Complement-Fixing Antigens in Burkitt Lymphoma Cell Lines

The complement-fixing (CF) activity of antigens from cultured Burkitt lymphoma cells was determined by using normal American sera as the source of antibody. Approximately 75% of the sera fixed complement with the positive cell lines. These lines contained the herpes-like virus detectable by electron microscopy. The content of CF antigen depended on the cell line used but appeared to be independent of the number of cells which produced Henle's immunofluorescence (IF) antigen. Only sera that reacted in the IF test also contained CF antibodies to the crude cell extracts.     

Detection and differentiation of antibodies to human T-cell lymphotropic virus types I and II by the immunofluorescence method.

We compared the sensitivities of the prototype human T-cell lymphotropic virus type I (HTLV-I)- and HTLV-II-transformed cell lines, MT2 and Mo-T, with that of an HTLV-II-infected cell line, clone 19, established in our laboratory, in the immunofluorescence (IF) test for detection of antibody to HTLV-I and HTLV-II. In addition, IF antibody titers with the three antigens were determined, and the results were compared with HTLV-I and HTLV-II typing by polymerase chain reaction (PCR). The MT2 cell line was more sensitive than the two HTLV-II cell lines for detecting HTLV-I antibody by IF, and clone 19 was more sensitive than Mo-T or MT2 for measuring HTLV-II antibody. In the titration study, the antigen that gave the highest titer correlated completely with the HTLV type determined by PCR, indicating that the relatively simple IF titration method can be used for differentiating HTLV-I and HTLV-II antibody in sera and plasmas.     

Expression of Epstein-Barr virus nuclear antigens 3, 4, and 6 are altered in cell lines containing B-type virus

A high proportion of HIV-positive sera were found to react with 130- and 180-kDa antigens which were present in the Jijoye cell line. The majority of the HIV-positive sera which detected these antigens also contained antibodies to Epstein-Barr virus (EBV) nuclear antigen 2B (EBNA2B) suggesting a relationship between B-type EBV strains and the expression of the 130K/180K antigens. Cell lines were established by infection of B lymphocytes with different A- and B-type strains of EBV. Incubation of these lines with sera from individuals infected with either A-type or B-type EBV strains demonstrated that the 130K and 180K antigens were only expressed by cell lines containing B-type virus. Sera from individuals infected with A-type EBV did not react with the 180K antigen in any cell lines but could detect EBNAs 3, 4, and 6 antigens in the A-type cell lines. Restriction enzyme analysis of the BamHI E region of the EBV genome revealed marked differences between the A and B types of the virus. These results demonstrate that expression of antigens encoded from the BamHI E region of EBV (EBNAs 3, 4, and 6) are altered in cell lines transformed by B-type strains of EBV.     

Mode of integration of Epstein-Barr virus genome into host DNA in Burkitt lymphoma cells.

The ebv dna integrated into the host genome from cell of an African Burkitt lymphoma biopsy has been compared to the corresponding fraction of EBV DNA from the cell line SU-AmB-2 which is of American Burkitt lymphoma origin. It is shown that while, in the case of the African biopsy, large pieces of EBV DNA sequences are integrated into the host DNA, much smaller pieces of virus DNA are integrated into the DNA of the SU-AmB-2 cells. The possibility that this difference might be related to the fact that EBV is rarely associated with Burkitt lymphomas outside East Africa is discussed.     

A comparison of human papovavirus T antigens.

A comparison was made of the T antigens induced in transformed cells or infected permissive cells by representatives of three categories of human papovavirus. The transformed hamster cell lines employed contained T antigen induced by either the BK or RF strains of papovavirus associated with human renal allografts; the JC strain of papovavirus from progressive multifocal leukoencephalopathy (PML), or a variant of SV40 virus isolated from PML. The human papovavirus T antigens were also compared with that of a human cell line transformed by SV40 of simian origin. Anti-T antibody prepared in hamsters against each of the hamster cell lines was absorbed with crude T antigen from each cell line, and the unabsorbed and absorbed antisera were tested for residual T antibody against each cell line, or against infected permissive cells by immunoperoxidase (IP) staining and complement-fixation (CF) tests. In unabsorbed antisera, T antibodies from each cell line cross-reacted with all T antigens in IP tests, and CF tests showed that T antisera reacted preferentially with T antigen induced by homologous virus. Absorpminants. T antigens of the two urine-derived strains, BK and RF, were identical or nearly so, but were clearly separable from T antigens of JC virus, PML-derived SV40 or simian-derived SV40. JC T antigen was intermediate, being more closely related to T antigens both of BK virus and SV40 virus than the latter were to each other. The T antigen of PML-derived SV40 could be distinguished from the T antigen of simian-derived SV40 and the T antigen of the SV40 variant from human brain was more closely related to those of the other human-derived papovaviruses than was the T antigen of SV40 from monkey kidney.     

Neutralization of lymphocyte immortalization by different strains of Epstein-Barr virus with a murine monoclonal antibody.

A murine monoclonal antibody was raised against the B95-8 strain of Epstein-Barr virus (EBV), which was isolated from a case of mononucleosis after blood transfusion (Hoffman et al. Proc. Natl. Acad. Sci. U.S.A. 77:2979, 1980). We provide evidence that neutralization of immortalization by this monoclonal antibody is virus specific, since its potency was inversely related to the dose of challenge virus. Furthermore, the monoclonal antibody recognized antigens on viruses grown in human as well as in marmoset cells. We show that this monoclonal antibody neutralized three other transforming strains of EBV originating, respectively, from American patients with mononucleosis and fatal polyclonal lymphoma and from an African child with Burkitt lymphoma. However the antibody did not neutralize or detect antigens by immunofluorescence in the W91 strain of EBV. The hybridoma antibody did neutralize other EBV strains derived from the same Burkitt lymphoma cell line (Nyevu), as was the case with the W91 strain. This monoclonal antibody provides clear evidence of antigenic differences on the surface of EBVs and will ultimately prove useful in defining the antigenic site on EBV which elicits neutralizing antibody.     

Epstein–Barr virus-associated antibody patterns in carcinoma of the post-nasal space

Sera from African patients with tumours of the post-nasal space, Hong Kong Chinese patients with carcinomas of the post-nasal space and from Indian donors with buccal, oro- and hypopharyngeal carcinomas, as well as African control sera from healthy persons and individuals with various non-neoplastic and neoplastic diseases were tested for anti-Epstein–Barr virus titres and for antibodies capable of blocking the direct membrane immunofluorescence reaction obtained between Epstein-Barr virus carrying lymphoblastoid cell lines and two different fluorescein conjugated reference sera; one from a patient with Burkitt lymphoma (F-Mutua conjugate) and the other from a patient with carcinoma of the post-nasal space (F-Kipkoech conjugate). Ninety per cent or more of the African and Chinese patients with carcinoma of the post-nasal space gave at least one high test, and 60% were high in all three assays. In contrast, African patients with post-nasal space tumours other than carcinomas, African controls and Indian buccal, oro- and hypopharyngeal carcinomas gave high reactions in all three assays in less than 20%.     

Immunoelectron microscopic study on the cross-reactivity of antibodies to adult T-cell leukemia-associated antigens in human and monkey sera

The cross-reactivity of antibodies to adult T-cell leukemia (ATL)-associated antigens (ATLA) in human and monkey sera was investigated by indirect immunoperoxidase and immunoferritin methods using a human cell line (MT-2) carrying a type C virus (HTLV), two monkey cell lines (Si-1 and Si-3) carrying HTLV, and a monkey cell line (Si-2) carrying a type C virus isolated from an anti-ATLA-positive monkey. Anti-ATLA-positive but not-negative human and monkey sera gave positive immunoperoxidase reaction with all four virus-positive cell lines when studied by light microscopy. Electron microscopic findings revealed ferritin or peroxidase labeling of virus particles and plasma membranes of these four cell lines with antibody-positive but not-negative human and monkey sera. These results clearly indicate the cross-reactivity of anti-ATLA antibodies in human and monkey sera at light and electron microscopic levels, and the presence of antigenic determinants common to the surface of type C virus particles of human and monkey origin.     

Discovery of the sequences of herpes simplex virus type 1 in the genomes of normal and transformed mouse and hamster cell lines

Sequences capable of hybridization with cloned fragments of herpes simplex virus type I (HSVI) DNA were found in genome DNA of Syrian hamster fibroblast lines transformed either by intact HSVI genome (cell line 14.012.81) or this virus DNA fragments (cell line EH/A44). The method of pinpoint hybridization demonstrated the presence in DNA of the cell lines under study of sequences capable of hybridization both with unique areas and with HSVI genome replicas. A correlation was established between the tumorigenic properties of the transformed lines and the number of HSVI sequences present in genomes of these lines. The genome of mouse cell line BaLB/3T3 was found to contain sequences homologous to HSVI replica areas, their number exceeding that of virus sequences present in the genome of nontransformed hamster cells (BHK/21 line).     

Immunocytochemical and ultrastructural characterization of human T-lymphotropic virus type I (HTLV-I)-producing rabbit lymphoid cell lines

Summary Fine structural and immunocytochemical characterization of rabbit lymphoid cell lines transformed by human T-lymphotropic virus type I (HTLV-I) was carried out. All nine cell lines tested were reactive with anti-HTLV-I-positive human, monkey, and rabbit sera and monoclonal antibody to HTLV-Ip 19, but not with anti-HTLV-I-negative sera and monoclonal antibodies to human Ia and pan-T antigens. All cell lines were strongly positive for monoclonal antibodies to rabbit Ia and pan-T antigens. Ultrastructurally, each cell line contained C-type virus particles in varying numbers in the extracellular space. These particles showed replication patterns similar to those in HTLV-I or simian T-lymphotropic virus type I (STLV-I)-producing human or monkey cells. In addition, anti-HTLV-I-positive rabbit serum gave positive immunoreactivity to HTLV-I or STLV-I by indirect immunoferritin method.These results indicate that the ultramorphology and replication patterns of HTLV-I in rabbit cell lines are indistinguishable from those of HTLV-I in human and monkey cell lines, HTLV-I in rabbit cells shares the common surface antigenic determinants with HTLV-I or STLV-I in human or monkey cells, and that these cells are definitely rabbit T cells bearing their own Ia antigens.     

IMMUNOELECTRON MICROSCOPIC STUDY ON THE CROSS-REACTIVITY OF ANTIBODIES TO ADULT T-CELL LEUKEMIA-ASSOCIATED ANTIGENS IN HUMAN and MONKEY SERA

The cross-reactivity of antibodies to adult T-cell leukemia (ATL)-associated antigens (ATLA) in human and monkey sera was investigated by indirect immunoperoxidase and immunoferritin methods using a human cell line (MT-2) carrying a type C virus (HTLV), two monkey cell lines (Si-1 and Si-3) carrying HTLV, and a monkey cell line (Si-2) carrying a type C virus isolated from an anti-ATLA-positive monkey. Anti-ATLA-positive but not-negative human and monkey sera gave positive immunoperoxidase reaction with all four virus-positive cell lines when studied by light microscopy. Electron microscopic findings revealed ferritin or peroxidase labeling of virus particles and plasma membranes of these four cell lines with antibody-positive but not-negative human and monkey sera. These results clearly indicate the cross-reactivity of anti-ATLA antibodies in human and monkey sera at light and electron microscopic levels, and the presence of antigenic determinants common to the surface of type C virus particles of human and monkey origin. ACTA PATHOL. JPN. 35: 863–870, 1985     

Studies on the defectiveness of adeno-associated virus (AAV). II. Effect of growth in CV-1 cells on the replication of adeno-associated virus.

The mechanisms by which helper viruses can promote AAV macromolecular synthesis have been investigated. The third system used was a continuous line of African green monkey kidney cells (CV-1) which is permissive for AAV when a simian adenovirus helper is used. When HSV was used as a helper, no AAV DNA, structural proteins, or IF antigens were detected, indicating the absence of HSV helper activity. HSV production and plaqueing in CV-1 cells were found to be comparable to those seen in Hep-2 cells (an epidermoid cancer line) and Vero cells (another line of African green monkey kidney cells). In both of these cell lines, HSV expresses its AAV helper functions. It was also observed that AAV had no effect on HSV replication in either Vero or CV-1 cells. Comparison of HSV protein production in CV-1 and Hep-2 cells demonstrated the absence of HSV proteins ICPO and ICP46 in the CV-1 line following the release of a cycloheximide block.     

Analysis of restriction endonuclease fragments of intracellular Epstein-Barr virus DNA and isoenzymes indicate a common origin of the Raji, NC-37, and F-265 human lymphoid cell lines

Circular Epstein-Barr virus DNA molecules isolated from different virus-transformed cell lines usually show similar but distinct restriction enzyme cleavage patterns. It is shown here that EBV DNA from the cell line Raji, which is of African Burkitt lymphoma origin, has the same cleavage pattern on digestion with either EcoRI, HindIII, or BamHI as EBV DNA from two more recently established human lymphoid cell lines, NC-37 and F-265, which are presumed to be of nontumor origin. On the other hand, these three EBV DNA isolates are clearly different from DNA of the standard B95-8 strain of the virus as judged by the same criteria. A restriction enzyme cleavage map of the circular EBV DNA from Raji cells has been made to provide a basis for the comparison. The isoenzyme patterns of 14 different marker enzymes were also investigated for the Raji, NC-37, and F-265 lines and found to be closely similar. These data indicate that the NC-37 and F-265 lines are not independent isolates of human lymphoid cell lines.     

Complement-fixing reactivity of Varicella-Zoster virus subunit antigens with sera from homotypic infections and heterotypic Herpes simplex virus infections.

Various subunit antigens of varicella-zoster (V-Z) virus were examined for complement-fixing (CF) activity with sera from homotypic infections and from herpes simplex virus (HSV) infections in which a CF antibody titer rise was demonstrated with crude V-Z antigen. The subunit antigens included nucleocapsids, envelopes, a soluble antigen produced from infected culture fluids by sucrose density gradient centrifugation, a soluble antigen produced by reducing the volume of clarified infected culture fluids, a soluble antigen derived from infected cell lysates, a "viral" antigen consisting largely of enveloped particles with a few nucleocapsids, and a cell membrane-associated antigen. None was more suitable than crude V-Z antigen for serodifferentiation of V-Z virus and HSV infections. The envelope antigen, cell membrane antigen, and the soluble antigen prepared by density gradient centrifugation showed little reactivity with sera from varicella and HSV infections, but gave high antibody titers with sera from zoster infections, suggesting that a secondary V-Z virus infection is required to produce an antibody response to these subunit antigens. Patients with varicella and zoster infections and the selected patients with HSV infections all showed significant CF antibody responses to the other V-Z subunit antigens.     

Antibodies to Surface Antigens of Lymphocytes and Lymphoblastoid Cells in Cold-Agglutinin-Positive Sera from Patients with Mycoplasma pneutnoniae Infection

IgM antibodies reacting in the cold with surface antigens of normal human blood lymphocytes were demonstrated by indirect immunofluorescence (IFL) in cold-agglutinin-positive sera from 21 patients with Mycoplasma pneumonias (MP) infection. Twenty to fifty per cent of the lymphocytes were stained. MP sera reacted also with 75%-100% of cells from two lymphoblastoid cell lines and one Burkitt lymphoma line and with about 80% of peripheral blood lymphocytes from one patient with chronic lymphatic leukaemia. The IFL reaction was negative with cells from a leukaemic lymphoid T-cell line (Molt-4). Lymphocyte fractionation experiments gave no evidence of a selective reactivity of MP sera with B or T lymphocytes of peripheral blond. Removal of cold agglutinins from the MP sera by absorption with human O erythrocytes significantly reduced the IFL reactivity with lymphocytes and lymphoblastoid cells, indicating the presence of I antigen on these cells. Furthermore, lymphoblastoid cells but not Molt lymphoid cells absorbed out most of the cold erythroagglutinins.     

Isolation and characterization of two new herpes-like viruses from capuchin monkeys.

Two herpes-like viruses were isolated from capuchin monkey (Cebus apella) brain and (Cebus albifrons) spleen cell cultures, respectively. Both isolates induced similar cytopathic effects consisting of rounded and ballooned cells in the original monkey cell cultures and in a wide range of permissive cell types. Neutralizing antibody to each virus was present in serum from the capuchin monkey from which it was isolated, but the two viruses did not cross-react by neutralization. Fluorescein isothiocyanate conjugates of hyperimmune rabbit serum to one of the isolates showed an antigenic cross relationship between the two isolates. By electron microscopy, herpes-like virus particles were observed in the nucleus and cytoplasm of infected human diploid fibroblast cell cultures. Virus-infected cell cultures stained with acridine orange revealed small deoxyribonucleic acid-containing intranuclear inclusion bodies. Both viruses were inhibited by 5-fluorodeoxyuridine and inactivated by chloroform or exposure to 56 degrees C for 30 min. Antisera prepared against 16 prototype herpesviruses and cytomegaloviruses did not neutralize approximately 100 50% tissue culture infective doses of either capuchin isolate. Neutralizing antibody to the capuchin isolates was detected in sera from 8 of 17 capuchin monkeys but not in sera from 16 humans, 15 chimpanzees, and 10 spider, 6 rhesus, and 5 squirrel monkeys.     

Attempts to induce interferon production by IdUrd induction and EBV superinfection in human lymphoma lines and their hybrids

Inducers of the Epstein-Barr virus (EBV) cycle, 5-iodo-2-deoxyuridine (IdUrd), phorbol myristate acetate (TPA) and sodium butyrate were tested for their ability to induce EBV-determined antigens, early antigen (EA) and virus capsid antigen (VCA), and to stimulate interferon (IF) production in a variety of EBV-carrying lymphoid cell lines. IdUrd and TPA induced IF production to various extents in the different lines, whereas sodium butyrate did not. There was no relationship between induction of the EBV cycle and production of IF; the two appear to be independent characteristics. Superinfection with the transforming B95-8 virus substrain of EBV induced IF production, whereas superinfection with the abortively cytopathic, non-transforming P3HR-1 substrain had little or no IF-inducing effect, in spite of its highly potent effect on virus antigen (particularly EA) synthesis. Analysis with specific antisera against IF showed that IF preparations produced by three different lymphoid cell lines in response to IdUrd treatment were composed of a mixture of the Le and F antigenic types, with the latter forming a minor species. In contrast, no detectable F interferon was present in spontaneously produced IF preparations from Namalwa cells, or after induction with B95-8 virus.     

Guinea Pig Herpes-Like Virus Infection I. Antibody Response and Virus Persistence in Guinea Pigs After Experimental Infection

Guinea pigs, experimentally infected with guinea pig herpes-like virus produced antibodies which were detectable by indirect hemagglutination (IHA), complement-fixation (CF), and neutralization tests. The IHA test appeared to be a more sensitive method than the CF or neutralization test for determining antibody response in guinea pigs immediately after infection, but the IHA method was not suitable for the detection of antibody in animals receiving small doses of virus. Antibody titers obtained by CF tests were generally higher than those obtained by the neutralization test, and they followed the same time course when individual animals were studied serially. Intracardiac inoculation produced the best antibody response in guinea pigs when compared with other routes of infection. Guinea pigs infected by the intraperitoneal, intranasal, or oral route showed rising antibody titers but the levels were low. Infectious virus was isolated from and persisted in all inoculated animals in the presence of antibody regardless of the route of inoculation. Recovery of infectious virus required cultivation or cocultivation of tissue cells containing virus. The administration of antilymphocyte sera delayed the appearance of IHA antibody but had no effect on antibodies determined by the CF- and neutralization tests.     

Antigen expression of metastasizing and non-metastasizing human melanoma cells xenografted into nude mice

In order to study differences in antigen expression related to the different stages of the process of metastasis of human melanoma cell lines, we determined the expression pattern of a series of well-characterized genes in a set of human melanoma cell lines with different metastatic behavior in nude mice. This set included non-metastatic (IF6, 530), sporadically metastatic (M14, Mel 57), and frequently metastatic (BLM, MV3) cell lines after subcutaneous inoculation. To study the phenotype of these cell lines both the cultured cells and representative samples of local tumors at the inoculation site and their metastases in the lungs were immunostained with a panel of monoclonal antibodies directed against melanocytic differentiation or progression antigens. Although most cell lines (IF6, 530, M14 and Mel 57) showed HLA-DR expressionin vitro, these antigens were lacking in all xenografted lesions studied with exception of the 530 cell line. 530 Xenografts, however, showed a dramatic down-regulation of HLA-DR compared with the cell linein vitro. The same phenomenon was seen with respect to ICAM-1 expression. The expression of all other antigens studied in xenografts, both in subcutaneous tumors and in lung lesions, was in general comparable to that in the melanoma cell linesin vitro, with exception of the 530 cell line. In all melanoma cell lines except 530 the degree of intra- and interlesional heterogeneity regarding the expression of all antigens studied was limited. Remarkably, comparison of the immunophenotype of the frequently metastasizing (BLM, MV3) and the sporadically (M14, Mel 57) or non-metastasizing (IF6, 530) cell lines showed that the two frequently metastasizing cell lines had marked expression of the progression antigens VLA-2 and epidermal growth factor receptor, and lack of expression of the differentiation antigen NKI-beteb. These findings warrant further studies on the role of these antigens in the process of metastasis of human melanoma cells in nude mice.