Ingrid Jochmus

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 L1ΔCE71–55 and L1ΔCE71–60 (carboxy‐terminal deletion of L1 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 L1ΔCE71–60 and L1ΔCE71–55 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 L1ΔCE71–60 CVLPs specifically lysed the RMA‐E7 transfectants as well as RMA cells loaded with the peptide E749–57, 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. Int. J. Cancer 81:881–888, 1999.

Human Papillomavirus Type 16 L1 Capsomeres Induce L1-Specific Cytotoxic T Lymphocytes and Tumor Regression in C57BL/6 Mice

ABSTRACT We analyzed capsomeres of human papillomavirus type 16 (HPV16) consisting of the L1 major structural protein for their ability to trigger a cytotoxic T-cell (CTL) response. To this end, we immunized C57BL/6 mice and used the L1165-173 peptide for ex vivo restimulation of splenocytes prior to analysis (51Cr release assay and enzyme-linked immunospot assay [ELISPOT]). This peptide was identified in this study as a Db-restricted naturally processed CTL epitope by HPV16 L1 sequence analysis, major histocompatibility complex class I binding, and 51Cr release assays following immunization of C57BL/6 mice with HPV16 L1 virus-like particles (VLPs). HPV16 L1 capsomeres were obtained by purification of HPV16 L1 lacking 10 N-terminal amino acids after expression in Escherichia coli as a glutathione S-transferase fusion protein (GST-HPV16 L1ΔN10). Sedimentation analysis revealed that the majority of the purified protein consisted of pentameric capsomeres, and assembled particles were not observed in minor contaminating higher-molecular-weight material. Subcutaneous (s.c.) as well as intranasal immunization of C57BL/6 mice with HPV16 L1 capsomeres triggered an L1-specific CTL response in a dose-dependent manner as measured by ELISPOT and 51Cr release assay. Significant reduction of contaminating bacterial endotoxin (lipopolysaccharide) from the capsomere preparation did not diminish the immunogenicity. Antibody responses (serum and vaginal) were less robust under the experimental conditions employed. In addition, s.c. vaccination with HPV16 L1 capsomeres induced regression of established tumors expressing L1 determinants (C3 tumor cells). Our data demonstrate that capsomeres are potent inducers of CTL responses similar to completely assembled T=7 VLPs. This result is of potential relevance for the development of (combined prophylactic and therapeutic) HPV-specific vaccines, since capsomeres can be produced easily and also can be modified to incorporate heterologous sequences such as early HPV proteins.

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.

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.

EPIDEMIOLOGIE, ATIOLOGIE UND PRAVENTION DES ZERVIXKARZINOMS

Durch molekulare und epidemiologische Studien wurde bewiesen, daß die humanpathogenen Papillomviren (HPV) Typ 16 und 18 ursächlich an der Entstehung des Zervixkarzinoms beteiligt sind. Diese Aussage gilt auch in bisher noch eingeschränktem Maße für andere sog. High-risk-HPV-Typen wie HPV 31, 33, 35, 39, 45, 51, 52, 56 und 58. Etwa 1/2 Mio. Frauen erkranken jährlich weltweit am Zervixkarzinom und zwischen 1–4% aller jüngeren Frauen leiden an einer Präkanzerose der Cervix uteri. Das Wissen um die virale Genese anogenitaler Neoplasien wird jedoch bisher nicht für die primäre und sekundäre Prävention dieser Erkrankungen eingesetzt. Dies liegt vor allem daran, daß die Infektion mit genitalen HPV-Typen bei jungen Frauen hoch prävalent ist, jedoch nur selten zum Karzinom führt. In der folgenden Übersicht werden daher die wichtigsten vorliegenden epidemiologischen Daten zum Zervixkarzinom und seinen Vorstufen auf ihre Assoziation mit HPV analysiert. Im weiteren werden die molekularbiologischen Daten von HPV für die ano- genitale Karzinogenese dargestellt. Abschließend wird auf die Bedeutung von HPV für die Prävention des Zervixkarzinoms eingegangen.

Immunological Aspects of the E6 and E7 Oncogenes: Tools for Diagnosis and Therapeutic Intervention

The antibody response against structural HPV proteins that was shown to develop during the natural course of HPV infections is likely the consequence of “mucosal immunization” (i.e., uptake and presentation by M cells), or depends on “secondary infection” through small wounds resulting in the exposure of these proteins to antigen presenting cells (APC) such as macrophages or dendritic cells. There is good evidence that antibodies against HPV capsid proteins are relevant in controlling HPV infections.1