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For investigation of proteins possessing assigned immunological properties, plasmids pPS31-42, pPS1-5, pPS2-17, and pPS1P-30 were constructed encoding the hepatitis B core protein (HBcAg) with N-terminally inserted immunodominant epitopes of preS regions (amino acids 31-36 or 94-105 of preS1, or 133-143 of preS2). Analysis of the hybrid proteins with the use of ELISA and immunoelectron microscopy showed that the insertions did not prevent specific aggregation of the protein molecules, the inserted sequences being exposed on the surface of the particles obtained, and both HBcAg and the corresponding preS determinants were antigenically active.

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A set of plasmids was constructed determining synthesis of hybrid proteins with insertions of antigenic determinants of preS1 and preS2 regions of HBV in the middle part of HBcAg. The polypeptides containing the 31-36 or monomeric 94-105 preS1 epitopes were water-soluble and formed particles similar to the 27-nm ones of native HBcAg, possessing antigenic properties of both HBcAg and the inserted epitope, while the hybrids containing 133-143 of preS2 or a trimeric form of the 94-105 preS1 epitope became membrane-bound. Another hybrid encoded by plasmid pPS2M31 contained two regions of HBcAg modification: insertion of amino acids 133-143 a.a. (preS2 region) in the N-terminal part and 31-36 (preS1) in the middle part of the carrier. The immunogenicity of the epitope inserted into the middle part of the HBcAg molecule was an order of magnitude higher than that of the same epitope in the N-part of the protein; the fact might be important for constructing artificial immunogens.

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The plasmids have been constructed permitting expression of hepatitis B viral polymerase and its functional domains, the catalytic and end ones, as hybrid proteins containing 12 alien amino acids in the N-terminal. Immunoblotting with the rabbit antisera to N- and C-terminals of hepatitis B polymerase amino acid sequence has demonstrated that constructed plasmids determine the synthesis of the corresponding proteins in bacterial cells. HBV polymerase is processed in Escherichia coli cells. The successful expression of HBV polymerase and its functional domains makes possible the detailed study of this protein structure and function.

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Hepatitis delta virus (HDV), a recently discovered infectious agent, participates in severe, often lethal forms of acute and chronic hepatitis and liver cirrhosis. Based on theoretical analysis of secondary structure, hydrophilicity and acrophilicity data, several regions of HDV antigen, presumably containing B-epitopes, have been revealed and the corresponding peptides have been synthesized by the solid phase method. All the peptides obtained reacted with the respective antipeptide rabbit sera. The peptides and their conjugates with BSA or KLH were used for ELISA with individual and pooled anti-HD-positive sera from patients with chronic delta hepatitis. The high antigenicity of the peptide 65-80 shows that one of the antigenically active regions of HDAg is situated between these amino acid residues and that the peptide may be used for detection of anti-HD antibodies in patients blood sera.

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We synthesized the 24-41, 30-36, 31-36, 24-30 fragments of the preS1-region of the hepatitis B (subtype ayw) envelope. The peptides were prepared by the solid phase synthesis on perfluorpolyethylene polymer grafted with polystyrene. The peptide chains were elongated from C-terminus using activated esters and symmetrical anhydrides of Boc-amino acids, cleaved off the solid phase by HBr or TFMSA in TFA, purified by gel filtration, and, after conjugation with protein carriers, inoculated into test animals. The resultant antibodies were shown to react with peptides. The blood sera from patients with acute hepatitis B reacted with the conjugates of peptides 24-41, 30-36, 31-36 in the immunoenzymic solid phase assay. The monoclonal antibodies for the preS1-polypeptide were shown to react with peptides 24-41, 30-36, 31-36 and with their conjugates. The results obtained were proved by the data of the epitope-mapping with overlapping hexapeptides.

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Polyclonal anti-idiotypic antibodies (anti-Id-AT or AT2) were produced to AT idiotopes of virion antigens--structural proteins of herpes simplex virus (HSV) and of early virus-induced antigens (VIA). The method of SPRIA established that anti-Id-AT seemed to be associated with AT active centers. Besides, immunization with anti-Id-AT produced anti-anti-Id-AT (AT3) similar in their specificity with antiviral AT or anti-VIA. It was shown that AT2 could be used as a diagnostic preparation instead of specific antigen for the detection of antibody to HSV or VIA in human sera by indirect RIA.

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Immunological methods (complement fixation test, immunofluorescence) demonstrated that the antigen associated with cytomegalovirus can be detected in some malignant and benign tumors and lymphoid cells in human leukemia. No antigen of the same specificity was found in tissues from the same patients and in normal donor lymphocytes.

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It is very difficult to produce specific antibody to individual antigens comprising the multicomponent antigenic system of a cell. It is particularly true of the antigens present in the cell in small amounts and characterized by low immunogenic potency, among them early antigens of herpes simplex virus (HSV). To overcome these difficulties the authors propose a method of preparation of a specific serum using specific immune complexes as immunogens. The use of the radioimmune binding method and a new specific serum to early HSV antigen has proved that HSV could be associated not only with cervical carcinoma, as believed by many research workers, but also with tumors of other localization, in particular, lung carcinoma and malignant melanomas.

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Leukocytes of 19 leukemic patients and 10 healthy donors were tested for the presence of Thomsen's antigen, using a method of specific adsorption of isogenic sera. Before the adsorption the cells were sonicated. Thomsen's antigen was found in leukocytes of leukemic patients but not in cells of healthy donors.

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Immunological methods (CFT, indirect IF) detected herpes simplex virus-associated antigen in leukocytes of 21 out of 56 leukemia patients. The antigen was more frequently found in leukocytes of patients with chronic lympho- and myeloleukemias, less frequently in acute forms of these diseases. No antigen was detectable in leukocytes of normal donors.

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Solid-phase radioimmunoassay (SPRIA) was used for examinations of human malignant tumors for the presence of retrovirus D-induced antigen and of blood sera from cancer patients for the presence of antibody to this antigen. Various tumor specimens differently reacted to anti-HEp-2 serum, the background values of binding of this serum with normal tissues (liver, lung, spleen) and leukemic cells from leukemia patients being low. The majority of the sera from cancer patients were found to contain antibody to HEp-2 antigen. No correlation was established between high levels of the antigen in tumors and high antibody titres in the blood sera of the same patients.

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Immunological methods of investigation (CFT, immunofluorescence and radioimmunoassay) showed that the antigen induced by retrovirus D was discovered in some malignant and benign human tumors of various localizations. No association of retrovirus D with human hemoblastoses could be established.

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A method of specific adsorption of appropriate antibodies from human serum was used to examine tumors of 15 patients with cancer and of 6 subjects with benign neoplasms. Tissues of cancerous tumors were found to contain Thomsen's antigen. Normal tissues of the same organ taken from the same patient did not contain the antigen nor was it contained by benign tumors. The titer of specific antibodies to Thomsen's antigen in the sera of patients suffering from cancer was considerably reduced as compared to that detected in normal subjects or in patients with benign tumors.

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The role of intracellular DNA from HEp-2 cell culture chronically infected with retrovirus D in generation of new non-virion antigen associated with this virus was studied. In transfection experiments, 23 DNA samples obtained from cells of variously localized human tumors were studied for the capacity to induce a new antigen of the same specificity. The DNA obtained from HEp-2 cells was found to be capable of inducing the new antigen production in a number of cells systems. The original cells contained no such antigen before inoculation. DNAs from 14 cancer tumors of the mammary glands, lungs, and gastrointestinal tract induced the synthesis of the new antigen in vitro and other cells indicating the presence of these tumor cells of at least a portion of viral genome responsible for the synthesis of the new retrovirus D-associated antigen. No production of this antigen was induced by any of the 6 specimens of normal tissues.

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The complement fixation test was used to examine 80 human mammary tumors for the presence of heterogeneous Forssman antigen: 62 cases of cancers of different malignancy (adenocarcinoma, solid tumors), 13 cases of fibroadenomas and 5 cases of fibrocystic mastopathies. Subject to study was also normal tissue of the mammary gland. Forssman antigen was not found in all the tumors tested, as it was not detected in tissues of the normal mammary gland. Therefore the process of transformation of normal cells to tumor ones is not accompanied by the occurrence in them of heterogenous Forssman antigen.

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The study showed the new antigen induced by herpes simplex virus type 2 (HSV-2) to be present not only in cancer tumors of the cervix and corpus uteri, and ovaries but also in malignant tumors of the mammary glands, kidneys, urinary bladder, as well as in tissues of fibrous-cystic mastopathy and fibroadenoma of the mammary glands. In malignant tumors of the cervix, however, the HSV-2-induced antigen was found more frequently and had a higher serological activity than in other tumors. In contrast to tumors, this antigen was not detected in normal tissues. Among 25 specimens of malignant cervical tumors containing the virus-induced antigen, 8 specimens contained virus antigen and 4 yielded biologically active virus. The virus-induced antigen is found in the tumors much more frequently then viral antigen or virus. Detection in the tumors of this new antigen demonstrates the presence in them of viral genetic information coding for the synthesis of this antigen as confirmed by transfection experiments.

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Tissues of malignant and benign tumors from 254 patients were examined for the presence in them of a new, retrovirus D-associated antigen. Most frequently, the virus-induced antigen was present in pulmonary carcinomas (54.5%) and in large intestine tumors (25%). This antigen was detected almost with the same frequency (11%-12.5%) in fibroadenomas and mammary carcinomas, only in 8% of gastric tumors and very rarely in fibrous-cystic mastopathies (2.6%). No virus-induced antigen was found in tissues not involved in the cancer process or in tissues of normal subjects. Detection of the retrovirus D-associated antigen in tumor cells indicates the presence in them of this virus or its genome.

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The additional evidence of the existence of antigenic differences between the early virus-induced antigen and structural retrovirus D antigens as well as antigens from human fetus normal tissues was obtained by quantitative complement fixation micromethod using radioactive chromium (51Cr-CFT). In examinations of 13 specimens of cancer tumours and fibroadenomas of the female mammary glands the new antigen was found in 8 cases, including 4 cancer tumours where this antigen could not be detected by the conventional CFT.

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Further study of a new oncornavirus D-induced antigen was done. Its immunological specificity and localization in cells was determined by a sensitive method of mixed hemadsorption. A continuous HEp-2 cell culture was used as the source of the new as well as virus antigen. Adsorbed specific sera to the new antigen and to oncornavirus D obtained by the previously developed method were used for the antigens detection. This method demonstrated localization of both virus antigens and oncornavirus D-induced antigens not included into the virion structure on the surface of oncornavirus D-infected cells. Mixed hemadsorption test as well as complement-fixation test gave the results identical in their specificity indicating the presence in HEp-2 cells of a new non-virion antigen.

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