Therapeutic vaccines for human papillomaviruses.

Although papillomavirus infections are not very immunogenic there is evidence that the immune system controls the spread of virus and the development of diseases associated with such infections. Certain types of human papillomaviruses (HPV) are the major cause of premalignant and malignant diseases of the anogenital tract, most notably cancer of the uterine cervix, a major health care problem worldwide. Since the viral oncoproteins E6 and E7 are constitutively expressed within the tumor cells, they are considered as suitable targets for attack by T lymphocytes. Several approaches to specifically trigger a cell-mediated immune response have been successful in experimental animals, leading to suppression of HPV-induced tumors. First clinical trials have been completed which raise hopes that a similar effect can also be achieved by therapeutic vaccination of humans.

A DNA vaccine based on a shuffled E7 oncogene of the human papillomavirus type 16 (HPV 16) induces E7-specific cytotoxic T cells but lacks transforming activity.

Vaccination with oncogene-derived DNA for anti-cancer treatment carries a risk of de-novo tumor induction triggered by the persisting recombinant DNA. We hypothesized that an oncoprotein whose primary sequence has been rearranged ('shuffled') to maintain all possible T cell epitopes still induces cytotoxic T cells against the authentic protein but is devoid of transforming properties. As a model antigen, we used the E7 oncoprotein of the human papillomavirus (HPV) type 16, the major cause of cervical cancer. We have generated an artificial E7 molecule in which four domains were rearranged and, in order to maintain all possible T cell epitopes, certain sequences were duplicated. Upon transfection of this shuffled E7 gene (E7SH) into RMA cells, presentation of an E7 Db-restricted T cell epitope was shown by an E7-specific CTL line in vitro. Immunization of C57BL/6 mice with E7SH DNA induced E7-specific CTL and also conveyed protection against E7-positive syngeneic tumor cells. No transforming activity of E7SH DNA in NIH3T3 cells was detected, as determined by focus formation, induction of S-phase under conditions of serum deprivation and degradation of endogenous pRB. Our results suggest that DNA shuffling may become a promising concept for DNA-based anti-cancer vaccines.

HPV16 L1E7 chimeric virus-like particles induce specific HLA-restricted T cells in humans after in vitro vaccination.

Cervical cancer has been shown to be highly associated with human papillomavirus (HPV) infection. The viral oncogenes E6 and E7 are constantly expressed by the tumor cells and are therefore targets for immunotherapy. In the present study we investigated the potential of HPV16 L1E7 chimeric virus-like particles (CVLP) to activate specific cytotoxic T lymphocytes in human blood donors. CVLP were expressed by recombinant baculovirus and purified. Direct incubation of freshly isolated peripheral blood lymphocytes (PBL) with CVLP resulted in induction of proliferation and growth of T cell lines. To enhance antigen presentation we also loaded dendritic cells with CVLP and used them to activate naive T cells. Growing cell lines were mainly CD3 positive (>95%) with a predominant CD4-positive and a minor CD8-positive component. Analysis of Tcell specificity was carried out by an interferon-gamma ELISpot assay. Dendritic cells pseudoinfected with CVLP or pulsed with human leukocyte antigen (HLA)-A*0201-restricted peptide E7(11-20) or with a newly identified HPV16 peptide L1(323-331) were used as stimulator cells. T cells responsive to CVLP were found in the cultures with frequencies of 0.5%-0.7%. Frequencies to peptides were around 0.1%. These T cells had cytolytic activity toward autologous B-lymphoblastic cell lines either pseudoinfected with CVLP or pulsed with HLA-A*0201-restricted peptides. They also lysed the HPV16- and HLA-A*0201-positive cervical cancer cell line CaSki, whereas HLA-A*0201-negative SiHa cells were not lysed. We conclude from our data that CVLP show promise for a therapeutic vaccine in patients with HPV16-positive cervical intraepithelial neoplasia lesions or cervical cancer.

Immunization against human papillomavirus infection and associated neoplasia.

BACKGROUND: Chimeric virus like particles (CVLPs) constructed by fusing human papillomavirus type 16 (HPV16) E7 sequences into the C-terminus of the viral L1 gene constitute the first generation of preventive and therapeutic HPV vaccines. Even though vaccination with DNA is highly efficient in the induction of a cytotoxic T-cell (CTL) response utilization of a DNA vaccine in the HPV context, it has been hampered by concern for the oncogenic potential of the E6 and E7 proteins encoded by the viral oncogenes. OBJECTIVE: To consider the use and impact of E7 DNA for immunization. EXPERIMENTAL: In addition to hemagglutination inhibition, a versatile assay to measure neutralization of yeast cell-derived pseudovirions carrying a green fluorescence reporter gene has now been developed. Mice immunized with the HPV16 CVLPs generate E7-specific CTLs, which kill E7 expressing or E7 peptide loaded RMA-cells, protect against tumor formation by syngeneic HPV transformed cells and also induce regression of already established tumors. Since generation of CTL response is achieved by presentation of epitopes as short peptides together with appropriate MHC class I molecules, complete proteins are not required. Instead a shuffled E7 protein has now been used successfully for generating CTL responses comparable to the CVLP responses in mice. CONCLUSIONS: Our preliminary results suggest that immunization with E7 shuffled DNA yields a response directed against the authentic E7 protein. Furthermore, booster immunization with E7 shuffled DNA would avoid inhibition by neutralizing antibodies, however, further studies are needed to guarantee that the shuffled E7 protein lacks oncogenic activity.

Chimeric virus-like particles of the human papillomavirus type 16 (HPV 16) as a prophylactic and therapeutic vaccine.

Infection by certain human papillomaviruses (HPV), most notably HPV types 16 and 18, is the major risk factor for cervical cancer. Worldwide, this disease represents the second most frequent malignant tumor in women; thus, there is urgent need for efficient therapy and prevention. The natural history of cervical cancer and its precursors (cervical intraepithelial neoplasias), as well as animal experiments, strongly suggest that the immune system controls both the primary infection (by neutralizing antibodies directed against the major structural protein L1) and the progression of the disease (via cytotoxic T cells specific for the viral oncoproteins expressed in transformed cells, e.g., E7). By the expression of an HPV 16 L1E7 fusion protein, we have generated chimeric virus-like particles (CVLP). Immunization of mice with CVLPs induces neutralizing antibodies directed against L1 virus-like particles (devoid of the E7 portion) and E7-specific T cells as measured in vitro. Vaccinated animals are protected against tumor growth following inoculation of syngeneic HPV 16-transformed cells. In addition, we observed a therapeutic effect of vaccination on pre-existing tumors. This data allowed us to conclude that CVLPs are suitable for prevention and therapy of HPV infection. A vaccine based on HPV 16 L1E7 CVLPs is currently under development.

E7-specific cytotoxic T cell tolerance in HPV-transgenic mice.

The "high-risk" human papillomavirus type 16 (HPV 16) is associated with the development of cervical cancer. Although the viral gene products E6 and E7 are constitutively expressed in HPV 16-associated lesions and therefore appear as candidate antigens for a specific immune response, the immune system fails to produce an efficient defence against tumor outgrowth in affected patients. Keratinocytes are the natural target cells of HPV infection. To investigate the E7-specific immune response in vivo, we used transgenic mice expressing the oncogenes E6 and E7 of HPV 16 under the control of the keratin 10 promoter in the suprabasal layers of the epidermis. This expression pattern closely reflects the viral early gene transcription that is observed in low grade cervical intraepithelial lesions (CIN). The transgene product E7 does not induce an immune response in these transgenic mice. However, upon vaccination anti-E7 antibodies were produced without causing signs of autoimmune disease. In contrast, E7-specific cytotoxic T lymphocytes (CTL) were not detected after immunization. From these results we conclude that in K10 HPV 16 E6/E7 transgenic mice the E7 transgene expression induces specific immunological tolerance on the CTL level.

Immune response to human papillomavirus 16 L1E7 chimeric virus-like particles: induction of cytotoxic T cells and specific tumor protection.

Expression of human papillomavirus type 16 (HPV 16) fusion proteins LI deltaCE7(1-55) and LI deltaCE7(1-60) (carboxy-terminal deletion of LI replaced by 55 or 60 amino-terminal amino acids of E7) leads to formation of chimeric papillomavirus-like particles (CVLPs). After "infection" of cells by CVLPs, the chimeric proteins can be detected in the cytosol and the endoplasmic reticulum (ER), suggesting that they are intracellularly processed via the MHC class I pathway and, therefore, able to activate cytotoxic T lymphocytes (CTLs). To investigate the cytotoxic immune response against HPV 16 LI deltaCE7(1-60) and LI deltaCE7(1-55) CVLPs, we immunized C57Bl/6 mice with various CVLP doses without adjuvant. Two weeks after immunization, spleen cells were prepared and stimulated in vitro using HPV 16 E7-expressing transfectants of the tumor cell line RMA. In 51Cr-release cytotoxicity assays, spleen cells of mice vaccinated with LI deltaCE7(1-60) CVLPs specifically lysed the RMA-E7 transfectants as well as RMA cells loaded with the peptide E7(49-57), which represents an H2-Db-restricted CTL epitope. This demonstrates that CVLPs induce an E7-specific CTL response in mice in the absence of an adjuvant. Furthermore, immunization with CVLPs prevented outgrowth of E7-expressing tumor cells even if inoculation of cells was performed 2 weeks before vaccination. We conclude from our data that CVLPs show promise for therapy of HPV-associated lesions.

Specificity of human cytotoxic T lymphocytes induced by a human papillomavirus type 16 E7-derived peptide.

In order to establish tumour-specific cytotoxic T lymphocyte (CTL) cell lines, T cells from a human papillomavirus (HPV) type 16-positive patient with a cervical carcinoma in situ and from a healthy volunteer were stimulated in vitro with autologous dendritic cells loaded with peptides derived from the viral transforming proteins E6 and E7 and corresponding to potential HLA-A*0201-restricted T cell epitopes. From each donor a small number of low-affinity CTL lines against the peptide E7/86-93 was obtained, which specifically lysed HLA-A*0201-expressing B-lymphocytes (cell line 721) loaded with this peptide. Cytotoxicity was also observed against two HLA-A*0201-E7-positive epithelial cell lines, the cervical carcinoma cell line CaSki and the HPV-16-immortalized foreskin-keratinocyte cell line HPK IA. However, since none of the CTL recognized both cell lines, and E7-expressing 721 transfectants were never lysed, it was concluded that the reactivity against CaSki and HPK IA cells was due to cross-reactivity on allogeneic HLA molecules rather than to E7 recognition, which emphasizes that the specificity of tumour cell lysis by peptide-induced CTL has to be interpreted with caution.

Human papillomavirus type 16 (HPV 16) E7 and major histocompatibility complex (MHC) class I and II expression in human keratinocytes in culture.

The low expression of major histocompatibility complex (MHC) class I antigens on human papillomavirus (HPV)-infected cervical carcinoma cells may be responsible for an insufficient cytotoxic T cell response against these cells. To investigate in vitro whether the HPV type 16 early gene product E7 influences cell surface expression of MHC class I and II molecules the HPV negative keratinocyte cell line HaCaT was either stably transfected with the E7 gene or infected with E7-recombinant vaccinia viruses. No difference in MHC class I transcription was detected between E7-transfected and untransfected HaCaT cells. MHC class I cell surface expression as determined by FACS analysis was stronger in some of the transfectants and less intensive in others when compared to untransfected HaCaT cells. In wildtype as well as in E7-recombinant vaccinia virus infected HaCaT cells downregulation of MHC class I molecules on protein and transcriptional level was observed. The alterations in MHC class I expression were independent of the presence and amount of E7-specific transcripts. None of the transfectants or infected HaCaT cells had MHC class II molecules on their cell surface. Hence, our data did not show a correlation between HPV 16 E7 and MHC expression in vitro.

Relation between skin cancer, humoral responses to human papillomaviruses, and HLA class II molecules in renal transplant recipients.

Human papillomaviruses (HPV), especially the epidermodysplasia verruciformis (EV)-associated HPV 5, 8, 14, 17, 20, and 47, are thought to play a role in the pathogenesis of some skin cancers in recipients of renal allografts. MHC class I and class II genes are involved in the cellular immune response to viral and tumor Ag. Little is known about humoral responses to HPV in recipients with and without skin cancer. We investigated the prevalence of antibodies to the early (E) protein E7 and the major capsid late (L) protein L1 of HPV 8. In addition, we studied the association of HLA class II molecules with these antibody responses. The E7 and L1 open reading frames of HPV 8 were bacterially expressed as beta-galactosidase fusion proteins, which were purified by preparative gel electrophoresis. Serum samples from 36 renal transplant recipients with and 91 recipients without skin cancer were screened for the presence of IgG and IgM antibodies to HPV 8 E7 and L1, by Western blot analysis. The detection of anti-HPV 8 L1 antibodies represents the immune response to HPV 8 and possibly other EV-associated HPV, because cross-reactivity between the representatives of this HPV subgenus can occur. The antibody responses to HLA Ag were used as controls. Recipients who had IgM antibodies but no IgG antibodies to L1 of HPV 8 (patients with no apparent class switch from IgM to IgG) had skin cancer in 50% of cases, whereas recipients who produced IgG antibodies (patients with an apparently good humoral response to L1 of HPV 8) had skin cancer in only 18% of cases. The estimated relative risk of skin cancer in recipients with no class switch, compared with the risk in those with a good humoral response, was 4.5 (95% confidence interval, 1.1 to 18.1). We found no association between the antibody response to HLA Ag and the occurrence of skin cancer. A strong linkage between the absent class switch of antibody production in response to L1 of HPV 8 and HLA-DR7 was observed (relative risk, 26.2). Renal transplant recipients who have no apparent class switch from IgM to IgG production in response to Ag encoded by L1 of HPV 8 or possibly other EV-associated HPV are at an increased risk of skin cancer. The association with HLA-DR7 indicates a genetic control of skin cancer development or regression, involving genes in the class II region of the MHC.

Major histocompatibility complex and human papillomavirus type 16 E7 expression in high-grade vulvar lesions.

To determine whether expression of the human papillomavirus (HPV) type 16 E7 open reading frame influences expression of major histocompatibility complex (MHC) antigens on the surface of squamous epithelial cells, serial frozen sections from seven HPV type 16-positive, high-grade vulvar intraepithelial neoplasia (VIN 2-3) lesions were tested for viral transcription by RNA-RNA in situ hybridization, for MHC expression by immunohistochemical staining with antibodies to MHC class I and II molecules, and for keratinocyte differentiation by immunohistochemical staining with anti-filaggrin and cytokeratin 10 antibodies. Despite the histologic appearance of high-grade VIN lesions, expression patterns of cytokeratin 10 and filaggrin suggested a certain degree of keratinocyte differentiation in all specimens. These differentiation markers were especially prominent in parakeratotic and hyperkeratotic superficial areas, which did not express MHC antigens or contain E7 mRNA. Expression of MHC class I molecules within dysplastic tissues was greater than within HPV type 16-negative, normal vulvar epithelium from the same patients. In five of the VIN 2-3 specimens anti-MHC class I antibodies reacted more strongly with cells of the basal and suprabasal layers than with cells of the epithelial surface. In one lesion basal cells stained less intensively than surface cells, whereas in another specimen all epithelial layers were equally MHC class I positive. Staining with anti-MHC class II antibodies was generally restricted to isolated foci, representing invading lymphocytes, tissue macrophages, and Langerhans cells. In two lesions, however, there was heterogeneous keratinocyte expression of MHC class II proteins, perhaps due to inflammation. Major histocompatibility complex antigen detection was independent of the presence or distribution pattern of E7-specific transcripts. Hence, a correlation between MHC and E7 expression appears unlikely in warty VIN lesions.

Detection of antibodies to the E4 or E7 proteins of human papillomaviruses (HPV) in human sera by western blot analysis: type-specific reaction of anti-HPV 16 antibodies.

To determine the cross-reactivity between early (E) proteins of different human papillomavirus (HPV) types, 346 serum samples were tested with E4 and E7 of HPV 16. Two hundred and sixteen of them were also tested with HPV 1 E4, 21 with HPV 11 E4 and E7, and 109 with HPV 18 E4 and E7. Viral fusion proteins were expressed in Escherichia coli and used as antigens in Western blot experiments. The sera were obtained from patients with HPV-associated genital lesions or cervical cancer, from renal transplant recipients and from patients hospitalized for reasons unrelated to HPV infections (the controls). In contrast to findings relating to HPV 16 E4 specific antibodies, the prevalence of anti-HPV 1 E4 antibodies was not greater in renal transplant recipients than in the controls. In each age group of the control population more sera reacted with HPV 1 E4 than with HPV 16 E4. Sera of patients with HPV-associated cervical diseases and cervical cancer reacted less frequently with HPV 11 E4 or E7 and HPV 18 E4 or E7, respectively, than with the corresponding HPV 16 proteins. Thirty of 117 HPV 16 E4 or E7 positive sera showed reactivity to the corresponding protein of either HPV 1, 11 or 18. As demonstrated by cross-absorption experiments performed with 26 of the double-reacting sera, 24 contained two populations of antibodies reacting with proteins of different HPV types whereas only two contained cross-reacting antibodies. We concluded that in the majority of sera antibodies to the HPV 16 E4 and E7 proteins are type-specific.

Comparison of ELISA and western blotting for human papillomavirus type 16 E7 antibody determination.

A total of 140 sera originating from healthy women and women with either cervical intraepithelial neoplasia or cervical cancer were tested for the presence of IgG antibody against E7 of human papillomavirus type 16 (HPV-16) by ELISA using a synthetic icosapeptide, denoted 16/E7-2, representing amino acids 11 to 30, and by Western blotting (WB) using a genetically engineered HPV-16 E7 fusion protein. Eighteen sera were found positive in either one or the other test. Positive reactions were more frequently detected in cervical carcinoma patients (12 of 34, 35.2%) than in the other individuals (six of 106, 5.7%). Ten children's (1 to 3 years of age) sera reacted in neither ELISA nor WB with HPV-16 E7. A high degree of concordance between the two tests was found suggesting that both tests detect the same or similar activity. To locate the reacting epitopes in the E7 protein, absorption tests were performed with peptides corresponding to various sections of the protein. Based on the results obtained, sera possessing antibody to HPV-16 E7 could be differentiated into those reactive with only the 16/E7-2 peptide and those reactive with other HPV-16 E7 epitopes.

Psychosocial adaptation of children and their parents to hospital and home hemodialysis.

Psychosocial adaptation of 20 children and adolescents treated by regular hemodialysis and their parents was analyzed by detailed semistructured interviews and questionnaires. The results in 10 patients treated in the centre and 10 followed at home were compared. The burdens of patients and parents induced by therapy as well as compliance, educational aspects and school activity in the treated children are described. Home dialysis usually provoked more fears of complications and aggressive feelings in patients and stress in parents but was superior to centre treated patients regarding social contacts and school activity of patients. From the data obtained, a comprehensive programme of psychosocial care for children with end-stage kidney disease was derived, including detailed instruction for parents and teachers of children on regular dialysis treatment.

[Behaviour therapy of autoagression with the Lesch-Nyhan-syndrome ( author's transl)]. / Verhaltenstherapie der Autoaggressionen beim Lesch-Nyhan-Syndrom.

Successful behaviour therapeutic treatment of autoagression in a child with Lesch-Nyhan-Syndrome was combined with the establishment of alternative behaviour at the same time. Treatment applying negative stimuli could be avoided.