Polypeptide semicarbazide glass slide microarrays: characterization and comparison with amine slides in serodetection studies

We have described in the accompanying article the preparation of peptide-protein semicarbazide microarrays and their use for the simultaneous serodetection of antibodies directed against different pathogens. Here, we present a comparative study between semicarbazide and amine glass slides in an immunofluorescent serodetection assay using HIV (Gp120, Gp41), HCV (mix-HCV, core, NS3, and NS4), and HBV (HBs) recombinant antigens. Amine and semicarbazide surfaces displayed the same sensitivity for antibodies detection just after printing. However, the reactivity of protein antigens changed rapidly upon aging on amine slides but not on semicarbazide slides. Peptide or protein semicarbazide microarrays were found to be remarkably stable for months. Additional data concerning the characterization of the semicarbazide surface (homogeneity of the slides, chemical stability, contact angle measurements, atomic force microscopy studies, reproducibility of serodetection results) are also presented and discussed.     

Preparation of monoclonal antibodies against duck hepatitis B core antigen and analysis of the monoclonal antibodies

Mice were immunized against duck hepatitis B virus core (DHBc) particles isolated from the liver of asymptomatic carrier ducks of duck hepatitis B virus (DHBV) by ultracentrifugation. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 12 clones of hybridoma cells secreting antibodies against DHBc (anti-DHBc) were isolated. According to the reactivity to core particles and core peptide obtained from DHBc particles treated with SDS-2ME, the 12 antibodies were classified into two groups. Two monoclonal antibodies reacted against both core particles and core peptide (B-type), the other ten monoclonal antibodies reacted against core particles but did not react against core peptide obtained from DHBc particles treated with SDS-2 ME. (A-type). Solid phase enzyme immuno assay (EIA) using these two types of antibodies could detect core antigenisity not only in the liver homogenate but also in the DHBV infected serum. Sucrose gradient analysis and gel filtration analysis revealed this DHBc antigenisity in the serum is not carried by core particles but carried by core peptide, equivalent to HBe antigen in the serum of Hepatitis B virus (HBV) carrier. This EIA may provide sensitive test monitoring both serum DHBe antigen levels and DHBc antigen levels in the liver during DHBV infection.     

A simple, rapid, and sensitive integrated protein microarray for simultaneous detection of multiple antigens and antibodies of five human hepatitis viruses (HBV, HCV, HDV, HEV, and HGV)

Protein microarrays for parallel detection of multiple viral antigens and antibodies have not yet been described in the field of human hepatitis virus infections. Here, we describe a simple, rapid and sensitive integrated protein microarray with three different reaction models. The integrated protein microarray could simultaneously determine in human sera two viral antigens (HBsAg, HBeAg) and seven viral antibodies (HBsAb, HBcAb, HBeAb, HCVAb, HDVAb, HEVAb, HGVAb) of human hepatitis viruses within 20 min. The results of the protein microarray were assessed directly by the naked eye but can also be analyzed by a quantitative detector. The detection limit of this protein microarray was 0.1 ng/ml for HBsAg. Overall, >85% concordance was observed between the integrated protein microarrays and an enzyme-linked immunosorbent assay for above hepatitis viral antigen and antibody detections in human sera. This integrated protein microarray can be easily optimized for clinical use and epidemiological screening for multiple hepatitis virus infections.     

Evidence for hepatitis C viral infection in patients with primary hepatocellular carcinoma.

In testing for antibodies to the hepatitis C virus (anti-HCV) in 112 patients with primary hepatocellular carcinoma, 10 of 33 white patients (30%) and 15 of 79 Asian patients (19%) had a positive response to the antibody. The antibody profile to individual hepatitis C viral antigens and the presence of circulating hepatitis C viral RNA were determined in the 25 patients. The anti-HCV antibodies most frequently detected were toward the antigens from the core (C22) and NS3 regions. Serum hepatitis C viral RNA was present in 17 of the 25 patients (68%), and these patients tended to have serum levels of alanine and aspartate aminotransferases higher than those patients without viremia (136 +/- 22 U per liter versus 64 +/- 11 U per liter and 161 +/- 26 U per liter versus 79 +/- 14 U per liter, respectively, both P < .05). Of the 15 Asian patients with hepatocellular carcinoma and anti-HCV, 4 (27%) had coexisting hepatitis B surface antigen (HBsAg) and 13 (87%) had antibodies to either hepatitis B core or surface antigen. Of the 10 white patients with anti-HCV, however, only 1 (10%) had hepatitis B virus antibodies (P < .01). Among 4 Asian patients with coexisting anti-HCV and HBsAg, 1 was found to have serum hepatitis B viral DNA and the other 3 had hepatitis C viral RNA. A history of blood transfusion was obtained from 12 of the 25 patients with anti-HCV (48%); 20 (80%) had coexisting cirrhosis. Our findings support the hypothesis that hepatitis C virus is an important etiologic agent in the development of primary hepatocellular carcinoma in both white and Asian patients in the United States.     

A Sensitive Serodiagnosis of Hepatitis C Virus (HCV) Infection with Two Non-Fused Peptides: Comparison of Antibody Responses Detected with a Newly Developed Assay and a Commercial Second-Generation Test

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of anti-HCV antibody. We assayed for antibodies against either oligopeptide (S29-1) deduced from the nucleocapsid gene or the product of nonstructural region (NS3) synthesized in a recombinant Escherichia coli (S4). To reduce false-positive results induced by non-specific binding of antibodies with a carrier protein and to increase the sensitivity of an immunoassay, non-fused S4 peptide was prepared by the recombinant DNA technique and site-specific proteolysis (by factor Xa). In 71 non-A, non-B hepatitis patients with chronic liver disease, 70 (98.5%) were positive by S29-1/S4 ELISA as well as by a second-generation test (Abbott II). On the other hand, of 40 serum samples from blood donors, in which anti-N14 (core) and C100-3 antibodies were not detected but hepatitis C virus (HCV) RNA was detectable by polymerase chain reaction (PCR), 24 (60%) were positive by S29-1/S4 ELISA, whereas only 18 (45%) were diagnosed by Abbott II. In addition, based on results in a small group of 92 blood donors, detection of anti-S29-1/S4 antibody correlated well with HCV viremia as confirmed by PCR. These results indicated that the preparation of non-fused protein (S4) by recombinant DNA technique and a combination of S29-1 and S4 as immobilized antigens in an ELISA provide a sensitive and specific diagnosis for HCV infection with good correlation with the presence of viral RNA as confirmed by PCR.     

Relation between Reactivity to the NS-4 Region Peptides of Hepatitis C Virus (HCV) and Clinical Features among Patients Infected with HCV Genotype 1b

Nearly all patients infected with hepatitis C virus (HCV) genotype 1b have reactivity to the core (c22-3) or non-structural (NS)-3 region (c33c) protein in a second-generation recombinant immunoblot assay (RIBA-2). However, reactivities to the NS-4 region antigens (5-1-1, c100-3) vary among patients. To clarify whether differences in serological reactivities to the NS-4 antigens are associated with the clinical features or response to interferon (IFN) therapy of patients infected with hepatitis C virus (HCV) genotype 1b, we clinically investigated 115 such patients. Positive reactions to 5-1-1 and c100-3 were seen in 75.7 and 79.1%, respectively, of the patients. There were no differences between the patients with and those without antibodies to NS-4 region antigens (5-1-1, c100-3) with regard to age, duration of HCV infection, severity of liver disease and virus load. Fifty-one of the patients were treated with recombinant IFN-α, and 17 of the 51 patients showed sustained response to the therapy. The sustained response was more frequently seen in the patients positive for antibodies to both 5-1-1 and c100-3 as compared with those negative for either or both antibodies (41.0% vs. 8.3%, P < 0.05).     

Atomic force microscopy detection of serological markers of viral hepatites B and C

The possibility of detection of serological markers, containing the hepatitis B surface antigen (HBsAg) and hepatitis C virus core-antigen (HCVcoreAg) in human serum, by a new atomic force microscopy (AFM)-based nanotechnological approach has been demonstrated. The antibodies against the hepatitis B virus surface antigen (anti-HBsAg) and the antibodies against the hepatitis C virus core antigen (anti-HCVcoreAg) were immobilized on an AFM-chip. It was shown that such approach enables to detect HBsAg, HCVcoreAg and the viral fragments containing these antigens in the serum. The comparative analysis of detection of HBsAg- and HCVcoreAg-containing particles by the AFM method versus traditional methods (ELISA, PCR) has demonstrated the 75% coincidence of results between the AFM and two other methods.     

Core antigen and antibody in woodchucks after infection with woodchuck hepatitis virus

The woodchuck hepatitis virus is a naturally occurring hepatitis B-like virus that infects the eastern woodchuck. Direct immunofluorescence staining for woodchuck hepatitis virus core antigen in liver biopsies demonstrated the presence of this antigen in 14 of 17 chronically infected woodchucks, and in 8 of 10 woodchucks undergoing acute infections. Fluorescent localization of woodchuck hepatitis virus core antigen was typically cytoplasmic, and this was confirmed further by electron microscopy. Experimental infection with woodchuck hepatitis virus was achieved in four of four woodchucks inoculated with serum from chronic carrier woodchucks. All infected animals developed a self-limited disease characterized by seroconversion to antibodies against the major viral antigens (core and surface antigens); naturally acquired acute infection demonstrated a similar course. A chimpanzee seronegative for all markers of hepatitis B virus developed a subclinical infection after inoculation with woodchuck hepatitis virus.     

Protective vaccination with hepatitis C virus NS3 but not core antigen in a novel mouse challenge model

BACKGROUND: Efficient vaccines against hepatitis C virus (HCV) infection are urgently needed. Vaccine development has been hampered by the lack of suitable small animal models to reliably test the protective capacity of immmunization. METHODS: We used recombinant murine gammaherpesvirus 68 (MHV-68) as a novel challenge virus in mice and tested the efficacy of heterologous candidate human vaccines based on modified vaccinia virus Ankara or adenovirus, both delivering HCV non-structural NS3 or core proteins. RESULTS: Recombinant MHV-68 expressing NS3 (MHV-68-NS3) or core (MHV-68-core) were constructed and characterized in vitro and in vivo. Mice immunized with NS3-specific vector vaccines and challenged with MHV-68-NS3 were infected but showed significantly reduced viral loads in the acute and latent phase of infection. NS3-specific CD8+ T cells were amplified in immunized mice after challenge with MHV-68-NS3. By contrast, we did neither detect a reduction of viral load nor an induction of core-specific CD8+ T cells after core-specific immunization. CONCLUSIONS: Our data suggest that the challenge system using recombinant MHV-68 is a highly suitable model to test the immunogenicity and protective capacity of HCV candidate vaccine antigens. Using this system, we demonstrated the usefulness of NS3-specific immunization. By contrast, our analysis rather discarded core as a vaccine antigen.     

Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease.

In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation.     

Antibodies to different virus antigens in patients with chronic hepatitis C

A total of 176 hospital patients with chronic hepatitis C (CHC), among them 110 males and 66 females, were examined. The spectrum of antibodies to four hepatitis C virus (HCV) proteins (core, NS3, NS4, NS5) and in 142 patients --IgM antibodies to HCV (anti-HCV IgM) were determined. In 92% of the CHC patients antibodies to core, NS3 and NS4 proteins were simultaneously detected. Differences in the detection of antibodies to HCV in males and females were not statistically reliable. In CHC patients aged up to 20 years anti-NS4 and anti-NS5 were less frequently detected. Among males of different age groups reliable differences in the detection rate of anti-NS5 were registered, while among females of different age groups no such differences were observed. With the increase of age these antibodies were detected somewhat more often. In females over 60 years anti-HCV IgM occurred more often than in males of the same age. The levels of alanine aminotransferase (ALT) were higher in persons with the presence of anti-NS5 and anti-HCV IgM than in persons with their absence. In all groups of CHC patients with biochemical activity and liver cirrhosis the detection rate of anti-HCV IgM was significantly higher than in patients with normal ALT activity. The antibody spectrum with the simultaneous absence of HCV IgM and anti-NS5, while found to contain antibodies to other HCV antigens, was registered significantly less frequently in patients with moderate and high CHC activity and the liver cirrhosis induced by HCV infection.     

Identification of Peptide Leads to Inhibit Hepatitis C Virus: Inhibitory Effect of Plectasin Peptide Against Hepatitis C Serine Protease

The emerging of hepatitis C virus (HCV) resistant strains has been considered as a main drawback of the available drugs. Since HCV has a large inactive surface, we would like to hypothesis that the mutation occur in HCV is minimal and causing less resistance against inhibition. In this study, a short peptide inhibitor of HCV namely plectasin was identified by HCV NS3-4A serine protease assay. Plectasin peptide showed considerable inhibition against HCV NS3-4A serine protease. Enzymatic activity of the recombinant NS3-4Apro was analysed by fluorescence release from several fluorogenic peptide substrates which resembling the dibasic cleavage site sequences of the flavivirus polyprotein precursor. Of all amc-labelled peptides, Pyr-RTKR-amc was the most efficiently cleaved substrate with the lowest Km value of 20 µM. The kinetic assay showed that plectasin peptide inhibited NS3-4Apro activity with an IC₅₀ value of 4.3 μM compared to the aprotinin as a standard proteases inhibitor with an IC₅₀ of 6.1 μM. From the results, plectasin peptide also demonstrated a dose-dependent inhibition of HCV replication with a considerable reduction in RLuc activity at 15 µM using HCV replicon- containing Huh-7 cells. Our study has identified a unique natural peptide that can be used to highlight novel structures for the development of drug derivatives with high efficacy of HCV NS3-4A protease inhibitors.     

Serodiagnosis of infectious diseases with antigen microarrays

AIMS: To generate protein microarrays by printing microbial antigens on slides to enable the simultaneous determination in human sera of antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus (HSV) types 1 and 2. METHODS AND RESULTS: Antigens were printed on activated glass slides using high-speed robotics. The slides were incubated with serum samples and subsequently with fluorescently labelled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected using confocal scanning microscopy and quantified with internal calibration curves. The microarray assay could detect as little as 0.5 pg of both IgG and IgM bound onto the glass surface. Precision profiles ranged from 1.7 to 18.5% for all the antigens. Microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. Overall >80% concordance was obtained between microarray and ELISA kits in the classification of sera. CONCLUSIONS: These results indicate that the microarray is a suitable assay format for the serodiagnosis of infectious diseases. SIGNIFICANCE AND IMPACT OF STUDY: Antigen microarrays can be optimized for clinical use, their performance is equivalent to ELISA but they offer significant advantages in throughput, convenience and cost.     

Intrahepatic hepatitis C virus replication correlates with chronic hepatitis C disease severity in vivo

The role of viral factors in the pathogenesis of chronic hepatitis C is unknown. The objective of the present study was to characterize markers of hepatitis C virus (HCV) infection and replication in liver biopsy specimens obtained from 65 genotype 1-infected subjects, including 31 who were coinfected with human immunodeficiency virus (HIV), and to analyze associations between intrahepatic viral markers and hepatitis C disease severity. The percentages of liver cells harboring HCV genomes (%G) and replicative-intermediate RNAs (%RI) were evaluated using strand-specific in situ hybridization, while HCV core and NS3 antigens were assessed by immunocytochemistry. HIV-positive and HIV-negative subjects had similar mean grades and stages of liver disease and had similar indices of HCV infection and replication in liver, even though coinfected subjects had significantly shorter mean disease duration (P = 0.0003). Multivariate analysis showed that %G was not associated with grade or stage of liver disease (P = 0.5 and 0.4, respectively), while %RI was strongly associated with liver inflammation (P < 0.001), liver fibrosis (P < 0.001), and serum alanine aminotransferase levels (P = 0.01). NS3 antigen (but not core) was more frequently detected in HCV RI-positive versus RI-negative specimens (P = 0.028). These findings demonstrate a link between HCV proliferation and hepatitis C disease severity and suggest similar pathogenic mechanisms in HIV-positive and HIV-negative individuals.     

Carbohydrate antigens of human megakaryocytes and platelet glycoproteins: a comparative study

Until now, carbohydrate antigens of human megakaryocytes have not been studied very extensively. For this reason, we investigated the staining pattern of 25 lectins and carbohydrate-specific monoclonal antibodies on paraffin-embedded trephine biopsies and acetone-fixed smears from patients with reactive and neoplastic bone marrow lesions. A biotin-streptavidin-alkaline phosphatase assay was used to visualize the binding of lectins or antibodies. Ulex europaeus agglutinin I (UEA-I) stained megakaryocytes in all cases tested. Monoclonal antibodies detecting fucosylated Lewis type 2 chain antigens (19-OLE, 12-4LE and LeuM1) were also reactive. Several lectins detecting backbone and core oligosaccharides [Helix pomatia agglutinin (HPA), peanut agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), soybean agglutinin (SBA)] bound to megakaryocytes only after neuraminidase digestion. Moreover, we investigated human platelet lysates to gain some information about the carbohydrate residues of platelet glycoproteins which are synthesized by megakaryocytes. The carbohydrate expression of platelets showed striking similarities to that of megakaryocytes. Immunoblotting experiments revealed a strong binding of UEA-I, 19-OLE and 12-4LE to a band isographic to glycoprotein (gp) Ib. After desialylation of glycoproteins transblotted to nitrocellulose, ECA and PNA also reacted with a band of this molecular weight. Gp Ib is known to contain a mucin-like peptide core with a great number of potential O-glycosylation sites. Therefore, it is tempting to speculate that carbohydrate residues characterized in this study are involved in the complex biological interactions of gp Ib.     

A novel recombinant multiepitope protein as a hepatitis C diagnostic intermediate of high sensitivity and specificity

A novel recombinant multiepitope protein has been designed that consists of six linear, immunodominant, and phylogenetically conserved epitopes from hepatitis C virus. Five of these antigens (core, NS3, NS4I, NS4II, and NS5) are being used in many of the third-generation kits while sixth epitope (core3g) is an additional sequence from a newly identified Indian isolate. The genes for these epitopes have been joined together to code for a single multiepitope protein that has been evaluated for its diagnostic potential for the detection of anti-HCV antibodies in human plasma. Two separate synthetic genes have been designed, both encoding the same six epitopes in a single open reading frame along with spacers having additional amino acids to function as flexible (r-HCV-F-MEP) or rigid (r-HCV-R-MEP) linkers. High-level expression of hepatitis C multiepitope protein in Escherichia coli has been achieved. The protein has been purified using a single affinity step yielding >25 mg pure protein/liter culture and used as the coating antigen in anti-HCV EIA. The use of this multiepitope protein eliminates the requirement for multiple diagnostic intermediates for the development of anti-HCV diagnostic kit. The sensitivity and specificity of the HCV multiepitope protein was evaluated by Boston Biomedica Worldwide Performance Panels, HCV Seroconversion Panels and Viral Co-infection Panels, and was found to be comparable with commercially available anti-HCV EIA kits. This analysis indicated its unequivocal performance as capture antigen in anti-HCV EIA. The high epitope density, careful choice of epitopes and use of E. coli system for expression, coupled with simple purification protocol provides the potential for the development of an inexpensive diagnostic test with high degree of sensitivity and specificity.     

Assessment of synthetic chimeric multiple antigenic peptides for diagnosis of GB virus C infection

The use of synthetic peptides of both structural and nonstructural proteins of GB virus C (GBV-C) has been studied for the development of new systems to diagnose infection caused by this virus. In an attempt to increase the antigenicity of linear peptide sequences, chimeric multiple antigenic peptides (MAPs) containing epitopes from E2, NS4, and NS5 GBV-C proteins have been synthesized. The synthetic constructs were evaluated by ELISA to establish whether the epitopes in chimeric branched peptides are more efficiently recognized by the specific antibodies compared to the monomeric linear sequences. Moreover, we have investigated the application of a commercial biosensor instrument for the detection of antibodies against the GBV-C in human serum samples. The results of the immunoassays reported in this work highlight the usefulness of synthetic tetrameric branched peptides containing sequences from envelope and nonstructural GBV-C proteins for the diagnosis of GBV-C infection. The potential clinical value of the MAP₄(E2-NS5a) for the serodiagnosis of GBV-C infection was demonstrated, thus providing the basis for performing prevalence studies of the infection among the hemodialyzed and hepatitis C virus (HCV)-infected population.     

Chips from chips: application to the study of antibody responses to methylated proteins

Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named "chips from chips", which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen.     

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.     

Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response

Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, na?ve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the na?ve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-gamma), protein-specific lymphoproliferative responses, IFN-gamma, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.