate the anti-papillomavirus activity of novel epigenetic targeting drugs, BET bromodomain inhibitors. Bromodomain and Extra-Terminal domain (BET) proteins are host proteins which regulate gene transcription, they bind acetylated lysine residues in histones and non-histone proteins via bromodomains, functioning as scaffold proteins in the formation of transcriptional complexes at gene regulatory regions. The BET protein BRD4 has been shown to be involved in the papillomavirus life cycle, as a co-factor for viral E2 and also mediating viral partitioning in some virus types. We set out to study the activity of small molecule BET bromodomain inhibitors in models of papillomavirus infection. Several BET inhibitors reduced HPV11 E1ˆE4 mRNA expression in vitro and topical therapeutic administration of an exemplar compound I-BET762, abrogated CRPV cutaneous wart growth in rabbits, demonstrating translation of anti-viral effects to efficacy in vivo. Additionally I-BET762 markedly reduced viability of HPV16 infected W12 cells compared to non-infected C33A cells. The molecular mechanism for the cytotoxicity to W12 cells is unknown but may be through blocking viral-dependent cell-survival factors. We conclude that these effects, across multiple papillomavirus types and in vivo, highlight the potential to target BET bromodomains to treat HPV infection.

Mouse papillomavirus infection persists in mucosal tissues of an immunocompetent mouse strain and progresses to cancer.

Mouse papillomavirus has shown broad tissue tropism in nude mice. Previous studies have tested cutaneous infections in different immunocompromised and immunocompetent mouse strains. In the current study, we examined mucosal infection in several immunocompetent and immunocompromised mouse strains. Viral DNA was monitored periodically by Q-PCR of lavage samples. Immunohistochemistry and in situ hybridization were used to determine viral capsid protein and viral DNA respectively. All athymic nude mouse strains showed active infections at both cutaneous and mucosal sites. Interestingly, NOD/SCID mice, which have a deficiency in T, B, and NK cells, showed minimal disease at cutaneous sites but developed persistent infection at the mucosal sites including those of the anogenital region and the oral cavity. Three strains of immunocompetent mice supported mucosal infections. Infections of the lower genital tract in heterozygous (immunocompetent) mice of the NU/J strain progressed to high grade dysplasia and to carcinoma in situ. Anti-MmuPV1 neutralizing antibodies were detected in the sera of all immunocompetent animals. Our findings demonstrate that the mucosae may be the preferred sites for this virus in mice. The mouse model is expected to be a valuable model for the study of mucosal papillomavirus disease, progression, and host immune control.

High-Resolution Structure Analysis of Antibody V5 and U4 Conformational Epitopes on Human Papillomavirus 16.

Cancers attributable to human papillomavirus (HPV) place a huge burden on the health of both men and women. The current commercial vaccines are genotype specific and provide little therapeutic benefit to patients with existing HPV infections. Identifying the conformational epitopes on the virus capsid supports the development of improved recombinant vaccines to maximize long-term protection against multiple types of HPV. Fragments of antibody (Fab) digested from the neutralizing monoclonal antibodies H16.V5 (V5) and H16.U4 (U4) were bound to HPV16 capsids and the structures of the two virus-Fab complexes were solved to near atomic resolution using cryo-electron microscopy. The structures reveal virus conformational changes, the Fab-binding mode to the capsid, the residues comprising the epitope and indicate a potential interaction of U4 with the minor structural protein, L2. Competition enzyme-linked immunosorbent assay (ELISA) showed V5 outcompetes U4 when added sequentially, demonstrating a steric interference even though the footprints do not overlap. Combined with our previously reported immunological and structural results, we propose that the virus may initiate host entry through an interaction between the icosahedral five-fold vertex of the capsid and receptors on the host cell. The highly detailed epitopes identified for the two antibodies provide a framework for continuing biochemical, genetic and biophysical studies.

Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17-36.

The currently available nonavalent human papillomavirus (HPV) vaccine exploits the highly antigenic L1 major capsid protein to promote high-titer neutralizing antibodies, but is limited to the HPV types included in the vaccine since the responses are highly type-specific. The limited cross-protection offered by the L1 virus-like particle (VLP) vaccine warrants further investigation into cross-protective L2 epitopes. The L2 proteins are yet to be fully characterized as to their precise placement in the virion. Adding to the difficulties in localizing L2, studies have suggested that L2 epitopes are not well exposed on the surface of the mature capsid prior to cellular engagement. Using a series of competition assays between previously mapped anti-L1 monoclonal antibodies (mAbs) (H16.V5, H16.U4 and H16.7E) and novel anti-L2 mAbs, we probed the capsid surface for the location of an L2 epitope (aa17-36). The previously characterized L1 epitopes together with our competition data is consistent with a proposed L2 epitope within the canyons of pentavalent capsomers.

Ranpirnase eradicates human papillomavirus in cultured cells and heals anogenital warts in a Phase I study.

BACKGROUND: Human papillomaviruses (HPV), the causative agents of anogenital warts, are the most prevalent sexually transmitted infectious agents, and wart treatment poses a persistent challenge. We assessed the safety and efficacy of treating HPV with ranpirnase, an endoribonuclease from the northern leopard frog that has been used extensively in Phase III oncology trials. METHODS: As initial verification of ranpirnase antiviral activity, we assessed its ability to eliminate papillomaviruses in cultured cells. To further assess its feasibility for treating anogenital warts in humans, we performed a Phase I study. Forty-two male volunteers with genital/perianal warts were treated topically with three different formulations of 1 mg/ml ranpirnase. Patients were monitored for 8 weeks or until healing. Four patients with HIV were treated in accordance with the compassionate programme but were not evaluated. RESULTS: In cultured cells, ranpirnase showed specific activity against HPV-11 with low toxicity (selectivity index >88). The broad applicability of ranpirnase for treating papillomaviruses was verified using the cottontail rabbit papillomavirus. In the clinical study, eight participants were lost-to-follow-up or discontinued due to protocol violation or non-compliance. Among 30 evaluable participants, topical ranpirnase was moderately well-tolerated, with discontinuation by 5 (16.7%) due to adverse reactions. Clinical healing was achieved by 25 participants (83.3%) and 50% improvement by the 5 discontinued participants (16.7%). The median time to clinical healing was 30 days. CONCLUSIONS: This study provides the first in vitro and clinical evidence of the antiviral efficacy of ranpirnase against HPV and supports assessment of ranpirnase in expanded clinical studies.

Cryoelectron Microscopy Maps of Human Papillomavirus 16 Reveal L2 Densities and Heparin Binding Site.

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. The current commercial vaccines are genotype specific and provide little therapeutic benefit to patients with existing HPV infections. Host entry mechanisms represent an excellent target for alternative therapeutics, but HPV receptor use, the details of cell attachment, and host entry are inadequately understood. Here we present near-atomic resolution structures of the HPV16 capsid and HPV16 in complex with heparin, both determined from cryoelectron micrographs collected with direct electron detection technology. The structures clarify details of capsid architecture for the first time, including variation in L1 major capsid protein conformation and putative location of L2 minor protein. Heparin binds specifically around the capsid icosahedral vertices and may recapitulate the earliest stage of infection, providing a framework for continuing biochemical, genetic, and biophysical studies.

Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17-36 epitopes.

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.

The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy.

UNLABELLED: The human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers. IMPORTANCE: Human papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.

Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization.

Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays.

HPV binding assay to Laminin-332/integrin α6ß4 on human keratinocytes.

Human papillomaviruses (HPVs) have been shown to bind to Laminin-332 (Ln-332) on the extracellular matrix (ECM) secreted by human keratinocytes. The assay described here is an important tool to study HPV receptor binding to the ECM. The assay can also be modified to study the receptors required for HPV infection and for binding to tissues. We previously showed that Ln-332 is essential for the binding of HPV11 to human keratinocytes and that infectious entry of HPV11 requires α6ß4 integrin for the transfer of HPV11 from ECM to host cells (Culp et al., J Virol 80:8940-8950, 2006). We also demonstrated that several of the high-risk HPV types (16, 18, 31 and 45) bind to Ln-332 and/or other components of the ECM in vitro (Broutian et al., J Gen Virol 91:531-540, 2010). The exact binding and internalization mechanism(s) for HPV are still under investigation. A better understanding of these mechanisms will aid in the design of therapeutics against HPVs and ultimately help prevent many cancers. In this chapter, we describe the HPV binding assay to Ln-332/integrin α6ß4 on human keratinocytes (ECM). We also present data and suggestions for modifying the assay for testing the specificity of HPV for receptors (by blocking receptors) and binding to human tissues (basement membrane, BM) in order to study binding mechanisms.

A cryo-electron microscopy study identifies the complete H16.V5 epitope and reveals global conformational changes initiated by binding of the neutralizing antibody fragment.

UNLABELLED: Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid-Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called "invading-arm" structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. IMPORTANCE: Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and "invading-arm" structures. This study advances the understanding of the neutralization mechanism used by H16.V5.

Pathogenesis of infection by human papillomavirus.

Human papillomaviruses (HPVs) are associated with benign lesions known as warts and several cancer types including cancer of the cervix, penis, anus and oral cavity. HPVs are classified by their oncogenic potential and are divided into high-risk oncogenic HPVs and low-risk HPVs. Tissue tropism is used as another means of classifying the virus, and HPVs are divided into types that infect mucosal or cutaneous tissues. Several risk factors have been identified that elevate an individual's likelihood of becoming infected with HPV including cigarette smoking, a large number of lifetime sexual partners and immunosuppression. Most HPV infections are cleared naturally, although persistent infection with oncogenic HPV types can lead to the cancers mentioned above. HPV has employed several mechanisms to avoid detection by the host immune system. Virus is released along with shedding skin cells in a nonlytic manner, and the virus has an altered codon usage leading to reduced expression of viral proteins. Infections from high-risk oncogenic HPV types that progress cause neoplasias that are defined as CIN1-CIN3 depending on the amount of abnormal cell growth and the level of cellular differentiation.

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.

Differential binding patterns to host cells associated with particles of several human alphapapillomavirus types.

The focus of this research was to compare the binding profiles of human papillomavirus (HPV) 11, 16, 18 and 45 virus-like particles (VLPs) to HaCaT cells and to the extracellular matrix (ECM) secreted by these cells. All four HPV types tested bind to a component(s) of the ECM. HPV11 VLP binding is blocked when the ECM is pretreated with an anti-laminin 5 (LN5) polyclonal antibody. A series of treatments utilizing heparins and heparinase revealed that HPV18 VLPs are dependent on heparan sulfates (HS) for binding to cells and ECM. HPV16 and HPV45 VLPs are dependent on HS for binding to HaCaT cells and dependent on both HS and LN5 for binding to ECM. These studies emphasize the need to study the binding characteristics of different HPV types before applying universal binding principles to all papillomaviruses.