Incorporation of RG1 epitope into HPV16L1-VLP does not compromise L1-specific immunity.

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.

The Kühtai data set: 25 years of lysimetric, snow pillow, and meteorological measurements.

Snow measurements at the Kühtai station in Tirol, Austria, (1920 m.a.s.l.) are described. The data set includes snow water equivalent from a 10 m2 snow pillow, snow melt outflow from a 10 m2 snow lysimeter placed at the same location as the pillow, meteorological data (precipitation, incoming shortwave radiation, reflected shortwave radiation, air temperature, relative air humidity, and wind speed), and other data (snow depths, snow temperatures at seven heights) from the period October 1990 to May 2015. All data have been quality checked, and gaps in the meteorological data have been filled in. The data set is unique in that all data are available at a temporal resolution of 15 min over a period of 25 years with minimal changes in the experimental setup. The data set can therefore be used to analyze snow pack processes over a long-time period, including their extremes and long-term changes, in an Alpine climate. Analyses may benefit from the combined measurement of snow water equivalent, lysimeter outflow, and precipitation at a wind-sheltered alpine site. An example use of data shows the temporal variability of daily and 1 April snow water equivalent observed at the Kühtai site. The results indicate that the snow water equivalent maximum varies between 200 and more than 500 mm w.e., but there is no statistically significant temporal trend in the period 1990-2015.

Setback distances between small biological wastewater treatment systems and drinking water wells against virus contamination in alluvial aquifers.

Contamination of groundwater by pathogenic viruses from small biological wastewater treatment system discharges in remote areas is a major concern. To protect drinking water wells against virus contamination, safe setback distances are required between wastewater disposal fields and water supply wells. In this study, setback distances are calculated for alluvial sand and gravel aquifers for different vadose zone and aquifer thicknesses and horizontal groundwater gradients. This study applies to individual households and small settlements (1-20 persons) in decentralized locations without access to receiving surface waters but with the legal obligation of biological wastewater treatment. The calculations are based on Monte Carlo simulations using an analytical model that couples vertical unsaturated and horizontal saturated flow with virus transport. Hydraulic conductivities and water retention curves were selected from reported distribution functions depending on the type of subsurface media. The enteric virus concentration in effluent discharge was calculated based on reported ranges of enteric virus concentration in faeces, virus infectivity, suspension factor, and virus reduction by mechanical-biological wastewater treatment. To meet the risk target of <10-4infections/person/year, a 12 log10 reduction was required, using a linear dose-response relationship for the total amount of enteric viruses, at very low exposure concentrations. The results of this study suggest that the horizontal setback distances vary widely ranging 39 to 144m in sand aquifers, 66-289m in gravel aquifers and 1-2.5km in coarse gravel aquifers. It also varies for the same aquifers, depending on the thickness of the vadose zones and the groundwater gradient. For vulnerable fast-flow alluvial aquifers like coarse gravels, the calculated setback distances were too large to achieve practically. Therefore, for this category of aquifer, a high level of treatment is recommended before the effluent is discharged to the ground surface.

Human papillomavirus vaccination induces neutralising antibodies in oral mucosal fluids.

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.

Safety and immunogenicity of BPV-1 L1 virus-like particles in a dose-escalation vaccination trial in horses.

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 µg 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.

[HPV infections of the oral and genital mucosa. Possibilities for primary prevention]. / HPV-Infektionen der Mund- und Genitalschleimhaut. Möglichkeiten der primären Prävention.

It is well accepted today that almost all cases of carcinoma of the cervix are caused by persistent infections with high-risk human papilloma viruses (HPV), especially through types HPV16 and HPV18 (ca. 70%). These same types are also responsible for some carcinomas of the vulva, anus, penis and oropharynx. This knowledge indicates that it should be possible to prevent these carcinomas if a majority of the causstive HPV infections can be prevented.

Hydrological modeling in alpine catchments: sensing the critical parameters towards an efficient model calibration.

For the Tyrolean part of the river Inn, a hybrid model for flood forecast has been set up and is currently in its test phase. The system is a hybrid system which comprises of a hydraulic 1D model for the river Inn, and the hydrological models HQsim (Rainfall-runoff-discharge model) and the snow and ice melt model SES for modeling the rainfall runoff form non-glaciated and glaciated tributary catchment respectively. Within this paper the focus is put on the hydrological modeling of the totally 49 connected non-glaciated catchments realized with the software HQsim. In the course of model calibration, the identification of the most sensitive parameters is important aiming at an efficient calibration procedure. The indicators used for explaining the parameter sensitivities were chosen specifically for the purpose of flood forecasting. Finally five model parameters could be identified as being sensitive for model calibration when aiming for a well calibrated model for flood conditions. In addition two parameters were identified which are sensitive in situations where the snow line plays an important role.

Rapid progression of an anal Buschke-Lowenstein tumour into a metastasising squamous cell carcinoma in an HIV-infected patient.

BACKGROUND: Buschke-Löwenstein tumour (BLT) of the anogenitalia is a locally invasive, destructively growing verrucous carcinoma that does not metastasise. Histologically BLT resembles benign condylomata acuminata. Nevertheless, the tumour grows relentlessly and may rarely progress into squamous cell cancer (SCC). RESULTS: A human immunodeficiency virus (HIV)-infected immunosuppressed patient developed (peri)anal warts accompanied by recurrent abscesses and fistulae. Histology revealed condylomata acuminata, and low-risk genital human papillomavirus (HPV) type 11b was detected. Six months later, the tumour had progressed into an ulcerated SCC that destroyed the rectum and perineum, with metastases to the inguinal lymph nodes. Whereas highly active antiretroviral therapy (HAART) effectively suppressed HIV replication, radiochemotherapy plus anti-EGFR antibody did not halt tumour progression, and the patient died from tumour-cachexia. DISCUSSION: As far as is known, this is the first report demonstrating rapid progression of a BLT into a metastasising SCC in an HIV-infected patient.

A subset of equine sarcoids harbours BPV-1 DNA in a complex with L1 major capsid protein.

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 <125 immunoprecipitable viral genomes/50 microl extract for the majority of specimens. Moreover, full-length BPV-1 genomes were detected in a complex with L1 proteins. These complexes may correspond to virion precursors or intact virions.

Human papillomavirus type 26 infection causing multiple invasive squamous cell carcinomas of the fingernails in an AIDS patient under highly active antiretroviral therapy.

Squamous cell carcinoma (SCC) of the nail unit is a rare disorder. An association with high-risk genital human papillomavirus (HPV) infection has been reported. We report a 28-year-old human immunodeficiency virus (HIV)-infected bisexual man who had multiple invasive SCC of the fingers, infected with the rare type HPV 26. Classification of HPV 26 as high- or intermediate-risk type has been uncertain, due to its rare presence in cervical cancer. Despite successful treatment with highly active antiretroviral therapy (HAART), the patient developed extensive hyperkeratotic nailbed proliferations of all fingers. Tumours were refractory to treatment and invaded into adjacent tissues. X-rays of the hands demonstrated bone invasion, necessitating amputation of distal phalanges of several fingers. Histologically, highly differentiated preinvasive and invasive verrucous SCCs were identified. Molecular DNA typing identified HPV 26 in the SCCs and in some premalignant lesions. By in situ hybridization HPV 26 DNA was detected in numerous tumour cells, indicating productive infection with high-level amplification of the viral genome. In the remaining proliferations, high-risk HPV type 58, cutaneous HPVs and a putative new HPV type were identified. HPV 26 infection appears to be causally involved in the development of SCC of the nail unit in this immunosuppressed patient. Timely evaluation of chronic verrucous nailbed tumours is recommended, especially in immunocompromised patients. Identification of HPV 26, besides known high-risk HPV types, may identify patients at risk for developing SCC of the nailbed and possibly at other locations.

[HPV vaccines. Prophylactic vaccines from virus-like particles]. / HPV-Vakzine. Prophylaktischer Spaltimpfstoff aus "virus-like particles" (VLP).

The new prophylactic HPV vaccines consist of virus-like particles self-assembled from recombinantly expressed L1 major capsid protein. Due to the absence of potentially oncogenic viral DNA, they have been proven safe and well tolerated in thousands of vaccinated individuals. Prophylactic HPV vaccination has demonstrated 100% efficacy in preventing type-specific persistent infections and associated genital dysplasias.

230-kDa and 190-kDa proteins in addition to desmoglein 1 as immunological targets in a subset of pemphigus foliaceus with a combined cell-surface and basement membrane zone immune staining pattern.

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.

Seroreactivity to human papillomavirus types 16, 18, 31, and 45 virus-like particles in a case-control study of cervical squamous intraepithelial lesions.

Serum IgG antibodies to human papillomavirus (HPV) types 16, 18, 31, and 45 virus-like particles were measured in a nested case-control study of cervical squamous intraepithelial lesions. HPV-16 seroreactivity was strongly associated with HPV-16 DNA detection (odds ratio, 9.0; 95% confidence interval, 4.4-19.4), and similar type specificity was observed for HPV-31 and -45. In contrast, seroreactivity to any type was associated with elevated seroreactivity to all others. Among cases and controls, HPV-16 showed the highest seroprevalence, with 23.8% of 80 cases and 10.5% of 258 controls seroreactive to HPV-16 alone, and another 27.5% and 5.4%, respectively, seroreactive to HPV-16 plus other types. Overall, 24 (30.0%) cases and 17 (6.6%) controls were seroreactive to multiple types. These data suggest that seroreactivity to a given type reflects mainly type-specific HPV infection as measured by DNA detection and may also signal past exposure to other types that are now only serologically detected.

Anaplastic squamous cell carcinoma (SCC) in a patient with chronic cutaneous graft-versus-host disease (GVHD).

We describe an allogeneic bone marrow (BM) recipient who developed aggressive, metastasizing squamous cell cancer (SCC) of the skin, and discuss possible risk factors in the development of this secondary solid tumor. The patient had been treated with cyclosporine (CsA), methyl-prednisolone and thalidomide for 3 years because of extensive de novo chronic cutaneous GVHD occurring 1 year after BMT. Ten years after BMT a locally invasive and metastasizing SCC occurred on the patient's neck, and diagnosis was confirmed by H&E histopathology and cytokeratin-immunohistochemistry. Analysis of genomic DNA did not reveal p53 mutations nor were HPV sequences detectable. Risk factors included conditioning for BMT with total body irradiation (TBI) and cyclophosphamide (Cy), immunosuppressive treatment for GVHD, and extensive exposure to UV radiation before and after BMT. Despite surgery and adjuvant chemotherapy with 5-fluorouracil (5-FU) the patient died 1 year after the diagnosis of SCC.

Desmoglein 3-ELISA: a pemphigus vulgaris-specific diagnostic tool.

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune-blistering disease of the skin and mucous membranes caused by autoantibodies against desmoglein 3 (Dsg3), an epidermal desmosomal adhesion protein of the cadherin family. Cloning of the Dsg3 gene and expression of the protein in a native conformation enabled the recent development of a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of PV autoantibodies. OBJECTIVES: To evaluate serum samples from patients with PV and other dermatologic diseases for anti-Dsg3 antibodies. To compare ELISA values with autoantibody titers obtained by classic indirect immunofluorescence (IIF). DESIGN: Serum samples from patients with PV and various other bullous and nonbullous skin diseases were tested for anti-Dsg3 reactivity by ELISA. SETTING: Ambulatory and hospitalized patients from a university hospital. PATIENTS: Fifty-two serum samples from 11 patients with PV, and serum samples from 11 patients with bullous pemphigoid, 12 patients with other bullous diseases, 22 patients with various nonbullous skin disorders, and 10 healthy individuals were tested. RESULTS: Forty-seven (98%) of 48 serum samples from patients with PV that were positive by IIF on monkey esophagus were also reactive by Dsg3-ELISA, whereas 4 of 4 IIF-negative PV serum samples showed no reactivity by ELISA. In addition, negative ELISA results were obtained from 11 of 11 serum samples from patients with bullous pemphigoid, 10 of 12 serum samples from patients with other bullous skin disorders, 7 of 9 serum samples from patients with autoimmune-connective tissue diseases, and 13 of 13 serum samples from patients with other nonbullous skin diseases. Interestingly, 1 patient with paraneoplastic pemphigus had positive ELISA results. There was a positive correlation (r = 0.654) between ELISA values and IIF titers within the whole population with PV. In addition, when multiple serum samples from 1 patient with PV sampled over a 2-year period were tested, ELISA reactivity paralleled both the IIF titers and the clinical course. CONCLUSION: The Dsg3-ELISA is a sensitive, objective, and PV-specific test that should be considered as an adjunct test for the management of patients with PV.

[Cicatricial pemphigoid with autoantibodies to laminin 5 (epiligrin) in a patient with metastatic endometrial carcinoma]. / Vernarbendes Pemphigoid mit Autoantikörpern gegen Laminin-5 (Epiligrin) bei einer Patientin mit metastasierendem Endometriumkarzinom.

A 71-year-old female patient presented with erosions of the oral, genital and ocular mucosa, but without skin lesions. One year prior to the onset of lesions, a metastatic adenocarcinoma of the endometrium had been diagnosed. In perilesional skin, direct immunofluorescence showed linear deposits of C3 and IgG along the basement membrane zone. Indirect immunofluorescence demonstrated anti-basement membrane zone antibodies which bound exclusively to the dermal side of 1M NaCl-split skin. In immunoprecipitation studies, the autoantibodies bound a set of proteins characteristic of epiligrin/laminin-5, and they specifically immunoblotted the alpha-subunit of this protein. These results confirmed our presumed diagnosis of anti-epiligrin cicatricial pemphigoid. Combined treatment with methylprednisolone and dapsone resulted in prompt remission, despite progression of her malignancy. So far, only eight patients with anti-epiligrin cicatricial pemphigoid have been reported worldwide. Our patient represents the first case from Austria and is clinically and immunopathologically similar to previously reported cases.

Chimeric papillomavirus virus-like particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model.

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 beta2-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.

Absence of p53 protein overexpression in precancerous lesions of the vulva.

BACKGROUND: Recently the authors reported the prognostic value of p53 protein overexpression in invasive squamous cell carcinoma of the vulva. The aim of this study was to evaluate the status of p53 overexpression and human papillomavirus (HPV) infection in patients with precancerous lesions of the vulva. METHODS: Biopsy specimens of 28 women (mean age, 44.2 years; range, 19-71 years) with warty and/or basaloid type vulvar intraepithelial neoplasia (VIN) of Grade 1 to 3 were examined retrospectively for p53 protein overexpression by immunohistochemistry. The presence of the HPV genome was assessed using a nested polymerase chain reaction (PCR) method with consensus primers directed against the L1 coding region. RESULTS: Neither the preoperative punch biopsy specimen nor the subsequent surgical specimen contained immunohistochemically detectable levels of p53 in this study of a group of younger women with preinvasive vulvar lesions. These results are in contrast to those obtained previously in older women with keratinizing squamous cell carcinoma demonstrating p53 protein overexpression in approximately 50% of patients. HPV DNA was detected in the vast majority of VIN cases (92.8%) using a highly sensitive nested PCR method. The current data indicate that p53 protein is not overexpressed in basaloid/warty VIN when evaluated by immunohistochemistry. In addition, this study confirms previous reports demonstrating the presence of HPV DNA in the majority of these lesions. CONCLUSIONS: These data suggest that p53 protein overexpression is not an early event in the pathogenesis of basaloid/warty type vulvar dysplasia and that HPV infection may contribute to the development of VIN.

Characterization of a human papillomavirus type 16 variant-dependent neutralizing epitope.

We have determined that three type-specific and conformationally dependent monoclonal antibodies, H16.E70, H16.U4, and H16.V5, neutralize pseudotype human papillomavirus type 16 (HPV16) virions in vitro. H16.U4 and H16.V5 neutralized pseudotype virions derived from the German HPV16 variant 114K and the Zairian variant Z-1194 with equal efficiency. In contrast, neutralization of Z-1194 pseudotype virions by H16.E70 was two orders of magnitude weaker than neutralization of 114K pseudotype virions. This difference correlated with enzyme-linked immunosorbent assay reactivity of H16.E70 to L1 virus-like particles of the two variants. A substitution at residue 282 of L1 was responsible for this differential reactivity, suggesting that this residue constitutes part of the H16.E70 epitope.