The effect of helper epitopes and cellular localization of an antigen on the outcome of gene gun DNA immunization.

In DNA vaccination, CD4(+) T-cell help can be enhanced by fusion of a gene encoding an immunization protein with a foreign gene or its part providing T(h) epitopes. To study the effect of helper epitope localization in a protein molecule, the influence of the vicinity of the helper epitope, and the impact of chimeric protein cellular localization, we fused the helper epitope p30 from tetanus toxin (TT, aa 947-967) with the N- or C-terminus of the mutated E7 oncoprotein (E7GGG) of human papillomavirus type 16, enlarged the p30 epitope with the flanking residues containing potential protease-sensitive sites and altered the cellular localization of the fusion constructs by signal sequences. The p30 epitope enhanced the E7-specific response, but only in constructs without added signal sequences. After localization of the fusion proteins into the endoplasmic reticulum and endo/lysosomal compartment, the TT-specific T(h)2 response was increased. The synthetic Pan DR epitope (PADRE) induced a stronger E7-specific response than the p30 epitope and its stimulatory effect was not limited to nuclear/cytoplasmic localization of the E7 antigen. These results suggest that in the optimization of immune responses by adding helper epitopes to DNA vaccines delivered by the gene gun, the cellular localization of the antigen needs to be taken into account.

In vitro and in vivo effects of reovirus on HPV16-transformed mice cells.

UNLABELLED: Oncolytic viruses are examined to serve as anticancer therapeutics. It is expected that in addition to direct oncolytic effect their action will also help eliciting a solid antitumor immunity. In presented series of experiments we have employed two HPV16-transformed mouse (strain C57/B6) cell lines, TC-1 and MK16/III/ABC (MK16), and reovirus type 3, strain Dearing (RV). Both cell lines are highly susceptible to RV and produce large amounts of infectious virus in vitro while normal human are not susceptible to RV. Still, some differences were encountered. TC-1 cells produced moderately lesser amounts of infectious virus, but, paradoxically, were more efficient producers of delta1 antigen of RV and as a consequence of virus infection died more rapidly than simultaneously infected MK16 cells. Minor differences between the cell lines were observed in the percentage of cells arrested in theG2/M phase of the cell cycle and in some markers of apoptosis. When inoculating high doses (5x106) of infected cells (MOI 10 PFU/cell) into syngeneic animals their oncogenic activity was strongly suppressed, nearly completely in the case of MK16 cells and somewhat less efficiently in the case of more oncogenic TC-1 cells. Immunizing experiments in which non-oncogenic doses (106) of RV infected TC-1 cells were tested in parallel with the same doses of irradiated cells brought surprising results. When immunized animals were challenged with TC-1 cells, the irradiated cells proved to be a much better immunogen that the infected cells. However, when challenged with MK16 cells the opposite was true. It is believed that this difference was associated with the different biological properties of the cell lines tested. KEYWORDS: reovirus type 3, HPV16-transformed mouse cell lines, apoptosis, cell cycle, immunization/challenge experiments.

Human papillomavirus infection and tumours of the anal canal: correlation of histology, PCR detection in paraffin sections and serology.

Human papillomavirus infection is an important etiological factor in squamous cell carcinoma of the anus (SCCA). Different histological variants of anal carcinomas displaying squamous differentiation, previously classified as separate tumours, were recently reclassified as SCCA by the WHO. In our recent study the presence of HPV was detected by PCR in biopsy specimens of 42 different anal tumours, including SCCA and its histological variants (n=22), adenocarcinomas (n=5), tubulovillous adenomas (n=5) and anal condylomas (n=10). HR HPV16 (high risk - HR) was detected in 18 of SCCA specimens (81.8%). All histological variants, i.e. tumours with basaloid, squamous and mixed histological patterns, were represented among the HPV-positive cancers. Four tumours (18.2%) were HPV negative. Low-risk (LR) HPV types were not detected within the SCCA group. HPV16 was identified in one adenocarcinoma, while four cases were HPV negative. Two adenomas showed presence of HPV16; one showed simultaneous positivity for HPV33. The remaining three tumours were HPV negative. Seven anal condylomas (70%) were LR HPV 6 and/or 11 positive, while three were HPV negative. The presence of HR HPV types was not observed in anal condylomas. Our results provide further evidence in support of the etiological role of HR HPV infection in the development of SCCA regardless of its histological appearance.

Longitudinal study of patients after surgical treatment for cervical lesions: detection of HPV DNA and prevalence of HPV-specific antibodies.

The principal aims of this study were to test whether persistence of human papillomavirus (HPV) DNA is predictive of recurrent disease in women after surgical treatment for cervical lesions, to distinguish between persistent and newly acquired HPV infection, and to observe the effect of surgical treatment on levels of HPV-specific antibodies. A group of 198 patients surgically treated for low-grade and high-grade squamous intraepithelial lesions and 35 age-matched controls were monitored for 18 months at 6-month intervals. The presence of HPV DNA in cervical smears was detected by means of consensus polymerase chain reaction, and serum levels of HPV-specific antibodies to HPV types 16, 18, 31, 33, and 45 were measured. In ten patients positive for HPV type 16 in consecutive samples, the HPV 16 variants were identified using a polymerase chain reaction specific for the long control region. Data regarding demographics, risk factors for cervical cancer, and risks related to HPV exposure were collected through a patient questionnaire. Subjects persistently positive for HPV DNA were more likely to present with cytological and/or colposcopical abnormalities. A higher reactivity to HPV-specific antibodies was observed in these women at the 18-month follow-up visit. All ten patients with HPV 16 infection detected in consecutive samples showed persistence of either the same prototype or the same variant during the follow-up period. Risky sexual behavior and smoking were more common in patients than in controls. Persistent HPV infection as demonstrated by both HPV DNA detection and antibody detection appears to be a risk factor for the recurrence of pathological findings in women after surgery. An individually based approach to surgical treatment is an important factor in the outcome of disease at follow-up.

Use of polyclonal rabbit antibodies for detection of the bcr-abl fusion zone in cells transfected with experimental bcr-abl DNA vaccines.

Rabbits were immunized with peptides covering the fusion zone of the chimeric bcr-abl protein in order to prepare antibodies capable of detecting the expression of a selected portion of this fusion zone, by a variety of experimental genetic vaccines. Three peptides of different size covering the b3a2 fusion zone, either unmodified or modified by the omission of alanine at the N-terminal of the a2 section of the fusion zone, and one peptide covering the unmodified b2a2 fusion zone were used. All were capable of eliciting antibodies reactive with the respective immunizing peptides. Their cross-reactivities, especially the results of cross-absorption experiments, strongly suggested that the serum of the rabbit immunized with an octadekapeptide mimicking the b3a2 fusion zone contained antibodies against a novel antigenic determinant created by the chimeric protein, and also against an epitope present in the adjacent a2 section but no antibody reactive with the adjacent b3 region. In Western blotting, these antibodies were capable of detecting the p210bcr-abl or a portion of it (a 25 amino acid-long sequence covering the b3a2 fusion zone) in lysates of 293T cells transfected with plasmids that carried either the full cDNA of the bcr-abl gene or a fragment thereof fused with either the HSP70 gene or certain other genes.

Characteristic of two mouse bcr-abl-transformed cell lines: I. General properties of the cells.

In an effort to develop an experimental system suitable for immunological studies in which Bcr-Abl-positive cells are to be used as antigens, we examined the properties of two mouse (Balb/c) established cell lines that express the Bcr-Abl protein and are oncogenic for syngeneic animals. Under standard conditions the two cell lines, viz. Ba-p210 (B210) and 12B1, expressed comparable amounts of the Bcr-Abl protein. However, they differed in a number of characteristics. From the morphological point of view, B210 cells were the more homogeneous, being mainly represented by leukaemic blastic cells with a large number of AgNORs as markers indicating a high proliferative activity. 12B1 cells were more polymorphic and giant cells were detected within their populations. Many 12B1 cells exhibited nuclear segmentation and "band-like" structures. Markers of proliferation were less frequent in 12B1 and the tendency for aging was more pronounced in these cells. The 12B1 cells were slightly more sensitive to imatinib mesylate than B210 cells. In B210 cells, the expression of MHC class I was downregulated, which was not the case with 12B1 cells. Both cell lines induced leukaemia-like disease in mice after intravenous application but, as compared with B210, 12B1 cells were about 100 times more oncogenic and the disease they induced was more aggressive. Moreover, 12B1, but not B210, induced tumours after subcutaneous or intraperitoneal inoculation.

Effect of IFN-gamma receptor gene deletion on vaccinia virus virulence.

Two vaccina virus (VV) strains, WR and Praha, were selected for a study undertaken to determine whether the virus-encoded interferon-gamma receptor (IFN-gamma R) plays any role in virus virulence. Both of the viruses expressed the B8R gene coding for IFN-gamma R in infected cell cultures. The nucleotide sequence of the Praha virus B8R gene was determined, and, when compared with the published sequence of the WR virus, it only displayed one silent nucleotide substitution. Mutants of the WR and Praha viruses with deleted B8R gene were constructed. In rabbits, skin lesions produced by the WR B8R-deleted mutants were smaller and tended to disappear earlier than those caused by wild-type WR virus. Similar results were obtained with both independently prepared WR B8R-deleted mutants. These data strongly suggested that the product of B8R gene did play a role in virus virulence. A similar comparison of the wild-type Praha virus and its mutant could not be done because of the very low virulence of the parental virus for rabbits.

Modified HPV16 E7 Genes as DNA Vaccine against E7-Containing Oncogenic Cells.

Therapeutic vaccines against tumors associated with human papillomaviruses (HPV) should elicit cellular immune responses against early HPV antigens, primarily the oncoproteins E7 and E6. Because of safety concerns, the direct use of an unmodified oncogene is impossible in human DNA vaccination. Therefore, we introduced three point mutations into the pRb-binding site of HPV16 E7 oncogene to eliminate its transformation potential. The resultant gene was denoted E7GGG. The rates of expression and the cellular localization of E7 and E7GGG proteins were comparable. In immunization-challenge experiments, the efficacy of plasmids containing the E7, E7GGG, or fusion genes of HPV16 E7, viz. L1DeltaCE7(1-60) (M. Muller et al., 1997, Virology 234, 93-111), and Sig/E7/LAMP-1 (T. C. Wu et al., 1995, Proc. Natl. Acad. Sci. USA 92, 11671-11675), was compared. While tumors developed in all animals immunized with the wild-type E7 gene, a significant proportion of mice remained tumor-free after vaccination with the E7GGG gene. The fusion gene L1DeltaCE7(1-60) induced negligible protection, but Sig/E7/LAMP-1 conferred the highest protection. Intradermal immunization by gene gun proved superior to i.m. inoculation. In "therapeutic" experiments, a 1-day delay between inoculation of oncogenic cells and the start of DNA immunization resulted in partial therapeutic effect, but a 3-day delay produced a substantially lower immunization effect. A combination of Sig/E7/LAMP-1 and E7GGG genes did not enhance the immune response. These results demonstrate a significant enhancement of HPV16 E7 immunogenicity after mutagenesis of the pRb-binding site, but the mutated E7 gene did not excel the Sig/E7/LAMP-1 fusion gene.

Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16.

In the endeavour to develop a model for studying gene therapy of cancers associated with human papillomaviruses (HPVs), mouse cells were transformed with the HPV type 16 (HPV16) and activated H-ras oncogenes. This was done by cotransfection of plasmid p16HHMo, carrying the HPV16 E6/E7 oncogenes, and plasmid pEJ6.6, carrying the gene coding for human H-ras oncoprotein activated by the G12V mutation, into secondary C57BL/6 mouse kidney cells. An oncogenic cell line, designated MK16/1/IIIABC, was derived. The epithelial origin of the cells was confirmed by their expression of cytokeratins. No MHC class I and class II molecules were detected on the surface of MK16/1/IIIABC cells. Spontaneous metastases were observed in lymphatic nodes and lungs after prolonged growth of MK16/1/IIIABC-induced subcutaneous tumours. Lethally irradiated MK16/1/IIIABC cells induced protection against challenge with 10(5) homologous cells, but not against a higher cell dose (5 x 10(5)). Plasmids p16HHMo and pEJ6.6 were also used for preventive immunization of mice. In comparison with a control group injected with pBR322, they exhibited moderate protection, in terms of prolonged survival, against MK16/1/IIIABC challenge (P < 0.03). These data suggest that MK16/1/IIIABC cells may serve as a model for studying immune reactions against HPV16-associated human tumours.

Immune response to vaccinia virus recombinants expressing glycoproteins gE, gB, gH, and gL of Varicella-zoster virus.

Immunogenicity of Varicella-zoster virus glycoproteins gE, gB, gH, and gL expressed by recombinant vaccinia viruses (VV) separately or simultaneously was determined in mice and guinea pigs by ELISA, Western blotting, radioimmunoprecipitation, plaque reduction assay, and skin test. Single VV-gE and VV-gB recombinants and double VV-gH/gL recombinant elicited specific antibodies with VZV neutralizing activity in mice. Co-expression of gE and gB by one recombinant VV resulted in an increased antibody response in comparison with immunization with single recombinants or their mixtures. Unlike anti-gB and anti-gH/gL antibodies, the gE-specific antibodies had no virus neutralizing activity in absence of complement, and when used alone, they even caused considerable increase of VZV infectious units. Moreover, immune sera containing anti-gE antibodies antagonized complement independent virus-neutralizing activity of anti-gB- and anti-gH/gL-positive sera. The ability to induce delayed hypersensitivity reaction to VZV antigens was observed after immunization of guinea pigs with gE- and/or gB-expressing VVs.

Characterization of interaction of gH and gL glycoproteins of varicella-zoster virus: their processing and trafficking.

Varicella-zoster virus (VZV) glycoproteins gH and gL were examined in a recombinant vaccinia virus system. Single expression of glycoprotein gL produced two molecular forms: an 18 kDa form and a 19 kDa form differing in size by one endoglycosidase H-sensitive N-linked oligosaccharide. Coexpression of gL and gH resulted in binding of the 18 kDa gL form with the mature form of gH, while the 19 kDa gL form remained uncomplexed. The glycosylation processing of gL was not dependent on gH; however, gL was required for the conversion of precursor gH (97 kDa) to mature gH (118 kDa). Subsequent analyses indicated that gL (18 kDa) was a more completely processed gL (19 kDa). Screening of the culture media revealed that gH and gL were secreted, but only if coexpressed and complexed together. The secreted form of gL was 18 kDa while that of gH was 114 kDa. The fact that secreted gH was smaller than intracytoplasmic gH suggested a proteolytic processing event prior to secretion. The 19 kDa form of gL was never secreted. These findings support a VZV gL recycling pathway between the endoplasmic reticulum and the cis-Golgi apparatus.

Longitudinal follow-up of antibody response to selected antigens of human papillomaviruses and herpesviruses in patients with invasive cervical carcinoma.

A group of 21 cervical-carcinoma patients was followed longitudinally. All patients had undergone intensive radiotherapy. In the course of a more than 5-year observation period, 2 patients died of cervical cancer, 1 from other causes, 3 were lost from follow-up, and 15 survived without any signs of the disease. Sera taken before, up to 17 months after and more than 5 years after the start of therapy, were tested by ELISA for IgG antibodies reactive with a broad spectrum of HPV-derived antigens, glycoprotein G of HSV 2, whole virion antigen of HCMV, and a synthetic peptide corresponding to the immuno-dominant region of EBNA 1. The therapy was associated with a marked decrease in E2 and E7 antibodies in nearly all patients possessing pre-existing antibodies; the changes in VLP antibody levels in the treated women were more rare and less pronounced. In the course of the observation period, seroconversion to gG HSV2 positivity was seen in 5 patients, while, a marked increase in pre-existing gG HSV2 antibodies was observed in 5 out of 7 originally seropositive patients. At enrollment, only 2 patients were free of HCMV antibody and only 1 was free of EBNA1 antibody; no seroconversion relative to either antigen was seen during the observation period.

[Papillomaviruses and human tumors]. / Vztah papillomaviru k lidským nádorum.

The report summarizes the main results obtained in the course of our research project. The results of immunological and epidemiological studies provide further proofs that human papillomaviruses (HPV) are the etiological agents in cervical neoplasia. In addition, they raise hopes that immunological methods may be utilized in diagnostics of cervical cancer and for monitoring the clinical course of this disease in the near future. Since the etiological relationship between HPV and cervical carcinoma seems to be proven beyond reasonable doubt, the development of prophylactic and therapeutic vaccines has become the dominant of the contemporary HPV reseach. For studying immune reactions against HPV-induced tumours we developed a model of HPV16-transformed rodent cells.

Secondary vaccination with vaccinia virus recombinants: role of residual virulence of recombinants and immunogenicity of extrinsic antigens.

ICR mice were immunized intraperitoneally with two doses (10(6) PFU per dose) of vaccinia virus (VV) recombinants of variable virulence expressing either the strongly immunogenic glycoprotein E (gE) of varicella zoster virus (VZV) or weakly immunogenic hepatitis B virus (HBV) preS2-S (S) antigen. Recombinants expressing gE were able to elicit primary and secondary anti-gE antibody irrespective of their residual virulence; after the second dose they did so even in the presence of VV antibody resulting from primary vaccination dose or under other conditions limiting VV replication. As for the S-recombinants, pronounced anti-S antibody development was only observed in mice which had received the more virulent recombinant virus as the first dose. A repeated dose of S-recombinants was unable to elicit a secondary anti-S antibody response. The present findings do not support the assumption that the poor immunogenicity of some extrinsic antigens could be overcome by administering repeated doses of the particular VV recombinant.

Prospective study on cervical neoplasia IV. Presence of HPV antibodies.

Sera collected in the course of a prospective study carried out in Prague in 1975-1983 were assayed for the presence of human papillomavirus (HPV) antibodies. Women with cervical neoplasia proven by biopsy at enrollment possessed antibodies to peptides derived from E2, E4 and E7 proteins of HPV16 and to virus-like particles (VLPs) of HPV16, -18 and -33 significantly more frequently than matched controls. Women without cervical neoplasia at enrollment who developed the disease in the course of the study differed from matched controls by a higher prevalence of antibodies against VLPs of HPV16 and -18 but not against early antigens of HPV16. In 19 of the latter subjects, paired serum specimens were tested, the first samples having been taken at enrollment and the second at diagnosis. Development of the disease was associated with seroconversion from negativity to positivity to at least one HPV antigen in 11 (57.9%) women.

DNA vaccine against oncogenic hamster cells transformed by HPV16 E6/E7 oncogenes and the activated ras oncogene.

The capability of DNA to elicit anti-tumour immunity was studied using human papillomavirus type 16 (HPV16)-transformed Syrian hamster cells denoted K3/II. These cells had been derived after cotransfection of primary kidney cell cultures with p16HHMo plasmid containing E6/E7 oncogenes of HPV16 and pEJ6.6 plasmid containing the activated human H-ras oncogene; they express both the HPV16 and activated H-ras genes. As a DNA vaccine, the p16HHMo plasmid was used. Three doses of the plasmid (either 100 microg or 10-15 microg per dose) were administered intramuscularly at 3-week intervals. The animals were challenged with four different doses (10(3)-10(6) per animal) of K3/II cells 10 days after the last plasmid injection. In one experiment the lower dose of plasmid DNA was also given in a mixture with the cationic lipid DOTAP. In another experiment, the pEJ6.6 plasmid (100 microg per dose) was used either alone or in combination with p16HHMo. In all experiments animals inoculated with the same doses of pBR322 plasmid served as controls. A moderate protective effect was observed in animals inoculated with the 100-microg doses of p16HHMo, but not in those inoculated with 10-15 microg of the same plasmid, whether given with or without DOTAP. A protective effect was also observed after administration of the pEJ6. 6 plasmid. At the time of challenge a portion of the p16HHMo-immunized, but not the pBR322-treated, animals possessed antibodies reactive in ELISA with peptides derived from the N-terminal portion of HPV16 E7 protein and with one peptide derived from E6 protein, while two other E6 peptides exhibited non-specific reactivity.

The frequency of HLA-DRB1 and -DQB1 alleles in cervical cancer cases in the Czech Republic.

The distribution of HLA class II DRB1 and DQB1 alleles in cervical carcinoma patients was compared with the frequency of these alleles found in healthy population living in the Czech Republic. The RFLP analysis and PCR-SSP were used for DNA typing. Although the differences in the frequency of DRB1*03, DQB1*02 and DQB1*0303 alleles between the cases and the controls were rather large, corrected P values did not reach significance.

Prevalence of antibodies to human papillomaviruses in the general population of the Czech Republic.

Sera from 450 individuals between the age of 1 and 80 years, representing the general population of the Czech Republic, were tested for the presence of antibodies to human-papillomavirus(HPV)-derived antigens. The following antigens were used: (i) HPV1 virions; (ii) HPV16, -18 and -33-virus-like particles (VLP); (iii) peptides derived from L2 open reading frames (ORFs) of HPV16 and HPV6/11; (iv) peptides derived from HPV16 E2, E4 and E7 ORFs of HPV16. The prevalence of antibodies reactive with the capsid-derived antigens was age-dependent, while no clear age dependence was observed in the distribution of antibodies to peptides derived from HPV16 early proteins. In individual sera, high correlations between the presence of antibodies reactive with the 2 L2 peptides, also between the antibodies reactive with different VLPs, were found. While the simultaneous presence of the 2 L2 antibodies was frequently detected in individual sera in all age groups, the simultaneous occurrence of VLP antibodies was detected mostly in subjects older than 20 years. There were no significant differences in HPV-antibody distribution between men and women.

Influence of the parental virus strain on the virulence and immunogenicity of recombinant vaccinia viruses expressing HBV preS2-S protein or VZV glycoprotein I.

Five triple-plaque purified vaccinia virus (VV) lines generated from smallpox Sevac VARIE vaccine (strain Praha) and three VV virus lines similarly derived from Wyeth DRYVAX vaccine were used for preparation of recombinants expressing the hepatitis B virus preS2-S gene. The same five Praha-derived virus lines were used to construct recombinants expressing the varicella-zoster virus (VZV) glycoprotein I (gpI) gene. Recombinants and their parental viruses were tested for the residual neurovirulence in mice. The virus lines and the recombinants derived therefrom differed markedly in this respect. Immunization of mice resulted in high levels of anti-HBsAg antibodies only in the case of recombinants derived from the relatively virulent viruses. In contrast, the levels of VZVgpI antibodies in mice were similar with all VV-VZV recombinants irrespective of the virulence of the parental virus line.

Induction of varicella-zoster virus-neutralizing antibodies in mice by co-infection with recombinant vaccinia viruses expressing the gH or gL gene.

Recombinant vaccinia viruses (VV) expressing the varicella-zoster virus (VZV) glycoprotein H (gH) or glycoprotein L (gL) were constructed. The 94 kDa gH intermediate glycoprotein was synthesized in cell cultures infected with the VV-gH recombinant, but only co-infection with both recombinants resulted in the synthesis of the fully processed 118 kDa gH molecule. The VV-expressed gH and gL formed a complex that displayed the conformational neutralization epitope detectable by means of human VZV gH-specific monoclonal antibody V3. Formation of this epitope was inhibited by tunicamycin but not by monensin. Simultaneous intraperitoneal inoculation of mice with high doses of both VV-gH and VV-gL viruses resulted in the development of VZV-neutralizing, complement-independent antibodies; these antibodies were not detected in mice infected solely with either the VV-gH or the VV-gL recombinant.