Jörg R. Schlehofer

Update on the prevalence of serum antibodies (IgG and IgM) to adeno‐associated virus (AAV)

In view of presumed non‐pathogenicity, tumor suppressive properties, and site‐specific integration of the viral genome the human parvovirus, adeno‐associated virus (AAV) has gained great interest as a gene transduction vector. Data on the seroprevalence of antibodies to AAV vary between reports, probably due to the different serological methods used. In order to understand better the immune response to AAV during natural infection, sera from different age groups and various geographical regions were compared for AAV antibodies using an ELISA. The data show that the prevalence of antibodies to AAV is similar in Europe (Germany, France, and Switzerland), Brazil, and Japan, indicating worldwide infection. It was confirmed that infection takes place during childhood. However, declining seropositivity thereafter and a second increase of seropositivity after 30 years of age suggests reinfection or reactivation of latent virus in particular as the prevalence of IgM antibodies in adults is relatively high. Furthermore, pregnant women were found to be significantly more frequently seropositive than non‐pregnant controls, hinting at a reactivation of persistent AAV (up to 80% of women carry AAV in genital tissue) in specific hormonal conditions, e.g., pregnancy. Cross‐reaction of serum antibodies with the different AAV types (defined by complement fixation) was observed by ELISA and neutralization tests confirming earlier results. The results suggest an unstable AAV antibody response allowing lifelong reinfection or reactivation of persisting virus possibly due to partial immunotolerance after an infection in utero, at delivery or during early infancy. J. Med. Virol. 59:406–411, 1999.

Human genital tissues containing DNA of adeno-associated virus lack DNA sequences of the helper viruses adenovirus, herpes simplex virus or cytomegalovirus but frequently contain human papillomavirus DNA.

The detection of DNA of the helper virus-dependent adeno-associated virus type 2 (AAV-2) in biopsies of material from spontaneous abortion and in tissue samples from the uterus raises the question of whether sequences of known helper viruses can be detected simultaneously within the same specimen despite the lack of histological evidence for the presence of lytic viruses. Therefore, we performed PCR analyses with primers detecting DNA sequences of viruses (adenovirus, herpes simplex virus and human cytomegalovirus) known for their helper activity in the replication of adeno-associated viruses. In addition, PCR was performed to detect DNA of human papillomaviruses (HPV), which were recently shown to be able to help AAV replication in vitro. In no cases were sequences of the known helper viruses found. However, HPV DNA was detected in approximately 60% of paraffin sections from uterus biopsies and cervical lesions containing AAV DNA and in approximately 70% of material from early miscarriage. This finding suggests that HPV may be a helper virus for AAV.

Discrimination between different types of human adeno-associated viruses in clinical samples by PCR

Persistent infection of human tissues with the helper virus-dependent parvovirus, adeno-associated virus (AAV) was detected by polymerase chain reaction (PCR) using primer pairs detecting AAV types 2, 3 or 5. In order to develop PCR protocols which discriminate between the different serotypes of AAV, the DNA of AAV-5 was sequenced partially and compared with the published sequences of AAV-2 and -3. Type specific oligonucleotides and specific probes which allow the distinction between human AAV types by PCR are described.

Sero-epidemiological analysis of the risk of virus infections for childhood leukaemia

Virus infections have been thought to be involved in the development of childhood leukaemia. In order to address this issue we determined, in a case‐control study, the prevalence of antibodies to viruses infecting blood or bone‐marrow cells [Epstein‐Barr virus (EBV), human herpes virus type 6 (HHV‐6), parvovirus B19] as well as to the human virus known for its tumour‐suppressive properties, the adeno‐associated virus type 2 (AAV‐2), in the sera of 121 children with leukaemia in Germany, and in 197 control individuals, hospitalized for other reasons, and matched for age and gender to the cases. In addition, we developed a questionnaire to be answered by the childrens parents, in order to gain information on previous infections of the children as well as to calculate for factors which may influence serological findings. Comparative determination of the prevalence of antibodies against AAV‐2, B‐19 or HHV‐6 revealed no significant differences in cases and controls. However, antibodies to EBV were more frequently found in children with leukaemia younger than 6 years of age (age at the time of diagnosis of leukaemia) than in controls. Apparently, infection with AAV‐2 has no protective effect in childhood leukaemia, in contrast to results observed for other malignancies. Similarly, and in accordance with results on leukaemia in adults, we found no indication of a protective effect of infection with the parvovirus B‐19. The data suggest that EBV, which is known to be involved in various lymphomas, may play a role in the development of childhood leukaemia in young children.

Dose-dependent regression of HeLa cell-derived tumours in SCID mice after parvovirus H-1 infection.

Parvoviruses of rodents are endowed with oncosuppressive properties. In particular, parvoviral infections protect host animals from spontaneous and chemical‐ or virus‐induced tumour initiation in laboratory animals. The present study was undertaken to substantiate the capacity of parvovirus H‐1 to inhibit therapeutically the growth of established tumours originating from human carcinoma cells implanted in recipient mice. To this end, quickly growing s.c. carcinomas were established by injection of human cervical carcinoma cells (HeLa) into immunodeficient (SCID) mice. Tumour‐bearing mice subsequently were inoculated with H‐1 at various multiplicities of infection. H‐1 virus infection led to regression of tumours, the onset and efficiency of which were dose‐dependent. Int. J. Cancer 75:584–589, 1998.

Evidence of chromosomal integration of AAV DNA in human testis tissue.

Persistent infection with adeno-associated virus (AAV) has been demonstrated in human tissues, most frequently in the female and male genital tract. The clinical significance of latent AAV infection remains, however, uncertain to date. The mode of latency of AAV is not known, i.e., it is unclear whether the viral genome is integrated in the cellular genome, and if integration occurs site-specifically in chromosome 19 as has been observed in cell culture. Therefore we investigated if viral DNA in AAV DNA-positive human testis samples from two patients, is integrated in the cellular genome. Using two different molecular approaches, uni-directional PCR and Walking Primer PCR, we could demonstrate that AAV DNA is present in an integrated form in testis tissue. Virus-cell DNA junction fragments were cloned and sequenced. A detailed analysis revealed integration within sequences of the so-called AAVS1 region on chromosome 19. These data demonstrate that AAV DNA can integrate also after natural infection, and that integration occurs within the AAVS1 region, at least in some cases.

Selective killing of carcinogen-treated SV40-transformed Chinese hamster cells by a defective parvovirus.

SV40-transformed Chinese hamster cells (CO631) were treated with 7, 12-dimethylbenz[alpha]anthracene, 4-nitroquinoline-N-oxide, or benzo[alpha]pyrene, respectively, at sublethal concentrations. Infection of these cells with the helper-dependent parvovirus, AAV-5 immediately prior to, or at the time of exposure to the chemical carcinogens resulted in effective killing of cells exposed to the combination virus plus carcinogen, AAV infection without additional treatment did not have a significant effect on cell survival. AAV infections have recently been shown to efficiently inhibit gene amplification induced by various initiators and herpes simplex virus infections. Data may suggest that gene amplification is an adaptive cellular response to initiating events resulting in enhanced cell survival.

FUNCTIONAL ANALYSIS OF DIFFERENT LMP1 PROTEINS ISOLATED FROM EPSTEIN-BARR VIRUS-POSITIVE CARRIERS

The Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is implicated in the development of several human malignancies. Latent membrane protein 1 (LMP1), an EBV protein with known oncogenic properties, may be important in the pathogenesis of EBV-associated tumors, particularly nasopharyngeal carcinoma (NPC) and Hodgkins disease (HD). Several reports suggested that sequence variations in the LMP1 gene may define a more aggressive, geographically restricted EBV-genotype. Most mutations in the LMP1 gene described are located within the C-terminus of the protein. However, the effect of these mutations on the biological function of the protein remains widely unknown. Therefore, this study aimed in investigating whether mutations detected in LMP1 genes isolated from different EBV-positive carriers have an effect on the biological function of the protein. For this purpose the LMP1 genes were amplified by nested PCR from DNA out of bone marrow and peripheral blood lymphocytes and sequenced. Three functional assays were performed in order to evaluate the biological activity of the different isolates: activation of the transcription factors NF-kappaB and AP-1 as well as the anchorage independent growth of LMP1 transfected ratl cells in soft agar. The results suggested that whereas differences in the activation of NF-kappaB through the various LMP1 isolates correlated tightly with their different expression levels, the outgrowth of transfected cells in soft agar did not and the transcription factor NF-kappaB therefore appeared not to be the major effector for the transformation of the rodent cell line ratl by LMP1. The various LMP1-isolates also differed in their capacity in activating the transcription factor AP-1. We found no correlation between the transforming ability of the LMPI isolates and activation of AP-1 suggesting that other so far uncharacterized domains also influence the transforming ability of the protein.

Persistence of parvovirus H-1 DNA in human B- and T-lymphoma cells

Persisting DNA of parvovirus H-1 could be demonstrated in cells of two human lymphoma cell lines, the Burkitt lymphoma cell line BL2 and the T-cell leukemia cell line Jurkat which survived infection with parvovirus H-1. Persistence of H-1 DNA rendered the cells resistant to a second H-1 infection. This resistance to H-1 superinfection persisted even after loss of H-1 DNA occurring after approximately 150-200 cell generations. Resistance to H-1 superinfection was accompanied by reduced uptake of infectious particles and by a block of H-1 DNA replication. This suggests that persistent H-1 infection leads to modifications of cellular functions involved in the permissivity for H-1.

Adenovirus infection induces amplification of persistent viral DNA sequences (simian virus 40, hepatitis B virus, bovine papillomavirus) in human and rodent cells.

Adenoviruses, types 2 and 12 induce amplification of SV40 DNA sequences in cells of the SV40-transformed human newborn kidney cell line, NB-E. Similarly, integrated hepatitis B virus DNA sequences in the human hepatoma cell line, PLC/*PRF/5, and bovine papillomavirus (BPV) DNA sequences in BPV-transformed mouse cells (ID13) are amplified by adenovirus infection. Thus, similar to herpes group or vaccinia viruses or DNA damaging agents, adenoviruses are able to mediate selective DNA amplification in addition to their reported mutagenic and chromosome damaging effects. The role of amplification of integrated viral DNA sequences in development and progression of specific tumors (e.g. hepatocellular carcinoma) remains to be determined.