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Humoral immunity to immunodominant epitopes of Hepatitis C virus in individuals infected with genotypes 1a or 1b
Clinical Immunology 111 (2004) 22 – 27 www.elsevier.com/locate/yclim
Rapid Communication
Humoral immunity to immunodominant epitopes of Hepatitis C virus in individuals infected with genotypes 1a or 1b Maria P. Carlos, Yasuhiro Yamamura, Quynh Vu, Kendra Conzen, David E. Anderson, and Jose´ V. Torres * Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA 95616, USA AIDS Research Program, Ponce School of Medicine, Ponce, Puerto Rico Received 9 October 2002; accepted with revision 17 November 2003
Abstract Cellular immunity against multiple Hepatitis C virus (HCV) proteins is observed in patients acutely infected with HCV most of whom later resolve infection. We wished to assess humoral immunity in patients infected with HCV 1a or 1b genotypes in relation to viral load using plasma samples from HCV-infected individuals and a panel of peptides representing immunodominant epitopes of HCV structural and nonstructural proteins. Plasma from HCV 1a- and 1b-infected patients, respectively, were divided into two groups: patients with low viral load (V100,000 RNA copies/ml) and patients with high viral load (z10,000,000 RNA copies/ml). The antigens were peptides representing epitopes from immunodominant regions of HCV core, E2, NS3, and NS4 proteins, as well as the hypervariable (HVR) epitopes in E2 from genotypes 1a and 1b. Individuals infected with HCV 1a evoked a stronger immune response to many immunodominant epitopes of HCV relative to individuals infected with HCV 1b. Moreover, among individuals infected with HCV 1a, those with low viral loads mounted significantly greater responses against these epitopes than did individuals with high viral loads. Our observations demonstrate that quantitatively different antibody responses are elicited against HCV depending on the genotype of infecting virus, and suggest that humoral immunity directed against multiple immunodominant epitopes in HCV 1a-infected individuals may help lower viral load in vivo. D 2004 Elsevier Inc. All rights reserved. Keywords: Hepatitis C virus; Antibody; Hepatocellular carcinoma; Genotype; Humoral
Introduction Several studies have suggested that humoral immunity directed against Hepatitis C virus (HCV) can influence the course of disease. HCV from experimentally infected healthy chimpanzees was neutralized following in vitro incubation with autologous antibodies [1 – 3]. Our study compares humoral immunity against HCV in individuals infected with HCV 1a and 1b genotypes, and correlates the strength of the antibody response with the viral load in each individual. Antibody responses to HCV 1a and 1b were evaluated with a panel of peptides representing
* Corresponding author. Department of Medical Microbiology and Immunology, School of Medicine, University of California, Tupper Hall Room 3134, Davis, CA 95616. Fax: +1-530-752-8692. E-mail address: [email protected] (J.V. Torres). 1521-6616/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2003.11.012
conserved immunodominant epitopes derived from multiple HCV proteins. Comparing antibody responses to epitopes based on homologous HCV subtypes among individuals infected with HCV genotype 1a or genotype 1b revealed that individuals infected with genotype 1a mounted significantly greater antibody responses than did patients infected with genotype 1b. The enhanced humoral response was directed at most of the epitopes in our panel. Within genotype 1a, the strength of the antibody response was inversely correlated with viral load. In contrast, individuals infected with genotype 1b mounted poor antibody responses to these epitopes regardless of viral load. The results demonstrate quantitative differences in humoral immunity against HCV depending on the infecting viral genotype, and furthermore suggest that differences in the vigor of the antibody response may influence viral load and potentially the course of disease.
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Materials and methods Subjects Archived plasma samples from HCV 1a- and 1b-infected patients were obtained from the AIDS Research Program at the Ponce School of Medicine in Puerto Rico and were confirmed positive for HCV 1a or 1b by ELISA and RTPCR. Plasma samples were randomly selected from chronically infected HCV patients under Ribavirin treatment who had signed informed consent forms. All tests and procedures were approved by the IRB at the Ponce School of Medicine, Puerto Rico. All plasma samples were first screened by the Abbott HCV EIA 2.0 test (Abbott Laboratories, Abbott Park, IL) that utilizes recombinant antigens c100-3, HC-31, and HC-34. ELISA-positive samples were then assessed for their HCV viral loads (HCV 1a or 1b RNA copies/ml) by the Amplicor HCV Monitor procedure (Roche Diagnostics Corp., Indianapolis, IN). Those samples with viral loads above the linear range of the assay were repeated after 100-fold dilution. Low viral load (4783 – 154,816 RNA copies/ml) and high viral load (2,937,300 –25,667,700 copies/ml) of the HCV genotypes were determined by line probe assay (LiPA) utilizing the Inno-LiPA HCV II (Innogenetics, N.V., Zwijndrecht, Belgium). Amplification products of the HCV Amplicor procedure were used for the LiPA assay. The ALT levels (U/l) ranged from 28 to 130 for the low viral load group and 36 to 221 for the high viral load group. Peptide synthesis Peptides representing defined epitopes of the immunodominant regions of Core (aa 7 –18), E2 (aa 484 – 499), NS3 (aa 1248 –1265), and NS4 (aa 1767– 1786), as well as peptides from the hypervariable regions of E2 HVR1 genotype 1a (aa 386 –406), E2 HVR2 genotype 1a (aa 472 –485), E2 HVR1 genotype 1b (aa 386 –406), and E2 HVR2 genotype 1b (aa 472 –485) were synthesized (Table 1). These epitopes are present in the core, E2, NS3, and NS4 proteins, including two previously characterized hypervariable regions of the E2 protein. An epitope of the core region (aa 7– 18), characterized as a B-cell epitope [4], overlaps with a CTL epitope (aa 2 –10) [5]. A sequence in the E2 protein (aa 484– 499) is also
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an immunodominant B-cell epitope [6] and overlaps with a CTL epitope (aa 489– 496) [7]. Sequences in NS3 (aa 1248– 1265) and NS4 (aa 1767 – 1786) are immunodominant CD4+ T-cell epitopes [5]. All peptides were synthesized by 9fluoroenylmethoxycarbonyl (Fmoc) chemistry as described previously [8]. The synthesis was performed using an automatic peptide synthesizer (PS-3, Rainin-Protein Technologies, CA). The final peptides were then cleaved and deprotected using trifluoroacetic acid. Peptides were lyophilized and dialyzed in distilled water. Enzyme-linked immunosorbent assay (ELISA) Dry peptide enzyme-linked immunosorbent assay (ELISA) was performed to determine the titers of antibodies present in patient plasma that could bind to conserved immunodominant epitopes of HCV Core, E2, NS3, and NS4 proteins. Binding of antibodies to the peptide was assessed using a previously described protocol [8]. Briefly, all test plasma were heat-inactivated for 30 min at 56jC. Flat-bottomed microtiter plates (Corning; Corning, NY) were coated overnight at 37jC with the peptides at a concentration of 5 Ag/well in 0.05 M sodium bicarbonate buffer. The plates were blocked with blocking buffer (10% blotto) for 2 h at room temperature and washed with Wash Solution (KPL, Gaithersburg, MD) before the plasma were added and the plates were incubated for 1 h at 37jC. Patient plasma, in addition to five control samples negative for HIV-1, HCV, and HTLV-1, were tested at 1:100, 1:500, 1:1000, 1:5000, and 1:10,000 dilutions. Following incubation with the test plasma, the plates were washed again and alkaline phosphatase-labeled secondary antibody (goat-antihuman IgG AP, Fisher, CA) was added at a 1:1000 dilution. Following incubation for 1 h at 37jC and washing, p-nitrophenyl phosphate substrate (KPL, Gaithersburg, MD) was added. The OD was measured spectrophotometrically at 405 nm with a BioRad automatic plate reader according to the manufacturer’s instructions (Model 3550, Hercules, CA). Endpoint antibody titers were defined as the greatest dilution of sample that maintained an optical density at least twice that of the average of control sera tested at the same dilution. The assays were repeated at least twice. The mean and the standard deviations of the OD readings were calculated. Average standard deviation ranged from 0.001
Table 1 Synthesized peptides representing defined immunodominant epitopes of HCV proteins Protein
Genbank accession no.
Position (aa)
Sequence
References
Core E2 E2 HVR1 1a E2 HVR2 1a E2 HVR1 1b E2 HVR2 1b NS3 NS4
BAA01582 BAA01582 AAB67038 AAB67038 CAB53095 CAB53095 BAA01582 BAA01582
7 – 18 484 – 499 383 – 406 472 – 485 383 – 406 472 – 485 1248 – 1265 1767 – 1786
PQRKTKRNTNRR PYCWHYPPKPCGIVPA AETHVTGGNAGRTTAGLVGLLTPG ISYANGSGLDERPY GGTYVTGGTMAKNTLGITSLFSPG ITYNESHSSDQRPY GYKVLVLNPSVAATLGFG NFISGIQYLAGLSTLPGNPA
[12,15] [14] [16] [16] [17] [17] [5] [5]
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to 0.05. Student’s t test and ANOVA were performed (GraphPad InStat version 2.03 for Mac, GraphPad Software, San Diego CA USA, http://www.graphpad.com) to determine if there were statistically significant differences in epitope recognition among patient groups.
Results Archived plasma samples for HCV genotype 1a were selected and classified into two groups of low and high viral load. Individuals with low viral load were defined as those
Fig. 1. Endpoint antibody binding titers against immunodominant epitopes present among HCV proteins. (A) Comparison of antibody titers to HCV epitopes among sera from patients infected with HCV genotype 1a (solid circles) or genotype 1b (open circles). (B) Comparison of antibody titers among patients infected with HCV genotype 1a with low (solid circles) or high (open circles) viral loads. (C) Comparison of antibody titers among patients infected with HCV genotype 1b with low (solid circles) or high (open circles) viral loads.
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that had measurable levels of virus RNA less than approximately 100,000 copies/ml as determined by Amplicor HCV Monitor procedure. Patients with high viral load possessed virus RNA copies that were typically greater than 10,000,000 copies/ml. Archived plasma samples from patients infected with HCV genotype 1b were selected and stratified in the same fashion. Patient ALT levels were in the range of 28 – 221 U/l when infected with genotype 1a and 9 – 434 U/l for genotype 1b. Statistical analysis by the Student’s t test was used to compare ALT levels between patients with HCV genotypes 1a and 1b; HCV 1a low viral load and 1a high viral load; and HCV 1b low viral load and 1b high viral load. Differences in ALT levels did not reach statistical significance for any of the comparisons. Peptides representing immunodominant epitopes of HCV 1a and 1b core, E2, E2 HVR1, E2 HVR2, NS3, and NS4 proteins were synthesized and used in ELISA to determine endpoint antibody titers present in plasma from genotyped HCV-infected patients relative to a panel of samples from uninfected controls. Scatter dot plots of antibody reactivity in individuals infected with genotype 1a and 1b are shown in Fig. 1. Results with P V 0.05 were considered statistically significant. At the lowest plasma dilution tested (1:100), all samples were reactive with all HCV peptides. As samples were diluted further, significant differences became apparent in terms of genotype and viral load. As shown in Fig. 1, panel A, patients infected with genotype 1a (n = 23) had significantly higher antibody titers against both conserved and hypervariable epitopes in the E2 envelope protein as well as nonstructural NS3 and NS4 protein epitopes than did patients infected with genotype 1b (n = 23). A trend toward significance was observed with antibody titers against the E2 HVR2 epitope ( P = 0.056). These data demonstrate that individuals infected with genotype 1a possess higher concentrations of antibodies directed against multiple viral proteins than individuals infected with genotype 1b. We also determined that there is a correlation between antibody titers and viral loads among individuals infected with HCV genotype 1a (Fig. 1, panel B). HCV 1ainfected patients with low viral loads (n = 11) had significantly higher antibody titers against several of the immunodominant epitopes than did 1a patients with high viral loads (n = 12). Statistical analysis demonstrated an inverse correlation between viral load and antibody titers against epitopes from E2 ( P = 0.040), E2 HVR1 ( P = 0.012), and NS4 ( P = 0.008). Also, trends toward statistical significance were observed against the E2 HVR2 ( P = 0.061) and NS3 epitopes ( P = 0.070). In general, patients infected with HCV genotype 1b had much lower antibody titers to the immunodominant epitopes examined regardless of viral load. We observed a significant correlation between antibody titer to an epitope from HCV core and viral load among patients infected with genotype 1b ( P = 0.028) (Fig. 1, panel C). Those
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with low viral loads (n = 11) possessed higher titers of antibodies than those with high viral loads (n = 12). No difference in the magnitude of antibody responses was observed to other immunodominant regions of HCV in patients infected with genotype 1b regardless of viral load.
Discussion In this study, we compared the strength and breadth of the antibody response to immunodominant HCV epitopes present in regions of the core, E2, NS3, and NS4 proteins among individuals infected with HCV genotypes 1a and 1b. Reactivity in the study populations varied significantly depending on both HCV viral load and genotype. Previous studies have described viral load as a parameter related to the pathogenesis of HCV. In some reports, high titers of viremia were correlated with advanced stages of liver disease [9,10]. Contrastingly, others have found no correlation between viremia and either histological changes or alanine aminotransferase (ALT) activities [11,12]. Because the relationship between viral load and liver damage is unclear, the only assumption made in the present study was that viral load in the plasma would substantially reflect the amount of virus present in the liver. This notion is supported by the observation of a direct correlation between the level of HCV-RNA in liver biopsies and the level of plasma viremia in chronically infected patients [13]. A multicenter study of chronically infected patients without therapy evaluated HCV-RNA serum levels longitudinally on a monthly basis over the course of over 2 years [14]. Monthly measurements indicated that HCV-RNA levels fluctuated with a median change of approximately 1 log. Accordingly, we chose to stratify our patient cohorts into groups that had viral loads that differed by approximately 2 logs. In this way, despite fluctuations in viral loads over time, we could be reasonably confident that samples were derived from individuals with persistently high versus low levels of HCV viremia. Numerous studies have demonstrated that viral loads are higher and disease progression more rapid in immunosuppressed patients with HCV [15 –18]. These data indicate that immunity directed against HCV does impact viral load (i.e., viral infection, replication, or survival) in infected individuals. Indeed, individuals who are acutely infected with HCV and resolve infection possess strong CTL and T helper cell responses directed against multiple structural and nonstructural proteins early after infection [19 – 26]. These data reinforce the concept that the concerted efforts of antibody, T helper cell, and CTL responses directed against epitopes in multiple viral proteins are needed to control or resolve HCV infection [27 –29]. Consistent with these observations, we found that among individuals infected with HCV genotype 1a, significantly higher anti-
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body titers were present against multiple structural (E2) and nonstructural (NS3 and NS4) proteins in those with low viral loads relative to those with high viral loads. One interpretation of this association is that the antibody pool mounted against HCV infection contains not only binding antibodies but also virus-neutralizing antibodies [30], particularly those directed against the E2 protein. Similar conclusions have been drawn in several studies that evaluated neutralizing binding activity of sera from HCVinfected individuals and HCV-vaccinated chimpanzees [1 –3]. However, infection of humans with HCV genotype 1a results in immunity that is rarely protective. Moreover, while a strong E2-specific antibody response may help lower viral load, it may also create strong immune pressure, leading to selection of new viral variants in vivo [31 – 33]. Ultimately, a longitudinal study is necessary to determine if those patients with robust antibody responses observed against multiple epitopes eventually resolve infection or fail to develop hepatocellular carcinoma. Several reports have previously shown that humoral immune responses in HCV-infected individuals target epitopes within the core, envelope, NS3, and NS4 proteins [34 – 36]. In our study, all HCV 1a-infected patients, regardless of viral load, had antibodies that recognized epitopes in all of the structural and nonstructural proteins examined. Individuals with low viral loads had higher antibody titers directed against the envelope protein, and in particular against the HVR1 region which contains a neutralizing epitope. They also possessed higher antibody titers against NS3 and NS4 proteins. The manner in which antibodies directed against nonstructural proteins may decrease viral load is unclear. One explanation is that high antibody titers to NS3 and NS4 epitopes examined are indirect consequences of increased recognition and destruction of infected cells with subsequent release of NS3 and NS4 antigens. Regardless of the viral load in a given individual, patients infected with strains of HCV genotype 1a mounted much greater antibody responses against structural and nonstructural epitopes of HCV than did patients infected with strains of HCV genotype 1b. Among HCV genotype 1b-infected patients, those with low viral loads had significantly higher antibody titers to the epitope in the core protein than did those with high viremia. This contrasted with patients infected with HCV genotype 1a, in which there was no correlation between viral load and recognition of this epitope. Thus, the antibody response directed against HCV appears to be influenced by the genotype of the infecting virus.
Acknowledgments Funding was provided by the NIH NCRR-RCMI (G12RR03050), the UC Davis Academic Senate, and the National Cancer Center.
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Rapid Communication
Humoral immunity to immunodominant epitopes of Hepatitis C virus in individuals infected with genotypes 1a or 1b Maria P. Carlos, Yasuhiro Yamamura, Quynh Vu, Kendra Conzen, David E. Anderson, and Jose´ V. Torres * Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, CA 95616, USA AIDS Research Program, Ponce School of Medicine, Ponce, Puerto Rico Received 9 October 2002; accepted with revision 17 November 2003
Abstract Cellular immunity against multiple Hepatitis C virus (HCV) proteins is observed in patients acutely infected with HCV most of whom later resolve infection. We wished to assess humoral immunity in patients infected with HCV 1a or 1b genotypes in relation to viral load using plasma samples from HCV-infected individuals and a panel of peptides representing immunodominant epitopes of HCV structural and nonstructural proteins. Plasma from HCV 1a- and 1b-infected patients, respectively, were divided into two groups: patients with low viral load (V100,000 RNA copies/ml) and patients with high viral load (z10,000,000 RNA copies/ml). The antigens were peptides representing epitopes from immunodominant regions of HCV core, E2, NS3, and NS4 proteins, as well as the hypervariable (HVR) epitopes in E2 from genotypes 1a and 1b. Individuals infected with HCV 1a evoked a stronger immune response to many immunodominant epitopes of HCV relative to individuals infected with HCV 1b. Moreover, among individuals infected with HCV 1a, those with low viral loads mounted significantly greater responses against these epitopes than did individuals with high viral loads. Our observations demonstrate that quantitatively different antibody responses are elicited against HCV depending on the genotype of infecting virus, and suggest that humoral immunity directed against multiple immunodominant epitopes in HCV 1a-infected individuals may help lower viral load in vivo. D 2004 Elsevier Inc. All rights reserved. Keywords: Hepatitis C virus; Antibody; Hepatocellular carcinoma; Genotype; Humoral
Introduction Several studies have suggested that humoral immunity directed against Hepatitis C virus (HCV) can influence the course of disease. HCV from experimentally infected healthy chimpanzees was neutralized following in vitro incubation with autologous antibodies [1 – 3]. Our study compares humoral immunity against HCV in individuals infected with HCV 1a and 1b genotypes, and correlates the strength of the antibody response with the viral load in each individual. Antibody responses to HCV 1a and 1b were evaluated with a panel of peptides representing
* Corresponding author. Department of Medical Microbiology and Immunology, School of Medicine, University of California, Tupper Hall Room 3134, Davis, CA 95616. Fax: +1-530-752-8692. E-mail address: [email protected] (J.V. Torres). 1521-6616/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2003.11.012
conserved immunodominant epitopes derived from multiple HCV proteins. Comparing antibody responses to epitopes based on homologous HCV subtypes among individuals infected with HCV genotype 1a or genotype 1b revealed that individuals infected with genotype 1a mounted significantly greater antibody responses than did patients infected with genotype 1b. The enhanced humoral response was directed at most of the epitopes in our panel. Within genotype 1a, the strength of the antibody response was inversely correlated with viral load. In contrast, individuals infected with genotype 1b mounted poor antibody responses to these epitopes regardless of viral load. The results demonstrate quantitative differences in humoral immunity against HCV depending on the infecting viral genotype, and furthermore suggest that differences in the vigor of the antibody response may influence viral load and potentially the course of disease.
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Materials and methods Subjects Archived plasma samples from HCV 1a- and 1b-infected patients were obtained from the AIDS Research Program at the Ponce School of Medicine in Puerto Rico and were confirmed positive for HCV 1a or 1b by ELISA and RTPCR. Plasma samples were randomly selected from chronically infected HCV patients under Ribavirin treatment who had signed informed consent forms. All tests and procedures were approved by the IRB at the Ponce School of Medicine, Puerto Rico. All plasma samples were first screened by the Abbott HCV EIA 2.0 test (Abbott Laboratories, Abbott Park, IL) that utilizes recombinant antigens c100-3, HC-31, and HC-34. ELISA-positive samples were then assessed for their HCV viral loads (HCV 1a or 1b RNA copies/ml) by the Amplicor HCV Monitor procedure (Roche Diagnostics Corp., Indianapolis, IN). Those samples with viral loads above the linear range of the assay were repeated after 100-fold dilution. Low viral load (4783 – 154,816 RNA copies/ml) and high viral load (2,937,300 –25,667,700 copies/ml) of the HCV genotypes were determined by line probe assay (LiPA) utilizing the Inno-LiPA HCV II (Innogenetics, N.V., Zwijndrecht, Belgium). Amplification products of the HCV Amplicor procedure were used for the LiPA assay. The ALT levels (U/l) ranged from 28 to 130 for the low viral load group and 36 to 221 for the high viral load group. Peptide synthesis Peptides representing defined epitopes of the immunodominant regions of Core (aa 7 –18), E2 (aa 484 – 499), NS3 (aa 1248 –1265), and NS4 (aa 1767– 1786), as well as peptides from the hypervariable regions of E2 HVR1 genotype 1a (aa 386 –406), E2 HVR2 genotype 1a (aa 472 –485), E2 HVR1 genotype 1b (aa 386 –406), and E2 HVR2 genotype 1b (aa 472 –485) were synthesized (Table 1). These epitopes are present in the core, E2, NS3, and NS4 proteins, including two previously characterized hypervariable regions of the E2 protein. An epitope of the core region (aa 7– 18), characterized as a B-cell epitope [4], overlaps with a CTL epitope (aa 2 –10) [5]. A sequence in the E2 protein (aa 484– 499) is also
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an immunodominant B-cell epitope [6] and overlaps with a CTL epitope (aa 489– 496) [7]. Sequences in NS3 (aa 1248– 1265) and NS4 (aa 1767 – 1786) are immunodominant CD4+ T-cell epitopes [5]. All peptides were synthesized by 9fluoroenylmethoxycarbonyl (Fmoc) chemistry as described previously [8]. The synthesis was performed using an automatic peptide synthesizer (PS-3, Rainin-Protein Technologies, CA). The final peptides were then cleaved and deprotected using trifluoroacetic acid. Peptides were lyophilized and dialyzed in distilled water. Enzyme-linked immunosorbent assay (ELISA) Dry peptide enzyme-linked immunosorbent assay (ELISA) was performed to determine the titers of antibodies present in patient plasma that could bind to conserved immunodominant epitopes of HCV Core, E2, NS3, and NS4 proteins. Binding of antibodies to the peptide was assessed using a previously described protocol [8]. Briefly, all test plasma were heat-inactivated for 30 min at 56jC. Flat-bottomed microtiter plates (Corning; Corning, NY) were coated overnight at 37jC with the peptides at a concentration of 5 Ag/well in 0.05 M sodium bicarbonate buffer. The plates were blocked with blocking buffer (10% blotto) for 2 h at room temperature and washed with Wash Solution (KPL, Gaithersburg, MD) before the plasma were added and the plates were incubated for 1 h at 37jC. Patient plasma, in addition to five control samples negative for HIV-1, HCV, and HTLV-1, were tested at 1:100, 1:500, 1:1000, 1:5000, and 1:10,000 dilutions. Following incubation with the test plasma, the plates were washed again and alkaline phosphatase-labeled secondary antibody (goat-antihuman IgG AP, Fisher, CA) was added at a 1:1000 dilution. Following incubation for 1 h at 37jC and washing, p-nitrophenyl phosphate substrate (KPL, Gaithersburg, MD) was added. The OD was measured spectrophotometrically at 405 nm with a BioRad automatic plate reader according to the manufacturer’s instructions (Model 3550, Hercules, CA). Endpoint antibody titers were defined as the greatest dilution of sample that maintained an optical density at least twice that of the average of control sera tested at the same dilution. The assays were repeated at least twice. The mean and the standard deviations of the OD readings were calculated. Average standard deviation ranged from 0.001
Table 1 Synthesized peptides representing defined immunodominant epitopes of HCV proteins Protein
Genbank accession no.
Position (aa)
Sequence
References
Core E2 E2 HVR1 1a E2 HVR2 1a E2 HVR1 1b E2 HVR2 1b NS3 NS4
BAA01582 BAA01582 AAB67038 AAB67038 CAB53095 CAB53095 BAA01582 BAA01582
7 – 18 484 – 499 383 – 406 472 – 485 383 – 406 472 – 485 1248 – 1265 1767 – 1786
PQRKTKRNTNRR PYCWHYPPKPCGIVPA AETHVTGGNAGRTTAGLVGLLTPG ISYANGSGLDERPY GGTYVTGGTMAKNTLGITSLFSPG ITYNESHSSDQRPY GYKVLVLNPSVAATLGFG NFISGIQYLAGLSTLPGNPA
[12,15] [14] [16] [16] [17] [17] [5] [5]
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to 0.05. Student’s t test and ANOVA were performed (GraphPad InStat version 2.03 for Mac, GraphPad Software, San Diego CA USA, http://www.graphpad.com) to determine if there were statistically significant differences in epitope recognition among patient groups.
Results Archived plasma samples for HCV genotype 1a were selected and classified into two groups of low and high viral load. Individuals with low viral load were defined as those
Fig. 1. Endpoint antibody binding titers against immunodominant epitopes present among HCV proteins. (A) Comparison of antibody titers to HCV epitopes among sera from patients infected with HCV genotype 1a (solid circles) or genotype 1b (open circles). (B) Comparison of antibody titers among patients infected with HCV genotype 1a with low (solid circles) or high (open circles) viral loads. (C) Comparison of antibody titers among patients infected with HCV genotype 1b with low (solid circles) or high (open circles) viral loads.
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that had measurable levels of virus RNA less than approximately 100,000 copies/ml as determined by Amplicor HCV Monitor procedure. Patients with high viral load possessed virus RNA copies that were typically greater than 10,000,000 copies/ml. Archived plasma samples from patients infected with HCV genotype 1b were selected and stratified in the same fashion. Patient ALT levels were in the range of 28 – 221 U/l when infected with genotype 1a and 9 – 434 U/l for genotype 1b. Statistical analysis by the Student’s t test was used to compare ALT levels between patients with HCV genotypes 1a and 1b; HCV 1a low viral load and 1a high viral load; and HCV 1b low viral load and 1b high viral load. Differences in ALT levels did not reach statistical significance for any of the comparisons. Peptides representing immunodominant epitopes of HCV 1a and 1b core, E2, E2 HVR1, E2 HVR2, NS3, and NS4 proteins were synthesized and used in ELISA to determine endpoint antibody titers present in plasma from genotyped HCV-infected patients relative to a panel of samples from uninfected controls. Scatter dot plots of antibody reactivity in individuals infected with genotype 1a and 1b are shown in Fig. 1. Results with P V 0.05 were considered statistically significant. At the lowest plasma dilution tested (1:100), all samples were reactive with all HCV peptides. As samples were diluted further, significant differences became apparent in terms of genotype and viral load. As shown in Fig. 1, panel A, patients infected with genotype 1a (n = 23) had significantly higher antibody titers against both conserved and hypervariable epitopes in the E2 envelope protein as well as nonstructural NS3 and NS4 protein epitopes than did patients infected with genotype 1b (n = 23). A trend toward significance was observed with antibody titers against the E2 HVR2 epitope ( P = 0.056). These data demonstrate that individuals infected with genotype 1a possess higher concentrations of antibodies directed against multiple viral proteins than individuals infected with genotype 1b. We also determined that there is a correlation between antibody titers and viral loads among individuals infected with HCV genotype 1a (Fig. 1, panel B). HCV 1ainfected patients with low viral loads (n = 11) had significantly higher antibody titers against several of the immunodominant epitopes than did 1a patients with high viral loads (n = 12). Statistical analysis demonstrated an inverse correlation between viral load and antibody titers against epitopes from E2 ( P = 0.040), E2 HVR1 ( P = 0.012), and NS4 ( P = 0.008). Also, trends toward statistical significance were observed against the E2 HVR2 ( P = 0.061) and NS3 epitopes ( P = 0.070). In general, patients infected with HCV genotype 1b had much lower antibody titers to the immunodominant epitopes examined regardless of viral load. We observed a significant correlation between antibody titer to an epitope from HCV core and viral load among patients infected with genotype 1b ( P = 0.028) (Fig. 1, panel C). Those
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with low viral loads (n = 11) possessed higher titers of antibodies than those with high viral loads (n = 12). No difference in the magnitude of antibody responses was observed to other immunodominant regions of HCV in patients infected with genotype 1b regardless of viral load.
Discussion In this study, we compared the strength and breadth of the antibody response to immunodominant HCV epitopes present in regions of the core, E2, NS3, and NS4 proteins among individuals infected with HCV genotypes 1a and 1b. Reactivity in the study populations varied significantly depending on both HCV viral load and genotype. Previous studies have described viral load as a parameter related to the pathogenesis of HCV. In some reports, high titers of viremia were correlated with advanced stages of liver disease [9,10]. Contrastingly, others have found no correlation between viremia and either histological changes or alanine aminotransferase (ALT) activities [11,12]. Because the relationship between viral load and liver damage is unclear, the only assumption made in the present study was that viral load in the plasma would substantially reflect the amount of virus present in the liver. This notion is supported by the observation of a direct correlation between the level of HCV-RNA in liver biopsies and the level of plasma viremia in chronically infected patients [13]. A multicenter study of chronically infected patients without therapy evaluated HCV-RNA serum levels longitudinally on a monthly basis over the course of over 2 years [14]. Monthly measurements indicated that HCV-RNA levels fluctuated with a median change of approximately 1 log. Accordingly, we chose to stratify our patient cohorts into groups that had viral loads that differed by approximately 2 logs. In this way, despite fluctuations in viral loads over time, we could be reasonably confident that samples were derived from individuals with persistently high versus low levels of HCV viremia. Numerous studies have demonstrated that viral loads are higher and disease progression more rapid in immunosuppressed patients with HCV [15 –18]. These data indicate that immunity directed against HCV does impact viral load (i.e., viral infection, replication, or survival) in infected individuals. Indeed, individuals who are acutely infected with HCV and resolve infection possess strong CTL and T helper cell responses directed against multiple structural and nonstructural proteins early after infection [19 – 26]. These data reinforce the concept that the concerted efforts of antibody, T helper cell, and CTL responses directed against epitopes in multiple viral proteins are needed to control or resolve HCV infection [27 –29]. Consistent with these observations, we found that among individuals infected with HCV genotype 1a, significantly higher anti-
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body titers were present against multiple structural (E2) and nonstructural (NS3 and NS4) proteins in those with low viral loads relative to those with high viral loads. One interpretation of this association is that the antibody pool mounted against HCV infection contains not only binding antibodies but also virus-neutralizing antibodies [30], particularly those directed against the E2 protein. Similar conclusions have been drawn in several studies that evaluated neutralizing binding activity of sera from HCVinfected individuals and HCV-vaccinated chimpanzees [1 –3]. However, infection of humans with HCV genotype 1a results in immunity that is rarely protective. Moreover, while a strong E2-specific antibody response may help lower viral load, it may also create strong immune pressure, leading to selection of new viral variants in vivo [31 – 33]. Ultimately, a longitudinal study is necessary to determine if those patients with robust antibody responses observed against multiple epitopes eventually resolve infection or fail to develop hepatocellular carcinoma. Several reports have previously shown that humoral immune responses in HCV-infected individuals target epitopes within the core, envelope, NS3, and NS4 proteins [34 – 36]. In our study, all HCV 1a-infected patients, regardless of viral load, had antibodies that recognized epitopes in all of the structural and nonstructural proteins examined. Individuals with low viral loads had higher antibody titers directed against the envelope protein, and in particular against the HVR1 region which contains a neutralizing epitope. They also possessed higher antibody titers against NS3 and NS4 proteins. The manner in which antibodies directed against nonstructural proteins may decrease viral load is unclear. One explanation is that high antibody titers to NS3 and NS4 epitopes examined are indirect consequences of increased recognition and destruction of infected cells with subsequent release of NS3 and NS4 antigens. Regardless of the viral load in a given individual, patients infected with strains of HCV genotype 1a mounted much greater antibody responses against structural and nonstructural epitopes of HCV than did patients infected with strains of HCV genotype 1b. Among HCV genotype 1b-infected patients, those with low viral loads had significantly higher antibody titers to the epitope in the core protein than did those with high viremia. This contrasted with patients infected with HCV genotype 1a, in which there was no correlation between viral load and recognition of this epitope. Thus, the antibody response directed against HCV appears to be influenced by the genotype of the infecting virus.
Acknowledgments Funding was provided by the NIH NCRR-RCMI (G12RR03050), the UC Davis Academic Senate, and the National Cancer Center.
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