Identification of the protein encoded by the E6 transforming gene of bovine papillomavirus.

Papillomaviruses (PV) contain several conserved genes that may encode nonstructural proteins; however, none of these predicted gene products have been identified. Papillomavirus E6 genes are retained and expressed as RNA in PV-associated human and animal carcinomas and cell lines. This suggests that the E6 gene product may be important in the maintenance of the malignant phenotype. The E6 open reading frame of the bovine papillomavirus (BPV) genome has been identified as one of two BPV genes that can independently transform mouse cells in vitro. A polypeptide encoded by this region of BPV was produced in a bacterial expression vector and used to raise antisera. The antisera specifically immunoprecipitated the predicted 15.5-kilodalton BPV E6 protein from cells transformed by the E6 gene. The E6 protein was identified in both the nuclear and membrane fractions of these transformed cells.

Efficacy of a Carrageenan gel Against Transmission of Cervical HPV (CATCH): interim analysis of a randomized, double-blind, placebo-controlled, phase 2B trial.

OBJECTIVES: To evaluate the efficacy of a carrageenan-based lubricant gel in reducing the risk of genital human papillomavirus (HPV) infections in women. METHODS: We conducted a planned interim analysis of a randomized, double-blind, placebo-controlled, phase 2B trial. Women aged 18 years and older were randomly assigned (1:1) to a carrageenan-based gel or a placebo gel to be self-applied every other day for the first month and before and after each intercourse during follow-up. Assessments were performed at 0.5, 1, 3, 6, 9 and 12 months. The primary outcome was incidence of a new infection by an HPV type that was not present at baseline. Intention-to-treat analyses were performed. RESULTS: Between January 2013 and June 2017, a total of 280 participants were randomly assigned to the carrageenan (n = 141) or the placebo (n = 139) arm. All participants were included in safety analyses, but three (1%) were excluded from efficacy analyses (HPV results unavailable for two participants in the carrageenan and one participant in the placebo arm). The median follow-up time was 9.2 months (interquartile range, 1.9-13.2 months). A total of 59 (42%) of 139 participants in the carrageenan arm and 78 (57%) of 138 participants in the placebo arm became infected by at least one new HPV type (hazard ratio = 0.64, 95% confidence interval = 0.45-0.89, p 0.009). A total of 62 (44%) of 141 participants in the carrageenan arm versus 43 (31%) of 139 participants in the placebo arm reported an adverse event (p 0.02), none of which was deemed related to the gels. CONCLUSIONS: Our trial's interim analysis suggests that using a carrageenan-based lubricant gel can reduce the risk of genital HPV infections in women.

Conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies.

High avidity and long-lasting autoantibodies to a self-polypeptide (TNF-alpha) were generated after parenteral vaccination of mice with low doses of virus-like particle-based (VLP-based) vaccines that were constructed by linking mouse TNF-alpha peptides to the surface of papillomavirus VLPs. High-titer autoantibodies were induced with or without coadministration of potent conventional adjuvants, but were enhanced by coadministration of CFA. Compared with immunization with the fusion protein alone, attachment to VLPs increased autoantibody titers 1,000-fold. A comparison of Ab responses against the self (TNF-alpha) and foreign components of the fusion protein showed that VLP conjugation abrogated the ability of the humoral immune system to distinguish between self and foreign. Similar levels of IgM were detected to self and foreign epitopes regardless of the assembly state of the antigen, suggesting that conjugation of self-peptides to VLPs promotes survival or expansion of mature autoreactive B cells. In a mouse model, vaccination with conjugated particles inhibited development of type II collagen-induced arthritis. Together, these results suggest a potentially flexible method to efficiently generate autoantibodies against specific self-proteins that mediate arthritis and other diseases.

The conserved C-terminal domain of the bovine papillomavirus E5 oncoprotein can associate with an alpha-adaptin-like molecule: a possible link between growth factor receptors and viral transformation.

The bovine papillomavirus E5 gene encodes an oncoprotein that can independently transform rodent fibroblasts. This small 44-amino-acid protein is thought to function through the activation of growth factor receptors. E5 activation of the epidermal growth factor receptor results in an increase in the number of activated receptors at the cell surface. This finding suggests that E5 may act by inhibiting the normal down regulation of activated epidermal growth factor receptor via coated pit-mediated endocytosis. We have constructed a fusion protein consisting of glutathione S-transferase and the conserved C-terminal domain of E5 (GST-E5) in order to identify E5-associated cellular proteins that may be involved in its transforming activity. We have identified a 125-kDa cellular protein with a strong associated serine kinase activity that specifically associated with GST-E5 in the reduced form but not with GST-E5 fusions that contained changes in several conserved amino acids. Microsequence and biochemical analyses suggest that p125 is a novel member of the alpha-adaptin family. Since alpha-adaptins have previously been shown to be involved in coated pit-mediated cell surface receptor endocytosis and down regulation, these results suggest that p125 may be an alpha-adaptin-like molecule involved in growth factor receptor down regulation and that E5 may act by inhibiting its activity.

Positive and negative regulation of cell proliferation by E2F-1: influence of protein level and human papillomavirus oncoproteins.

E2F-1 is a member of a family of transcription factors implicated in the activation of genes required for the progression through the S phase of the cell cycle. We have examined the biological activities of E2F-1 with short-term colony-forming assays and long-term immortalization assays. High levels of E2F-1, produced by transfection of the E2F-1 cDNA under the control of a strong promoter, reduced colony formation in normal human foreskin keratinocytes (NHFKs). This inhibition could not be overcome by wild-type human papillomavirus type 16 (HPV16) E6 and E7, two proteins which cooperate to immortalize NHFKs, or by a transdominant p53 mutant. High levels of E2F-1 also inhibited growth of primary and established fibroblasts. The growth-inhibitory activity required the DNA binding function of E2F-1 but not its transactivation or pRB binding activities. A positive role for lower levels of E2F-1 in NHFK immortalization was established by examining the ability of E2F-1 to complement HPV16 E7 mutants that were unable to cooperate with HPV16 E6 to immortalize NHFKs. Although E2F-1 was unable by itself to cooperate with E6, it did, in conjunction with E6, complement a p24GLY mutant of E7 that is defective for immortalization and binding of pRB and pRB-related proteins. By contrast, E2F-1 was unable to complement two other E7 mutants, p2PRO and p31/32ARG/PRO, which are also defective in the immortalization assay, although their proteins display wild-type binding of pRB in vitro. Since the binding of E7 to pRB results in disruption of pRB-E2F interaction and release of transcriptionally active E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E3F activity are important but not sufficient for E7-induced keratinocyte immortalization.

The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH.

We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass, and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming activity of c-rasH is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-rasH mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated by cooperation between the BPV E5 gene and ras. E5-dependent cooperation was seen for v-rasH as well as for c-rasH, which suggests that the major effect of E5 was to increase the susceptibility of the cell to transformation to a given level of ras activity. The cooperation assay was used to test the potential importance, in c-rasH, of codons 93 to 108, 123 to 130, and 166 to 183, which were nonessential for v-rasH transformation. Relative to the respective transforming activity of wild-type c-rasH and v-rasH, mutants with lesions in codons 102 and 103 were significantly less active in their c-rasH forms than in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype.

Association of serum immunoglobulin G antibodies against human papillomavirus type 16 capsids with anal epidermoid carcinoma.

BACKGROUND: Anal epidermoid carcinoma is a relatively rare tumor, but its incidence has been increasing rapidly during the past few years. Genetic material from the major oncogenic types of human papillomavirus (HPV), types 16 and 18, has regularly been demonstrated in a substantial proportion of anal cancers, suggesting an etiologic role of HPV infection. Recently, serum antibodies against HPV type 16 capsids were shown to be a serologic measure of HPV16 infection. PURPOSE: We investigated whether serum antibodies against HPV16 capsids are associated with an increased risk of developing anal cancer. METHODS: Serum samples from 64 patients (48 women and 16 men) with untreated anal epidermoid cancer and from 79 age- and sex-matched healthy blood donors were analyzed for the levels of serum immunoglobulin G (IgG) against capsids of HPV16 by the enzyme-linked immunosorbent assay. The levels of serum IgG against HPV type 6 and bovine papillomavirus (BPV) capsids, as well as against HPV16 peptide antigens, were also measured. RESULTS: Whereas antibodies against HPV6 or BPV capsids were not significantly associated with anal cancer, the presence of IgG against HPV16 capsids exceeding the anti-BPV antibody levels was demonstrated among 55% (35 of 64) of the case patients but only among 4% (three of 79) of the control subjects (odds ratio [OR] = 30.4; 95% confidence interval [CI] = 8.4-161.5). Antibodies against HPV16 E2 and E7 peptides were also more common among case patients (OR = 12.8 and 95% CI = 5.4-31.5 for E2; OR = 3.0 and 95% CI = 1.4-6.7 for E7). CONCLUSION: The results suggest that HPV16 capsid antibodies are serologic markers for anal cancer. IMPLICATION: Exposure to HPV16 or related viruses appears to be a major risk factor in the majority of anal cancers.

A virus-like particle enzyme-linked immunosorbent assay detects serum antibodies in a majority of women infected with human papillomavirus type 16.

BACKGROUND: Previous studies have demonstrated that genital infection with high-risk types of human papillomavirus (HPV), most often HPV16, is the most significant risk factor for the development of cervical cancer. However, serologic assays that have been developed to identify high-risk HPV infection have either failed to associate serum reactivity with other indicators of HPV infection or have identified only a minority of HPV-infected individuals. PURPOSE: Our purpose was to determine whether a specifically developed enzyme-linked immunosorbent assay (ELISA) could detect IgG anti-HPV16 virion antibodies in the sera of women who had tested positive for genital HPV16 infection by DNA-based methods. METHODS: An ELISA was developed using newly developed HPV16 virus-like particles as antigens to detect anti-HPV16 virion IgG antibodies. These particles are comprised of HPV16 structural proteins that are self-assembled in insect cells after expression by recombinant baculoviruses. The sera of 122 women, whose HPV status had been previously evaluated by nucleic acid-based methods, were tested by this ELISA. RESULTS: The sera of 59% of women (32 of 54) positive for genital HPV16 DNA by polymerase chain reaction (PCR) were positive in the ELISA assay compared with sera from women who had tested negative for HPV DNA (P < .0005). In contrast, 6% of HPV DNA-negative women (two of 31) and 9% of women positive for low-risk HPV6/11 DNA (one of 11) were ELISA positive by this criterion. The sera of women who were DNA positive for two additional high-risk HPV types were evaluated; the sera of 31% of HPV18-positive (four of 13) and 38% of HPV31-positive women (five of 13) were positive in the HPV16 particle ELISA. The sera of 75% of HPV16 DNA-positive women with severe dysplasias (12 of 16) gave positive ELISA results. The sera of 67% of women (28 of 42) who tested positive for HPV16 DNA by both PCR and the less sensitive ViraType assay tested positive in the ELISA compared with 33% of women (four of 12) who were positive by PCR but negative by ViraType (P < .05). CONCLUSION: The majority of women with cervical HPV16 infection generate an IgG antibody response to conformationally dependent epitopes of HPV16 L1 that can be detected by ELISA. IMPLICATION: This particular ELISA, or a similar one incorporating virus-like particles of additional HPV types, may be useful in determining the natural history of high-risk HPV infection and perhaps help to identify women at risk for developing cervical cancer.

Serologic association between human papillomavirus type 16 infection and esophageal cancer in Shaanxi Province, China.

BACKGROUND: The existence of large geographic variations in the prevalence of esophageal cancer in some countries, such as China, indicates that environmental risk factors may be important in the development of this disease. Some studies have implicated genital-mucosal strains of human papillomaviruses (HPVs) in the etiology of this cancer. PURPOSE: We conducted a case-control study in Shaanxi Province, China, an area with a population at high risk for esophageal cancer, to assess the association of this disease with infection by HPV type 16 (HPV16), the most common cancer-associated genital-mucosal HPV type. METHODS: Ninety individuals with esophageal cancer and 121 cancer-free control subjects were identified among the patients in two hospitals in Xi'an, Shaanxi Province. The control subjects were matched to the case patients on the basis of age and sex. Blood specimens were drawn from all study subjects, and serum was isolated by routine methods. The presence of HPV16 antibodies in serum samples was determined by use of an enzyme-linked immunosorbent assay (ELISA) that used baculovirus-derived HPV16 virus-like particles as the antigen. A similar ELISA that used bovine papillomavirus type 1 (BPV1) virus-like particles as the antigen controlled for the specificity of HPV16 seroreactivity. Data from the HPV16 and the BPV1 assays were normalized with respect to results obtained in each assay with a control serum of known HPV16 seroreactivity. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to examine the association between HPV16 seroreactivity and esophageal cancer. Reported P values are two-sided. RESULTS: The mean seroreactivity to HPV16 virus-like particles was significantly higher for the cancer patients than for the control subjects (mean value +/- standard deviation = 0.85 +/- 0.22 versus 0.74 +/- 0.18; P<.0001). When the cancer patients and control subjects were compared by sex and age groups, the differences in mean seroreactivity remained statistically significant. The difference in mean seroreactivity to BPV1 virus-like particles between cancer patients and control subjects was not statistically significant (0.81 +/- 0.28 versus 0.88 +/- 0.32; P = .12); this result was not altered when sex and age groups were compared. By use of a cutoff point for HPV16 seropositivity that was established in studies of cervical neoplasia, 24% of the cancer patients were seropositive compared with 7% of the control subjects, yielding a sex- and age-adjusted OR of 4.5 (95% CI = 1.8-11.9). In general, the OR for esophageal cancer increased with increasing HPV16 seroreactivity. CONCLUSIONS AND IMPLICATIONS: HPV16 infection may be a risk factor for esophageal cancer. Further studies of the association between HPV16 infection and the incidence of esophageal cancer are needed.

Prospective seroepidemiologic study of human papillomavirus infection as a risk factor for invasive cervical cancer.

BACKGROUND: Major risk factors for invasive cervical cancer include infection with human papillomavirus (HPV), infection with other sexually transmitted pathogens (e.g., Chlamydia trachomatis), and smoking. Since exposures to these risk factors can be related, the contribution of any single factor to cervical carcinogenesis has been difficult to assess. We conducted a prospective study to define the role of HPV infection in cervical carcinogenesis, with invasive cancer as an end point. METHODS: A nested case-control study within a joint cohort of 700,000 Nordic subjects was performed. The 182 women who developed invasive cervical cancer during a mean follow-up of 5 years were matched with 538 control women on the basis of age and time of enrollment. Serum samples taken at enrollment were analyzed for evidence of tobacco use (i.e., cotinine levels); for antibodies against HPV types 16, 18, and 33; and for antibodies against C. trachomatis. Relative risks (RRs) were estimated by use of conditional logistic regression. RESULTS: Presence of antibodies against HPV in serum (seropositivity) was associated with an increased risk of cervical cancer, and adjustment for smoking and for C. trachomatis seropositivity did not affect this finding (RR = 2.4; 95% confidence interval [CI] = 1.6-3.7). HPV16 seropositivity was associated primarily with an increased risk of squamous cell carcinoma (RR = 3.2; 95% CI = 1.7-6.2). In contrast, risk associated with HPV18 seropositivity tended to be higher for cervical adenocarcinoma (RR = 3.4; 95% CI = 0.8-14.9). In populations with a low prevalence of antibodies against C. trachomatis, the HPV16-associated risk of cervical cancer was very high (RR = 11.8; 95% CI = 3.7-37.0); in contrast, in populations with a high prevalence of antibodies against C. trachomatis, no excess risk was found. CONCLUSION: Past infection with HPV16 increases the risk of invasive cervical squamous cell carcinoma, most clearly seen in populations with a low prevalence of sexually transmitted diseases.

Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine.

BACKGROUND: Studies in animal models have shown that systemic immunization with a papillomavirus virus-like particle (VLP) vaccine composed of L1, a major structural viral protein, can confer protection against subsequent experimental challenge with the homologous virus. Here we report results of a double-blind, placebo-controlled, dose-escalation trial to evaluate the safety and immunogenicity of a human papillomavirus (HPV) type 16 (HPV16) L1 VLP vaccine in healthy adults. METHODS: Volunteers were given intramuscular injections with placebo or with 10- or 50-microg doses of HPV16 L1 VLP vaccine given without adjuvant or with alum or MF59 as adjuvants at 0, 1, and 4 months. All vaccine recipients were monitored for clinical signs and symptoms for 7 days after each inoculation. Immune responses were measured by an HPV16 L1 VLP-based enzyme-linked immunosorbent assay (ELISA) and by an HPV16 pseudovirion neutralization assay. The antibody titers were given as the reciprocals of the highest dilution showing positive reactivity in each assay. All statistical tests were two-sided. RESULTS: The prevaccination geometric mean ELISA titer for six seropositive individuals was 202 (range, 40--640). All vaccine formulations were well tolerated, and all subjects receiving vaccine seroconverted. Serum antibody responses at 1 month after the third injection were dose dependent in recipients of vaccine without adjuvant or with MF59 but were similar at both doses when alum was the adjuvant. With the higher dose, the geometric means of serum ELISA antibody titers (95% confidence intervals) to purified VLP 1 month after the third injection were as follows: 10,240 (1499 to 69 938) without adjuvant, 10,240 (1114 to 94 145) with MF59, and 2190 (838 to 5723) with alum. Responses of subjects within each group were similar. Neutralizing and ELISA antibody titers were highly correlated (Spearman correlation =.85), confirming that ELISA titers are valid proxies for neutralizing antibodies. CONCLUSIONS: The HPV16 L1 VLP vaccine is well tolerated and is highly immunogenic even without adjuvant, with the majority of the recipients achieving serum antibody titers that were approximately 40-fold higher than what is observed in natural infection.

Immunoglobulin G responses against human papillomavirus type 16 virus-like particles in a prospective nonintervention cohort study of women with cervical intraepithelial neoplasia.

BACKGROUND: Infection with cancer-linked human papillomavirus (HPV) types such as HPV type 16 (HPV16) is the most important risk factor in the development of cervical cancer. It has been shown that immunoglobulin G (IgG) antibody responses against HPV16 virus-like particles (VLPs) are specifically associated with genital HPV16 infection. PURPOSE: The aim of this study was to determine the temporal relationships between the presence of HPV16 VLP-specific IgGs, HPV16 infection patterns, and the course of premalignant cervical disease. METHODS: Plasma samples from 133 women who had been diagnosed originally with mild to moderate cervical dyskaryosis and enrolled in a prospective non-intervention cohort study conducted in Amsterdam, The Netherlands, from 1991 through 1996 were analyzed for the presence of HPV16 VLP-specific IgGs by use of an enzyme-linked immunosorbent assay. A detailed analysis was performed on 43 women with different HPV16 infection patterns during a follow-up period of 10-34 months. Progression or regression of cervical intraepithelial neoplasia (CIN) lesions was monitored by cytologic and colposcopic testing at intervals of 3-4 months. HPV typing in cervical smears was performed by use of a polymerase chain reaction-based assay. Statistical analysis of the serologic data was performed by use of the Mann-Whitney U test or 2 x 2 table analyses. RESULTS: The presence of HPV16 VLP-specific IgGs in the plasma of the patients was found to be associated with the presence of HPV16 DNA in the cervical smear. Significantly higher proportions of patients with persistent HPV16 infections (i.e., who were polymerase chain reaction positive in three to 11 consecutive tests) than of patients with cleared HPV16 infections were found to be positive for the presence of HPV16 VLP-specific IgGs (18 [69.2%] of 26 versus nine [28.1%] of 32, respectively; P = .003). HPV16 VLP-specific IgGs were consistently detected in all women (n = 11) who were persistently HPV16 DNA positive during follow-up and whose disease ultimately progressed to CIN III (histologically diagnosed severe dysplasia or carcinoma in situ). CONCLUSION: HPV16 VLP-specific IgG responses are present in the plasma of a majority of patients with persistent HPV16 infections and histologically confirmed high-grade lesions but only in a smaller subset of patients with cleared HPV16 infections and either normal cervical histology or low-grade CIN lesions. IMPLICATIONS: These results suggest that HPV16 VLP-specific antibodies are not responsible for the clearance of virally induced CIN lesions but that they might, in patients with persistent HPV16 infections, be indicative of an increased cervical cancer risk.

Transformation by human papillomavirus 16 E6 and E7: role of the insulin-like growth factor 1 receptor.

Human papillomavirus-16 E6 and E7 inactivate the tumor suppressors p53 and pRB, respectively, and cooperate during malignant transformation, but the downstream molecular events remain incompletely understood. Using fibroblast cell lines derived from mice with a homozygous disruption of the insulin-like growth factor-1 receptor (IGF-1R) gene (R- cells) and their wild-type (WT) littermates, we have stably transfected plasmids encoding E6 and E7 proteins and examined their transforming potential in these cells. Consistent with previous studies using NIH3T3 cells, pooled cultures of E7-transfected WT cells readily formed colonies after suspension in soft agar. In contrast, R- cells were not transformed by E7. E6 had little transforming activity in WT (WT/E6) or R- (R-/E6) cells. However, transfection of R- cells with E6 plus E7 resulted in extensive colony formation. Because IGF-1R and E6 appear to be functionally equivalent in this transformation assay and both have been implicated in antiapoptotic responses, we investigated the apoptotic responses of the cells after exposure to the potent protein kinase C inhibitor, staurosporine. Compared to WT cells, R- cells were relatively resistant to staurosporine-induced apoptosis, but susceptibility to staurosporine was decreased in both WT/E6 and R-/E6 cells relative to WT and R- cells transfected with mock vector, respectively. In fibroblast cells from p53 gene knockout mice, transfection with E6 also conferred relative resistance to staurosporine-induced apoptosis. Our data suggest that transformation by E7 requires the participation of the IGF-1R and that E6 may assist E7 in transforming R- cells by functionally substituting for the IGF-1R. Because IGF-1R activated by its ligands (IGF-1 and IGF-2) protects cells from apoptosis, the role of the IGF-1R and E6 in transformation by E7 is probably related to the recruitment of survival pathways. In addition, because E6 suppressed apoptosis in p53 knockout cells, our data also suggest that E6 may participate in a p53-independent process that protects cells from apoptosis.

Papillomaviruses: prophylactic vaccine prospects.

Identification of a subset of HPV types as etiologic agents of cervical cancer and other malignancies implies that development of an effective vaccine against HPV infection could have a major impact on tumors attributable to these viruses. The ability of the L1 major capsid protein of papillomaviruses to self-assemble into VLPs that can, when inoculated systemically, induce high levels of neutralizing antibodies and protect animals against experimental viral challenge makes L1 VLPs an excellent candidate subunit vaccine. VLPs have the limitation of inducing type-specific immunity. Studies in humans are required to determine whether systemic vaccination with L1 VLPs will prevent sexually transmitted HPV infection. Since prospective efficacy trials will take several years to complete, considering alternative approaches is also worthwhile.

Two antibodies that neutralize papillomavirus by different mechanisms show distinct binding patterns at 13 A resolution.

Complexes between bovine papillomavirus type 1 (BPV1) and examples of two sets of neutralizing, monoclonal antibodies (mAb) to the major capsid protein (L1) were analyzed by low-dose cryo-electron microscopy and three-dimensional (3D) image reconstruction to 13 A resolution. mAb #9 is representative of a set of neutralizing antibodies that can inhibit viral binding to the cell surface, while mAb 5B6 is representative of a second set that efficiently neutralizes papillomaviruses without significantly inhibiting viral binding to the cell surface. The 3D reconstructions reveal that mAb #9 binds to L1 molecules of both pentavalent and hexavalent capsomeres. In contrast, 5B6 binds only to hexavalent capsomeres, reflecting the significant structural or environmental differences for the 5B6 epitope in the 12 pentavalent capsomeres. Epitope localization shows that mAb #9 binds monovalently to the tips of capsomeres whereas 5B6 binds both monovalently and bivalently to the sides of hexavalent capsomeres approximately two-thirds of the way down from the outer tips, very close to the putative stabilizing intercapsomere connections. The absence of mAb 5B6 from the pentavalent capsomeres and its inability to prevent viral binding to the cell surface suggest that receptor binding may occur at one or more of the 12 virion vertices.

TGF alpha enhances locomotion of cultured human keratinocytes.

A cDNA sequence coding for the full-length human transforming growth factor alpha (TGF alpha) precursor protein was introduced into and transcribed in cultured human keratinocytes, using a high-titer, high-expression, murine amphotropic retrovirus. Keratinocytes were shown capable of post-translationally modifying the TGF alpha primary translation product, with the subsequent formation of several cell-associated and secreted forms of TGF alpha, at least five of which, including the 50 amino acid mature species, can potentially bind and activate the epidermal growth factor receptor. Cells overexpressing the TGF alpha gene assumed a spindled morphology with long, bipolar filamentous processes and displayed increased locomotion. The soluble, mature form of TGF alpha alone also could induce the observed changes in cell shape and motility when added to keratinocyte cultures exogenously. The effects were dose dependent, and up to fourfold increases in locomotion were caused by TGF alpha in the absence of bovine pituitary extract (BPE). The addition of BPE to high concentrations of TGF alpha further enhanced keratinocyte motility to eightfold over baseline, suggesting a synergistic interaction between the two factors. These experiments demonstrate that keratinocytes can synthesize several forms of TGF alpha and that TGF alpha, besides being mitogenic, may have other important regulatory functions in keratinocytes.

Papillomaviruses and cervical cancer: pathogenesis and vaccine development.

A subset of human papillomaviruses (HPVs) has been implicated as the principal etiologic agents of cervical cancer. Cervical cancers consistently retain and express two of the viral genes, E6 and E7. Although infection with HPV seems to be necessary, other factors, such as cellular immune function, play an important role in determining whether cervical infection will regress, persist, or progress to cancer. The close relationship between viral infection and cancer makes HPV an attractive target for prophylactic and therapeutic vaccines. Candidate vaccines have been shown to have efficacy in animal models, and human clinical trials are planned or in progress.

Papillomavirus-like particle vaccines.

Papillomavirus-like particle (VLP)-based subunit vaccines have undergone rapid development over the past 8 years. Three types are being investigated. The most basic type is composed of only the L1 major capsid protein and is designed to prevent genital human papillomavirus (HPV) infection by inducing virus-neutralizing antibodies. On the basis of positive results in animal models, clinical trials of this type of vaccine for HPV16, and other types, are currently under way. Preliminary results have been encouraging in that systemic immunization with the L1 VLPs induced high serum titers of neutralizing antibodies without substantial adverse effects. The second type of vaccine incorporates other papillomavirus polypeptides into the VLPs as L1 or L2 fusion proteins. These chimeric VLPs are designed to increase the therapeutic potential of an HPV vaccine by inducing cell-mediated responses to nonstructural viral proteins, such as E7. Studies in mice indicate that these vaccines generate potent antitumor cytotoxic lymphocyte (CTL) responses while retaining the ability to induce high-titer neutralizing antibodies. It is likely that prophylactic and therapeutic clinical trials of chimeric VLPs will be initiated in the near future. The third type of VLP-based vaccine is designed to induce autoantibodies against central self-antigens by incorporating self-peptides into the outer surface of VLPs, a process that could have therapeutic potential in various disease settings unrelated to HPV infection. In a recent proof of concept study, a peptide from an external loop of mouse CCR5 protein was inserted into a neutralizing epitope of L1. In mice, the particles generated by this chimeric L1 were able to induce high titers of CCR5 antibodies that specifically recognized the surface of CCR5-transfected cells and blocked in vitro infection of an M-tropic human immunodeficiency virus strain.

Human papillomavirus immunology and vaccine prospects.

As a result of several recent advances in molecular biology, the association between human papillomavirus (HPV) infection and cervical cancer has been firmly established and the oncogenic potential of certain HPV types has been clearly demonstrated. These observations provide the impetus for the development of novel vaccines to prevent or treat HPV-associated cervical cancer. Because there is no effective culturing system to propagate HPV, traditional approaches for studying HPV and developing vaccines have been hampered. However, recent studies using recombinant subunit preparations in animals have yielded promising results and encourage their investigation in human trials. Strategies currently under investigation focus on the induction of effective humoral immune responses for prophylaxis against subsequent HPV infection; for the treatment of existing HPV infections, techniques to improve cell-mediated immunity by enhancing viral antigen recognition are being studied. Small-scale human trials using several different vaccine approaches should be completed within the next few years and field trials of the most promising one(s) could begin within a decade. The development of successful therapeutic and/or prophylactic vaccines offers an attractive alternative to existing screening and treatment programs for cervical cancer and may result in a substantial reduction in the worldwide morbidity from this disease.

High-risk human papillomavirus is sexually transmitted: evidence from a follow-up study of virgins starting sexual activity (intercourse).

Genital human papillomavirus (HPV) infection is generally considered to be sexually transmitted. However, nonsexual spread of the virus has also been suggested. The goal of this study was to assess: (a) the role of sexual intercourse in the transmission of HPV; (b) the determinants for seroconversion; and (c) the correlation between HPV DNA, abnormal cervical cytology, and serological response to HPV16. One hundred virgins and 105 monogamous women were randomly selected from a population-based cohort study in Copenhagen, Denmark, in which the women were examined twice with 2-year interval (interview, cervical swabs, Pap smear, blood samples). The presence of HPV DNA was determined by GP5+/6+ primers based HPV-PCR-EIA. HPV 16 virus-like particles (VLP) antibodies were detected by ELISA. All of the virgins were both HPV DNA negative and seronegative to VLP16, except for one woman who was weakly HPV 6 DNA positive. Only those virgins who initiated sexual activity became HPV DNA positive and/or VLP16 positive. The most important determinant of HPV DNA acquisition was the number of partners between the two examinations. The only significant risk factor for HPV 16 VLP seroconversion among women acquiring HPV DNA was HPV type. Our results show that sexual intercourse is important in the transmission of HPV, and that HPV 16 VLP seroconversion and the development of cervical lesions only occur after HPV transmission. Remarkably, no cervical lesions were found in HPV 16 DNA positive women who had seroconverted. Although based on small numbers, this may suggest that the development of antibodies had a protective effect.