Biomedical subjects

A Gigler

Publications and source records attributed to A Gigler.

5 recordsLinked to original sources

PubMed1999

Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins.

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 microgram/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 micrograms/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.

Amino Acid Sequence↗

PubMed1998

Characterization of cis-acting and NS1 protein-responsive elements in the p6 promoter of parvovirus B19.

Parvovirus B19 infections are associated with diverse clinical manifestations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, possibly due to the activity of the only functional promoter (p6) of the B19 virus genome in combination with both cell- and cell cycle-specific factors and the trans-activator protein NS1. As presented here, p6 promoter sequences derived from several B19 virus isolates proved to be highly conserved. Furthermore, mutations did not affect any of the potential binding sites for transcription factors. One variation of the base at position 223 was identified only in B19 virus isolates derived from patients with persistent infection or chronic arthritis. To determine promoter activity and to characterize regulatory elements, sequences spanning the total p6 promoter and subfragments of them were introduced into a eukaryotic expression vector upstream of the luciferase gene (from Photinus pyralis). After transfection into HeLa, CEM, BJAB, and K562 cells, the p6 promoter was found to be highly active. When introduced into the erythroid cell line K562, p6-controlled transcription exceeded that of the simian virus 40 promoter-enhancer used as a control by more than 25-fold. Sequence elements relevant for promoter activity mapped to the regions from nucleotides (nt) 100 to 190 and 233 to 298. Also, the segment from nt 343 to 400 downstream of the TATA box was important for transcriptional activity in HeLa and K562 cells. By transfecting the promoter-luciferase constructs into a HeLa cell line stably carrying the viral NS1 gene under the control of an inducible promoter, transcriptional activity mediated by the p6 promoter rose significantly after induction of NS1 expression. The region from nt 100 to 160 proved to be essential for NS1-mediated transcriptional activation. Furthermore, NS1-mediated transactivation was dependent on the presence of two GC-rich elements arranged in tandem upstream of the TATA box. These data indicate that NS1-mediated p6 transactivation is dependent on a multicomponent complex combining NS1 with ATF, NF-kappaB/c-Rel, and GC-box binding cellular factors.

Base Sequence↗

PubMed1996

Sequence variability among different parvovirus B19 isolates.

Parvovirus B19 is the causative agent of a variety of clinical manifestations, ranging from asymptomatic to severe infection. The basis for this complex pattern of B19-associated diseases is as yet poorly understood. In general there are two different possibilities: firstly, the infected individuals may have a genetic or acquired predisposition, which renders them susceptible for a certain course of infection; secondly, differences in the B19 genome may result in different outcomes of infection. In order to investigate this second possibility we have partially sequenced the genomes of 20 different B19 isolates derived from serum samples from patients with various B19-associated diseases. Four distinct regions, which cover nearly half of the genome and include parts of the coding regions of all three major B19 proteins-NS1, VP1 and VP2, were selected for sequencing. Comparisons between the different extracted virus isolates at the DNA and protein levels revealed that isolates from patients with persistent parvovirus B19 infection show a tendency towards higher genome variability with respect to isolates derived from persons with acute infection.

Base Sequence↗

PubMed1995

Antibodies to the nonstructural protein of parvovirus B19 in persistently infected patients: implications for pathogenesis.

Three patients with persistent parvovirus B19 infection, as documented by the prolonged presence of IgM directed to the viral capsid proteins and detection of viral DNA in serum by dot-blot hybridization or polymerase chain reaction (PCR), were investigated for the presence of antibodies to the nonstructural protein NS-1 of parvovirus B19. This was done by using an ELISA based on recombinant NS-1 protein. Whereas control sera displayed no reactivity, sera from persistently infected patients showed a strong specific antibody response to NS-1. Patients were followed for 3-18 months, during which IgM titers declined but IgG directed to the nonstructural protein remained detectable. The appearance of NS-1-specific antibodies might indicate an altered course of viral infection leading to the establishment of persistently active infection and subsequent destruction of cells of nonerythroid lineage.

Adult↗

PubMed1995

Antibodies to parvovirus B19 NS-1 protein in infected individuals.

Human parvovirus B19 is the aetiological agent of the common childhood disease erythema infectiosum (fifth disease). The infection is usually benign and self-limiting, but in adults cases of severe arthritis which may persist for years have been reported. Neutralizing antibodies directed against the structural proteins are usually produced shortly after the infection. The immune response against the third major protein, the non-structural protein NS-1, of parvovirus B19 has not been characterized so far. We cloned and expressed the full-length NS-1 protein and fragments thereof in Escherichia coli. The purified recombinant proteins were used to investigate the presence of antibodies to the NS-1 protein in sera from patients with parvovirus B19 infection. Specific antibodies could be detected in sera from patients suffering from severe parvovirus B19-associated arthritis using Western blot analysis and an ELISA. Sera from patients with acute or past infection without complications did not contain detectable levels of immunoglobulin to NS-1. The use of subfragments of the NS-1 protein allowed localization of the antigenic domains in the carboxy-terminal region of the protein.

Antibodies, Viral↗