Reinhard Höpfl

Assembled baculovirus-expressed human papillomavirus type 11 L1 capsid protein virus-like particles are recognized by neutralizing monoclonal antibodies and induce high titres of neutralizing antibodies.

Baculovirus-expressed human papillomavirus type 11 (HPV-11) major capsid protein (L1) virus-like particles (VLPs) were produced in insect cells and purified on CsCl density gradients. The VLPs retained conformational neutralizing epitopes that were detected by a series of HPV-11-neutralizing monoclonal antibodies. Electron microscopy determined that the HPV-11 L1 VLPs were variable in size with a surface topography similar to that of infectious HPV-11. The VLPs were very antigenic, and induced high titres of neutralizing antibodies in rabbits and mice when used as an immunogen without commercial preparations of adjuvant. These VLP reagents may be effective vaccines for protection against HPV infections.

Spontaneous regression of CIN and delayed-type hypersensitivity to HPV-16 oncoprotein E7

We investigated delayed-type hypersensitivity to human papillomavirus (HPV) in women with cervical dysplasia or cancer. Women were challenged by skin tests with synthetic HPV-16 E7 oncoprotein peptides. 11 women were regressors (cleared disease without treatment) and 37 were progressors (required surgery). Antibodies to early antigens (markers for progression) were detectable in a higher proportion of cancer patients than all other patients, particularly progressors with cervical intraepithelial neoplasia (CIN). By contrast, cellular immunity to HPV-16 E7, measured by skin test, was significantly (p=0.0001) associated with clinical and cytological resolution of HPV-induced CIN, indicating that E7-specific T-helper cells have a role in control of HPV.

Buschke—Loewenstein tumour infiltrating pelvic organs

Summary We report a 42‐year‐old HIV‐negative patient with a 12‐year history of exceptionally extensive genital warts and coexisting verrucous carcinoma of the anogenital region (Buschke–Loewenstein tumour). Masses of both tumour and viral papillomas infiltrated the external genitalia, perineum and buttocks, pelvic diaphragm and parts of the lesser pelvis, as well as the urethra, prostate and parts of the urinary bladder, necessitating repeated surgical intervention and plastic reconstruction. Adjuvant interferon‐α therapy was given without any lasting effects. Human papillomavirus type 6 was detected by DNA in situ hybridization and Southern blot analysis.

Detection of new human papillomavirus sequences in skin lesions of a renal transplant recipient and characterization of one complete genome related to epidermodysplasia verruciformis-associated types

Human papillomavirus (HPV) DNA, originally isolated from patients suffering from the skin disease epidermodysplasia verruciformis (EV), and a growing number of related sequences have recently been detected in a high percentage of benign and malignant skin lesions of both immunosuppressed and immunocompetent people. HPV L1 DNA fragments (374-389 bp long) from a solar keratosis and a squamous cell carcinoma (SCC) of a renal transplant recipient were amplified, cloned and sequenced. In 54 clones, six different HPV sequences were identified. One of these six corresponded to the known type HPV-8 and two (RTRX3 and RTRX7) have been described previously in cutaneous lesions of immunosuppressed patients. The remaining three sequences were different from all known HPV types: an HPV-9-related sequence (77.4% identity), an RTRX2-related sequence (82.6% identity), and an HPV-22-related sequence (83.7% identity). These three sequences, representing putatively new HPV types, were named RTRX8, RTRX9 and RTRX10, respectively. RTRX7 was found in the majority of clones from both lesions. The complete genome of RTRX7 (7731 bp) was cloned as six overlapping subgenomic fragments, generated by nested PCR with DNA extracts from the SCC. RTRX7 showed a genome organization typical of HPVs associated with EV. The L1 DNA sequence differed by 15% from the corresponding region of its closest known relative, HPV-12; thus, RTRX7 can be regarded as a new HPV type. RTRX7 DNA could not be detected by Southern blot hybridization with the homologous probe, indicating that the DNA concentration was below one copy per 10 cells in the investigated SCC.

Specific serum IgG, IgM and IgA antibodies to human papillomavirus types 6, 11, 16, 18 and 31 virus-like particles in human immunodeficiency virus-seropositive women.

To evaluate the humoral immune response to human papillomavirus (HPV) in women infected with human immunodeficiency virus (HIV), serum samples of 83 HIV-positive individuals were analysed by ELISA for specific antibodies of the isotypes IgG, IgA and IgM recognizing HPV-6, -11, -16, -18 and -31 L1 virus-like particles (VLPs). Papillomavirus-related lesions were present in 30 of 83 HIV-positive women. Twenty-one women (25%) presented with high-/intermediate-grade anogenital squamous intraepithelial lesions. PCR analysis and sequencing for HPV typing was done from biopsy specimens of 18 women; PCR-positive results were obtained in 90% of cases. In addition, HPV DNA hybrid capture assays were performed from cervical swabs of 58 HIV-positive women, 53% of whom had a positive result for high-risk HPV. Overall, positive IgG reactivity to HPV-6/-11 and HPV-16/-18/-31 was seen in 19%/31% and 49%/30%/24% of HIV-positive women, respectively. HPV-seropositivity was even higher than in 48 HIV-negative cervical intraepithelial neoplasia/cancer patients with percentages as follows: 8%/2% and 31%/15%/15%. This difference was significant for HPV-16 (P=0.046). IgA responses were comparable to IgG. IgM responses were low. The extraordinarily high rate of antibodies to the capsid protein L1 of high-risk HPVs (HPV-16, -18 and/or -31) in 58% of HIV-positive women compared to 19% (P=0.00001) of 102 healthy HIV-negative control women suggests a high lifetime cumulative exposure to HPV and increased expression of capsid proteins due to cellular immunodeficiency in HIV-infected women.

Spontaneous or interferon-γ-induced t-cell infiltration, hla-dr and icam-1 expression in genitoanal warts are associated with th1 or mixed th1/th2 cytokine mrna expression profiles

The purpose of the study was to investigate the cytokine gene expression patterns and immunohistochemical characteristics of genitoanal warts in order to obtain a clue as to the immunological mechanisms possibly relevant for wart regression or persistence. We analysed surgically removed warts from 11 patients, 2 of whom were immunosuppressed. Lesions of five of the nine otherwise healthy individuals were additionally treated with intralesional interferon-γ (IFNγ) prior to surgery. Invasion of CD4+ T cells into the papillomas and HLA-DR and ICAM-1 expression on keratinocytes were found in two otherwise healthy patients and were intensified by intralesional IFNγ in four of five patients. The mRNA expression patterns in seven of eight nonrecurrent warts were compatible with a predominant TH1 or mixed TH1/TH2 cytokine profile. In contrast, in recalcitrant warts of three patients (one healthy, two immunocompromised) histological signs of immunoreactivity and TH1-like cytokine mRNA expression were not detected. In recurrent warts of a renal transplant patient, IL-4 and IL-5 mRNA expression was repeatedly found suggesting a predominant TH2 response. In conclusion, immunoreactivity to genitoanal warts such as T-cell infiltration, HLA-DR and ICAM-1 expression was associated with a predominant TH1 or mixed TH1/ TH2 cytokine mRNA expression profile.

High prevalence of high risk human papillomavirus-capsid antibodies in human immunodeficiency virus-seropositive men: a serological study

BackgroundSerological study of human papillomavirus (HPV)-antibodies in order to estimate the HPV-prevalence as risk factor for the development of HPV-associated malignancies in human immunodeficiency virus (HIV)-positive men.MethodsSera from 168 HIV-positive men and 330 HIV-negative individuals (including 198 controls) were tested using a direct HPV-ELISA specific to HPV-6, -11, -16, -18, -31 and bovine PV-1 L1-virus-like particles. Serological results were correlated with the presence of HPV-associated lesions, the history of other sexually transmitted diseases (STD) and HIV classification groups.ResultsIn HIV-negative men low risk HPV-antibodies were prevailing and associated with condylomatous warts (25.4%). Strikingly, HIV-positive men were more likely to have antibodies to the high-risk HPV types -16, -18, -31, and low risk antibodies were not increased in a comparable range. Even those HIV-positive heterosexual individuals without any HPV-associated lesions exhibited preferentially antibody responses to the oncogenic HPV-types (cumulative 31.1%). The highest antibody detection rate (88,8%) was observed within the subgroup of nine HIV-positive homosexual men with anogenital warts. Three HIV-positive patients had HPV-associated carcinomas, in all of them HPV-16 antibodies were detected. Drug use and mean CD4-cell counts on the day of serologic testing had no influence on HPV-IgG antibody prevalence, as had prior antiretroviral therapy or clinical category of HIV-disease.ConclusionHigh risk HPV-antibodies in HIV-infected and homosexual men suggest a continuous exposure to HPV-proteins throughout the course of their HIV infection, reflecting the known increased risk for anogenital malignancies in these populations. The extensive increase of high risk antibodies (compared to low risk antibodies) in HIV-positive patients cannot be explained by differences in exposure history alone, but suggests defects of the immunological control of oncogenic HPV-types. HPV-serology is economic and can detect past or present HPV-infection, independently of an anatomical region. Therefore HPV-serology could help to better understand the natural history of anogenital HPV-infection in HIV-positive men in the era of antiretroviral therapy.

Presentation of Tetanus Toxoid to Autologous T Cells by Dendritic Cells Generated from Human Blood

Dendritic cells (DC) are highly specialised to initiate primary immune responses and may therefore serve as natural adjuvant in future strategies for specific immunotherapy, e. g. with tumor antigens. The originally developed culture system to generate DC from peripheral human blood with GM-CSF and IL-4 was dependent on the use of fetal calf serum. We employed such DC as antigen presenting cells in a modified lymphocyte proliferation assay to measure the response of autologous T cells to tetanus toxoid. However, a substantial proliferative response of T cells was also observed in control wells without antigen, i.e. in the setting of a syngeneic mixed leukocyte reaction. This makes it difficult, if not impossible, to monitor antigen-specific responses in vitro. In a recently developed improved method fetal calf serum was replaced by 1% autologous human plasma. Using such DC in our lymphocyte proliferation assay background proliferation was markedly reduced. T cell responses to tetanus toxoid were strongest when the antigen was added to DC three days before cocultivation with T cells. We conclude that DC cultured in FCS-free autologous systems, suitable for clinical use, can process and present tetanus protein to autologous T cells. Using such DC in a lymphocyte proliferation assay may facilitate the measurement of antigen-specific T cell responses.

Leukocyte proliferation in vitro against cottontail rabbit papillomavirus in rabbits with persisting papillomas/cancer or after regression.

Leukocyte proliferation responses to cottontail rabbit papillomavirus (CRPV) were measured in vitro with fresh whole blood as well as with ammonium chloride lysis-separated leukocytes. The antigens used were (1) CRPV particles produced in the athymic (nu/nu) mouse xenograft system and (2) purified bacterial fusion proteins of the CRPV major and minor capsid proteins L1 and L2. CRPV-infected domestic rabbits with persistent papillomas or after papilloma regression, as well as uninfected controls were studied. There was a clearcut difference between infected and uninfected animals. We demonstrated antigen-specific leukocyte proliferation to at least one CRPV antigen in 12 of 21 infected rabbits but there was no positivity in 9 control animals (P=0.004). There was whole-blood reactivity preferentially to intact CRPV particles in regressors. Specific but weak leukocyte proliferation against CRPV particles was detected in 6 of 9 regressor rabbits (66%) but only in 1 of 12 progressors (8%; P=0.0158). This trend of greater reactivity to intact CRPV particles in regressors as compared with progressors was not seen with peripheral blood leukocytes isolated by ammonium chloride lysis. We conclude that specific leukoproliferative responses against capsid CRPV proteins exist in rabbits experimentally infected with CRPV.

Anale HPV-Infektionen

Anogenitale Infektionen mit humanen Papillomaviren (HPV) verzeichnen in den letzten Jahren gerade in jüngeren Generationen durch frühe sexuelle Aktivität eine steigende Inzidenz. Neue Erkenntnisse der ätiologischen, epidemiologischen und pathophysiologischen Zusammenhänge erfordern eine Überarbeitung der proktologischen Position HPV-assoziierter Erkrankungen der Analregion. Dies ist auch aufgrund der ansteigenden Inzidenz präkanzeröser und invasiver Neubildungen nötig. In Ermangelung großer, kontrollierter Studien stellt dieser Konsensusbericht über weite Strecken die Expertenmeinung der auf dem Gebiet der HPVInfektion erfahrenen Autoren aus den Gebieten Proktologie, Dermatologie, Gynäkologie, Radiologie, Strahlentherapie und Pathologie dar. Anale HPV-Infektionen1