Recombinant Viral Antigens Research Articles

Detection of human parvovirus B19-specific IgM and IgG antibodies using a recombinant viral VP1 antigen expressed in insect cells and estimation of time of infection by testing for antibody avidity

Sera from patients with symptoms of recent human parvovirus B19 (B19) infection were tested for B19-specific IgM in an immunofluorescence assay (IFA) using insect cells expressing B19 recombinant VP1 coat protein as an antigen. A highly significant correlation ( P< 0.001) was found between titres obtained in the IgM IFA and the units obtained in an IgM antibody-capture RIA using plasma derived native B19 antigen. An IgG IFA using the recombinant antigen was performed on 57 sera and the antibody avidity determined. There was a highly significant correlation ( P< 0.001) between the relative amounts of low avidity B19-specific IgG antibodies and time after onset of illness. This finding allows the detection of IgG to be used for diagnosing acute infection.

Diagnostics of persistent viruses: human cytomegalovirus as an example.

Infections with persistent viruses such as herpesviruses have become of significant clinical importance with the increasing number of immunocompromised patients at risk to suffer from severe disease. As antiviral chemotherapy is available for herpesvirus infections, the diagnostic methods for rapid and sensitive detection of symptomatic infection have been developed and recently refined. In human cytomegalovirus (HCMV), the use of recombinant viral antigens provides a rationale to improve serological assays. This may be of use for the discrimination of primary versus secondary infection. Early diagnosis of symptomatic HCMV infection in immunosuppressed patients can be most effectively achieved by the detection of a viral tegument protein, pp65, in peripheral blood leukocytes. This early diagnosis has been shown to be of major importance for the effective treatment of these patients. HCMV infection in solid organs can be demonstrated by immunohistochemistry using monoclonal antibodies against viral proteins. HCMV involvement in diseases of the central nervous system in AIDS patients can be verified by the detection of very small amounts of HCMV DNA in cerebrospinal fluid by polymerase chain reaction. This method may prove useful for monitoring HCMV encephalitis and neuropathy.

Detection of hepatitis C virus antibodies with new recombinant antigens: assessment in chronic liver diseases

A new serological assay to detect antibodies against hepatitis C. based on a recombinant protein (BHC10) which incorporates structural and non-structural viral antigens, was tested in 67 healthy subjects and 409 patients with various forms of liver disease. Results were compared with the current assay based on the recombinant non-structural viral antigen c100 and with the recently introduced second-generation assay, Ortho2. None of the healthy subjects was positive by any of the assays. In patients with chronic non-A, non-B hepatitis the prevalence of anti-BHC10 was 96.8%, higher than anti-c100 (83.3%, p < 0.001) and similar to Ortho2 (94.3%). False-positive results were less frequently found when BHC10 was used. These findings show that assays incorporating structural and non-structural antigens provide higher sensitivity to detect hepatitis C virus infection and they define an almost exclusive role of hepatitis C virus in the genesis of chronic non-A, non-B hepatitis.

Reliable confirmation and quantitation of human immunodeficiency virus type 1 antibody using a recombinant‐antigen immunoblot assay

The recombinant DNA-derived, human immunodeficiency virus (HIV) antigen-based immunoblot assay (RIBA-HIV216) is a new supplemental (confirmatory) test developed to detect antibodies to HIV-1. The assay employs four recombinant viral antigens, corresponding to HIV-1 p24, p31, p41 and gp120 proteins, in an immunoblot format. With this assay, HIV-1 antigen reactivity was detected in all 683 infected patient serum or plasma specimens evaluated; 665 (97.6%) of these sera met the criteria for a positive interpretation, and 18 (2.6%) were classified as indeterminate. All 683 samples reacted with the recombinant gp41-equivalent protein. The first sequential enzyme immunoassay (EIA)-reactive samples collected from 33 seroconverting homosexual men reacted on RIBA-HIV216. Eleven (1.1%) of 999 EIA-negative blood donor sera reacted weakly with a single recombinant antigen (p24 or p31), whereas 13 to 48 percent had indeterminate reactions on viral lysate Western blots. One (1.5%) of 66 EIA-positive, Western blot-negative blood donor samples and 19 (29%) of 66 EIA-positive, Western blot-indeterminate blood donor samples scored indeterminate on RIBA-HIV216. Nonspecific reactivity was seen with only 1 (0.8%) of 114 patient sera containing possible interfering antibodies, whereas 33 percent of these samples had indeterminate reactions on Western blot and 35 percent had equivocal reactions on immunofluorescence assay (IFA). We conclude that the RIBA-HIV216 is approximately as sensitive as and significantly more specific than virus-derived Western blot and IFA. The RIBA-HIV216 also allows for semiquantitation of specific antibodies that may be of value in clinical staging and therapeutic monitoring.

Parenterally transmitted non-A, non-B hepatitis: Virus-specific antibody response patterns in hepatitis C virus-infected chimpanzees

An established chimpanzee model of parenterally-transmitted non-A, non-B hepatitis was used to define virus-specific immune response patterns in acutely and persistently infected animals. Serial bleedings were obtained from 23 chimpanzees that had been experimentally infected with an isolate of hepatitis C virus, originally recovered from contaminated lots of factor VIII (antihemophilic) materials. Sera were assayed for the presence of antihepatitis C virus by a newly developed radioimmunoassay procedure that incorporated recombinant DNA-expressed viral antigen as a reagent. Twenty-one of 23 hepatitis C virus infected animals were shown to acquire antihepatitis C virus, most within 2–8 weeks after the major peak of alanine aminotransferase activity. All chimpanzees with biochemical, electron microscopic, and histological evidence of chronic disease clearly acquired antibody; 14 of 16 animals observed through the acute phase of disease were also shown to acquire antibody. A booster effect or anamnestic response was noted in two chimpanzees (one of which was negative for antihepatitis C virus following the acute phase of disease) after challenge with hepatitis C virus. Antihepatitis C virus was not neutralizing, because some animals with high levels of antibody were also shown to have high titers of circulating hepatitis C virus. The development and maintenance of anti-hepatitis C virus appears to reflect concomitant virus replication and high potential for infectivity.

An immunofluorescence assay for the detection of parvovirus B19 IgG and IgM antibodies based on recombinant viral antigen

An indirect immunofluorescence assay for serum IgG and IgM antibodies to human parvovirus B19 was established using recombinant B19 viral antigen, the capsid protein VP1, which had been produced in a baculovirus expression system. This protein gives a strong and characteristic signal in the immunofluorescence assay, making it a suitable candidate for this test system. The test results showed a good correlation with results obtained with a solid-phase capture radioimmunoassay (Cohen et al., 1983). 76% of sera from a random selection of blood donors were positive for B19 IgG which agrees with previous findings. The course of the IgM and IgG antibody response to B19 infection could be followed with the immunofluorescence assay by determining the titers of series of sera taken after a recent B19 infection. Investigation of 24 sera containing rubella-specific IgM showed no cross-reactivity with the recombinant B19 VP1 used in this test system. The test described here has the advantage of being based on a renewable source of antigen and will be further evaluated for routine diagnostic use in comparison with radioimmunoassay.

Specific enzyme-linked immunosorbent assay for the detection of antibodies to the human spumavirus

Recombinant plasmid clones were constructed harbouring the central domains of the outer membrane protein and the transmembrane protein of the env gene of human spumaretrovirus (HSRV). The corresponding fusion proteins were expressed in E. coli, purified and used subsequently to produce antibodies against the HSRV env proteins in rabbits. The authenticity of the bacterially produced domain of the HSRV env proteins was shown by radioimmunoprecipitation of the viral env glycoprotein from HSRV-infected human cells with rabbit antibodies raised against the recombinant antigens. The recombinant viral antigens were used to establish a sensitive and spumavirus-specific enzyme-linked immunosorbent assay (ELISA). This anti HSRV antibody ELISA makes it possible to screen human sera for the presence of spumavirus infections.

Hepatitis C virus: The major causative agent of viral non-A, non-B hepatitis

A 'blind' recombinant immunoscreening approach, of general application to studies of infectious diseases, was used to clone and identify the genome of the previously uncharacterized non-A, non-B hepatitis (NANB) virus. This agent is a positive-stranded RNA virus that appears to be distantly related to the flaviviridae family. A recombinant viral antigen (C100-3) was used to develop a capture assay for circulating antibody. Data obtained using this assay indicate that this agent, termed the hepatitis C virus (HCV), is the major cause of post-transfusion, community-acquired and cryptogenic, NANB. Anti-C100-3 antibody appears to be directed towards dominant, non-structural viral epitopes. It is a non-neutralising antibody that develops generally late in infection and is a particularly good marker of chronic, persistent viraemia. Many asymptomatic but infectious blood donors can now be detected using this antibody assay. HCV is associated with the development of hepatocellular carcinoma and possibly, other liver diseases.

Cleavage of procaryotically expressed human immunodeficiency virus fusion proteins by factor Xa and application in western blot (immunoblot) assays

The proteins p15 and p24 of the human immunodeficiency virus (HIV) type 1 gag gene were expressed as fusion proteins in Escherichia coli for detecting antibodies against the acquired immunodeficiency virus by Western blot (immunoblot) analysis. These fusion proteins contain amino acids 1 to 375 of the E. coli beta-galactosidase linked to the viral protein(s) by a recognition sequence for the specific protease factor Xa. They are obtained in large amounts in insoluble inclusion bodies. To avoid ambiguous results caused by cross-reaction of sera with bacterial proteins in Western blots, we purified the recombinant fusion proteins and subsequently removed the bacterial part of the fusions by cleavage with factor Xa. The cleavage mixtures were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The viral proteins obtained by this method did not contain any bacterial proteins or protein fragments. Thus, false-positive results in HIV Western blot analysis with bacterially expressed HIV proteins can be excluded with these purified recombinant viral antigens.

Detection of Antibodies to Human T-Cell Lymphotropic Virus-III (HTLV-III) with an Immunoassay Employing a Recombinant Escherichia coli-Derived Viral Antigenic Peptide

A peptide containing 82 amino acid residues encoded by a gene segment in the env-lor region of HTLV-III has been produced in large quantities in Escherichia coli. Solid phase immunoassays employing the recombinant peptide as an antigenic adsorbent can reproducibly detect antibodies in sera of patients with HTLV-III infection. In several representative serum panels, the assay detected the presence of antibodies in 131 of 132 sera from patients with AIDS or AIDS-related complex (ARC), and in none of 471 normal blood donors. These results indicate that a sensitive, specific and quantitative immunoassay for HTLV-III infection can be developed with a recombinant viral antigen as the immunoreactive agent.