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Current human papillomavirus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain for which vaccine coverage is lacking. In addition, all current HPV vaccines rely on aluminum salt-based adjuvant formulations that function through unclear mechanisms with few substitutes available. In an effort to expand the toolbox of available adjuvants suitable for HPV vaccines, we compared the immunogenicity of the RG1-VLP (virus-like particle) vaccine in BALB/c mice when formulated with either the aluminum hydroxide adjuvant Alhydrogel or the novel polyphosphazene macromolecular adjuvant poly[di (carboxylatoethylphenoxy) phosphazene] (PCEP). PCEP-formulated RG1-VLPs routinely outperformed VLP/Alhydrogel in several measurements of VLP-specific humoral immunity, including consistent improvements in the magnitude of antibody (Ab) responses to both HPV16-L1 and the L2 RG1 epitope as well as neutralizing titers to HPV16 and cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39. Dose-sparing studies indicated that RG1-VLPs could be reduced in dose by 75% and the presence of PCEP ensured activity comparable to a full VLP dose adjuvanted by Alhydrogel. In addition, levels of HPV16-L1 and -L2-specific Abs were achieved after two vaccinations with PCEP as adjuvant that were equivalent to or greater than levels achieved with three vaccinations with Alhydrogel alone, indicating that the presence of PCEP resulted in accelerated immune responses that could allow for a decreased dose schedule. Given the extensive clinical track record of polyphosphazenes, these data suggest that substitution of alum-based adjuvants with PCEP for the RG1-VLP vaccine could lead to rapid seropositivity requiring fewer boosts, the dose-sparing of commercial VLP-based vaccines, and the establishment of longer-lasting humoral responses to HPV.

Current human papilloma virus (HPV) vaccines provide substantial protection against the most common HPV types responsible for oral and anogenital cancers, but many circulating cancer-causing types remain that lack vaccine coverage. The novel RG1-VLP (virus-like particle) vaccine candidate utilizes the HPV16-L1 subunit as a backbone to display an inserted HPV16-L2 17–36 a.a. “RG1” epitope; the L2 RG1 epitope is conserved across many HPV types and the generation of cross-neutralizing antibodies (Abs) against which has been demonstrated. In an effort to heighten the immunogenicity of the RG1-VLP vaccine, we compared in BALB/c mice adjuvant formulations consisting of novel bacterial enzymatic combinatorial chemistry (BECC)-derived toll-like receptor 4 (TLR4) agonists and the aluminum hydroxide adjuvant Alhydrogel. In the presence of BECC molecules, consistent improvements in the magnitude of Ab responses to both HPV16-L1 and the L2 RG1 epitope were observed compared to Alhydrogel alone. Furthermore, neutralizing titers to HPV16 as well as cross-neutralization of pseudovirion (PsV) types HPV18 and HPV39 were augmented in the presence of BECC agonists as well. Levels of L1 and L2-specific Abs were achieved after two vaccinations with BECC/Alhydrogel adjuvant that were equivalent to or greater than levels achieved with 3 vaccinations with Alhydrogel alone, indicating that the presence of BECC molecules resulted in accelerated immune responses that could allow for a decreased dose schedule for VLP-based HPV vaccines. In addition, dose-sparing studies indicated that adjuvantation with BECC/Alhydrogel allowed for a 75% reduction in antigen dose while still retaining equivalent magnitudes of responses to the full VLP dose with Alhydrogel. These data suggest that adjuvant optimization of HPV VLP-based vaccines can lead to rapid immunity requiring fewer boosts, dose-sparing of VLPs expensive to produce, and the establishment of a longer-lasting humoral immunity.

Equine sarcoids are common therapy-resistant skin tumours induced by bovine papillomavirus type 1 or 2 (BPV1, BPV2) infection. We have previously shown that prophylactic vaccination with BPV1 L1 virus-like particles (VLPs) efficiently protects horses from experimental BPV1-induced pseudo-sarcoid development. Here, we assessed BPV1 L1 VLP vaccine-mediated long-term protection from experimental tumour formation in seven horses 5 years after immunization with three different doses of BPV1 L1 VLPs, and three unvaccinated control animals. Horses were challenged by intradermal inoculation with infectious BPV1 virions at 10 sites on the neck (106 virions per injection). In vaccinated horses, BPV1 challenge did not result in any apparent lesions irrespective of vaccine dosage and BPV1-neutralizing antibody titres that had dropped considerably over time and below the detection limit in one individual. Control horses developed pseudo-sarcoids at all inoculation sites. We conclude that immunization of horses with BPV1 L1 VLPs induces long-lasting protection against experimental BPV1 virion-induced disease.

The candidate pan-Human Papillomavirus (HPV) vaccine RG1-VLP are HPV16 major capsid protein L1 virus-like-particles (VLP) comprising a type-common epitope of HPV16 minor capsid protein L2 (RG1; aa17-36). Vaccinations have previously demonstrated efficacy against genital high-risk (hr), low-risk (lr) and cutaneous HPV. To compare RG1-VLP to licensed vaccines, rabbits (n = 3) were immunized thrice with 1 µg, 5 µg, 25 µg, or 125 µg of RG1-VLP or a 1/4 dose of Cervarix®. 5 µg of RG1-VLP or 16L1-VLP (Cervarix) induced comparable HPV16 capsid-reactive and neutralizing antibodies titers (62,500/12,500–62,500 or 1000/10,000). 25 µg RG1-VLP induced robust cross-neutralization titers (50–1000) against hrHPV18/31/33/45/52/58/26/70. To mimic reduced immunization schedules in adolescents, mice (n = 10) were immunized twice with RG1-VLP (5 µg) plus 18L1-VLP (5 µg). HPV16 neutralization (titers of 10,000) similar to Cervarix and Gardasil and cross-protection against hrHPV58 vaginal challenge was observed. RG1-VLP vaccination induces hrHPV16 neutralization comparable to similar doses of licensed vaccines, plus cross-neutralization to heterologous hrHPV even when combined with HPV18L1-VLP.

Poly[di(carboxylatomethylphenoxy)phosphazene] (PCMP), a new member of polyphosphazene immunoadjuvant family, is synthesized. In vitro assessment of a new macromolecule revealed hydrolytic degradation profile and immunostimulatory activity comparable to its clinical stage homologue PCPP; however, PCMP was characterized by a beneficial reduced sensitivity to the ionic environment. In vivo evaluation of PCMP potency was conducted with human papillomavirus (HPV) virus-like particles (VLPs) based RG1-VLPs vaccine. In contrast with previously reported self-assembly of polyphosphazene adjuvants with proteins, which typically results in the formation of complexes with multimeric display of antigens, PCMP surface modified VLPs in a composition dependent pattern, which at a high polymer-to VLPs ratio led to stabilization of antigenic particles. Immunization experiments in mice demonstrated that PCMP adjuvanted RG1-VLPs vaccine induced potent humoral immune responses, in particular, on the level of highly desirable protective cross-neutralizing antibodies, and outperformed PCPP and Alhydrogel adjuvanted formulations.

Background: Mucosal human papillomaviruses (HPV) are a major cause of cancers and papillomas of the anogenital and oropharyngeal tract. HPV-vaccination elicits neutralising antibodies in sera and cervicovaginal secretions and protects uninfected individuals from persistent anogenital infection and associated diseases caused by the vaccine-targeted HPV types. Whether immunisation can prevent oropharyngeal infection and diseases and whether neutralising antibodies represent the correlate of protection, is still unclear. Methods: We determined IgG and neutralising antibodies against low-risk HPV6 and high-risk HPV16/18 in sera and oral fluids from healthy females (n=20) before and after quadrivalent HPV-vaccination and compared the results with non-vaccinated controls. Results: HPV-vaccination induced type-specific antibodies in sera and oral fluids of the vaccinees. Importantly, the antibodies in oral fluids were capable of neutralising HPV pseudovirions in vitro, indicating protection from infection. The increased neutralising antibody levels against HPV16/18 in sera and oral fluids post-vaccination correlated significantly within an individual. Conclusions: We provide experimental proof that HPV-vaccination elicits neutralising antibodies to the vaccine-targeted types in oral fluids. Hence, immunisation may confer direct protection against type-specific HPV infection and associated diseases of the oropharyngeal tract. Measurement of antibodies in oral fluids represents a suitable tool to assess vaccine-induced protection within the mucosal milieu of the orophayrynx.

Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17-36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous HPV infections.

We show that minor capsid protein L2 is full length in clinical virion isolates and prepare furin-cleaved pseudovirus (fcPsV) as a model of the infectious intermediate for multiple human papillomavirus (HPV) types. These fcPsV do not require furin for in vitro infection, and are fully infectious in vivo. Both the γ-secretase inhibitor XXI and carrageenan block fcPsV infection in vitro and in vivo implying that they act after furin-cleavage of L2. Despite their enhanced exposure of L2 epitopes, vaccination with fcPsV particles fails to induce L2 antibody, although L1-specific responses are similar to PsV with intact L2. FcPsV can be applied in a simple, high-throughput neutralization assay that detects L2-specific neutralizing antibodies with >10-fold enhanced sensitivity compared with the PsV-based assay. The PsV and fcPsV-based assays exhibit similar sensitivity for type-specific antibodies elicited by L1 virus-like particles (VLP), but the latter improves detection of L1-specific cross-type neutralizing antibodies.

Licensed human papillomavirus (HPV) vaccines, based on virus-like particles (VLPs) self-assembled from major capsid protein L1, afford type-restricted protection against HPV types 16/18/6/11 (or 16/18 for the bivalent vaccine), which cause 70% of cervical cancers (CxCas) and 90% of genital warts. However, they do not protect against less prevalent high-risk (HR) types causing 30% of CxCa, or cutaneous HPV. In contrast, vaccination with the minor capsid protein L2 induces low-level immunity to type-common epitopes. Chimeric RG1-VLP presenting HPV16 L2 amino acids 17–36 (RG1 epitope) within the DE-surface loop of HPV16 L1 induced cross-neutralizing antisera. We hypothesized that RG1-VLP vaccination protects against a large spectrum of mucosal and cutaneous HPV infections in vivo. Immunization with RG1-VLP adjuvanted with human-applicable alum-MPL (aluminum hydroxide plus 3-O-desacyl-4′-monophosphoryl lipid A) induced robust L2 antibodies (ELISA titers 2,500–12,500), which (cross-)neutralized mucosal HR HPV16/18/45/37/33/52/58/35/39/51/59/68/73/26/69/34/70, low-risk HPV6/11/32/40, and cutaneous HPV2/27/3/76 (titers 25–1,000) using native virion- or pseudovirion (PsV)-based assays, and a vigorous cytotoxic T lymphocyte response by enzyme-linked immunospot. In vivo, mice were efficiently protected against experimental vaginal challenge with mucosal HR PsV types HPV16/18/45/31/33/52/58/35/39/51/59/68/56/73/26/53/66/34 and low-risk HPV6/43/44. Enduring protection was demonstrated 1 year after vaccination. RG1-VLP is a promising next-generation vaccine with broad efficacy against all relevant mucosal and also cutaneous HPV types.

Infections with sexually transmitted high-risk Human Papillomavirus (hrHPV), of which there are at least 15 genotypes, are responsible for a tremendous disease burden by causing cervical, and subsets of other ano-genital and oro-pharyngeal carcinomas, together representing 5% of all cancer cases worldwide. HPV subunit vaccines consisting of virus-like particles (VLP) self-assembled from major capsid protein L1 plus adjuvant have been licensed. Prophylactic vaccinations with the 2-valent (HPV16/18), 4-valent (HPV6/11 /16/18), or 9-valent (HPV6/11/16/18/31/33/45/52/58) vaccine induce high-titer neutralizing antibodies restricted to the vaccine types that cause up to 90% of cervical carcinomas, a subset of other ano-genital and oro-pharyngeal cancers and 90% of benign ano-genital warts (condylomata). The complexity of manufacturing multivalent L1-VLP vaccines limits the number of included VLP types and thus the vaccines’ spectrum of protection, leaving a panel of oncogenic mucosal HPV unaddressed. In addition, current vaccines do not protect against cutaneous HPV types causing benign skin warts, or against beta-papillomavirus (betaPV) types implicated in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. In contrast with L1-VLP, the minor capsid protein L2 contains type-common epitopes that induce low-titer yet broadly cross-neutralizing antibodies to heterologous PV types and provide cross-protection in animal challenge models. Efforts to increase the low immunogenicity of L2 (poly)-peptides and thereby to develop broader-spectrum HPV vaccines are the focus of this review.

Bovine papillomavirus types 1 and 2 (BPV-1 and BPV-2) are known to induce common equine skin tumours, termed sarcoids. Recently, it was demonstrated that vaccination with BPV-1 virus-like particles (VLPs) is safe and highly immunogenic in horses. To establish a BPV-1 challenge model for evaluation of the protective potential of BPV-1 VLPs, four foals were injected intradermally with infectious BPV-1 virions and with viral genome-based and control inocula, and monitored daily for tumour development. Blood was taken before inoculation and at weekly intervals. BPV-1-specific serum antibodies were detected by a pseudo-virion neutralization assay. Total nucleic acids extracted from tumours, intact skin and PBMCs were tested for the presence of BPV-1 DNA and mRNA using PCR and RT-PCR, respectively. Intralesional E5 oncoprotein expression was determined by immunofluorescence. Pseudo-sarcoids developed exclusively at sites inoculated with virions. Tumours became palpable 11–32 days after virion challenge, reached a size of ≤20 mm in diameter and then resolved in ≤6 months. No neutralizing anti-BPV-1 serum antibodies were detectable pre- or post-challenge. BPV-1 DNA was present in lesions but not in intact skin. In PBMCs, viral DNA was already detectable before lesions were first palpable, in concentrations correlating directly with tumour growth kinetics. PBMCs from two of two foals also harboured E5 mRNA. Immunofluorescence revealed the presence of the E5 protein in tumour fibroblasts, but not in the apparently normal epidermis overlying the lesions. Together with previous findings obtained in horses and cows, these data suggest that papillomavirus infection may include a viraemic phase.

Summary Reasons for performing study: Infection with bovine papillomaviruses types 1 and 2 (BPV-1, BPV-2) can lead to the development of therapy-resistant skin tumours termed sarcoids and possibly other skin diseases in equids. Although sarcoids seriously compromise the welfare of affected animals and cause considerable economic losses, no prophylactic vaccine is available to prevent this common disease. In several animal species and man, immunisation with papillomavirus-like particles (VLP) has been shown to protect efficiently from papillomaviral infection. Hypothesis: BPV-1 L1 VLPs may constitute a safe and highly immunogenic vaccine candidate for protection of horses against BPV-1/-2-induced disease. Methods: Three groups of 4 horses each received 50, 100 or 150 mg of BPV-1 L1 VLPs, respectively, on Days 0, 28 and 168. Three control horses received adjuvant only. Horses were monitored on a daily basis for one week after each immunisation and then in 2 week intervals. Sera were collected immediately before, 2 weeks after each vaccination and one and 2 years after the final boost and analysed by pseudovirion neutralisation assay. Results: None of the horses showed adverse reactions upon vaccination apart from mild and transient swelling in 2 individuals. Irrespective of the VLP dose, all VLP-immunised horses had developed a BPV-1-neutralising antibody titre of 1600 plaque forming units (pfu)/ml 2 weeks after the third vaccination. Eight of 10 trial horses still available for follow-up had neutralising antibody titres 1600 pfu/ml one year and 800 pfu/ml 2 years after the last immunisation. Conclusion: Intramuscular BPV-1 L1 VLP vaccination in horses is safe and results in a long-lasting antibody response against BPV-1. Neutralisation titres were induced at levels that correlate with protection in experimental animals and man. Potential relevance: BPV-1 L1 VLPs constitute a promising vaccine candidate for prevention of BPV-1/-2-induced disease in equids.

The amino (N) terminus of the human papillomavirus (HPV) minor capsid protein L2 can induce low-titer, cross-neutralizing antibodies. The aim of this study was to improve immunogenicity of L2 peptides by surface display on highly ordered, self-assembled virus-like particles (VLP) of major capsid protein L1, and to more completely characterize neutralization epitopes of L2. Overlapping peptides comprising amino acids (aa) 2 to 22 (hereafter, chimera or peptide 2-22), 13 to 107, 18 to 31, 17 to 36, 35 to 75, 75 to 112, 115 to 154, 149 to 175, and 172 to 200 of HPV type 16 (HPV16) L2 were genetically engineered into the DE surface loop of bovine papillomavirus type 1 L1 VLP. Except for chimeras 35-75 and 13-107, recombinant fusion proteins assembled into VLP. Vaccination of rabbits with Freund's adjuvanted native VLP induced higher L2-specific antibody titers than vaccination with corresponding sodium dodecyl sulfate-denatured proteins. Immune sera to epitopes within residues 13 to 154 neutralized HPV16 in pseudovirion neutralization assays, whereas chimera 17-36 induced additional cross-neutralization to divergent high-risk HPV18, -31, -45, -52, and -58; low-risk HPV11; and beta-type HPV5 (titers of 50 to 10,000). Aluminum hydroxide-monophosphoryl lipid A (Alum-MPL)-adjuvanted VLP induced similar patterns of neutralization in both rabbits and mice, albeit with 100-fold-lower titers than Freund's adjuvant. Importantly, Alum-MPL-adjuvanted immunization with chimeric HPV16L1-HPV16L2 (peptide 17-36) VLP induced neutralization or cross-neutralization of HPV16, -18, -31, -45, -52, and -58; HPV6 and -11; and HPV5 (titers of 50 to 100,000). Immunization with HPV16 L1-HPV16 L2 (chimera 17-36) VLP in adjuvant applicable for human use induces broad-spectrum neutralizing antibodies against HPV types evolutionarily divergent to HPV16 and thus may protect against infection with mucosal high-risk, low-risk, and beta HPV types and associated disease.

Licensed human papillomavirus (HPV) vaccines are expected to prevent high-risk (hr) HPV-infections (most notably types 16 and 18). Whether HPV vaccination will change the distribution of hrHPVs at the population level is open, since competition between HPV types is not well understood. Two stratified random subcohorts (1983–1997 and 1995–2003) of 7,815 and 3,252 women with a minimum of 2 pregnancies (

The expression pattern of human papillomavirus (HPV) capsid antigen L2 is poorly described, and the significance of its localization with both promyelocytic leukemia protein (PML) and Daxx in a subnuclear domain, nuclear domain 10 (ND-10), when ectopically expressed in tissue culture cells is controversial. To address whether ND-10 localization of L2 occurs in natural cervical lesions, we used a HPV16 and HPV18 L2-specific monoclonal antibody (RG-1), in addition to rabbit antiserum to HPV6 L2, to localize L2. Immunohistochemical staining with RG-1 produced diffuse staining in the nuclei of some cells located within the superficial epithelial layers in eight of nine cases of HPV16/18(+) cervical intraepithelial neoplasia grade 1 (CIN1); however, no staining was observed in HPV16/18(+) high-grade CIN (0 of 8 cases), normal cervical epithelium (0 of 20 cases), cervical squamous cell carcinoma (0 of 102 cases), adenocarcinoma (0 of 51 cases), or adenosquamous carcinoma (0 of 6 cases). HPV16/18(+) cervical lesions that express L2 exhibit higher HPV16/18 genome copies per cell compared with those that do not positively stain with RG-1 (P = 0.04). RG-1 staining of HeLa cells transfected with L2 expression constructs was frequently concentrated in the ND-10, particularly in cells expressing high levels of L2, and co-localized with the cellular markers of ND-10, PML, and Daxx. In contrast, L2 was primarily diffuse within the nucleus and distinct from ND-10 as defined by PML immunofluorescent staining in CIN lesions, condylomata, and HPV16-transduced organotypic cultures.

Bovine papillomavirus type 1 or 2 (BPV-1, BPV-2) are accepted causal factors in equine sarcoid pathogenesis. Whereas viral genomes are consistently found and expressed within lesions, intact virions have never been detected, thus permissiveness of sarcoids for BPV-1 replication remains unclear. To reassess this issue, an immunocapture PCR (IC/PCR) was established using L1-specific antibodies to capture L1-DNA complexes followed by amplification of the viral genome. Following validation of the assay, 13 sarcoid-bearing horses were evaluated by IC/PCR. Samples were derived from 21 tumours, 4 perilesional/intact skin biopsies, and 1 serum. Tissue extracts from sarcoid-free equines served as controls. IC/PCR scored positive in 14/24 (58.3%) specimens obtained from sarcoid-patients, but negative for controls. Quantitative IC/PCR demonstrated

Background: Since 2003, an ongoing outbreak of lymphogranuloma venereum (LGV), caused by Chlamydia trachomatis biovar L2b, has been reported among men who have sex with men. Methods: Twenty-four samples positive for C. trachomatis were analyzed for specific biovars and genovariants by genotyping of the variable segment (VS) 4, VS2 and VS1 regions of the outer membrane protein (omp) A. In addition we assessed the patients’ sociodemographic background and clinical signs and symptoms. Results: Twenty-four men who have sex with men presented with either anorectal or inguinal symptoms and tested positive for C. trachomatis DNA. Of these, the L2 genotype accounted for 15 patients, with a high coinfection rate with HIV (73.3%) and other sexually transmitted infections (53.4%). Analysis of the VS1, VS2, and VS4 regions of the ompA gene revealed the variant L2b in 8 patients. In 4 patients, 3 new L2 sequences were identified with nucleotide changes in the VS1, VS2, and VS4 region, respectively, defining new strains designated L2c, d, e. Conclusions: This outbreak of LGV represents the further spread of C. trachomatis L2 infection. Sequence analysis of ompA regions shows heterogeneity of L2 variants, suggesting more than 1 source of the LGV infections diagnosed in Vienna.

Prion diseases are fatal neurodegenerative disorders caused by proteinaceous infectious pathogens termed prions (PrP(Sc)). To date, there is no prophylaxis or therapy available for these transmissible encephalopathies. Passive immunization with monclonal antibodies recognizing the normal host-encoded prion protein (PrP(C)) has been reported to abolish PrP(Sc) infectivity and to delay onset of disease. Because of established immunologic tolerance against the widely expressed PrP(C), active immunization appears to be difficult to achieve. To overcome this limitation, papillomavirus-like particles were generated that display a nine amino acid B-cell epitope, DWEDRYYRE, of the murine/rat prion protein in an immunogenic capsid surface loop, by insertion into the L1 major capsid protein of bovine papillomavirus type 1. The PrP peptide was selected on the basis of its previously suggested central role in prion pathogenesis. Immunization with PrP-virus-like particles induced high-titer antibodies to PrP in rabbit and in rat, without inducing overt adverse effects. As determined by peptide-specific ELISA, rabbit immune sera recognized the inserted murine/rat epitope and also cross-reacted with the homologous rabbit/human epitope differing in one amino acid residue. In contrast, rat immune sera recognized the murine/rat peptide only. Sera of both species reacted with PrP(C) in its native conformation in mouse brain and on rat pheochromocytoma cells, as determined by immunoprecipitation and fluorescence-activated cell sorting analysis. Importantly, rabbit anti-PrP serum contained high-affinity antibody that inhibited de novo synthesis of PrP(Sc) in prion-infected cells. If also effective in vivo, PrP-virus-like particle vaccination opens a unique possibility for immunologic prevention of currently fatal and incurable prion-mediated diseases.

Genomes of human papillomaviruses (HPV) are common in biopsies from non-melanoma skin cancers but are also found on healthy skin and it is possible that HPV positivity in tumor biopsies by PCR may merely reflect contamination of the lesion surface. To investigate this issue, 229 immunocompetent patients were tested for HPV DNA in swab samples collected on top of skin tumors and in biopsies of the same tumors, obtained after stripping with tape to remove superficial layers. HPV DNA was detected on top of 69% (159 of 229) of the lesions, and in 12% (28 of 229) of the stripped biopsies (p

Pemphigus erythematosus, initially described as a combination of pemphigus with lupus erythematosus, and pemphigus foliaceus are now frequently considered localized and generalized variants of superficial pemphigus. Yet diagnostic criteria for pemphigus erythematosus remain controversial. Distinct from pemphigus foliaceus, pemphigus erythematosus displays immune depositions at the dermal-epidermal junction, which suggests additional immunopathological mechanisms. We present three patients with clinical and histopathologic signs of superficial pemphigus, who all exhibited an immunomorphology characteristic of pemphigus erythematosus. Complement depositions in a granular-linear fashion were consistently found at the dermal-epidermal junction besides in vivo bound and circulating antikeratinocyte cell-surface autoantibodies. Histopathology showed subcorneal acantholysis, and all sera contained antidesmoglein 1 but not antidesmoglein 3 autoantibodies detected by enzyme-linked immunosorbent assays (ELISA). Additional autoantibodies against a 230-kDa protein and against a 190-kDa protein comigrating with bullous pemphigoid antigen 1 (BP230) and periplakin, respectively, were present in all the patients' sera. As two sera specifically reacted with BP230 by ELISA, the presence of BP230-specific autoantibodies could be associated with dermal-epidermal immune staining in these patients. In pemphigus erythematosus, dermal-epidermal immune staining is generally attributed to the deposition of immune complexes, while the presence of BP230-specific autoantibodies has not been reported in this disease previously. Perhaps, the unique autoantibody profile of the patients in the study permits discrimination between patients with superficial pemphigus that display additional dermal-epidermal immune staining from those with conventional pemphigus foliaceus on a molecular basis. Further studies will be required to substantiate the frequency of this occurrence and to unravel its pathogenic significance.

Neutralization capsid epitopes are important determinants for antibody-mediated immune protection against papillomavirus (PV) infection and induced disease. Chimeric L1 major capsid proteins of the human PV type 16 (HPV-16) and the bovine PV type 1 (BPV-1) with a foreign peptide incorporated into several capsid surface loops self-assembled into pentamers or virus-like particles (VLP). Binding patterns of neutralizing monoclonal antibodies (MAb) and immunization of mice confirmed (i) that regions around aa 282–286 and 351–355 contribute to neutralization epitopes and identified the latter region as an immunodominant site and (ii) that placing a foreign peptide in the context of an assembled structure markedly enhanced its immunogenicity. Pentamers disassembled from wild-type HPV-16 and BPV-1 VLPs displayed some of the neutralization epitopes that were detected on fully assembled VLPs, but were deficient for binding a subset of neutralizing MAb that inhibit cell attachment.

Papillomavirus-like particles (VLPs) are a promising prophylactic vaccine candidate to prevent human papillomavirus (HPV) infections and associated epithelial neoplasia. However, they are unlikely to have therapeutic effects because the virion capsid proteins are not detected in the proliferating cells of the infected epithelia or in cervical carcinomas. To increase the number of viral antigen targets for cell-mediated immune responses in a VLP-based vaccine, we have generated stable chimeric VLPs consisting of the L1 major capsid protein plus the entire E7 (11 kDa) or E2 (43 kDa) nonstructural papillomavirus protein fused to the L2 minor capsid protein. The chimeric VLPs are indistinguishable from the parental VLPs in their morphology and in their ability to agglutinate erythrocytes and elicit high titers of neutralizing antibodies. Protection from tumor challenge was tested in C57BL/6 mice by using the tumor cell line TC-1, which expresses HPV16 E7, but not the virion structural proteins. Injection of HPV16 L1/L2-HPV16 E7 chimeric VLPs, but not HPV16 L1/L2 VLPs, protected the mice from tumor challenge, even in the absence of adjuvant. The chimeric VLPs also induced protection against tumor challenge in major histocompatibility class II-deficient mice, but not in β2-microglobulin or perforin knockout mice implying that protection was mediated by class I-restricted cytotoxic lymphocytes. These findings raise the possibility that VLPs may generally be efficient vehicles for generating cell-mediated immune responses and that, specifically, chimeric VLPs containing papillomavirus nonstructural proteins may increase the therapeutic potential of VLP-based prophylactic vaccines in humans.

The epidemiologic determinants of seroreactivity to human papillomavirus (HPV) type 16 L1/L2 virus-like particles (VLPs) were assessed separately in HPV-16 DNA-positive and -negative women participating in a nested case-control study of incident cervical neoplasia. Seventy-four women with cervical HPV-16 DNA and 656 cytologically normal HPV-16 DNA-negative subjects were interviewed and tested at two time points for viral DNA and once (at the later time) for VLP seroreactivity. Among subjects who were currently HPV-16 DNA-negative, seroreactivity odds ratios increased from 2.9 for 2-5 male sex partners (vs. 0 or 1) to 5.4 for 6-9 partners and 14.0 for > or = 10. Thus, prior cervical infection may be a major determinant of seroreactivity in HPV-16 DNA-negative women. This trend was not observed in HPV-16 DNA-positive subjects. Seroreactivity was independently associated with oral contraceptive use, particularly in HPV-16 DNA-negative subjects with use for > or = 10 years. Consequently, a possible role for virus-steroid hormone interactions in seroconversion is suggested.

We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific.

Virus-like particles were produced in insect cells containing either the L1 and L2 capsid proteins of bovine papillomavirus type 4 (BPV-4) or only the L1 protein. Both preparations of VLPs proved to be extremely effective prophylactic vaccines. Thirteen of 15 calves immunised with either L1–L2 VLPs or L1–VLPs were refractory to experimental challenge with high doses of BPV-4 and did not develop papillomas, while 9 of 10 control animals developed multiple oral papillomas. VLPs were not efficient as therapeutic vaccine in calves with established papillomas, although VLP-vaccinated animals appeared to undergo tumour regression more rapidly than nonvaccinated control animals. Antibody responses in VLP-vaccinated calves were associated with prevention of disease but not with regression of papillomas. Thus prophylactic VLP vaccination is effective in preventing disease in this model of mucosal papillomavirus infection. VLPs and native virus share at least some conformational epitopes, as shown by the cross-reactivity of their antibodies.

It is not known whether DNA sequence variants of human papillomavirus type 16 (HPV-16) are distinct serotypes. To examine this question, the reactivities of women's sera from Zaire (n = 97) and Denmark (n = 123) were compared in IgG-specific ELISAs based on virus-like particles (VLPs) composed of the L1 major capsid protein derived from an HPV-16 variant common in central Africa (Z-1194) or one common in northern Europe (114K). These L1s differ in seven amino acids. There was a strong correlation between reactivity in the two assays for both sets of sera (correlation coefficients, 0.73 and 0.85 for Zairian and Danish sera, respectively). In only 1 serum was there evidence for a specific reaction to one but not the other VLP variant. The results support the conclusion that the virions of strains Z-1194 and 114K are serologically cross-reactive.

Virus-like particles (VLPs) composed of L1 derived from bovine papillomavirus type 1 (BPV-1), several human papillomavirus types, or cottontail rabbit papillomavirus (CRPV) agglutinated mouse but not human or rat erythrocytes. Treatment of mouse erythrocytes with trypsin prevented hemagglutination (HA) by BPV-1. Sera from rabbits immunized with native CRPV VLPs, which protect against experimental CRPV infection, exhibited high titers of antibodies that inhibited CRPV VLP HA activity, while sera from rabbits immunized with denatured CRPV VLPs or native BPV VLPs, which do not protect against CRPV infection, were not inhibitory. Testing for HA inhibition is a rapid and simple method for examining the serological relatedness of papillomaviruses and measuring protective antibody titers after VLP vaccination.

The NCI Division of Cancer Prevention's PREVENT Cancer Preclinical Drug Development Program supports preclinical development and clinical translation of novel cancer preventive interventions. One of the projects currently supported came from principal investigators, Drs. Reinhard Kirnbauer and Richard Roden, who requested cGMP production of a novel chimeric virus-like particle (VLP)-based human papillomavirus (HPV) vaccine, comprising HPV16 L1 VLP with HPV16 L2-RG1 epitope repetitively displayed on the capsid surface (RG1-VLP). The vaccine is designed to prevent infection by a broad range of HPV types, including mucosal and cutaneous types not targeted by current vaccines (J Invest Dermatol 2013, 133:2706), and thus has the potential to become a broadly effective next generation HPV vaccine. The cGMP process is currently being developed and validated by Paragon Bioservices in Baltimore, MD, under NCI contract with MRIGlobal. RG1-VLP is expressed in Sf9 insect cells infected with a triple-plaque-purified recombinant baculovirus. After cell culture clarification and capsid maturation steps, VLPs are purified by a series of chromatography steps to remove host cell derived impurities. The purified VLPs are analyzed by ELISA and Western blot immunoassays. Removal of host cell protein and DNA is confirmed by ELISA and qPCR, respectively. Assembly of a chimeric RG1-VLP protein into full size VLP structures (approximately 50-60 nm in diameter) is verified by transmission electron microscopy. cGMP-produced VLPs should be available during the first half of 2016. The PREVENT Program will perform the required toxicology studies and will assist with IND filing, for a first-in-human Phase I study of RG1-VLP vaccine. While the clinical protocol is still under development, it is anticipated that 15-20 healthy adult volunteers will be enrolled into each of three to four escalating dose groups. While the primary objective of the Phase I study will be to establish the safety of RG1-VLP, preliminary immunogenicity studies will be included. Immunogenicity of RG1-VLP will be evaluated by serum anti-L1 and anti-RG1 antibody levels as well as protective and cross-neutralizing antibodies against HPV16 and a broad panel of HPV types including at least one type not covered by marketed vaccines. Citation Format: George W. Buchman, Brian P. Howard, Nadia Abdallah, Bala Medicherla, Mary Anne Fisher, Jonathan M. White, Michelle L. Kennedy, Shizuko Sei, Richard B.S. Roden, Christina Schellenbacher, Reinhard Kirnbauer, Robert H. Shoemaker. cGMP production of a chimeric virus-like particle vaccine for prevention of HPV-associated cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2367.

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.

We have generated four mouse monoclonal antibodies (MAbs) to bovine papillomavirus virions that bound type-specific, adjacent, and conformationally dependent epitopes on the L1 major capsid protein. All four MAbs were neutralizing at ratios of 1 MAb molecule per 5 to 25 L1 molecules, but only three effectively blocked binding of the virus to the cell surface. Therefore, antibodies can prevent papillomavirus infection by at least two mechanisms: inhibition of cell surface receptor binding and a subsequent step in the infectious pathway. The neutralizing epitopes of the bovine papillomavirus L2 minor capsid protein were mapped to the N-terminal half of L2 by blocking the neutralizing activity of full-length L2 antiserum with bacterially expressed peptides of L2. In addition, rabbit antiserum raised against amino acids 45 to 173 of L2 had a neutralizing titer of 1,000, confirming that at least part of the N terminus of L2 is exposed on the virion surface.

Reasons for performing study We have already demonstrated that vaccination with empty BPV1 capsids termed virus-like particles (VLP) protects horses from experimental infection with BPV1 virion. Long-term monitoring of antibody titres in experimental horses and data from other species suggest that this protection is long lasting. Objectives To test protection against experimental infection with BPV-1 virion in horses that were vaccinated with BPV-1 L1 VLP approximately 5 years earlier. Study design Controlled experiment. Methods Seven horses, vaccinated 3 times (boosters after 4 weeks and 6 months) in 2007/2008 with doses of BPV1 L1 VLP ranging from 50 μg to 150 μg/dose and 3 unvaccinated control horses were challenged by intra-dermal inoculation with cow wart derived BPV1 virion (5 × 107 BPV-1 virions per wheal, 10 wheals per horse). Inoculation sites were monitored for 10 weeks. Blood for serum antibody titre determination by pseudovirion neutralisation assay was taken on the day of challenge and after 6 months. Results Six of 7 vaccinated horses had measurable serum antibody titres (1:50 to 1:400). These titres were boosted by inoculation (about one step of dilution). Two of 3 unvaccinated controls remained sero-negative. One control horse showed sero-conversion. All control horses developed tumours at all 10 inoculation sites. Tumours appeared approximately 2 weeks after inoculation and reached maximum sizes of up to 8 mm. Regression was complete by 8 weeks after their first appearance in all horses. All vaccinated horses remained completely free from tumours. No influence of dose rate or antibody titre on the level of protection could be determined. Conclusions BPV-1 L1 VLP vaccination proves to be fully effective in protecting horses from experimental infection and tumour formation 5 years post immunisation. The protection was complete even in horses with low or unmeasurable antibody titres. This is another step towards establishing a vaccination against equine sarcoids. Ethical animal research: This experiment was approved by the institutional ethics committee and the national authority under license No: GZ: 68.205/0144-II/3b/2012. Sources of funding: Veterinary University Vienna, Austrian Research Fund (FWF). Competing interests: None.

Although infections with multiple human papillomavirus (HPV) types have been reported widely, more information is needed on the occurrence of the different types. We determined the distribution of seroprevalences to multiple HPV types in Finland and Uganda to compare the epidemiology of the different HPV types in the 2 populations. Serum samples were obtained from 2784 Finnish and 1964 Ugandan women (mean ages 22 y and 25 y, respectively) of whom 44% and 57%, respectively, had antibodies to at least 1 of the 7 HPV types (6, 11, 16, 18, 31, 33, 45) tested (p < 0.001). Multiple HPV antibody positivity was common. HPV45-seropositive Finns had a higher risk of having antibodies to other high-risk HPV types: HPV18 (odds ratio (OR) = 10.9), HPV31 (OR 6.1), HPV33 (OR 12.2), than their Ugandan counterparts: HPV18 (OR 3.4), HPV31 (OR 2.2), HPV33 (OR 3.3). Increased estimates for being double antibody-positive were also noted among HPV18- and HPV16-seropositive women, but there were no major differences between HPV16-seropositive Finns and Ugandans. In addition to biological and behavioural factors, iatrogenic and societal factors (screening vs no screening) may also result in the different occurrence of infections with the high-risk HPV types in Finland and Uganda.

Intercellular adhesion molecule-1 (ICAM-1) is involved in cell-cell interactions of leukocytes and parenchymal cells and thus plays an important role in immunologic and inflammatory reactions. The expression of ICAM-1 that is found on many different cells such as melanocytes and melanoma cells is induced by various cytokines, including interferon-gamma (IFN gamma), interleukin (IL)-1 and tumor necrosis factor alpha (TNF alpha). Because expression of ICAM-1 in melanoma was found to correlate with increased risk of metastasis, the regulation of ICAM-1 expression on human melanocytes and melanoma cells was investigated. Foreskin-derived melanocytes and melanoma cell lines (A375, G361) were incubated with different cytokines and ICAM-1 expression was evaluated by fluorescence-activated cell sorter. IFN gamma, IL-1, IL-7, TNF alpha, and TNF beta significantly upregulated ICAM-1 expression in a dose-dependent manner. Most interestingly, the cytokine IL-6, which does not influence adhesion-molecule expression on other cells, significantly upregulated melanocyte and melanoma cell ICAM-1 expression. This effect was dose dependent and could be blocked by an IL-6 antibody. Irradiation with ultraviolet (UVB) light did not influence constitutive ICAM-1 expression on melanoma cells and melanocytes, but suppressed cytokine-induced ICAM-1 expression when cells were harvested 16 h after irradiation. These findings were further confirmed by Northern blot analysis, showing a marked accumulation of ICAM-1 mRNA after cytokine treatment, which was reduced by irradiation with UVB light. However, when UVB-exposed melanoma cells were cultured for at least 48 h induction of ICAM-1 expression was observed. These data indicate that, similar to other cells, ICAM-1 expression on melanoma cells and melanocytes is regulated by cytokines and that UVB light affects ICAM-1 expression on melanocytic cells in a biphasic manner.

WHIM-syndrome is an inherited immunodeficiency disorder with abnormal susceptibility to human papillomavirus (HPV) infection and diseases. We determined safety and immunogenicity to a quadrivalent HPV vaccine in WHIM-syndrome by detection of HPV-specific antibodies and lymphoproliferation. In virus-like-particle (VLP)-ELISA, a WHIM patient showed antibody titers up to 400 for HPV-6/11/16/18, whereas immuno-competent controls developed titers of 6400-25,600. In pseudovirion assays, the patient's neutralization titers ranged from 20 to 400 to the four HPV vaccine types, while titers of 1600-25,600 were detected in healthy vaccinees. Specific proliferation of PBMC of the WHIM patient to the HPV vaccine was demonstrated. This first report on response to HPV vaccination in WHIM-immunodeficiency highlights that patients with WHIM-syndrome, and probably other immunodeficiencies, may benefit from HPV immunoprophylaxis.

Interleukin-6 (IL-6) is a multifunctional inflammatory cytokine that is produced by monocytes and keratinocytes upon stimulation. Because psoriasis is a skin disease characterized by a hyperproliferative activity of keratinocytes and an inflammatory infiltrate, in the present study IL-6 production of monocytes and keratinocytes of patients with psoriasis was investigated. Peripheral blood mononuclear cells (PBMC) derived from psoriatics, atopics, and healthy controls were incubated for 24 h and, subsequently, supernatant IL-6 activity was measured using an IL-6-dependent hybridoma cell line (B9). Compared to controls and atopics, PBMC of psoriatics produced significantly increased amounts of biologically active IL-6. These findings were also confirmed by Western blot analysis using a specific antiserum directed against IL-6. Moreover, when the sera of the same patients were tested for IL-6 activity, sera of psoriatics contained significantly elevated amounts of circulating IL-6 in comparison to samples from atopics and healthy controls. In contrast to normal or uninvolved skin, keratinocytes in psoriatic lesions were remarkably positive for IL-6 as detected by immunohistochemistry and in situ hybridization. In addition, IL-6 also was found to induce its own synthesis and release by monocytes. These findings indicate that keratinocytes and monocytes in psoriasis are activated to produce increased amounts of IL-6, which may be one of the mediators involved in the regulation of both local and systemic inflammatory reactions occurring in skin diseases such as psoriasis.

Evidence of a possible association of cutaneous human papillomavirus (HPV) types, especially members of the genus Betapapillomavirus, and the development of non-melanoma skin cancer (NMSC) is accumulating. Vaccination with virus-like particles (VLPs) consisting of self-assembled L1, the major capsid protein, has been introduced to control anogenital HPV infection. This study examined the serological relationship between betapapillomavirus (beta-PV) types 5 and 8 and the new type HPV-92, which has recently been isolated from a basal cell carcinoma containing a high number of viral genomes. Following expression by recombinant baculoviruses, the L1 protein of HPV-92 self-assembled into VLPs that elicited high-titre antibodies after immunization, similar to VLPs from HPV-5 and -8. Haemagglutination inhibition (HAI) assays were used as a surrogate method for the detection of virion-neutralizing antibodies, which correlates with protection from infection. Antisera raised against HPV-5 and -8 VLPs displayed HAI activity not only against the homologous type, but also against heterologous HPV types 5, 8 and 92, whereas HAI activity of antisera against HPV-92 VLP was restricted to the homologous type. The results of neutralization assays using HPV-5 pseudovirions were consistent with those from HAI assays. Cross-neutralizing immune responses by VLP vaccination against heterologous HPV types may provide broader protection against the multiplicity of HPV types detected in NMSC. If a close link to HPV infection can be conclusively established, these results may provide a basis for further evaluation of VLPs of beta-PVs as candidates for a prophylactic skin-type HPV vaccine, aimed at reducing the incidence of NMSC.

Bovine papillomaviruses of types 1 and 2 (BPV-1 and -2) chiefly contribute to equine sarcoid pathogenesis. However, the mode of virus transmission and the presence of latent infections are largely unknown. This study established a PCR protocol allowing detection of ≤10 copies of the BPV-1/-2 genes E5 and L1. Subsequent screening of peripheral blood mononuclear cell (PBMC) DNA derived from horses with and without BPV-1/2-induced skin lesions demonstrated the exclusive presence of E5, but not L1, in PBMCs of BPV-1/2-infected equines. To validate this result, a blind PCR was performed from enciphered PBMC DNA derived from 66 horses, revealing E5 in the PBMCs of three individuals with confirmed sarcoids, whereas the remaining 63 sarcoid-free animals were negative for this gene. L1 could not be detected in any PBMC DNA, suggesting either deletion or interruption of this gene in PBMCs of BPV-1/-2-infected equines. These results support the hypothesis that PBMCs may serve as host cells for BPV-1/-2 DNA and contribute to virus latency.

Cutaneous human papillomaviruses (HPV) are common in nonmelanoma skin cancers, benign skin lesions, and healthy skin. Increased seroprevalences for cutaneous HPV among nonmelanoma skin cancer patients have been described. To determine whether antibodies to cutaneous HPV are related to presence of the virus and/or to skin disease, we collected serum and biopsies from both lesions and healthy skin from 434 nonimmunosuppressed patients (72 squamous cell carcinomas, 160 basal cell carcinomas, 81 actinic keratoses, and 121 benign lesions). Biopsies were analyzed for HPV DNA by PCR, cloning, and sequencing. Serum antibodies to the major capsid protein L1 of HPV 1, 5, 6, 8, 9, 10, 15, 16, 20, 24, 32, 36, 38, and 57 as well as to the oncoproteins E6 and E7 of HPV 8 and 38 were detected using a multiplexed fluorescent bead-based assay. Type-specific seroprevalence among patients with the same type of HPV DNA (sensitivity of serology) varied from 0% to at most 28%. Presence of HPV DNA and antibodies to the same HPV type was not significantly correlated. However, seropositivity to any HPV type was significantly more common among patients positive for HPV DNA of any HPV type (odds ratio, 1.90; 95% confidence interval, 1.55-2.34). Seroprevalences were similar among the different patient groups but was, for most HPV types, somewhat higher among squamous cell carcinoma patients than among basal cell carcinoma patients (P < 0.01). In conclusion, additional studies are required to clarify the biological meaning of seropositivity as a marker of cutaneous HPV infection and skin disease. (Cancer Epidemiol Biomarkers Prev 2008;17(1):189–95)

Tumor necrosis factor alpha (TNF-alpha), in addition to being cytotoxic for certain tumor cells, has turned out as a multifunctional cytokine that is involved in the regulation of immunity and inflammation. Since human keratinocytes have been demonstrated to be a potent source of various cytokines, it was investigated whether epidermal cells synthesize and release TNF-alpha. Supernatants derived from normal human keratinocytes (HNK) and human epidermoid carcinoma cell lines (KB, A431) were tested both in a TNF-alpha-specific ELISA and a bioassay. In supernatants of untreated epidermal cells, no or minimal TNF-alpha activity was found, while after stimulation with lipopolysaccharide (LPS) or ultraviolet (UV) light, significant amounts were detected. Western blot analysis using an antibody directed against human TNF-alpha revealed a molecular mass of 17 kD for keratinocyte-derived TNF-alpha. These biological and biochemical data were also confirmed by Northern blot analysis revealing mRNA specific for TNF-alpha in LPS- or ultraviolet B (UVB)-treated HNK and KB cells. In addition, increased TNF-alpha levels were detected in the serum obtained from human volunteers 12 and 24 h after a single total body UVB exposure, which caused a severe sunburn reaction. These findings indicate that keratinocytes upon stimulation are able to synthesize and release TNF-alpha, which may gain access to the circulation. Thus, TNF-alpha in concert with other epidermal cell-derived cytokines may mediate local and systemic inflammatory reactions during host defense against injurious events caused by microbial agents or UV irradiation.

Ultraviolet B (UVB) radiation (280-320 nm) is capable of suppressing selected cell mediated immune responses by inhibiting the function of antigen presenting/accessory cells. Human keratinocytes and carcinoma cell lines (A431) upon UVB radiation or treatment with PMA secrete a suppressor factor, which blocks IL 1 activity (hEC-contra-IL 1). Therefore, the capacity of this UVB-inducible cytokine to modulate human accessory cell function was tested. Human peripheral blood mononuclear cells were stimulated with the mitogenic anti-CD3 monoclonal antibody OKT3 and thymidine incorporation into proliferating T-cells was measured as an index for monocyte accessory cell activity. Addition of hEC-contra-IL 1 which was purified by HPLC chromatography partially decreased OKT3 induced T-cell proliferation in a dose dependent manner. Human EC-contra-IL 1, however, failed to inhibit blastogenesis when T-cells depleted of accessory cells were stimulated in an accessory cell independent fashion via OKT3 attached to the bottom of microtiter plates. Recombinant human (rh) IL 1, but not rhIL 6 was able to reconstitute hEC-contra-IL 1 suppressed blastogenesis in a dose dependent manner. Furthermore, the combined addition of h-EC-contra-IL 1 and an antibody against rhIL 6 to cultures resulted in an additive inhibitory effect which could not be observed when hEC-contra-IL 1 was added together with a monoclonal antibody against rhIL 1 alpha/beta. These studies indicate that hEC-contra-IL 1 is capable of suppressing human accessory cell function by specifically blocking IL 1 activity. This property of hEC-contra-IL 1 points to a novel mechanism by which UVB radiation may modulate human accessory cell function in an indirect manner.

Background. A spectrum of cutaneous human papillomaviruses (HPVs) is detectable in nonmelanoma skin cancers, as well as in healthy skin, but the significance that the presence of these types of HPV DNA has for the pathogenesis of skin cancer remains unclear. Methods. We studied 349 nonimmunosuppressed patients with skin lesions (82 with squamous cell carcinomas, 126 with basal cell carcinomas, 49 with actinic keratoses, and 92 with benign lesions). After superficial skin had been removed by tape, paired biopsy samples-from the lesion and from healthy skin from the same patient-were tested for HPV DNA. Risk factors for HPV DNA were analyzed in multivariate models. Results. Overall, 12% of healthy skin samples were positive for HPV DNA, compared with 26% of benign lesions, 22% of actinic keratoses, 18% of basal cell carcinomas, and 26% of squamous cell carcinomas. HPV DNA was associated with sites extensively exposed to the sun, both for the lesions (odds ratio [OR], 4.45 [95% confidence interval {CI}, 2.44-8.111) and for the healthy skin samples (OR, 3.65 [95% CI 1.79-7.44]). HPV types of Beta-papillomavirus species 2 predominate in squamous cell carcinomas (OR, 4.40 [95% CI, 1.92-10.06]), whereas HPV types of Beta-papillomavirus species 1 are primarily found in benign lesions (OR, 3.47 [95% CI, 1.72-6.99]). Conclusions. Cutaneous HPV types are primarily detected at sites extensively exposed to the sun. HPV types of Beta-papillomavirus species 2, but not of species 1, are associated with squamous cell carcinoma. (Less)

Persistent infection with oncogenic human papillomaviruses (HPV) types causes all cervical and a subset of other anogenital and oropharyngeal carcinomas. Four high-risk (hr) mucosal types HPV16, 18, 45, or 59 cause almost all cervical adenocarcinomas (AC), a subset of cervical cancer (CxC). Although the incidence of cervical squamous cell carcinoma (SCC) has dramatically decreased following introduction of Papanicolaou (PAP) screening, the proportion of AC has relatively increased. Cervical SCC arise mainly from the ectocervix, whereas AC originate primarily from the endocervical canal, which is less accessible to obtain viable PAP smears. Licensed (bivalent and quadrivalent) HPV vaccines comprise virus-like particles (VLP) of the most important hr HPV16 and 18, self-assembled from the major capsid protein L1. Due to mainly type-restricted efficacy, both vaccines do not target 13 additional hr mucosal types causing 30% of CxC. The papillomavirus genus alpha species 7 (α7) includes a group of hr types of which HPV18, 45, 59 are proportionally overrepresented in cervical AC and only partially (HPV18) targeted by current vaccines. To target these types, we generated a chimeric vaccine antigen that consists of a cross-neutralizing epitope (homologue of HPV16 RG1) of the L2 minor capsid protein of HPV45 genetically inserted into a surface loop of HPV18 L1 VLP (18L1-45RG1). Vaccination of NZW rabbits with 18L1-45RG1 VLP plus alum-MPL adjuvant induced high-titer neutralizing antibodies against homologous HPV18, that cross-neutralized non-cognate hr α7 types HPV39, 45, 68, but not HPV59, and low risk HPV70 in vitro, and induced a robust L1-specific cellular immune response. Passive immunization protected mice against experimental vaginal challenge with pseudovirions of HPV18, 39, 45 and 68, but not HPV59 or the distantly related α9 type HPV16. 18L1-45RG1 VLP might be combined with our previously described 16L1-16RG1 VLP to develop a second generation bivalent vaccine with extended spectrum against hr HPV.

Peptides of the papillomavirus L2 minor capsid protein can induce antibodies (Ab) that neutralize a broad range of human papillomavirus (HPV) genotypes. Unfortunately, L2 is antigenically subdominant to L1 in the virus capsid. To induce a strong anti-L2 Ab response with cross-neutralizing activity to other mucosal types, chimeric virus-like particles (VLP) were generated in which HPV16 L2 neutralization epitopes (comprising L2 residues 69-81 or 108-120) are inserted within an immunodominant surface loop (between residues 133 and 134) of the L1 major capsid protein of bovine papillomavirus type 1 (BPV1). These chimeras self-assembled into pentameric capsomers, or complete VLP similar to wild type (wt) L1 protein. Immunization of rabbits with assembled particle preparations induced L2-specific serum Ab with titers 10-fold higher than those induced by cognate synthetic L2 peptides coupled to KLH. Antisera to both chimeric proteins partially neutralized HPV16 pseudovirions, confirming that both HPV16 L2 peptides define neutralization epitopes. When analyzed for the ability to cross-neutralize infection by authentic HPV11 virions, using detection of early viral RNA by RT-PCR-assays as the readout, immune serum to chimeric protein comprising L2 residues 69-81, but not 108-120, was partially neutralizing. In addition, mouse-antiserum induced by vaccinations with synthetic L2 peptide 108-120, but not 69-81, was partially neutralizing in this assay. Induction of cross-neutralization Ab by L2 epitopes displayed on chimeric VLP represents a possible strategy for the generation of broad-spectrum vaccines to protect against relevant mucosal HPV and associated neoplasia.

Immunization with amyloid-β (Aβ) prevents the deposition of Aβ in the brain and memory deficits in transgenic mouse models of Alzheimer’s disease (AD), opening the possibility for immunotherapy of AD in humans. Unfortunately, the first human trial of Aβ vaccination was complicated, in a small number of vaccinees, by cell-mediated meningoencephalitis. To develop an Aβ vaccine that lacks the potential to induce autoimmune encephalitis, we have generated papillomavirus-like particles (VLP) that display 1–9 aa of Aβ protein repetitively on the viral capsid surface (Aβ-VLP). This Aβ peptide was chosen because it contains a functional B cell epitope, but lacks known T cell epitopes. Rabbit and mouse vaccinations with Aβ-VLP were well tolerated and induced high-titer autoAb against Aβ, that inhibited effectively assembly of Aβ1–42 peptides into neurotoxic fibrils in vitro. Following Aβ-VLP immunizations of APP/presenilin 1 transgenic mice, a model for human AD, we observed trends for reduced Aβ deposits in the brain and increased numbers of activated microglia. Furthermore, Aβ-VLP vaccinated mice also showed increased levels of Aβ in plasma, suggesting efflux from the brain into the vascular compartment. These results indicate that the Aβ-VLP vaccine induces an effective humoral immune response to Aβ and may thus form a basis to develop a safe and efficient immunotherapy for human AD.

Objective : To study human papillomavirus type 16 in the aetiology of cervical carcinoma. Design : Within a cohort of 18814 Finnish women followed for up to 23 years a nested case-control study was conducted based on serological diagnosis of past infection with human papillomavirus type 16. Subjects : 72 women (27 with invasive carcinoma and 45 with in situ carcinoma) and 143 matched controls were identified during the follow up. Main outcome measure : Relative risk of cervical carcinoma in presence of IgG antibodies to human papillomavirus type 16. Results : After adjustment for smoking and for antibodies to various other agents of sexually transmitted disease, such as herpes simplex virus type 2 and Chlamydia trachomatis, the only significant association was with infection with human papillomavirus type 16 (odds ratio 12.5; 95% confidence interval 2.7 to 57, 2P Conclusion : This prospective study provides epidemiological evidence that infection with human papillomavirus type 16 confers an excess risk for subsequent development of cervical carcinoma. Key messages Key messages Human papillomavirus type 16 is the main micro-organism linked to the development of cervical cancer Prospective studies of infection with this virus and cervical cancer have not been reported because of ethical and clinical difficulties and because diagnosis of past infections with the virus has not been possible In this nested case-control study in over 18000 Finnish women who donated blood to a serum bank 25 years ago we were able to measure past infection with human papillomavirus type 16 with new serological tools The results show that infection with the virus confers an increased risk of developing cervical cancer

Papillomaviruses replicate in stratified epithelia of skin and mucosa. Infection with certain human papillomavirus (HPV) types is the main cause of anogenital neoplasia, in particular cervical cancer. Early events of papillomavirus infectivity are poorly understood. While heparan sulfate proteoglycans (HSPGs) mediate initial binding to the cell surface, the class of proteins carrying heparan sulfates has not been defined. Here we examined two processes of papillomavirus infection, attachment of virus-like particles (VLP) to cells and infection with authentic HPV type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major epithelial form and is strongly upregulated in wound edge keratinocytes. We employed K562 cells, which lack HSPGs except minor amounts of endogenous betaglycan, and stable clones that express cDNAs of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated with levels of heparan sulfate on the cell surface. Parental K562 bound HPV16 VLP weakly, whereas all three K562 transfectants demonstrated enhanced binding, with the highest binding capacity observed for syndecan-1-transfected cells, which also expressed the most HSPG. For HPV11 infectivity assays, a high virion inoculum was required to infect K562 cells, whereas ectopic expression of syndecan-1 increased permissiveness eightfold and expression of syndecan-4 or glypican-1 fourfold. Infection of keratinocytes was eliminated by treatment with heparitinase, but not phospholipase C, further implicating the syndecan family of integral membrane proteins as receptor proteins. Human keratinocytes with a homozygous deletion of α6 integrin are permissive for HPV11 infection. These results indicate that several HSPGs can serve as HPV receptors and support a putative role for syndecan-1, rather than α6 integrin, as a primary receptor protein in natural HPV infection of keratinocytes.