Results are presented for the 50 HAVAB-negative samples, 46 postvaccination samples (vaccine), 15 convalescent-phase samples from subjects with chronic hepatitis C computer virus contamination, and 25 samples obtained from individuals with acute HAV contamination. individuals were evaluated by immunoassay that used either HAV or rV-ORF antigen. All samples (86 of 86) in which HAV antibody was detected by a commercial enzyme-linked immunosorbent assay (ELISA) also tested positive by the recombinant antigen-based immunoassay (VacRIA). Similarly, all samples (50 of 50) that were HAV antibody unfavorable also tested unfavorable by the VacRIA. The lower limit of detection of HAV antibody was comparable among immunoassays with either HAV or rV-ORF antigen. Thus, in the population studied, the sensitivity and specificity of the VacRIA were equivalent to those of the commercial ELISA. Since production of recombinant antigen is usually faster and less expensive than production of traditional HAV antigen, the development of diagnostic HAV antibody assessments with recombinant HAV antigen appears warranted. Hepatitis A computer virus (HAV) is the sole member of the hepatovirus genus within the picornavirus family (6,19). With a few notable exceptions, the computer virus does not cause cytopathic effects in cell culture and grows slowly and only to a low titer (9,11,23). Thus, production of HAV antigen for vaccine use or for diagnostic testing purposes is usually slow and expensive. The computer virus contains a positive-sense, single-stranded SB 242084 RNA genome which encodes a single polyprotein of 2,227 amino acids (7,8). The polyprotein is usually autocatalytically processed into three major structural proteins (VP0, VP1, and VP3; also referred to as 1AB, 1D, and 1C respectively) (for a review, see reference34). Each of the three viral structural proteins has been expressed as a recombinant protein in a variety of prokaryotic and eukaryotic expression systems (12,13,15,16,18,20,22,26,38); however, none of the individual recombinant proteins was able to elicit high-titer neutralizing antibody or to bind to neutralizing monoclonal antibodies. Although human antibodies binding to denatured HAV structural and nonstructural proteins have been documented (18,24,25,30,37), the diagnostic power of these nonneutralizing antigens has not been extensively studied. In some experimentally infected primates, the antibody response to nonstructural proteins is usually short-lived (25,37), and commercial HAV SB 242084 antibody assessments do not appear to detect antibody DLL4 directed against individual structural proteins (15,38; for reviews, see recommendations29and39). Consequently, individual recombinant HAV antigens do not appear promising as an antigen source for routine diagnostic immunoassays. These results, along with other data, have led to the understanding that the crucial epitopes within the immunodominant neutralization antigenic site on HAV are defined conformationally and require assembly of the structural proteins into subviral particles (33,35). HAV morphogenesis requires several actions, the first of which is SB 242084 the assembly of the promoter, a structure containing one copy each of VP0, VP1, and VP3 (1AB, 1C, and 1D, respectively). This structure has a sedimentation coefficient of 5S (2,4,27). Five promoters assemble into a pentamer (sedimentation coefficient of 14S), which contains most of the neutralization antigenic sites found on complete virions (35). Twelve pentamers form the viral vacant capsid (70S), and this particle is usually antigenically indistinguishable from infectious virions (35). To become infectious, the vacant capsid must encapsidate full-length, genomic RNA. The final infectious HAV particle (virion) has a sedimentation coefficient of 156S (2,4,8). We previously expressed the entire HAV open reading frame in recombinant baculoviruses and vaccinia viruses (18,26,36,38). We exhibited that this polyprotein is usually accurately processed into structural proteins that assemble into pentamers and vacant capsids (26,35,38). These particles contain the epitopes comprising the immunodominant HAV neutralization antigenic site and elicit HAV-neutralizing antibodies in experimental animals (35). The purpose of this study was to evaluate different cell culture systems to optimize recombinant HAV antigen production and to determine the suitability of this antigen for use in diagnostic immunoassays. (This work was presented in part at the American Gastroenterological Association and American Association for the Study of Liver Diseases Annual Getting together with, 11 May 1997, Washington, D.C.) == MATERIALS AND METHODS == == Computer virus and cells. == HAV HM-175 was propagated in BS-C-1 cells as described previously (30,31,38). This strain originated from the same strain used for the SmithKline Beecham HAV vaccine (3). Wild-type (WT) vaccinia computer virus and recombinant vaccinia computer virus expressing the HAV open reading frame (rV-ORF) were selected and propagated as described previously (38). Cells were maintained and vaccinia computer virus infections were carried out in BS-C-1 TK-143, HeLa, MRC-5, and Vero cells (38) and in Epstein-Barr computer virus (EBV)-transformed human B cells (40) as described previously. == Patients samples. == Serum or plasma samples were obtained from 25 well-characterized patients with acute hepatitis.