Engineering of the hepatitis C virus helicase for enhanced seroreactivity.

Hepatitis C Virus c33, a recombinant protein comprising residues 1192-1457 of NS3 helicase, has been a mainstay of HCV serology for decades. With seven unpaired cysteines, seroreactivity of E. coli expressed c33 is dependant on reductants. While engineering a c33 replacement for new anti-HCV serological tests, we sought to reduce oxidation sensitivity, a liability for immunodiagnostic reagent stability. A series of cysteine-to-serine substituted variants of a c33-like antigen was constructed and evaluated for reactivity against a panel of HCV-positive sera. Several variants were essentially nonreactive while others exhibited reactivity similar to or better than the wild-type construct. One demonstrated equivalent potency to wild-type but also diminished DTT dependence. To explore enhanced anti-NS3 reactivity, we constructed and examined an expanded series of antigens comprising individual helicase domains, the full-length helicase, additional cysteine-to-serine variants, and variants at positions critical to catalytic activity. Immunoassays using these latter NS3 helicase recombinants demonstrated that domain 1 possessed significantly more seroreactivity than previously believed, that the use of soluble full-length helicase protein enhanced sensitivity by several-fold over c33, and that anti-NS3 helicase seroreactivity was further enhanced by the introduction of point mutations which altered the catalytic activity or oxidation sensitivity of the antigen.

Detection of HCV core antigen in human serum and plasma with an automated chemiluminescent immunoassay.

BACKGROUND: Currently, the detection of HCV infection in blood donors relies on the ability of immunoassays to detect circulating HCV antibodies. However, a significant delay exists between the time of infection and the development of antibodies. This delay (window period) can last up to 70 days. The introduction of NAT for the detection of HCV RNA has reduced this window period dramatically. However, NAT is labor intensive, prone to contamination, and expensive as compared with standard serologic tests. STUDY DESIGN AND METHODS: An automated, microparticle-based chemiluminescent assay for the detection of HCV core antigen in human serum and plasma was developed. The specificity and sensitivity of this prototype assay were evaluated by testing a population of normal blood donors and commercially available seroconversion panels. RESULTS: The HCV core antigen assay exhibited a 99.9-percent specificity by detecting a single repeatably reactive sample out of 1004 normal donors tested. Assay sensitivity was determined by comparing the HCV core antigen detection rate with the antibody seroconversion profile and the rate of HCV RNA detection. Among 15 seroconversion panels examined, core antigen was detected in 69 of 70 antibody-negative and/or RNA-positive samples for a sensitivity relative to NAT of 98.6 percent. CONCLUSION: These data indicate that the automated, microparticle-based chemiluminescent HCV core antigen assay can reduce the window period for detection of potentially infected blood donors by 32.7 days, and it represents a viable alternative to HCV RNA testing.

Experimental infection of rhesus macaques with West Nile virus: level and duration of viremia and kinetics of the antibody response after infection.

Reports of transfusion-associated cases of West Nile virus (WNV) infection indicate the need for sensitive screening methods to identify WNV-infected blood products. We experimentally infected 5 rhesus macaques with WNV, to determine the level and duration of viremia, the kinetics of the humoral immune response, and the sensitivity of various assay systems for detecting WNV in blood. All macaques developed subclinical infections with low levels of viremia; nested reverse-transcription polymerase chain reaction was the most sensitive method for detecting virus or viral RNA in blood. Specific WNV antibodies appeared during the second week of infection; the results of an IgM enzyme-linked immunosorbent assay became positive on the ninth or tenth day after infection, followed in 1-2 days by hemagglutination-inhibiting and neutralizing antibodies. Our results suggest that both nucleic acid and serological testing may be needed to determine exposure to WNV and to identify potentially infected blood donors.

Combination HCV core antigen and antibody assay on a fully automated chemiluminescence analyzer.

BACKGROUND: HCV exposure among blood donors is serologically determined by detection of antibodies to HCV (anti-HCV); however, the recent development of an assay for the detection of HCV core antigen identifies infection before anti-HCV development. Simultaneous detection of HCV core antigen and anti-HCV would shorten the window period before seroconversion over conventional HCV antibody screening assays. STUDY DESIGN AND METHODS: A prototype chemiluminescent immunoassay was developed for simultaneous detection of HCV core antigen and anti-HCV in human sera and plasma. The assay was performed on a single-channel instrument representing an automated serologic analyzer (PRISM, Abbott Laboratories) system. Sensitivity and specificity were evaluated by testing 23 HCV seroconversion panels and plasma or sera from volunteer blood donors. RESULTS: The prototype HCV core antigen and antibody combination assay detected 80 of 89 (89.9% ) HCV RNA-positive and antibody-negative specimens from 23 panels, thereby reducing the seroconversion window period by an average of 34.3 days compared to PRISM HCV antibody detection. All PRISM HCV antibody-positive specimens were detected by the combination assay for a relative sensitivity of 100 percent. The repeatedly reactive rate was 0.20 percent based on testing of 3017 screened anti-HCV-negative sera and plasma. CONCLUSIONS: The prototype combination assay was shown to detect HCV core antigen and anti-HCV simultaneously and significantly closed the time gap between the initial detection of HCV RNA and the first appearance of detectable antibodies to HCV.