High-dimensional immune profiling of total and rotavirus VP6-specific intestinal and circulating B cells by mass cytometry.

In-depth phenotyping of human intestinal antibody secreting cells (ASCs) and their precursors is important for developing improved mucosal vaccines. We used single-cell mass cytometry to simultaneously analyze 34 differentiation and trafficking markers on intestinal and circulating B cells. In addition, we labeled rotavirus (RV) double-layered particles with a metal isotope and characterized B cells specific to the RV VP6 major structural protein. We describe the heterogeneity of the intestinal B-cell compartment, dominated by ASCs with some phenotypic and transcriptional characteristics of long-lived plasma cells. Using principal component analysis, we visualized the phenotypic relationships between major B-cell subsets in the intestine and blood, and revealed that IgM(+) memory B cells (MBCs) and naive B cells were phenotypically related as were CD27(-) MBCs and switched MBCs. ASCs in the intestine and blood were highly clonally related, but associated with distinct trajectories of phenotypic development. VP6-specific B cells were present among diverse B-cell subsets in immune donors, including naive B cells, with phenotypes representative of the overall B-cell pool. These data provide a high dimensional view of intestinal B cells and the determinants regulating humoral memory to a ubiquitous, mucosal pathogen at steady-state.

Localization of membrane permeabilization and receptor binding sites on the VP4 hemagglutinin of rotavirus: implications for cell entry.

The surface of rotavirus is decorated with 60 spike-like projections, each composed of a dimer of VP4, the viral hemagglutinin. Trypsin cleavage of VP4 generates two fragments, VP8*, which binds sialic acid (SA), and VP5*, containing an integrin binding motif and a hydrophobic region that permeabilizes membranes and is homologous to fusion domains. Although the mechanism for cell entry by this non-enveloped virus is unclear, it is known that trypsin cleavage enhances viral infectivity and facilitates viral entry. We used electron cryo-microscopy and difference map analysis to localize the binding sites for two neutralizing monoclonal antibodies, 7A12 and 2G4, which are directed against the SA-binding site within VP8* and the membrane permeabilization domain within VP5*, respectively. Fab 7A12 binds at the tips of the dimeric heads of VP4, and 2G4 binds in the cleft between the two heads of the spike. When these binding results are combined with secondary structure analysis, we predict that the VP4 heads are composed primarily of beta-sheets in VP8* and that VP5* forms the body and base primarily in beta-structure and alpha-helical conformations, respectively. Based on these results and those of others, a model is proposed for cell entry in which VP8* and VP5* mediate receptor binding and membrane permeabilization, and uncoating occurs during transfer across the lipid bilayer, thereby generating the transcriptionally active particle.

Proteolysis of monomeric recombinant rotavirus VP4 yields an oligomeric VP5* core.

Rotavirus particles are activated for cell entry by trypsin cleavage of the outer capsid spike protein, VP4, into a hemagglutinin, VP8*, and a membrane penetration protein, VP5*. We have purified rhesus rotavirus VP4, expressed in baculovirus-infected insect cells. Purified VP4 is a soluble, elongated monomer, as determined by analytical ultracentrifugation. Trypsin cleaves purified VP4 at a number of sites that are protected on the virion and yields a heterogeneous group of protease-resistant cores of VP5*. The most abundant tryptic VP5* core is trimmed past the N terminus associated with activation for virus entry into cells. Sequential digestion of purified VP4 with chymotrypsin and trypsin generates homogeneous VP8* and VP5* cores (VP8CT and VP5CT, respectively), which have the authentic trypsin cleavages in the activation region. VP8CT is a soluble monomer composed primarily of beta-sheets. VP5CT forms sodium dodecyl sulfate-resistant dimers. These results suggest that trypsinization of rotavirus particles triggers a rearrangement in the VP5* region of VP4 to yield the dimeric spikes observed in icosahedral image reconstructions from electron cryomicroscopy of trypsinized rotavirus virions. The solubility of VP5CT and of trypsinized rotavirus particles suggests that the trypsin-triggered conformational change primes VP4 for a subsequent rearrangement that accomplishes membrane penetration. The domains of VP4 defined by protease analysis contain all mapped neutralizing epitopes, sialic acid binding residues, the heptad repeat region, and the membrane permeabilization region. This biochemical analysis of VP4 provides sequence-specific structural information that complements electron cryomicroscopy data and defines targets and strategies for atomic-resolution structural studies.

Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood.

The functional status of virus-specific CD8+ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8+ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNgamma) or tumor necrosis factor-alpha (TNFalpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8+ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8+ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.

Protective intestinal anti-rotavirus B cell immunity is dependent on alpha 4 beta 7 integrin expression but does not require IgA antibody production.

Rotavirus (RV) is the main cause of severe gastroenteritis in young children; protection has been correlated with intestinal Ab responses. Using a mouse model of RV infection and beta(7)-deficient (beta(7)(-/-)) mice, which do not express alpha(4)beta(7) integrin, we demonstrated the importance of alpha(4)beta(7) integrin in B cell-mediated anti-RV immunity. beta(7)(-/-) mice acutely infected with murine RV resolved infection and developed normal serum IgG Abs but had diminished intestinal IgA responses. alpha(4)beta(7)(-/-) immune B cells did not resolve RV infection when adoptively transferred into RV-infected Rag-2-deficient mice. Fewer RV-specific B cells were found in the intestine of Rag-2-deficient mice transferred with beta(7)(-/-) B cells compared with wild type. The absence of alpha(4)beta(7) expression and/or a lower frequency of IgA-producing cells among transferred beta(7)(-/-) B cells could have accounted for the inability of these cells to resolve RV infection following passive transfer. To distinguish between these possibilities, we studied the importance of IgA production in RV infection using IgA-deficient (IgA(-/-)) mice. IgA(-/-) mice depleted of CD8(+) T cells were able to clear primary RV infection. Similarly, adoptive transfer of immune IgA(-/-) B cells into chronically infected Rag-2-deficient mice resolved RV infection. We further demonstrated in both wild-type and IgA(-/-) mice that, following oral RV infection, protective B cells reside in the alpha(4)beta(7)(high) population. Our findings suggest that alpha(4)beta(7) integrin expression is necessary for B cell-mediated immunity to RV independent of the presence of IgA.

CCR7 expression and memory T cell diversity in humans.

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag alpha(4)beta(7) integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.

Rotavirus VP6 expressed by PVX vectors in Nicotiana benthamiana coats PVX rods and also assembles into viruslike particles.

The rotavirus major inner capsid protein (VP6) has been expressed in Nicotiana benthamiana plants using vectors based on potato virus X (PVX). VP6 was expressed either as a fusion with the PVX coat protein or from an additional subgenomic promoter inserted to enable both VP6 and PVX coat protein to be expressed independently. Both approaches yielded VP6, which retained the ability to form trimers. VP6 expressed from the subgenomic promoter assembled into paracrystalline sheets and tubes. Expression as a fusion protein yielded PVX rods that presented an external "overcoat" of VP6, but unexpectedly, some rotavirus protein also assembled into icosahedral viruslike particles (VLPs). The assembly of viral protein into VLPs suggests that prior display of VP6 on the flexuous PVX rod facilitates the subsequent assembly of VP6 into stable icosahedral particles.

Protective immunity to rotavirus shedding in the absence of interleukin-6: Th1 cells and immunoglobulin A develop normally.

We investigated whether interleukin-6 (IL-6) was required for the development of immunoglobulin A (IgA)- and T-helper 1 (Th1)-associated protective immune responses to rotavirus by using adult IL-6-deficient mice [BALB/c and (C57BL/6 x O1a)F(2) backgrounds]. Naive IL-6(-) mice had normal frequencies of IgA plasma cells in the gastrointestinal tract. Consistent with this, total levels of IgA in fecal extracts, saliva, and sera were unaltered. In specific response to oral infection with rhesus rotavirus, IL-6(-) and IL-6(+) mice exhibited efficient Th1-type gamma interferon responses in Peyer's patches with high levels of serum IgG2a and intestinal IgA. Although there was an increase in Th2-type IL-4 in CD4(+) T cells from IL-6(-) mice following restimulation with rotavirus antigen in the presence of irradiated antigen-presenting cells, unfractionated Peyer's patch cells failed to produce a significant increase in IL-4. Moreover, virus-specific IgG1 in serum was not significantly increased in IL-6(-) mice in comparison with IL-6(+) mice. Following oral inoculation with murine rotavirus, IL-6(-) and IL-6(+) mice mediated clearance of rotavirus and mounted a strong IgA response. When IL-6(-) and IL-6(+) mice [(C57BL/6 x O1a)F(2) background] were orally inoculated with rhesus rotavirus and later challenged with murine rotavirus, all of the mice maintained high levels of IgA in feces and were protected against reinfection. Thus, IL-6 failed to provide unique functions in the development of IgA-secreting B cells and in the establishment of Th1-associated protective immunity against rotavirus infection in adult mice.

Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses.

Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.

Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate.

BACKGROUND & AIMS: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. Successful vaccine development is crucial in controlling global HCV infection. We have previously described the generation of HCV-like particles (HCV-LPs) in insect cells using a recombinant baculovirus containing the complementary DNA of the HCV structural proteins. These HCV-LPs had similar morphological and biophysical properties as the putative virions. In this study, we analyzed the structural features, antigenic composition, seroreactivity, and immunogenicity of purified HCV-LPs. METHODS: HCV-LPs were analyzed by electron microscopy and antibody immunolabeling and precipitation. An enzyme-linked immunosorbent assay (ELISA) using HCV-LPs was developed. The humoral response to HCV-LPs in mice was studies by core and envelope ELISAs, Western immunoblotting, and immunofluorescence. RESULTS: Structural and antigenic compositions of HCV-LPs were shown to be similar to those of putative HCV virions. Using the HCV-LP ELISA, high-titer anti-HCV antibodies were detected in individuals infected with various HCV genotypes. In vivo, HCV-LPs elicited a humoral response broadly directed against HCV structural proteins. CONCLUSIONS: HCV-LPs resemble HCV virions and are capable of inducing a humoral response targeted against various regions of HCV structural proteins, suggesting that HCV-LPs may be promising as a potential vaccine candidate.

The epithelial cell response to rotavirus infection.

Rotavirus is the most important worldwide cause of severe gastroenteritis in infants and young children. Intestinal epithelial cells are the principal targets of rotavirus infection, but the response of enterocytes to rotavirus infection is largely unknown. We determined that rotavirus infection of HT-29 intestinal epithelial cells results in prompt activation of NF-kappaB (<2 h), STAT1, and ISG F3 (3 h). Genetically inactivated rotavirus and virus-like particles assembled from baculovirus-expressed viral proteins also activated NF-kappaB. Rotavirus infection of HT-29 cells induced mRNA for several C-C and C-X-C chemokines as well as IFNs and GM-CSF. Mice infected with simian rotavirus or murine rotavirus responded similarly with the enhanced expression of a profile of C-C and C-X-C chemokines. The rotavirus-stimulated increase in chemokine mRNA was undiminished in mice lacking mast cells or lymphocytes. Rotavirus induced chemokines only in mice <15 days of age despite documented infection in older mice. Macrophage inflammatory protein-1beta and IFN-stimulated protein 10 mRNA responses occurred, but were reduced in p50-/- mice. Macrophage inflammatory protein-1beta expression during rotavirus infection localized to the intestinal epithelial cell in murine intestine. These results show that the intestinal epithelial cell is an active component of the host response to rotavirus infection.

Studies of the role for NSP4 in the pathogenesis of homologous murine rotavirus diarrhea.

A rotavirus (RV) nonstructural protein, NSP4, has recently been proposed to function as an enterotoxin in the pathogenesis of RV diarrhea. The role of NSP4 in the pathogenesis of RV diarrhea was examined by infecting cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice with virulent murine RV and by comparing deduced amino acid sequences of RV gene 10 encoding NSP4 from three distinct sets of virulent and tissue culture-adapted avirulent variant RVs. Homozygous CFTR (CFTR-/-) mice, which do not respond to any known intestinal secretagogues, experienced diarrhea comparable to that in normal CFTR+/+ littermates after RV challenge. Comparison of amino acid sequences of NSP4 from virulent and attenuated pairs of RVs failed to show consistent or significant changes. Together, these data suggest that enterotoxigenic properties of RV NSP4 are not critical in the pathogenesis of murine RV diarrhea and that attenuation of murine RVs is not usually mediated by mutations in the gene encoding NSP4.

Hepatitis G virus: is it a hepatitis virus?

Hepatitis G virus (HGV) and GB virus C (GBV-C) are two newly discovered viral agents, different isolates of a positive-sense RNA virus that represents a new genus of Flaviviridae. The purpose of this review is to analyze new data that have recently been published on the epidemiology and associations between HGV and liver diseases such as posttransfusion hepatitis, acute and chronic non-A-E hepatitis, fulminant hepatitis, cryptogenic cirrhosis, and hepatocellular carcinoma. The role of HGV in coinfection with other hepatitis viruses, the response to antiviral therapy, and the impact of HGV on liver transplantation are also discussed. HGV is a transmissible blood-borne viral agent that frequently occurs as a coinfection with other hepatitis viruses due to common modes of transmission. The prevalence of HGV ranges from 0.9 to 10% among blood donors throughout the world and is found in 1.7% of volunteer blood donors in the United States. The majority of patients infected with HGV by blood transfusion do not develop chronic hepatitis, but hepatitis G viremia frequently persists without biochemical evidence of hepatitis. Serum HGV RNA has been found in 0 to 50% of patients with fulminant hepatitis of unknown etiology and 14 to 36% of patients with cryptogenic cirrhosis. The association between HGV and chronic non-A-E hepatitis remains unclear. Although HGV appears to be a hepatotrophic virus, its role in independently causing acute and chronic liver diseases remains uncertain.

Production and characterization of murine IgA monoclonal antibodies to the surface antigens of rhesus rotavirus.

Rotavirus is the single most important cause of severe diarrhea in humans and is diffuse in most animal species worldwide, and an understanding of the antigenic properties of the virus is essential to the design of rational vaccine strategies. To better understand the localization of viral epitopes involved in antibody-mediated neutralization of virus infectivity, we have orally immunized mice with live rhesus rotavirus (RRV) and generated a panel of hybridoma cell clones secreting IgA class monoclonal antibodies. A total of 12 neutralizing IgA MAbs to VP4 and VP7 proteins were studied for their epitope specificity and topographical relationships by hemagglutination-inhibition assays, neutralization assays, and competitive-binding assays with previously mapped MAbs. In addition, neutralization-escape virus mutants were selected and gene segments for each variant were cloned and sequenced. Two IgA MAbs were found to be directed to the antigenic region A of the VP7 protein at amino acid 94, and 10 MAbs were directed at the VP8 trypsin cleavage fragment of VP4. Five of the VP4-specific MAbs identified the same neutralization epitope on the RRV VP8 protein, not previously associated with RRV neutralization. All neutralization-escape variants selected by this antibody group contained mutations at amino acids 132- 135 of VP4. One IgA MAb selected for a mutation at amino acid 190 of VP4, and the corresponding viral mutant failed to agglutinate erythrocytes. This MAb mapped to an epitope recognized by 2 additional IgA MAbs. These results suggest that oral immunization of mice with RRV elicits an IgA immune response which is predominantly directed toward antigenic determinants on the VP8 portion of VP4. As a consequence, the route of immunization may alter immunodominant neutralization responses elicited to rotavirus.

Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity.

Rotaviruses are the leading cause of severe gastroenteritis and dehydrating diarrhea in young children and animals worldwide. A murine model and "backpack tumor" transplantation were used to determine the protective effect of antibodies against VP4(an outer capsid viral protein) and VP6(a major inner capsid viral protein). Only two non-neutralizing immunoglobulin A (IgA) antibodies to VP6 were capable of preventing primary and resolving chronic murine rotavirus infections. These antibodies were not active, however, when presented directly to the luminal side of the intestinal tract. These findings support the hypothesis that in vivo intracellular viral inactivation by secretory IgA during transcytosis is a mechanism of host defense against rotavirus infection.

Role of B cells and cytotoxic T lymphocytes in clearance of and immunity to rotavirus infection in mice.

The immune mechanisms involved in clearance of and immunity to rotavirus infection are poorly understood. Although mice with severe combined immunodeficiency (SCID mice) become chronically infected, nude mice have been reported to clear rotavirus infection similarly to immunocompetent controls. To better characterize the role of cytotoxic T lymphocytes (CTLs) in clearance of and immunity to rotavirus infection, we infected naive or previously infected beta 2-microglobulin (beta 2m) knockout mice with murine rotavirus. Naive beta 2m knockout mice shed rotavirus antigen 2 days longer than did normal control mice but completely resolved primary infection. beta 2m knockout naive mice treated with depleting doses of an anti-CD8 monoclonal antibody before infection shed viral antigen for an additional day. Upon rechallenge, beta 2m knockout mice, either treated with the anti-CD8 antibody or not treated, were completely resistant to reinfection. Clearance of rotavirus infection in naive beta 2m knockout mice correlated with the development of intestinal rotavirus-specific immunoglobulin A. Before rechallenge, beta 2m knockout mice had high levels of intestinal rotavirus-specific immunoglobulin A. These findings suggest that CTLs mediate rotavirus clearance but are not required for this function and that CTLs are not necessary for development of immunity to rotavirus reinfection. To further characterize the effector mechanisms involved in clearance and prevention of rotavirus infection, similar studies were performed with B-cell-deficient JHD knockout mice. After primary infection, most naive JHD mice had similar virus-shedding clearance curves as did control mice and completely resolved primary infection. However, 2 of 29 became chronically infected. All JHD mice treated with anti-CD8 antibody became chronically infected with murine rotavirus. Upon rechallenge, JHD mice which had cleared primary infection were all susceptible to reinfection. These findings suggest that B cells also play a role in clearance of primary infection but are absolutely necessary for development of immunity against rotavirus reinfection.

Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses.

Hepatitis C virus (HCV) encodes two putative virion glycoproteins (E1 and E2) which are released from the polyprotein by signal peptidase cleavage. In this report, we have characterized the complexes formed between E1 and E2 (called E1E2) for two different HCV strains (H and BK) and studied their intracellular localization. Vaccinia virus and Sindbis virus vectors were used to express the HCV structural proteins in three different cell lines (HepG2, BHK-21, and PK-15). The kinetics of association between E1 and E2, as studied by pulse-chase analysis and coprecipitation of E2 with an anti-E1 monoclonal antibody, indicated that formation of stable E1E2 complexes is slow. The times required for half-maximal association between E1 and E2 were 60 to 85 min for the H strain and more than 165 min for the BK strain. In the presence of nonionic detergents, two forms of E1E2 complexes were detected. The predominant form was a heterodimer of E1 and E2 stabilized by noncovalent interactions. A minor fraction consisted of heterogeneous disulfide-linked aggregates, which most likely represent misfolded complexes. Posttranslational processing and localization of the HCV glycoproteins were examined by acquisition of endoglycosidase H resistance, subcellular fractionation, immunofluorescence, cell surface immunostaining, and immunoelectron microscopy. HCV glycoproteins containing complex N-linked glycans were not observed, and the proteins were not detected at the cell surface. Rather, the proteins localized predominantly to the endoplasmic reticular network, suggesting that some mechanism exists for their retention in this compartment.

Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein.

We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase.

Characterization of hepatitis C virus structural proteins with a recombinant baculovirus expression system.

We cloned and expressed the sequences encoding the structural proteins of the hepatitis C virus in a baculovirus eukaryotic expression system. Four recombinant constructs expressed sufficient hepatitis C virus-specific proteins in insect cell culture to allow analysis of protein cleavage, glycosylation and immunoreactivity. Using immunoblot analysis, we detected a 22-kD protein corresponding to the hepatitis C virus capsid protein cleaved from a larger precursor. Recombinant constructs encoding the presumptive envelope (E1) protein produced products ranging from 30 to 35 kD, whereas constructs encoding the presumptive E2/NS1 protein expressed products ranging in size from 68 to 73 kD. The recombinant envelope proteins were glycosylated, as shown by sensitivity to endoglycosidase F digestion, whereas the capsid was not. We examined the immunoreactivity of these recombinant proteins using sera from 50 patients chronically infected with HCV. Forty-seven of 50 of these sera contained antibodies against the capsid, 14 (28%) also had antibodies against E1 and at least 5 (10%) had antibody against E2/NS1. Forty-seven of 50 sera (94%) were viremic, as determined on hepatitis C virus polymerase chain reaction. The three sera that were hepatitis C virus polymerase chain reaction negative did not have envelope antibodies, whereas all sera that had envelope antibodies were also hepatitis C virus polymerase chain reaction positive. Thus antibodies to baculovirus-expressed hepatitis C virus structural proteins, including E1 and E2/NS1, are found in the presence of viremia.