CD4+ T cell immunity against the human papillomavirus-18 E6 transforming protein in healthy donors: identification of promiscuous naturally processed epitopes

Infection by 'high-risk' human papillomaviruses (HPV) is associated with the development of neoplastic lesions. HPV-18 is responsible for a very aggressive form of cancer and poor survival. As for other HPV types, immune surveillance has probably a role in the control of the infection. However, very little is known on HPV-18 immunogenicity. CD4(+) T cells from 16 healthy donors were tested ex vivo for reactivity to synthetic peptides corresponding to 3 sequences on the HPV-18 E6 transforming protein predicted by bioinformatics as promiscuous HLA-DR ligands, and to the recombinant E6 protein. We found 3 donors with CD4(+) T cells that specifically proliferated in the presence of HPV-18 E6 antigens and produced IFN-gamma in the presence of the E6 protein. We then propagated CD4(+) T cell lines and clones from the responsive subjects to better characterize the recognized sequences. We show that E6(52-66) and E6(97-111) are indeed promiscuous and, most importantly, they contain naturally processed epitopes. Collectively, our data indicate that healthy donors may develop spontaneous CD4 immunity against HPV-18 E6 epitopes, thus strongly suggesting the potential for this protein to elicit in the host a natural productive immune response.     

Frequency of antibodies against E4 and E7 from human papillomavirus type 16 in Mexican soldiers

Summary. The high prevalence of HPV in men’s genitalia and the low frequency of virus-associated lesions gave rise to questions on the influence of infection-site on the HPV antibody profile. In a cross-sectional study, HPV infection in penis and urethra, and serum antibodies against HPV-16 E4 and E7 proteins were evaluated in 288 Mexican soldiers. The results showed that HPV prevalence was 31% (51% in penis, 11% in urethra and 38% in both sites), while 47% were multiple infections. Overall, seroprevalence was 13% for anti-E4 antibodies and 6% for anti-E7. However, the highest prevalence of anti-E4 antibodies was observed in men with HPV infection in urethra (30%), while for E7 antibodies, the highest prevalence (10%) was found in men who tested positive for HPV in penis. The prevalence of IgG and IgA anti-E4 was related to HPV-16 urethral infection, while detection of HPV-16 in penis was related to IgG anti-E7 prevalence. In conclusion, the high-risk sexual behavior observed in this population might be responsible for high HPV prevalence and multiple infections. However, the seroprevalence of E4 and E7 was similar to that observed in healthy Mexican women. These results suggest that the humoral immune response against HPV infection in men differs, depending on the site of infection.     

Prevalence and stability of antibodies to 37 human papillomavirus types — A population-based longitudinal study

Information about serostability of cutaneous HPV types over time is very limited. We investigated seroprevalence and serostability of 37 different HPV types over 4½ years in an Australian population-based study. Sera and data were analyzed for 390 people who had never been diagnosed with SCC and had blood collected in 1992, 1993 and 1996.Eighty-six percent of participants were seropositive to at least one of the 37 HPV types at baseline. HPV-4 was the type with the highest seroprevalence (41%), followed by HPV-38 and HPV-8 (both 33%). Over 90% of people retained their baseline serostatus during the 4½ year follow-up. Highest serostability was observed for HPV-88 (99.7% stayed seropositive or seronegative), while HPV-65 was least stable with 17% altering their serostatus during follow-up.Seroprevalence to cutaneous HPV types are relatively stable over time, and a single measure can be used as a reasonable marker of long-term antibody status.     

Serological study in Tunisian cervical cancer patients

Objective

The aim of this study is to use a novel ELISA, based on five recombinant HPV-16 and HPV-18 proteins, for detection HPV-specific antibodies in a case-control study.

Patients and methods

L1, E6 and E7 genes have been over expressed in Escherichia coli as double fused proteins. These recombinant proteins were used in a GST-capture ELISA as coating antigens. Human sera were collected from women with cervical cancer. Negative human sera were collected from patients apparently healthy and may be affected by other infectious agents.

Results

Most of the sera showed a positive reactivity to at least one of the HPV-16 or HPV-18 proteins (52/71). A percentage of 39.50% of the sera from HPV-16 infected women and 21.12% of the sera from women infected by HPV-18 genotype recognised at least one of the HPV-16 or HPV-18 proteins. Sera showed different reactivity to L1, E6 and E7 antigens, and only a few serum samples reacted to L1, E6 and E7 HPV-16, E6 and E7 HPV-18 (co-infection). Differences of reactivity between cases and controls were significant (P < 0.0001).

Conclusion

This novel ELISA, based on recombinant HPV-16 and HPV-18 antigens, is able to detect antibodies in women infected by HPV genotypes. The assay is easy to perform and has low cost, making it suitable for monitoring the natural history of HPV infections as well as for detecting pre-existing HPV antibodies in women who receive vaccination.     

Human papillomavirus-16 E6 variants in cervical squamous intraepithelial lesions from HIV-negative and HIV-positive women.

We studied 48 human papillomavirus (HPV)-16-positive squamous intraepithelial lesions (SILs) from HIV-negative patients (16 low-grade SILs [LSILs]; 32 high-grade SILs [HSILs]) and 13 HPV-16-positive SILs from HIV-positive patients with AIDS (1 LSIL; 12 HSILs). After HPV typing, the entire HPV-16 E6 coding region was amplified and sequenced in all samples. We detected 12 HPV-16 E6 prototypes and 4 variants among the LSILs in HIV-negative patients, and 15 HPV-16 E6 prototypes and 17 HPV-16 variants in the HSIL group. The most prevalent variant of SIL types was European 350G, present in 3 and 13 cases, respectively. In 3 HSILs and no LSILs we found mixed infection by an HPV-16 E6 prototype and a variant. Two variants (1 each in LSIL and HSIL) were of non-European lineage. The only LSIL in HIV-positive patients had an HPV-16 E6 prototype; in the HSILs, we found 8 HPV-16 E6-prototypes, 4 with mixed infection with HPV-31 and 4 variants, all European 350G. The higher proportion of HPV-16 E6 variants in HSIL than in LSIL in HIV-negative patients suggests a greater risk of progression. However, further studies are needed.     

Detection of human papillomavirus genotypes with liquid bead microarray in cervical lesions of northern Chinese patients

Human papillomavirus (HPV) is the main cause of cervical cancer. Blending multiplex polymerase chain reaction (PCR) amplification and multiplex hybridization to liquid bead microarray (LBMA), we detected and identified 25 common HPV genotypes using type-specific primers for HPV E6 and E7 genes in cervical lesions of northern Chinese patients. Of the 511 cervical samples, 349 (68.3%) were found to be HPV positive by HPV-LBMA. The distribution was 22 HPV positive of 100 in the control group (22%), 41 of 80 with chronic cervicitis (51%), 80 of 99 with cervical intraepithelial neoplasia (CIN) I (81%), 46 of 56 with CIN II (82%), 67 of 74 with CIN III (90%), and 93 of 102 with invasive cervical carcinoma (91%). HPV-16 was the most frequent genotype in the CIN and cervical cancer groups. The most common genotypes were HPV-16 (28%), HPV-58 (14%), HPV-52 (14%), HPV-18 (8%), and HPV-33 (7%) in the CIN group, and HPV-16 (63%), HPV-52 (9%), HPV-18 (7%), HPV-58 (7%), and HPV-33 (5%) in the cervical cancer group. HPV-LBMA found multiple genotypes in 1 of 22 control (4%), 64 of 193 CIN (33%), and 22 of 93 cervical cancer (24%). The HPV-LBMA results were compatible with those of PCR and DNA sequencing. HPV-LBMA is a simple, high-throughput method that provides useful information on viral genotype and multiple HPV infections in cervical lesions. In northern China, the most common high-risk HPV genotypes seem to be HPV types 16, 58, 52, 18, and 33. Genetic information on HPV in cervical specimens could provide particular benefits in the management of cervical lesions.     

The prevalence of HPV-18 and variants of E6 gene isolated from cervical cancer patients in Taiwan.

BACKGROUND: Cervical cancer is the second most common cancer in women worldwide. It has been considered that human papillomavirus (HPV) is associated with cervical cancer. Currently, more than 80 different serotypes of HPV have been characterized and they are divided into low- and high-risk groups. The most common types that lead to cervical cancer are HPV-16 and -18. The viral oncogenes E6 and E7 are associated with the development of cervical cancer. In previous study, the variants of HPV-16 E6 gene have been reported. It suggests that variants may influence the morbidity of carcinogenesis, but the variant study on HPV-18 remains unknown. OBJECTIVES: To identify the variants of integrated HPV-18 E6 gene in the prevalent infection of HPV-18 of cervical cancer patients. STUDY DESIGN: 25 cervical cancer patients were clinically identified and the biopsies were obtained. The infectious HPV types were identified by PCR and Southern blotting analysis. The DNA fragments of the integrated HPV-18 E6 were amplified by PCR and cloned. The nucleotide sequences were obtained by sequencing. RESULTS: The prevalence of HPV infection in our 25 cases was HPV-18 (100%) and 7 out of these 25 cases (28%) were co-infected with HPV-16. The most dominant mutation among 25 tested patients was a silence mutation C183G of the E6 coding region. CONCLUSIONS: The prevalent HPV infectious serotype is HPV-18, which differs from the worldwide prevalent type. The identified HPV-18 E6 variants had a unique silence mutation located on C183G in E6 coding region.     

Sourcing of the WHO human papillomavirus type 18 international standards for HPV antibody levels

BackgroundHPV serology is important for studies of vaccine immunogenicity, but can not be performed in a comparable manner without international standardisation.ObjectivesTo find suitable candidate sera from naturally infected persons for use as International Standards (IS) for antibodies to high-risk HPVs, with priority for HPV-18.Study design946 healthy Thai women (median age 44, range 18–83) and 61 cervical cancer patients were screened using an HPV pseudovirion-Luminex assay to detect antibodies to genital (HPV-6,-11,-16,-18,-31,-33,-45,-52,-58,-68) and non-genital HPV types (HPV-5,-15,-32,-38 and -76). Suitable candidate sera should ideally be mono-specific (have reactivity against only one genital HPV) and have high antibody levels that are stable over time.ResultsSeroprevalences of HPV-16,-31,-52 and -58 were at least twice as high among cancer patients compared to healthy individuals. Thirteen healthy women who met the IS inclusion criteria in initial testing also consented to blood-bag donations. Donations from 2 women with high HPV-18 Ab titers were pooled to the HPV-18 candidate IS, later established as the WHO official IS for HPV antibodies. Sera that could potentially be used as candidate IS for other oncogenic HPVs have also been identified.ConclusionsIn the Thai population, seroepidemiology implicated HPV types HPV-16,-31,-52 and -58 as particularly associated with cervical cancer. A well characterized cohort study has allowed sourcing of materials for an IS for HPV-18 antibodies and could conceivably be used for IS for other HPV types as well.     

Koilocytic changes are not elicited by human papillomavirus genotypes with higher oncogenic potential

Koilocytes are considered a common cytopathological effect in patients with human papillomavirus (HPV) infection. Thus, we aimed to elucidate whether koilocytes are common to all HPV infections. Liquid-based cytology samples from 651 patients with abnormal Papanicolaou (Pap) test results were used to analyze the presence of koilocytes and HPV genotype. HPV genotype was determined in complete liquid cytology samples and microdissected cell samples from Pap smear slides using the uniplex E6/E7 polymerase chain reaction method, which can detect 39 mucosal HPV genotypes. Koilocytes were found in 29.3% (191) of all patients. Logistical regression analysis of diverse HPV genotypes revealed that infections with low-risk HPV types (HPV-6b, HPV-40, HPV-42, HPV-61, HPV-74, HPV-89, and HPV-90), probably high-risk HPV types (HPV-53 and HPV-66), and high-risk types (HPV-39 and HPV-56) were significantly associated with the presence of koilocytes. However, HPV-16, HPV-18, and HPV-52, which have higher oncogenic potential, were not found to be associated with koilocytes. These results were confirmed by HPV genotyping using microdissected koilocytes in 27 patients.Most common high-risk types belonging to α-9 and α-7 genotypes appear to rarely induce koilocytic changes. Therefore, koilocytes may provide additional useful information for predicting the risk of progression to high-grade lesions.     

Cervical human papillomavirus (HPV) infection and HPV type 16 antibodies in South African women

There is a high incidence of cervical cancer in South African women. No large studies to assess human papillomavirus virus (HPV) infection or HPV type 16 (HPV-16) exposure have occurred in the region, a requirement for policy making with regards to HPV screening and the introduction of vaccines. Control women (n = 1,003) enrolled in a case control study of hormonal contraceptives and cervical cancer were tested for 27 cervical HPV types by reverse line blot analysis. The seroprevalence of HPV-16 immunoglobulin G (IgG) and IgA antibodies was assessed by a virus-like particle-based enzyme-linked immunoassay of 908 and 904 control women, respectively, and of 474 women with cervical cancer. The cervical HPV prevalence was 26.1%. The HPV-16 IgG seroprevalence was 44.4% and the HPV-16 IgA seroprevalence was 28.7% in control women, and these levels were significantly higher (61.8% and 52.7%, respectively) for women with cervical cancer (odds ratio [OR], 2.1 and 2.8, respectively). The cervical HPV prevalence showed an association with cervical disease, and the HPV-16 IgG prevalence decreased while the HPV-16 IgA prevalence increased with increasing age (P < 0.05). The prevalence of oncogenic HPV types (including HPV-16) decreased with age, whereas nononcogenic HPV types showed limited association with age. Multivariate analysis revealed cervical HPV infection to be associated with herpes simplex virus type 2 infection (OR, 1.7) and increasing years of education (OR, 1.9). HPV-16 IgG antibodies were inversely associated with current smoking status (OR, 0.6), and the presence of HPV-16 IgA antibodies was inversely associated with the use of alcohol (OR, 2.1) and inversely associated with the use of oral contraceptives (OR, 0.6). High levels of exposure to HPV, and particularly HPV-16, were evident in this population. The apparent increase of serum HPV-16 IgA with increasing age requires further investigation.     

Epitope-mapped monoclonal antibodies against the HPV16E1--E4 protein.

The human papillomavirus (HPV) E1--E4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing monoclonal antibodies to the E1--E4 protein of HPV16, which is the HPV type most frequently associated with cervical cancer. The three antibodies (TVG 401, 402, and 403) detect adjacent epitopes within the major seroreactive region of the molecule and show no reactivity against the E4 proteins of HPV1, HPV2, HPV4, or HPV6. The E1--E4 protein migrates as a 10K species on SDS-gel electrophoresis and forms cytoplasmic inclusion granules in infected cells in vitro similar in appearance to those produced by HPV1 in benign warts. In naturally occurring HPV16-induced tumors the E1--E4 protein was detected in the cytoplasm of cells in the upper layers of the lesion in areas in which HPV16 DNA replication was occurring, as determined by in situ hybridization. Although the epitopes recognized by these monoclonal antibodies survive brief fixation in 5% formaldehyde, reactivity was destroyed by prolonged fixation. These monoclonal antibodies represent the first against HPV16 E1--E4 and should complement those already available to E7 and L1 for the screening of frozen sections of clinical biopsies and will be of value in monitoring the progression of HPV infection from benign lesions to invasive cancer.     

Antibodies against human papillomavirus type 16 and 18 E6 and E7 proteins in cervicovaginal washings and serum of patients with cervical neoplasia.

Serum antibodies against the E6 and E7 proteins of human papillomavirus (HPV) 16 and 18 are associated with cervical cancer. The aim of this study was to investigate the presence of local antibodies against HPV in cervicovaginal washings (CWs). In this study antibodies against the native HPV16 and HPV18 E6/E7 proteins were detectable in CWs (48%) and sera (29%) from patients with cervical cancer (n = 21) utilizing a sandwich protein enzyme-linked immunosorbent assay (ELISA). In paired CWs and sera from patients with cervical intraepithelial neoplasia (n = 38) and from healthy women (n = 22) no antibodies against these proteins were found. In 10 of 11 patients, the antibody response corresponded with the HPV type in the cervical smear and/or tumor tissue, which indicates the HPV type specificity of the assay. In 7 of 11 patients with antibody reactivity against HPV16 or HPV18 E6 and/or E7 proteins a higher level of antibody reactivity in the CWs than in the paired serum samples was found at similar inputs of total IgG. This suggests that the antibodies in the CWs against the investigated HPV proteins in these patients were locally produced.     

The E5 Protein of HPV-6, but Not HPV-16, Associates Efficiently with Cellular Growth Factor Receptors

The transforming activity of the prototype E5 protein of bovine papillomavirus type 1 (BPV-1) is associated with its binding to, and activation of, both the platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. The E5 proteins of human papillomavirus types 6 and 16 (HPV-6, HPV-16) also transform rodent cells in the presence of the EGF receptor. In this study we examined whether epitope-tagged HPV E5 proteins could associate with three different tyrosine kinase-containing growth factor receptors: the EGF receptor, the erbB2 receptor, and the PDGF receptor. The HPV-6 E5 protein was found to associate efficiently with all three of these growth factor receptors, while the HPV-16 E5 protein did not. These findings suggest either that the in vitro transforming activities of HPV-6 and HPV-16 E5 proteins involve a similar mechanism unrelated to receptor binding (e.g., binding to the 16-kDa membrane pore protein) or that they proceed along distinct pathways, with receptor binding being important for HPV-6. Regardless of the ultimate mechanisms, the differences between the HPV-6 and HPV-16 E5 proteins in binding to growth factor receptors may potentially contribute to the distinctive morphologies of their respective neoplastic lesions.     

Human papillomavirus-16 and -18 replication in esophagus squamous cancer cell lines does not require sheterologous E1 and E2 proteins

Human papillomaviruses (HPVs) are doublestranded DNA viruses that replicate in the nuclei of squamous epithelial cells. HPVs can be classified into high-risk (e.g., types 16, 18, 31, and 33) or low-risk (e.g., types 6, 11, and 30), depending on their association with benign or malignant tumors. We recently described the association of HPV-16 and -18 with esophagus squamous cell cancer. HPV replication was studied in representative cell lines derived from esophagus cancers. HPV-16 and -18 genomes were independently transiently transfected into HCE-4 and HCE-7 cell lines with and without E1 and E2 genes under heterologous promoters. Southern blot analysis demonstrated that these cell lines support viral replication. However, heterologous E1 and E2 are not required for HPV replication. These findings suggest that specific host nuclear factors in esophageal squamous epithelial cells may support HPV replication. © 1995 Wiley-Liss, Inc.     

Comparison of peptide enzyme-linked immunosorbent assay and radioimmunoprecipitation assay with in vitro-translated proteins for detection of serum antibodies to human papillomavirus type 16 E6 and E7 proteins.

Antibodies to human papilloma virus (HPV) type 16 (HPV-16) E6 and E7 proteins in serum are markers for HPV-associated invasive cervical carcinoma. We compared two assays, a radioimmunoprecipitation assay with in vitro-translated HPV-16 E6 and E7 proteins and an enzyme-linked immunosorbent assay (ELISA) with E6 and E7 synthetic peptides, for their abilities to discriminate serologically between patients with invasive cervical cancer and controls. Among the patients, antibody prevalences were higher by the E6 radioimmunoprecipitation assay (55.7%) than by the E6 peptide ELISA (15.5%), but among the controls, they were lower by the radioimmunoprecipitation assay (1.7%) than by the E6 peptide ELISA (5%). For E7, antibody prevalences among the patients were comparable by the radioimmunoprecipitation assay (43%) and the peptide ELISA (41%), but among the controls they were higher by the E7 peptide ELISA (17.4%) than by the radioimmunoprecipitation assay (4.1%). There was good agreement between the E7 radioimmunoprecipitation assay and the E7 peptide ELISA among patients but not among controls. In tests with representative sera, heat denaturation of the translated proteins resulted in a complete loss of reactivity to the E6 protein and a marked decrease in reactivity to the E7 protein. Our study showed that the radioimmunoprecipitation assay discriminates better than the peptide ELISA between patients with invasive cervical cancer and controls and that this is related to the ability of the radioimmunoprecipitation assay to detect conformational epitopes.     

A genotype distribution of human papillomaviruses detected by polymerase chain reaction and direct sequencing analysis in a large sample of common warts in Japan

There have been no large-scale epidemiological studies of human papillomavirus (HPV) genotype distribution of common warts in Japan. A total of 213 patients with common warts (104 males and 109 females) in Japan were studied to detect HPV genotype distribution by polymerase chain reaction (PCR) and direct sequencing analysis. The results were as follows: 94 HPV-1a (44.1%), 35 HPV-4 (16.4%), 30 HPV-65 (14.1%), 13 HPV-27 (6.1%), 13 HPV-2a (6.1%), 9 HPV-57b (4.22%), 3 HPV-16 (1.41%), 2 HPV-6a (0.94%), 2 HPV-63 (0.94%), and 1 case for each of HPV-3, -5, -5b, -7, -10, -21, -29, -47, -56, -57, -62, and -92 (0.47%, respectively). Four cases (1.88%) were found in which two different HPV types were detected within the lesions: one case of HPV-1a with HPV-16, one case of HPV-1a with HPV-65, one case of HPV-6a with HPV-8, and one case of HPV-65 with HPV-16. There were seven cases of mucosal types (3.3%), that is, two HPV-6a, three HPV-16, one HPV-56, and one HPV-62, and three cases of epidermodysplasia verruciformis (EV)-related types (1.41%), that is, one HPV-5, one HPV-5b (both of which belonged to a high-risk group), and one HPV-47 (which belonged to a low-risk group). To date, this is the largest sequencing-based study of HPV for common warts in Japan. It is said that common warts are induced predominantly by HPV-2, -27, and -57 in European population. However, the present results showed that in Japan they were induced mostly by HPV-1, -4, and -65. This suggests that regional differences in HPV genotype distribution may exist between European and Japanese populations.     

Down-regulation of MHC class I is a property common to papillomavirus E5 proteins

The E5 protein family of papillomaviruses comprises small hydrophobic proteins which are associated with the cell endomembrane compartments. The functions of the E5 proteins, particularly those of HPV, are still far from clear. We have reported that the E5 proteins of BPV-1, BPV-4, HPV-16 and HPV-6 down-regulate MHC class I, potentially helping the virus evade the host immune response. Others have described MHC class I down-regulation by HPV-2 E5. We report here that another E5 protein, HPV-83 E5, likewise down-regulates MHC class I and propose that interference with expression, assembly and/or transport of MHC class I is a common property of all E5 proteins evolved by the virus to circumvent host immunosurveillance and thus establish productive infection.     

MVA E2 Recombinant Vaccine in the Treatment of Human Papillomavirus Infection in Men Presenting Intraurethral Flat Condyloma

BACKGROUND: Human papillomavirus (HPV) is the etiologic agent for warts and cervical cancer. In Mexico, the death rate from cervical cancer is extremely high, and statistical data show that since 1990 the number of deaths is increasing. Condylomas and cancer of the penis are the most common lesions presented in men; bladder and prostate cancer in men are also associated with the presence of HPV. Since HPV is transmitted by sexual intercourse, treating both partners is necessary in order to eliminate the virus in the population. Approaches to this include preventative vaccines such as Gardasil, and therapeutic vaccines to treat established infections in both men and women. This will be the only way to decrease the numbers of deaths due to this malignancy. PATIENTS AND METHODS: We conducted a phase I/II clinical trial to evaluate the potential use of the recombinant vaccinia viral vaccine MVA E2 (composed of modified vaccinia virus Ankara [MVA] expressing the E2 gene of bovine papillomavirus) to treat flat condyloma lesions associated with oncogenic HPV in men. Fifty male patients with flat condyloma lesions were treated with either MVA E2 therapeutic vaccine or fluorouracil (5-fluorouracil). Thirty men received the therapeutic vaccine, at a total of 10(6) virus particles per dose, administered directly into the urethra once every week over a 4-week period. Twenty control patients were treated with 5% fluorouracil 1mL twice weekly over a 4-week period directly into the urethra. Reduction of lesions or absence of papillomavirus infection was monitored by colposcopy and histologic analysis. The immune response after MVA E2 treatment was determined by measuring the antibodies against the MVA E2 virus and by analyzing the lymphocyte cytotoxic activity against cancer cells bearing oncogenic papillomavirus. Presence of papillomavirus was determined by the Hybrid Capture method. RESULTS: Twenty-eight of 30 patients showed no lesion or presence of papillomavirus as diagnosed by colposcopy and brush histologic examination after 4 weeks of MVA E2 treatment. These patients showed complete elimination of flat condyloma in the urethra and no acetowhite spots were detected over the prepuce. In two other patients the acetowhite spots and flat condyloma did not diminish. All patients developed antibodies against the MVA E2 vaccine and E2 protein, and generated a specific cytotoxic response against papilloma-transformed cells. Viral DNA was not detected in MVA E2-treated patients. In the control group, 13 of 20 patients were free of lesions. Three of these patients had recurrence of lesions after 3 months of treatment and none of the patients developed specific antibodies against cancer cells. In contrast, patients treated with MVA E2 did not show any recurrence of lesions after 1 year of treatment. In addition, none of the patients had local or systemic adverse effects according to the WHO classification 1-4. CONCLUSIONS: Therapeutic vaccination with MVA E2 proved to be very effective in stimulating the immune system against papillomavirus, and in generating regression of flat condyloma lesions in men.