An easy-to-prepare, highly efficient antigen for cervical cancer serology.
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Antibodies, Neoplasm↗
Biomedical subjects
V Vonka
Publications and source records attributed to V Vonka.
Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger.
Using Escherichia coli system expressing papilloma virus HPV16 E7MS2 fusion protein as a model system, a novel procedure was applied to solubilize, purify and refold recombinant proteins from E. coli inclusion bodies. The necessity to reactivate proteins at low protein concentrations (owing to their tendency to aggregate at high concentrations) was overcome by solubilization of inclusion bodies in alkaline solution and immobilization of proteins on a strong and resistant anion exchanger. This procedure has an inherent advantage of combining refolding and purification procedures in one step. The solubilization of the fusion protein in an alkaline reagent with the use of an anion exchanger resulted in considerable purification of the recombinant protein at a fairly high concentration. The protein was soluble under mild conditions and reacted with antibodies against the "native" papilloma virus.
Anion Exchange Resins↗
Production and simple purification of a protein encoded by part of the gag gene of HIV-1 in the Escherichia coli HB101F+ expression system inducible by lactose and isopropyl-beta-D-thiogalactopyranoside.
The development of the Escherichia coli expression system, which was prepared by transferring the F' episome from strain 71/18 to a highly to a transformable F- strain HB101, is described. These new HB101 (F+) cells, which produced high levels of lac repressor, were capable of taking up lactose and grew under strict selection conditions. A relatively simple two-step purification of part of a protein (M(r) 27,000) encoded by the gag gene of HIV-1 in this expression system is described. The supernatant prepared by removal of cell debris was precipitated by 30% saturation of ammonium sulphate. The protein spectrum was characterized by gel electrophoresis, immunoblotting and ion-exchange titration curves. Optimum separation was achieved using a strong anion exchanger (Mono Q) at pH 8.0. The purified protein did not cross-react with antibodies to E. coli.
Antibodies, Bacterial↗
Antibody response to a synthetic peptide derived from the human papillomavirus type 6/11 L2 protein in recurrent respiratory papillomatosis: correlation between Southern blot hybridization, polymerase chain reaction, and serology.
Recurrent respiratory papillomatosis (RRP) is the most common benign tumour of the larynx, affecting both children and adults. We present a series of 25 patients, including 10 cases of juvenile multiple, 8 cases of adult solitary, and 7 cases of adult multiple RRP. Biopsy tissue from each patient was screened by Southern blot hybridization and polymerase chain reaction for the presence of human papillomavirus (HPV) DNA. Sera from patients and age- and sex-matched controls were tested for the presence of HPV-specific antibodies using a synthetic peptide derived from the minor capsid protein (L2) of HPV 6/11. By Southern blot hybridization and/or polymerase chain reaction, biopsies from all patients were positive for HPV 6/11 DNA. There was no difference in antibody response between cases and controls. Female cases and controls had significantly higher antibody titers than male subjects. A correlation was observed between the HPV-specific antibody level and the number of surgery-necessitating recurrences.
Adolescent↗
Comparative sensitivity of the immunoelectronmicroscopy test and ELISA with intact virions in detecting HPV-1 antibodies and age distribution of these antibodies among general population and in children's homes.
A high degree of concordance between the results of immunoelectronmicroscopy and ELISA tests with purified human papillomavirus (HPV-1) particles was observed. The prevalence of HPV-1 antibodies in children living in children's homes was higher than in the general population.
Adolescent↗
Presence of antibodies to seven human papillomavirus type 16-derived peptides in cervical cancer patients and healthy controls.
Sera from 133 cervical cancer patients and 154 healthy women (controls) from Spain and Colombia were tested in IgG-specific ELISAs for the presence of antibodies against seven peptides derived from five open-reading frames of human papillomavirus (HPV) type 16. Three of the peptides corresponded to overlapping regions of the N-terminal half of E7 protein; the other peptides corresponded to selected regions of E2, E4, L1, and L2 proteins. The prevalence of antibodies against E2 and E7 peptides was significantly different between patients and controls. The most marked differences were for E7 peptides. HPV DNA polymerase chain reaction diagnoses of cervical scrapes were available; these were correlated with serologic findings. In HPV-16 DNA-positive patients, E7 antibodies were more broadly and more strongly reactive than in other patient groups.
Age Factors↗
[Correlation of clinical and virologic diagnosis of cervical infection with human papillomaviruses].
The presented investigation is concerned with contemporary diagnostic possibilities of HPV Infection of the Cervix. The authors present the results of virological examinations of 228 female patients in the Centre for Oncological Prevention. The examination was made by hybridization techniques, using probes specific for HPV 6, 11, 16 and 18 and by serological methods where IgG antibodies were assessed against synthetic peptides, corresponding to several HPV epitopes, as antigens. 156 women (68.4%) were virologically positive, 72 (31.6%) were negative. Subsequently the authors investigated the diagnostic accuracy of HPV changes of the cervix by clinical methods, i.e. colposcopy and cytology, as compared with virological methods. On colposcopic examination uncertain--i.e. insignificant--results were recorded in 24.6%, on cytological examination in 19.7%. In patients where these methods gave unequivocal results (either + or-) a correct forecast of the presence of HPV during colposcopic examination was recorded in 71.1%, in cytological examinations in 66.9%. At least one of the clinical methods assessing papilloma virus infection was prognostically correct in 90.4%. From the investigation ensures that prebioptic methods provide the clinician with relatively reliable information on the presence of HPV infection and enable him to select a therapeutic and dispensarization procedure adequate to the finding. However, they cannot replace virological examination among other reasons also because they cannot assess the HPV type.
Colposcopy↗
Identification of seroreactive epitopes of human papillomavirus type 18 E7 protein by synthetic peptides.
Nine everlapping peptides covering the entire sequence of early protein E7 of human papillomavirus type 18 (HPV-18) were synthesized and tested as antigens with pools of selected human sera in ELISA. Peptides denoted 18/E7-2, 18/E7-3, and 18/E7-5 (amino acid positions 11-33, 21-40, and 41-60, respectively) were reactive with pooled sera originating from HPV-18 DNA-positive cervical cancer patients but not with sera from HPV-16 DNA-positive cervical cancer patients or from condyloma acuminata patients. This suggested that the epitopes contained in these peptides were HPV-18 type-specific, relative to HPV types 16, 11, and 6. On the other hand, 18/E7-1 (aa 1-23) and 18/E7-6 (aa 51-70) peptides were cross-reactive. The prevalence of antibodies reactive with 18/E7-2, 18/E7-3, and 18/E7-5 peptides in cervical carcinoma patients was very low. Thus, the utilization of these peptides for monitoring HPV-18 infection seems to be rather limited.
Amino Acid Sequence↗
[New methods of isolating recombinant proteins].
Recombinant proteins are isolated from very complex protein mixtures present in the producing cell. The isolation process involves in general four mutually interconnected stages: 1. release of the recombinant protein from the cellular environment, 2. preparation of the specimen for separation, 3. separation, 4. qualitative and quantitative analysis of the preparation. Each of these stages is formed by a complex series of methods which destroy the cellular wall, solubilize the specimen and involve the use of suitable precipitation, chromatographic, electrophoretic, immunochemical and other techniques for separation and analysis. In the submitted paper the authors describe a relatively simple isolation of recombinant peptide used for the preparation of a diagnostic kit for AIDS. From knowledge of the sequence of nucleotides in cDNA antisense peptides can be derived which have a high affinity with the isolated protein and they can be also used for affinity chromatography. An effective isolation technique is the use of mimetic ligands on the basis of textile dyes. By their combination and possible modification it is also possible to achieve separation of the required protein from contaminating substances. The mentioned highly specific methods can be combined with classical chromatographic techniques. During every step individual chromatographic fractions are tested by SDS electrophoresis in polyacrylamide gel incl. possible use of immunoblotting a specific staining.
Escherichia coli↗
[Problems associated with the development of vaccines based on recombinant vaccinia virus].
A collection of single and double vaccinia virus recombinants was prepared. The recombinants contained either genes for different forms of surface protein or core protein of HBV or the gene for glycoprotein I of varicella-zoster virus. Cells infected with the recombinants produced the respective foreign antigens. Specific antibodies against the heterologous antigens were induced in mice immunized with the recombinants. The insertion of the second foreign gene into the genome of single recombinants did not influence either the extent of production of the first protein in vitro or its immunogenicity for mice.
Animals↗
Range of HPV 16 E7 antibodies in cervical cancer patients and healthy subjects.
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Antibodies, Viral↗
Epitope mapping of the human papillomavirus type 16 E4 protein by means of synthetic peptides.
Eight overlapping icosapeptides covering the entire sequence of the E4 protein of human papillomavirus type 16 (HPV-16), were prepared and tested for their reactivity with human sera in IgG-specific ELISA. The strongest reactivity of sera from HPV-16 DNA-positive invasive cervical carcinoma (INCA) patients was detected with the peptide denoted 16/E4-6, covering amino acids 51 to 70. Subsequently nearly 200 sera were tested for the presence of the 16/E4-6-specific antibody. Reactivity was more frequent in cervical intraepithelial neoplasia patients and INCA patients than in matched control subjects. Sera from INCA patients were also tested for antibody reactive with peptide 16/E7-2 covering the major type-specific reactive region of the HPV-16 E7 protein. Only four of 13 sera possessing the 16/E4-6-specific antibody were reactive with the 16/E7-2 peptide.
Adult↗
Synthesis and immunogenicity of hepatitis B virus envelope antigen expressed by recombinant vaccinia virus. Finding of retention signal in the C-terminal portion of the preS1 domain of subtype adyw.
Five different recombinant vaccinia viruses expressing the envelope antigen of hepatitis B virus (HBsAg) under the control of the P7.5 promoter were constructed. Cell cultures infected with some of the recombinant viruses synthesized both middle (M) and major surface (S) protein of HBsAg. It was shown that the length of the nontranslated sequence preceding preS2-ATG influenced the extracellular or intracellular HBV antigen distribution and the preS2:S antigen ratio. Some recombinants synthesized an M protein that was enlarged by additional 35 amino acids of preS1 domain and was entirely retained within the infected cells. Antibody responses to the S and preS2 antigens in mice revealed significant differences in the immunogenicity of individual recombinants.
Animals↗
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.
Adult↗
Immunogenicity in mice of varicella-zoster virus glycoprotein I expressed by a vaccinia virus-varicella-zoster virus recombinant.
Several vaccinia virus (VV)-varicella-zoster virus (VZV) recombinants expressing glycoprotein I (gpI) of VZV were isolated from the Prague strain of VV. One of these, v46, was inoculated intraperitoneally into mice. Groups of mice were bled 4 and 8 weeks later and their sera were examined for anti-VZV and anti-VV antibodies by ELISA. At 4 weeks, all mice inoculated with the three largest virus doses (10(7), 10(6) and 10(5) p.f.u.), and at 8 weeks all mice inoculated with the four highest virus doses (10(7), 10(6), 10(5), and 10(4) p.f.u.), had developed both anti-VV and anti-VZV antibodies. Antibodies were also detected in a high proportion of mice infected with lower doses of virus and in some instances VZV antibodies were present in the absence of VV antibodies. None of the animals inoculated in parallel with either a thymidine kinase-negative mutant of the original VV or diluent alone developed antibody reactive with VZV. The specificity of the reaction was assessed further by Western blotting using anti-gpI monoclonal antibodies as a positive control. Sera from animals immunized with v46 possessed antibody capable of neutralizing extracellular VZV in the presence of complement.
Animals↗
Selection of L cell sublines resistant to (E)-5-(2-bromovinyl)-2'-deoxyuridine.
A mouse thymidine kinase (TK) deficient L cell subline and L cell sublines biochemically transformed by herpes simplex virus TK were cultured in the presence of increasing concentrations of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) which inhibited the growth of all sublines, and resistant sublines were isolated. Their growth properties were dependent on the medium condition used for selection. One subline had lost viral TK activity, while another retained viral TK activity with altered sensitivity of TK to BVDU in comparison with that of the original subline. Growth characteristics and TK activity of sublines are discussed.
Animals↗
Priming effect of recombinant vaccinia virus coding for the middle hepatitis B surface antigen.
Recombinant vaccinia virus expressing the middle hepatitis B virus surface antigen was incapable of inducing marked antibody response against the S and pre-S2 antigenic specifities in mice. However, mice immunized with this virus produced antibodies to both these antigens after the following administration of subtreshold doses of plasmatic hepatitis B surface antigen.
Animals↗
Properties of a 9-(2-phosphonylmethoxyethyl)adenine (PMEA)-resistant herpes simplex virus type 1 virus mutant.
After repeated passages of herpes simplex type 1 (HSV-1) KOS virus in the presence of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) a mutant denoted PMEAr HSV-1 was isolated which grew well in the presence of 50-100 micrograms.ml-1 of the drug. PMEAr HSV-1 was still sensitive to the related phosphonate analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA). In fact, it was more susceptible to the action of HPMPA than the original virus. PMEAr HSV-1 also retained sensitivity to 5-bromo-2'-deoxyuridine and other, viral thymidine kinase-dependent substances such as (E)-5-(2-bromovinyl)-2'-deoxyuridine. However, PMEAr HSV-1 was much less sensitive to acyclovir, 1-(beta-D-arabinofuranosyl)cytosine and 1-(beta-D-arabinofuranosyl)thymine than the parental KOS virus.
Acyclovir↗