SV40 tumor rejection induced by vesicular stomatitis virus bearing SV40 tumor-specific transplantation antigen (SV40-TSTA). I. Specificity of immunoprotection and effect of enzyme treatment on TSTA activity.

Highly purified vesicular stomatitis virus (VSV) was obtained from VSV-infected SV40-transformed hamster cell lines. Immunization with this virus protected hamsters against challenge with SV40-transformed cells (TSV5-cl2). This protection was obtained regardless of the source of the SV40-transformed cells (e.g. cat, rat, hamster) used to produce VSV, and was therefore associated with the SV40 tumor-specific transplantation antigen (SV40-TSTA). Furthermore, when grown on spontaneously transformed cell lines or on cells transformed by a different oncogenic DNA virus, such as polyoma virus, the VSV failed to protect against the SV40-induced tumor. It was concluded that the SV40-TSTA activity of purified VSV is due to the incorporation of SV40-TSTA within the viral envelope. When VSV was treated with proteolytic enzymes (bromelain, trypsin) no loss of TSTA-induced tumor rejection was observed, although VSV had lost its ability to induce virus-neutralizing antibody. This clearly demonstrates that the TSTA activity is not related to the viral spikes. Phospholipase C suppressed the TSTA activity but neutralizing activity was still detectable in the anti-VSV sera. The results presented here demonstrate that the protection afforded by VSV is highly specific. It is particularly interesting that SV40-TSTA activity may be conveyed by the lipid core of the viral envelope.     

The SV40 S antigen; A carcinoembryonic-type antigen of the hamster?

In addition to hamster tumor cells induced or transformed by SV40, hamster anti-SV40 S (Surface) antiserum also reacted with non-SV40-exposed cell lines spontaneously induced (BHK 21) or transformed by heterologous oncogenic DNA and RNA viruses in the indirect membrane fluorescent antibody test. The antiserum titered equally with both BHK and SV40-transformed cells and the reaction could be absorbed with either of these cell lines, or with hamster embryo. The antiserum also reacted with early hamster embryo cells, although various organ cells from late fetuses, newborns and adults were negative. Mouse cell lines spontaneously induced (BALB/c-3T3) or transformed by SV40 and adenovirus 12 were also non-reactive. The results suggest that the antigen in question is a hamster somatic antigen present during embryonic life and derepressed in lines of actively growing cells.     

Isolation of a SV40-like Papovavirus from a human glioblastoma.

A human glioblastoma multiforme (M27) tested in early cell cultures by indirect immunofluorescence staining showed SV40-related tumor (T)-antigen, 95% of the cells being positive. SV40-related viral capsid (V)-antigen was absent in all cells tested. Experiments to rescue this virus were performed by fusing M27 cells with CV-I monkey cells, which were permissive for SV40, using polyethylene glycol (PEG) as fusion factor. We succeeded in isolating virus particles SV40-GBM which electron microscopy showed to correspond in size and morphology to papovaviruses. Serological tests (hemagglutination, neutralization, fluorescent antibody) revealed that the virus is indistinguishable from SV40. Despite this apparent antigenic identity SV40-GBM differs slightly from SV40 wild type. This virus can propagate and produce CPE in both CV-I cells and primary fetal human kidney cells. Furthermore digestion of SV40-GBM DNA with the HindII/III restriction endonucleases revealed minor differences compared with the SV40 DNA. Therefore the virus SV40-GBM obtained from glioblastoma cells seems to be closely related to the SV40-PML viruses described earlier.     

Analysis of minimal functions of simian virus 40. I. Oncogenic transformation of Syrian hamster kidney cells in vitro by photodynamically inactivated SV40

SV40 was photodynamically inactivated in the presence of Toluidine blue 0 by irradiation with white light for different periods of time. Different functions of SV40 were inactivated at different rates. The most sensitive function was the capacity of the virus to replicate followed by the capacity to induce T antigen and cell DNA synthesis. The transformation capacity was the most resistant. No evidence was obtained that the survival of transformation activity after large decrease in infectivity was due to multiplicity reactivation. Syrian hamster kidney cells transformed in vitro by photodynamically inactivated SV40 had the same properties as those transformed by control virus. They showed identical morphology, were T-antigen positive, contained the same number of SV40 DNA genome equivalents integrated in their DNA and were oncogenic when inoculated into adult hamsters.     

Carcinogen-mediated induction of SV40 DNA synthesis in SV40 transformed Chinese hamster embryo cells

Exposure of SV40-transformed Chinese hamster embryo cells (lineCO50) to a series of physical and chemical carcinogens (includingactivation-dependent and activation-independent varieties) resultedin the induction of viral DNA synthesis. The carcinogen mediatedamplification of SV40 DNA was demonstrated by a highly sensitivein situ hybridization procedure for the detection of cells synthesizingSV40 DNA. Treatment of CO50 cells with an inhibitor of polycyclichydrocarbon metabolism (7,8-benzoflavone) prior to the applicationof benzo[a]pyrene or 7,12-dimethyl-benz[a]anthracene preventedthe induction of SV40 DNA synthesis, indicating that the inductiondepends upon the metabolic activation of these compounds. Non-carcinogenichydrocarbons were inactive under this assay. Two different protocolsfor determining the inducing potential of a compound are presented.The properties of this test and its possible use as a short-termassay for potential carcinogens is discussed. The possibilitythat the induction of SV40 DNA synthesis is a reflection ofa general gene amplification phenomenon mediated by carcinogensis discussed.     

Induction of simian virus 40 (SV40) transplantation immunity in mice by SV40-transformed cells of various species.

Specific tumor rejection was obtained with the use of simian virus 40 (SV40)-transformed cells from several species including man, rat, ape, sheep, and hamster. Growth of the syngeneic sarcoma mKSA in BALB/c mice was strikingly inhibited following a single immunization with as few as 10(3) intact, viable cells. Non-SV40-transformed cells did not induce tumor rejection activity nor did SV40-transformed lines induce immunity against the 3-methylcholanthrene-induced sarcoma Meth A, syngeneic with BALB/c mice. A close relationship existed between the tumor rejection antigen, the tumor-specific transplantation antigen (TSTA) located on the plasma membrane, and the intranuclear tumor antigen (T-ag). Both were associated with the DNA sequence of the early region of the SV40 genome, and TSTA activity was found in the nucleus. However, we did not observe a close parallelism between T-ag activity and TSTA. Neverthesless, the results strongly suggested that TSTA, like T-ag, was encoded by the virus.     

New antigens in SV40 transformed cells. I. Demonstration of three new antigenic constituents in several cell lines of different species transformed by the SV40

New antigens in SV40 transformed cells. I. Demonstration of three new antigenic constituents in several cell lines of different species transformed by the SV40 The soluble components of different cell lines in various species (hamster, mouse, rat and dog) were compared by means of precipitation test in agar. Some lines were spontaneously transformed, others were transformed by SV40 virus and oncogenic viruses. The utilization of rabbit and hamster antisera, specific for a clone of SV40 transformed cells (TSV₅Cl₂) demonstrated the presence of three new antigens in the various cell strains transformed by SV40. These antigens are different from the nuclear T antigen and probably also from the transplantation antigen located on the cell membrane.     

Simian virus 40 (SV40) production from SV40-transformed human amnion cells of established lines.

Sixteen established cell lines of simian virus 40 (SV40)-transformed human amnion cells were examined for SV40 production. Many of these lines produced SV40 for extensive periods. Virus production had not ceased for 2 lines after 18 months, for 3 lines after 12 months, and for 3 lines at 3 months after recovery from "crisis". Three lines became virus-free in the first month, 1 line in the second month, 1 in the third month, and 1 in the fourth month, and 2 lines stopped virus production between 6 and 11 months after recovery. The virus titers were relatively low. Inclusion body-containing cells were infrequent. In contrast, in most cultures of SV40-transformed human fibroblasts rescued from crisis, no infectious virus was demonstrated, although exceptions have been reported. Virus was produced after heterokaryon formation of cells of the virus-free amnion lines with CV-1 cells in the presence of inactivated Sendai virus, as observed for SV40-transformed human fibroblasts. During the crisis period, some of the SV40-transformed amnion cells produced substantial amounts of virus. Titers decreased during the later periods of crisis. The most pronounced decrease in titers was in cultures from which established lines were recovered.     

Evidence for virus-specific transplantation antigen in cells of lymphoid neoplasms induced by papovavirus SV40

Hamster lymphocyte leukemia and lymphosarcoma induced in vivo by papova-virus SV40 were tested for the presence of SV40-specific transplantation antigen. The cells derived from lymphocytic leukemia (GD-248) blocked primary SV40 oncogenesis in vivo and also immunized adult hamsters against the transplantation of SV40 tumor cells which have been shown to carry SV40-specific transplantation antigen. Lymphosarcoma cells (GD-36), although immunosensitive, were not effective in blocking SV40 oncogenesis. GD-36 cells were also less immunogenic than GD-248 cells. These results indicate that the appearance of lymphoid cell tumors in adult hamsters by SV40 is not due to the absence of SV40-specific transplantation antigen in these tumors.     

Association of SV40 with human tumours

SV40 was discovered as a contaminant of poliovirus vaccines that were inadvertently administered to millions of people in Europe and the United States between 1955 and 1963. Shortly afterwards, SV40 was proven to be oncogenic in rodents and capable of transforming human and animal cells in vitro. The possibility that SV40 might cause tumours in humans thus became a subject of scientific and public interest and scrutiny. However, largely due to a lack of significant epidemiological evidence, interest in assessing SV40's potential carcinogenic role in humans diminished. Recently, many laboratories have reported the presence of SV40-like DNA in a high proportion of human mesotheliomas, ependymomas and osteosarcoma (the three main types of tumours caused by virus in hamsters), renewing the question whether SV40 might be a human tumour virus. Molecular data from these studies are reviewed to re-evaluate the potential role of SV40 as a human carcinogen.     

SV40 infection induces telomerase activity in human mesothelial cells

Mesotheliomas are malignant tumors of the pleural and peritoneal membranes which are often associated with asbestos exposure and with Simian virus 40 (SV40) infection. Telomerase activity is repressed in somatic cells and tissues but is activated in immortal and malignant cells. We evaluated telomerase activity in seven primary malignant mesothelioma biopsies and matched lung specimens and 20 mesothelioma cell lines and eight corresponding primary tumor cultures. All the tumor biopsies, and nearly all primary cell mesothelioma cultures and cell lines were telomerase positive. The findings in cell lines paralleled those observed in primary cultures in cases where paired samples were available. Next, we found that SV40, a DNA tumor virus present in approximately 50% of mesothelioma biopsies in the USA, induced telomerase activity in primary human mesothelial cells, but not in primary fibroblasts. Telomerase activity became detectable as early as 72 h following wild-type (strain 776) SV40 infection, and a clear DNA ladder was detectable 1 week after infection. The amount of telomerase activity increased during passage in cell culture and appeared to parallel increases in the cellular amounts of the SV40 large T-antigen. Thus, SV40 infection leads to telomerase activity before the infected mesothelial cells become transformed and immortalized. SV40 infection of human fibroblasts did not cause detectable telomerase activity. We also determined that the SV40 small t-antigen (tag) plays an important role in inducing telomerase activity because this activity was undetectable or minimal in mesothelial cells infected and/or transformed by SV40 tag mutants. Asbestos alone did not induce telomerase activity, and asbestos did not influence telomerase activity in mesothelial cells infected with SV40. Induction of telomerase activity by SV40 may be related to the very high rate of mesothelial cell immortalization that is characteristically associated with SV40 infection of mesothelial cells.     

Study of the induction mechanism in Syrian hamster cells transformed by SV40 virus. I. Properties of a clonal cell line

A clonal cell line (clone 2 TSV-5) has been derived from a primary SV40 Syrian hamster turmor. Association of clone 2 TSV-5 cells to kidney cells of some animal species (Cercopithecus monkey, Rhesus monkey, Man) allows to recover infectious SV40 virus from the mixed cultures. This phenomenon can be considered an induction phenomenon. The clone 2 TSV-5 is characterized by a mean frequency of induction by association of 6%, but the SV40 genome is entirely present in all these clonal cells. The induced SV40 virus is similar to the SV40 virus by which the clone 2 TSV-5 cells have been originally converted. The clone 2 TSV-5 is not inducible by rabbit kidney cells, by mouse embryo cells or by human KB cells. It is neither inducible by × or ultraviolet irradiations, nor by several chemical agents, such as mitomycin C, proflavine and hydrogen peroxide. Three other Syrian hamster cell lines converted by SV40 virus are not inducible by Cercopithecus monkey kidney cells. It is assumed that these malignant cells contain only a portion of the viral genome.     

Differences in the manifestation of virus-specific surface antigen between cells transformed by SV40 and UV-irradiated SV40

Marked differences in the manifestation of SV40-specific surface (S-) antigen were found between hamster cell lines transformed in vitro and in vivo by SV40 and UV-irradiated or photodynamically inactivated SV40. In the mixed hemagglutination reaction (MHA) cell lines induced by non-irradiated SV40 yielded positive results when tested against specific antisera up to dilutions of 1:1280. In comparison, cell lines induced by inactivated SV40 only showed positive reactions in the MHA-test in dilutions up to 1:80 or 1:160 respectively. The demonstration of small quantities of SV40-specific S-antigen in cell lines transformed by inactivated SV40 could only be achieved by use of hyperimmune sera produced by a special immunization procedure. However, if these cells were tested in the MHA reaction against anti-S-sera, produced according to conventional methods described in the literature, they yielded negative results. All cell lines with diminished quantities of demonstrable S-antigen caused malignant tumors when inoculated into adult hamsters. The capacity of UV-irradiated SV40 to induce a specific transplantation resistance in adult Syrian hamsters was investigated. The irradiated virus induced a markedly reduced transplantation resistance.     

SV40-positive brain tumor in scientist with risk of laboratory exposure to the virus

Simian virus 40 (SV40) is a DNA tumor virus known to induce cancers in laboratory animals. There are numerous reports of the detection of SV40 DNA and/or proteins in human malignancies of the same types as those induced by SV40 in animals, including brain cancers. However, known exposure to the virus has not yet been linked directly to cancer development in a specific individual. Here we describe the detection of SV40 sequences in the meningioma of a laboratory researcher who had a probable direct exposure to SV40 and subsequently developed a tumor positive for viral DNA sequences indistinguishable from those of the laboratory source. This case suggests a link between viral exposure and tumor development.     

Association between SV40 and non-Hodgkin's lymphoma

Millions of people worldwide were inadvertently exposed to live simian virus 40 (SV40) between 1955 and 1963 through immunization with SV40-contaminated polio vaccines. Although the prevalence of SV40 infections in humans is not known, numerous studies suggest that SV40 is a pathogen resident in the human population today. SV40 is a potent DNA tumor virus that is known to induce primary brain cancers, bone cancers, mesotheliomas, and lymphomas in laboratory animals. SV40 oncogenesis is mediated by the viral large tumor antigen (T-ag), which inactivates the tumor suppressor proteins p53 and pRb. During the last decade, independent studies using different molecular biology techniques have shown the presence of SV40 DNA, T-ag, or other viral markers in primary human brain and bone cancers and malignant mesotheliomas. Evidence suggests that there may be geographic differences in the frequency of these virus-positive tumors. Recent large independent controlled studies have shown that SV40 T-ag DNA is significantly associated with human non-Hodgkin's lymphoma (NHL). In our study, we analyzed systemic NHL from 76 HIV-1-positive and 78 HIV-1-negative patients, and nonmalignant lymphoid samples from 79 HIV-1-positive and 107 HIV-1-negative patients without tumors; 54 colon and breast carcinoma samples served as cancer controls. We used polymerase chain reaction (PCR) followed by Southern blot hybridization and DNA sequence analysis to detect DNAs of polyomaviruses and herpesviruses. SV40-specific DNA sequences were detected in 64 (42%) of 154 NHL, none of 186 nonmalignant lymphoid samples, and none of 54 control cancers. For NHL from HIV-1-positive patients, 33% contained SV40 DNA and 39% Epstein Barr virus (EBV) DNA, whereas NHLs from HIV-1-negative patients were 50% positive for SV40 and 15% positive for EBV. Few tumors were positive for both SV40 and EBV. Human herpesvirus type 8 was not detected. SV40 sequences were found most frequently in diffuse large B cell and follicular-type lymphomas. We conclude that SV40 is significantly associated with some types of NHL and that lymphomas should be added to the types of human cancers associated with SV40.     

In vivo induction of tumor-specific immunity by glycolipid extracts of SV40-transformed cells

Glycolipid extracts were prepared from various Syrian golden hamster cell lines, either SV40-transformed or spontaneously transformed. To detect possible SV40-TSTA activity of the glycolipid preparations, normal hamsters were inoculated with different glycolipid extracts and were subsequently challenged with an SV40 tumor-cell line. Significant immunoprotection against SV40 tumor challenge was induced with glycolipids obtained from SV40-transformed cell lines. This was expressed as complete tumor rejection or as a decrease in tumor growth rate, when compared to controls. No protective effects were induced with glycolipid extracts from spontaneously transformed cells. Results suggest that tumor-specific glycolipids synthesized in cells transformed by SV40 virus could act as tumor transplantation antigens responsible for specific tumor rejection in syngeneic hosts.     

SV40 transfection of human kidney epithelial cells and stability of chromosome 11

Simian virus 40 (SV40) transformation has been used in a variety of mammalian cells and has been shown to extend their life span. We therefore decided to apply these results to normal kidney and tumoral cells derived from Wilms' patients. Wilms' tumour, a nephroblastoma which presents in early childhood, has been linked to deletions and rearrangements in chromosome 11. Analysis of gene structure in the 11p13 locus involved in the development of the tumour has been restricted by the very short life-span of the tumoral cells in vitro. We transfected normal kidney WT/NK, tumoral WT/T cells and human foetal kidney HFK cells with 2 SV40-derived plasmids SV3neo (pBR322-SV40-containing neomycin bacterial gene) and SVori- (pMK-origin mutated SV40). We isolated a high number of SV40-transfected cell lines. The efficiency of transfection appeared to be extremely low in WT/T cells compared with HFK and even WT/NK. The life span of the cell lines was increased in relation to their untransfected homologues. However, in all of the cell lines except 3, senescence occurred, after crisis step or not. We looked at different markers associated with SV40 transformation of mammalian cells and specifically with the presence of SV40 T antigen in the cells and its consequences: AIG, tumorigenicity, expression and insertion in genomic DNA of SV40 T antigen. Genetic studies involving karyotypic and restriction fragment length polymorphism (RFLP) analysis demonstrated that, despite a frequent pseudo-diploidy, the cell lines derived have conserved the 11p13 locus.     

In vitro induction of tumor-specific immunity by highly purified VSV grown in SV40-transformed cells and tumor glycolipids incorporated into liposomes

Cytotoxic effector lymphocytes (CL) were induced by in vitro immunization of spleen cells from normal Syrian hamsters to syngeneic tumor cells, either SV40-transformed (EH-SV) or spontaneously transformed (EH-N). The lymphocyte reactivity was measured in a direct 51Cr release cytotoxicity assay performed with EH-SV- and EH-N-labelled targets. A specific cytotoxic effect against tumor cells carrying the sensitizing antigens was observed. Cytotoxic effector lymphocytes were also induced by in vitro immunization of hamster spleen cells to highly purified vesicular stomatitis virus (VSV) grown either in syngeneic SV40-transformed fibroblasts or in "normal" fibroblasts. Purified virus possessing an intact envelope or virus subparticles devoid of their glycoprotein spikes stimulated the cellular immune responses against host tumor antigens present within the viral envelope. Cytotoxicity assays have revealed two tumor-specific antigens (TSA), one induced by SV40 and present in SV40-transformed cell lines and the other present in "normal" cells. CL were also induced by in vitro sensitization of spleen cells from normal hamsters to liposomes containing the polar glycolipid fraction from EH-SV and/or EH-N cells. A specific cytotoxic effect against tumor cells that have supplied the glycolipid extract was observed, suggesting specific recognition of glycolipid antigens characteristic for each tumor line. This study supports the view that surface glycolipids act as tumor-specific antigens implicated in the destruction of SV40-induced tumors in Syrian hamsters.     

Properties of transformed hamster cells containing SV40 tumor antigen in the cytoplasm

The properties of hamster cells containing SV40 tumor (T) antigen in the cytoplasm, rather than the nucleus, were determined. Eight cell lines were established from eight tumors induced by hamster embryo fibroblasts transformed in vitro by PARA (2cT)-adenovirus 7. Six cell lines contained only cytoplasmic SV40 T-positive cells while two were a mixture of nuclear T-positive and cytoplasmic T-positive cells. All the cell lines contained SV40 S antigen, all caused the production of SV40 T antibody in vivo, and four elicited the production of adenovirus T antibody. The cell lines seem to have acquired an infinite life span in vitro. The localization of T antigen apparently can be a stable phenomenon because five of the cell lines have retained T antigen exclusively in the cytoplasm for over 40 passages in tissue culture. The cytoplasmic T antigen could be detected by complement fixation in addition to immunofluorescence. Cytoplasmic T-positive cells were readily transplantable in vivo and contained SV40 TSTA demonstrable by both immunogenicity and immunosensitivity procedures. Two of the three cytoplasmic variants of PARA appear to be weakly oncogenic in newborn hamsters.     

Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples

The rhesus monkey virus Simian Virus 40 (SV40) is a member of the polyomavirus family. It was introduced inadvertently to human populations through contaminated polio vaccine during the years 1956-1963, can induce experimental tumors in animals and transform human cells in culture. SV40 DNA has been identified in mesothelioma and other human tumors in some but not all studies. We tested prediagnostic sera from 49 mesothelioma cases and 147 matched controls for antibodies against the viral capsid protein VP1 and the large T antigen of SV40 and of the closely related human polyomaviruses BK and JC, and for SV40 DNA. Cases and controls were identified among donors to the Janus Serum Bank, which was linked to the Cancer Registry of Norway. Antibodies were analyzed by recently developed multiplex serology based on recombinantly expressed fusions of glutathione-S transferase with viral proteins as antigens combined with fluorescent bead technology. BKV and JCV specific antibodies cross- reactive with SV40 were preabsorbed with the respective VP1 proteins. Sera showing SV40 reactivity after preabsorption with BKV and JCV VP1 were further analyzed in SV40 neutralization assays. SV40 DNA was analyzed by SV40 specific polymerase chain reactions. The odds ratio for being a case when tested positive for SV40 VP1 in the antibody capture assay was 1.5 (95% CI 0.6-3.7) and 2.0 (95% CI 0.6-7.0) when only strongly reactive sera where counted as positive. Although some sera could neutralize SV40, preabsorption with BKV and JCV VP1 showed for all such sera that this neutralizing activity was due to cross-reacting antibodies and did not represent truly SV40-specific antibodies. No viral DNA was found in the sera. No significant association between SV40 antibody response in prediagnostic sera and risk of mesothelioma was seen.