Protective immune response to hepatitis C virus ... - Wiley Online Library

Protective Immune Response to Hepatitis C Virus in Chimpanzees Rechallenged Following Clearance of Primary Infection SUZANNE E. BASSETT,1 BERNADETTE GUERRA,2 KATHLEEN BRASKY,3 EMIL MISKOVSKY,4 MICHAEL HOUGHTON,5 GARY R. KLIMPEL,1 AND ROBERT E. LANFORD2

Hepatitis C virus (HCV) infections were evaluated in chimpanzees that had previously cleared HCV and were rechallenged. Animals that had previously cleared HCV infection rapidly cleared homologous and heterologous virus upon rechallenge, indicative of a strong protective immunity. In one animal, sterilizing immunity was observed with regard to viremia, although viral RNA was transiently detected in the liver. Accelerated viral clearance following rechallenge with HCV was observed in animals that had not been exposed to HCV for over 16 years, suggesting that long-lasting protective immunity may be possible. The ability of peripheral blood mononuclear cells (PBMC) to recognize HCV proteins was evaluated during the course of the rechallenge experiments. A very early and strong in vitro recall response to HCV nonstructural proteins appeared to be associated with viral clearance. In contrast, proliferative responses to HCV proteins were not observed in 4 persistently infected chimpanzees, and a weak proliferative response was observed in 1 of 2 animals during acute resolving infection. The results suggest that a strong T-cell proliferative response is induced upon rechallenge of chimpanzees with HCV and that this response is associated with rapid viral clearance. The antibody response to HCV proteins increased by over 1,000-fold in all animals following rechallenge as well. A more complete understanding of the role of the cellular immune response in the clearance of HCV and the nature of the protective immune response following viral clearance may aid in the generation of therapies and vaccines. (HEPATOLOGY 2001;33:1479-1487.)

Abbreviations: HCV, hepatitis C virus; CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cells; ALT, alanine transaminase; RT-PCR, reverse transcriptase-polymerase chain reaction; ge, genome equivalent; ELISA, enzyme-linked immunosorbent assay; SI, stimulation index. From the 1Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX; 2Department of Virology and Immunology, and 3Department of Laboratory Animal Medicine, Southwest Research Primate Research Center, Southwest Foundation for Biomedical Research, San Antonio, TX; 4Department of Internal Gastroenterology, University of Texas Medical Branch at Galveston, Galveston, TX; and 5Chiron Corporation, Emeryville, CA. Received November 20, 2000; accepted February 27, 2001. Supported by grants T32 AI07536, U19 AI40035, and P51 RR13986 from the National Institutes of Health. Address reprint requests to: Robert E. Lanford, Ph.D., Department of Virology and Immunology, Southwest Regional Primate Research Center, Southwest Foundation for Biomedical Research, 7620 NW Loop 410, San Antonio, TX 78227. E-mail: [email protected]; fax: 210-670-3329. Copyright © 2001 by the American Association for the Study of Liver Diseases. 0270-9139/01/3306-0018$35.00/0 doi:10.1053/jhep.2001.24371

Hepatitis C virus (HCV) infections represent a serious health problem. The majority of HCV infections develop into chronic infections that may progress to cirrhosis and hepatocellular carcinoma.1 HCV is classified in the Hepacivirus genus of the Flaviviridae family.2 The HCV genome is approximately 9.6 kb and consists of single-stranded RNA of positive polarity. The viral RNA has a single large open reading frame that encodes for a polyprotein of approximately 3,000 amino acids.3 The structural proteins are located at the amino terminal end of the polyprotein and include the capsid protein and 2 envelope glycoproteins, E1 and E2. The nonstructural proteins are preceded by a p7 domain of unknown function and include NS2-NS5. The NS2 domain forms an autoprotease with the amino-terminal portion of NS3. The amino terminus of NS3 encodes a serine protease and the carboxy terminus encodes a helicase, which plays a role in viral RNA replication. NS4A is a cofactor for the serine protease. The viral RNAdependent RNA polymerase is encoded by NS5B.4 The functions of NS4B and NS5A are unknown. The chimpanzee is the only animal model for studying HCV infection. Humans and chimpanzees with persistent HCV infections mount an antibody response to most HCV proteins.5 HCV-specific antibody does not appear to protect humans and chimpanzees from infection and is actually associated with active viremia rather than viral clearance. The kinetics of antibody production to HCV proteins and the pattern of antibodies to individual proteins do not appear to predict disease outcome (clearance versus persistence). The humoral immune response to the nonstructural HCV proteins appears to be similar in humans and chimpanzees.5 In contrast, antibody responses to HCV structural proteins are observed less frequently in chimpanzees than in humans for reasons not understood.5-11 Studies in chimpanzees have revealed that antibody neutralization of HCV is not easily attained.12,13 Recently, Cooper et al. observed that strong antibody responses to HCV proteins were not necessary for viral clearance in HCV-inoculated chimpanzees.14 Several investigators have also observed that circulating HCV-specific antibodies do not prevent reinfection of chimpanzees with HCV.15-18 Therefore, T cells may play a more critical role than antibodies in the resolution of HCV infection. HCV antigen-specific CD8⫹ T cells have been observed in the peripheral blood and liver of humans and chimpanzees during HCV infection.5,19 A multispecific cytotoxic T lymphocyte (CTL) response may control HCV replication to some extent, yet the cellular immune response appears to be incapable of clearing the virus in most individuals.20-23 Recently, the CTL responses from humans and chimpanzees that

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cleared HCV infection have been analyzed.14,24 Liver biopsies specimens were obtained during the course of acute infection in chimpanzees that either cleared HCV infection or became persistently infected.14 Viral clearance in these animals was associated with an early and broad CTL response to multiple HCV epitopes. Similar to the findings in chimpanzees, a strong and persistent T-cell response was associated with viral clearance in humans acutely infected with HCV and in HCVseronegative humans with exposure to HCV.24-27 CD4⫹ T cells appear to be important in the clearance of HCV infection. Several studies have suggested an association between a vigorous in vitro recall response to HCV proteins and viral clearance in humans during the acute phase of infection.28-32 In humans, CD4⫹ T-cell responses associated with viral clearance are targeted toward the nonstructural proteins. A strong response to NS3 may be particularly critical in the resolution of acute HCV infection.28-30 The cytokine profiles of HCV-specific CD4⫹ cells of individuals that clear the virus tend to display a T-helper type I profile.24,33 The HCV-specific in vitro recall response in chimpanzees has not been studied. In this study, the relationship of disease outcome in HCV-inoculated chimpanzees to the ability of peripheral blood mononuclear cells (PBMC) to recognize various HCV proteins was evaluated in a series of animals that had previously cleared HCV and were rechallenged. The results showed a strong, long-lasting protective immunity to HCV upon rechallenge that resulted in very rapid viral clearance and was associated with a T-cell proliferative response to nonstructural proteins and a dramatic increase in antibody titer to HCV proteins. In contrast, no T-cell proliferative response was noted in chimpanzees with chronic HCV infections, and a detectable response was observed in only 1 of 2 acutely infected animals. These findings suggest that long-term, protective immunologic memory may be induced in individuals who clear an initial challenge of HCV. MATERIALS AND METHODS Serum, PBMC, and Liver Biopsy Tissues From Chimpanzees. Serum samples, PBMC, and liver biopsy tissues were collected from HCVinoculated chimpanzees housed at the Southwest Regional Primate Research Center at the Southwest Foundation for Biomedical Research. Animals were cared for by members of the Department of Laboratory Animal Medicine in accordance with the Guide for the Care and Use of Laboratory Animals and all protocols were approved by the Institutional Animal Care and Use Committee. Percutaneous liver needle biopsy specimens were collected to obtain liver tissue specimens. TELAZOL (10 mg/kg) was used for immobilization before specimen collection. Alanine transaminase (ALT) values were assessed by standard laboratory procedures at the time the serum samples were collected. Chimpanzees. Chimpanzees x123, x130, x174, and x216 were persistently infected with HCV. The virologic status of these animals has been previously reported.34 Chimpanzee x123 was inoculated with a factor VIII concentrate contaminated with HCV genotype 1a in 1981. Chimpanzee x130 received an uncharacterized NANBH containing HCV genotype 1a inoculum in 1978. Chimpanzees x174 and x216 were inoculated with the genotype 1a H77 strain35,36 of HCV in 1988 and 1985, respectively. The 2 acutely infected chimpanzees, x329 and x363, were inoculated with the H77 strain of HCV. Chimpanzee x178 was initially inoculated with genotype 1b factor VIII in August 1984. Chimpanzee x178 received the same genotype 1b strain in April 2000. Chimpanzee x361 was initially inoculated with H77 in March 1998 and was rechallenged with the HCV-1 strain (genotype 1a) in September 1999. Chimpanzee x198 was inoculated with H77 in September 1989 and was rechallenged with the genotype 1b strain

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of HCV in February 2000. Chimpanzee x187 was inoculated with H77 in December 1993 and was rechallenged with HCV-1 in September 1999. Reverse Transcription Polymerase Chain Reaction Analysis of Serum From HCV-Inoculated Chimpanzees. HCV RNA was isolated from serum or

liver tissue using RNazol (Leedo, Houston, TX). HCV RNA was quantified by a real time, 5⬘ exonuclease reverse transcription polymerase chain reaction (RT-PCR) (TaqMan) assay using the ABI 7700 Sequence Detector (PE Biosystems, Foster City, CA).37 The primers and probe were derived from the 5⬘ noncoding region (NCR) and were selected using the Primer Express software designed for this purpose (PE Biosystems). The forward primer was from nucleotide 149 to 167 (5⬘-TGCGGAACCGGTGAGTACA-3⬘), the reverse primer was from nucleotide 210 to 191 (5⬘-CGGGTTTATCCAAGAAAGGA-3⬘), and the probe was from nucleotide 189 to 169 (5⬘CCGGTCGTCCTGGCAATTCCG-3⬘). The fluorogenic probe was labeled with FAM and TAMRA and was obtained from Synthegen (Houston, TX). The primers and probe were used at 10 pmol/50 ␮L reaction. The reactions were performed using a TaqMan Gold RTPCR kit (PE Biosystems) and included a 30-minute 48°C reverse transcription step, followed by 10 minutes at 95°C, and then 50 cycles of amplification using the universal TaqMan standardized conditions; 15 seconds at 95°C for denaturation and 1 minute at 60°C for annealing and extension. Standards to establish genome equivalents (ge) were synthetic RNAs transcribed from a clone of the 5⬘ NCR of the HCV-1 strain. Synthetic RNA was prepared using the T7 Megascript Kit (Ambion, Austin, TX) and was purified by DNase treatment, RNazol extraction, and ethanol precipitation. RNA was quantified by optical density and 10-fold serial dilutions were prepared from 1 million to 10 copies using tRNA as a carrier. These standards were run in duplicate in all TaqMan assays to calculate genome equivalents in the experimental samples. The calibration curves from one preparation of synthetic RNA to the next were essentially identical and yielded values comparable with commercially available assays. HCV Antibody Testing. Chimpanzee sera were analyzed for antiHCV antibody using a third-generation enzyme-linked immunosorbent assay (ELISA Testing System 3.0; Ortho Diagnostic Systems, Raritan, NJ). The ELISA was performed according to the manufacturer’s instructions. The assay kit contains the following recombinant antigens: c200 (aa 1192-1931, NS3, and NS4), c22-3 (aa 2 to 120, capsid) and NS5 (2054-2995). Antibody titers are represented as the reciprocal of the last 5-fold dilution of sera that was positive by ELISA. Proliferation Assays. PBMC were cultured in 96-well microtiter plates using 3 replicates for each condition with 2 ⫻ 105 cells/well. PBMC were cultured in RPMI 1640 supplemented with 10% (vol/ vol) heat-inactivated fetal calf serum at 37°C with 5% CO2 and 100% humidity for 7 days in the presence or absence of HCV proteins at a concentration of 5 ␮g/mL. The following purified recombinant proteins were provided by the Chiron Corporation (Emeryville, CA): c22-3 (aa 2-120, capsid), c100-3 (aa 1569-1931, NS3-NS4), NS5 (aa 2054-2995), c200 (aa 1192-1931), E2 and c25 (2-120 and 11921935). The HCV proteins were derived from HCV-1 genotype 1a and had greater than 99% homology with the H77 sequence, and ranged from 83% (NS5) to 97% (c22-3) homology to regions of the genotype 1b strain. The proteins were expressed in Saccharomyces cerviciae as human superoxide dismutase fusion proteins and were purified as previously described.38 The proliferative responses were evaluated by measuring the amount of [3H] thymidine taken up by the cells on day 6. The cells were harvested on day 7, and the counts per minute were measured using a ␤-counter (Matrix 9600; Packard Instruments Meridan, CT). The stimulation index (SI) was calculated as follows: (average counts per minute from the wells incubated with an antigen)/(average counts per minute from the wells without antigen). A biologically important SI in response was defined as greater than or equal to 2.5. An unpaired Student’s t test was also used to analyze the significance of the proliferative responses. The proliferative response to anti-CD3 (1 ␮g/mL) at day 3 was used as a positive

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control. Proliferative responses to recombinant human superoxide dismutase and yeast extract at concentrations of 5 ␮g/mL were used as negative controls. All of the controls had an SI lower than 2.5 and were insignificant by the Student’s t test. RESULTS Evaluation of Proliferative Responses to HCV Proteins in Chimpanzees Persistently Infected With HCV. Proliferative responses to

HCV proteins were analyzed in chimpanzees persistently infected with HCV. Chimpanzees x123, x130, x174, and x216 were inoculated with various strains (all genotype 1a) of HCV during the past 12 to 21 years (see Materials and Methods). Results of a cross-sectional study revealed that these chimpanzees were viremic based on RT-PCR analysis.34 A PBMC sample was collected to assess whether an in vitro recall response could be detected to different HCV proteins using standard proliferation assays. Proliferative responses to HCV capsid, E2, NS3, NS4, or NS5 were not observed in these viremic chimpanzees (data not shown). Infection Profiles and T-Cell Proliferative Responses in Acutely Infected Chimpanzees. Chimpanzees x363 and x329 were inoc-

ulated with plasma containing the H77 strain (genotype 1a) of HCV,35,36 and both animals cleared viral infection. HCV RNA, ALT levels, anti-HCV antibody, and proliferative responses were analyzed from closely spaced serial bleeds. HCV RNA was detected in serum and liver samples from x363 during the first 16 weeks postinoculation (Fig. 1A). At week 16, the serum and liver viral RNA levels had decreased by approximately 3 logs in comparison with the peak values at 8 to 10 weeks postinoculation. RNA was not detected in samples taken after 16 weeks. Seroconversion for anti-HCV antibody occurred at 10 weeks postinoculation. ALT values remained within the normal range throughout the analysis except for an increase to 91 U/L at 10 weeks postinoculation. Analysis of the proliferative responses of PBMC to HCV proteins revealed that chimpanzee x363 responded to c200 and c-25 at 10 weeks postinoculation (Fig. 1B). The proliferative response of PBMC from x363 declined rapidly and was undetectable 2 weeks later. Serum samples collected from chimpanzee x329 were positive for HCV RNA during the first 10 weeks postinoculation (Fig. 2A) at which time chimpanzee x329 cleared the virus. All serum samples collected after week 10 postinoculation were negative for HCV RNA, and HCV RNA was detected in the liver at weeks 2 and 8, but not at week 12. Seroconversion for anti-HCV antibody occurred at 8 weeks postinoculation. Peak ALT (179 U/L) occurred on week 8 and remained slightly elevated throughout the analysis. Analysis of the proliferative responses of PBMC revealed that chimpanzee x329 had a very weak response to c-25 at 10 and 12 weeks postinoculation (Fig. 2B), with an SI of 2.2 and 2.3, respectively. Although these values did not reach the cutoff of 2.5, the SIs were above 2.5 (2.6 and 3.0 for c-25 at weeks 10 and 12, respectively) when the calculations were preformed using another commonly used method (response to HCV antigen-response to recombinant human superoxide dismutase/response to recombinant human superoxide dismutase). We predicted that a secondary immune response to HCV would result in stronger proliferative responses and thus would better elucidate the proliferative responses associated with viral clearance. Thus, the following experiments involved 4 chimpanzees that had previously resolved HCV in-

FIG. 1. (A) HCV RNA, ALT, and anti-HCV antibody profiles and (B) HCV-specific proliferative responses of PBMC from x363, an acutely infected chimpanzee. Chimpanzee x363 was inoculated with the genotype 1a, H77 strain of HCV, and serum samples collected at various time points were analyzed for increases in serum ALT levels, HCV RNA by quantitative, realtime (TaqMan) RT-PCR, and seroconversion for anti-HCV antibodies. The presence (⫹) or absence (⫺) of HCV RNA in the serum or liver and serum anti-HCV antibody is indicated. The dashed line indicates the ALT upper normal limit (55 U/liter). The solid line indicates the serum ALT values. The solid bars represent the quantity of HCV RNA in the serum (ge/mL of serum), and open bars represent the level of viral RNA in the liver (ge/␮g of total liver RNA). (B) The PBMC proliferation response to HCV antigens was analyzed at various time points after inoculation. The proliferative responses to c100-3 (aa 1569-1931, NS3-NS4), c200 (aa 1192-1931, NS3-NS4), c-25 (aa 2-120 and aa 1192-1935, capsid, and NS3-NS4), and NS5 (aa 2054-2995) are represented as the stimulation index.

fection and were rechallenged with homologous or heterologous strains of HCV. Rapid Viral Clearance and T-Cell Proliferative Responses Following Rechallenge With a Nonhomolgous Genotype 1a Strain of HCV.

The initial rechallenge studies were performed with a chimpanzee that had recently cleared HCV under the assumption that an animal that had recently cleared infection would have the highest probability of displaying a memory response and

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ing that this animal had sterilizing immunity to HCV despite the relatively high dose of inoculum used in these studies (Fig. 3A). The lower threshold for HCV RNA in the TaqMan assay represents 10 copies, thus with the analysis of 10 ␮L of serum the final cutoff is 1,000 ge/mL. Although no RNA was detectable in the serum, viral RNA was detected in a liver biopsy specimen collected at week 4. However, the level of viral RNA present in the biopsy suggested that less than 0.001% of the hepatocytes were infected at this time (10 ge per ␮g of liver cell RNA or 5 ⫻ 105 cells). Liver biopsy specimens collected at weeks 0, 8, and 12 were negative for HCV RNA. HCV antibody was detected in all serum samples analyzed. A dramatic increase in the HCV-specific antibody titer was observed from a preinoculation titer of 100 (week 0) to a titer of 125,000 by 8

FIG. 2. (A) HCV RNA, ALT, and anti-HCV antibody profiles and (B) HCV-specific proliferative responses of PBMC from x329, an acutely infected chimpanzee. Chimpanzee x329 was inoculated with the genotype 1a, H77 strain of HCV, and serum samples that were collected at various time points were analyzed for increases in serum ALT levels, HCV RNA by quantitative, real-time (TaqMan) RT-PCR, and seroconversion for anti-HCV antibodies. The PBMC proliferation response to HCV antigens was analyzed at various time points after inoculation. For details, see the legend to Fig. 1.

protective immunity. Chimpanzee x361 was initially inoculated with the H77 strain of genotype 1a in March 1998. Serum samples collected for the first 10 weeks postinoculation were positive for HCV RNA (data not shown). Seroconversion for anti-HCV antibody occurred at week 11. The ALT levels were normal except for slight elevations between weeks 3 and 5 (peak ALT 91 U/L, week 4) and 8 and 11 (peak ALT 123 U/L, week 11). Chimpanzee x361 cleared HCV infection by 14 weeks postinoculation. All serum and liver samples analyzed beyond week 15 were negative for HCV RNA. Chimpanzee x361 was rechallenged with 1.7 ⫻ 106 ge of the HCV-1 prototype strain of genotype 1a in September 1999, approximately 1.5 years after the initial HCV infection. The HCV-1 strain is approximately 97% homologous to H77 (78 of 3,010 aa differ).37 HCV RNA was undetectable in all serum samples analyzed (weeks 0-18 and week 26), suggest-

FIG. 3. (A) HCV RNA, ALT, and anti-HCV antibody profiles and (B) HCV-specific proliferative responses of PBMC from x361, a chimpanzee that had previously cleared an infection with the genotype 1a H77 strain of HCV and was rechallenged with the genotype 1a HCV-1 strain of HCV. Serum samples collected at various time points were analyzed for increases in serum ALT levels, HCV RNA by quantitative, real-time (TaqMan) RT-PCR, and seroconversion for anti-HCV antibodies. The PBMC proliferation response to HCV antigens was analyzed at various time points after inoculation and is represented as the stimulation index. For details, see the legend to Fig. 1.


weeks postinoculation. The ALT levels remained within the normal range at all times after rechallenge. To assess the recall responses to HCV proteins standard proliferation assays with PBMC were performed using c22-3, c100-3, NS5, c200, E2, and c25, as well as the negative controls superoxide dismutase and yeast extract and anti-CD3 as a positive control (see Materials and Methods). A rapid, multiantigen proliferative response was observed by 2 weeks postinoculation including responses to c200, c25, and NS5 (Fig. 3B). The responses fell below the SI cutoff of 2.5 by week 3, but were again positive on weeks 6, 14, and 22. Next, we evaluated rechallenge of an animal that had cleared HCV infection 6 years previously to determine whether the protective immunity was long lasting. Similar to chimpanzee x361, chimpanzee x187 had previously cleared infection with the H77 strain of HCV and was rechallenged with HCV-1. Chimpanzee x187 was inoculated with H77 in December 1993. Serum samples collected for the first 15 weeks postinoculation were positive for HCV RNA by RTPCR analysis (data not shown). HCV RNA was not detected in serum or liver samples collected after 15 weeks postinoculation. Seroconversion for anti-HCV antibody occurred at 15 weeks postinoculation. The HCV-specific antibody was still detectable 6 years postinoculation. Except for a slight elevation (74 U/L) at 9 weeks postinoculation, ALT levels remained within the normal range throughout the analysis. Approximately 6 years after clearing the initial challenge with H77, x187 was rechallenged with 1.7 ⫻ 106 genome equivalents of HCV-1 in September 1999. HCV RNA was detectable by RT-PCR in serum samples collected on weeks 1, 2, and 4 after rechallenge and in liver tissue on week 4 (Fig. 4A). HCV RNA was not detected in the serum or in liver tissue after 4 weeks after rechallenge. Peak ALT (92 U/L) occurred on week 1 and then returned to normal by week 2. Thus, despite the high dose inoculum and the relatively long period of time that had elapsed from the primary infection, x187 rapidly cleared a heterologous but closely related challenge virus. Standard proliferation assays were performed to assess in vitro recall responses of PBMC to different HCV proteins (Fig. 4B). Vigorous proliferative responses were detected within 2 weeks of rechallenge. Proliferative responses to c100, c200, c-25, and NS5 were detected at 2 and 3 weeks after rechallenge in chimpanzee x187. The SI was high in comparison with the other animals examined with some values exceeding 20. Although the proliferative responses rapidly waned, weak proliferative responses to c200 and c-25 were still observed at 22 weeks after rechallenge. In addition, the HCV-specific antibody titer increased from 20 to 125,000 (6,250-fold) between 0 and 8 weeks postinoculation. Rapid Viral Clearance and T-Cell Proliferative Responses Following Rechallenge With a Homologous Genotype 1b Strain of HCV. To

extend the time frame between the time of primary infection and the time of rechallenged and to extend the observations to a different subtype of HCV, we performed a rechallenge experiment with an animal 16 years after the initial infection with genotype 1b. Chimpanzee x178 was inoculated with a genotype 1b strain of HCV in August 1984. An archived serum sample from October 1984 was analyzed for HCV RNA by RT-PCR. The presence of HCV RNA and an increase in ALT levels confirmed that x178 became infected (data not shown). Serum samples collected 11 and 16 years postinoculation were negative for HCV RNA by RT-PCR. Chimpanzee x178 was rechallenged with 1.7 ⫻ 106 genome equivalents of the

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FIG. 4. (A) HCV RNA, ALT, and anti-HCV antibody profiles and (B) HCV-specific proliferative responses of PBMC from x187, a chimpanzee that had previously cleared an infection with the genotype 1a H77 strain of HCV and was rechallenged with the genotype 1a HCV-1 strain of HCV. Serum samples collected at various time points were analyzed for increases in serum ALT levels, HCV RNA by quantitative, real-time (TaqMan) RT-PCR, and seroconversion for anti-HCV antibodies. The PBMC proliferation response to HCV antigens was analyzed at various time points after inoculation and is represented as the stimulation index. For details, see the legend to Fig. 1.

same genotype 1b strain in April 2000, 16 years after the primary infection. The inoculum was obtained in April 2000 from a chimpanzee (x183) that received the same inoculum at the same time as x178 but became chronically infected. Chimpanzee x178 rapidly cleared the virus upon rechallenge. Peak levels of viremia and viral RNA in the liver occurred on week 2 and declined thereafter to week 6 (Fig. 5A). HCV RNA was not detected in the serum or liver after week 6. The ALT levels

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well. The in vitro recall responses of PBMC to various HCV proteins revealed proliferative responses to c200 at weeks 2, 6, and 10 and to c25 at weeks 6, 10, and 12 after rechallenge (Fig. 5B). The amino acid sequences of the recall antigens were approximately 91% (c200) and 92% (c25) homologous to the genotype 1b challenge virus. Rapid Viral Clearance and T-Cell Proliferative Responses Following Rechallenge With Different HCV Subgenotype. To determine

FIG. 5. (A) HCV RNA, ALT, and anti-HCV antibody profiles and (B) HCV-specific proliferative response of PBMC from x178, a chimpanzee that had previously cleared an HCV infection and was rechallenged with a homologous genotype 1b strain of HCV. Serum samples collected at various time points were analyzed for increases in serum ALT levels, HCV RNA by quantitative, real-time (TaqMan) RT-PCR, and seroconversion for anti-HCV antibodies. The PBMC proliferation response to HCV antigens was analyzed at various time points after inoculation and is represented as the stimulation index. For details, see the legend to Fig. 1.

whether the protective immunity upon rechallenge extended to rechallenge with a more heterologous virus, a rechallenge experiment was performed in which an animal previously infected with genotype 1a was rechallenged with genotype 1b. Chimpanzee x198 was inoculated with H77 in September 1989. HCV RNA was detected by RT-PCR in several serum samples collected between 8 and 12 weeks postinoculation (data not shown). A serum sample collected 16 weeks postinoculation and all samples examined at later time points were HCV RNA negative. A serum sample collected 11 years postinoculation was still negative for HCV RNA, but was positive for anti-HCV antibodies by ELISA. Peak ALT values of 166 U/L occurred at week 10. Chimpanzee x198 was rechallenged in February 2000 with 1.7 ⫻ 106 ge of the same inoculum as used for x178. Although HCV RNA was detected by RT-PCR in serum and liver samples for the first 4 weeks after rechallenge, chimpanzee x198 cleared the virus prior to week 6 (Fig. 6A). Rapid viral clearance occurred after rechallenge despite the considerable divergence in amino acid sequence between the initial genotype 1a inoculum and the 1b challenge strain. Anti-HCV antibody was detected in all serum samples tested. The HCV-specific antibody titer increased from 20 to 125,000 between 0 and 8 weeks postinoculation. The ALT levels remained within the normal range throughout the analysis, except for a slight elevation (71 U/L) at week 2 after rechallenge. Again, viral clearance occurred much more rapidly upon rechallenge, despite 11 years having elapsed since the primary infection and the considerable divergence between the 2 inocula. PBMC collected from chimpanzee x198, 11 years after the initial HCV infection and before rechallenge, proliferated weakly in response to c-25 (Fig. 6B). A rapid increase occurred in the proliferative response to c200 and c25 on weeks 2 and 4. A proliferative response to c25 was detected at all sampling times out to 12 weeks after rechallenge. A proliferative response to c200 was observed at weeks 2, 4, 10, and 12 postinoculation, and a proliferative response to c100-3 was observed at week 4. DISCUSSION

remained within the normal range throughout the analysis. An antibody response to HCV was first detected 4 weeks after rechallenge, and the antibody titer increased from 100 to 125,000 between 4 and 8 weeks postinoculation. Although archived serial serum samples from x178 were not available to determine the precise time of viral clearance from the primary infection, a peak ALT value of 262 U/L was observed on week 12 postinoculation, suggesting that viral clearance from the primary infection occurred after this time. These data indicate that clearance from the rechallenge was much more rapid than from the primary infection despite the 16 years that had elapsed since exposure. In addition, the persistently normal ALT values following rechallenge in comparison to the moderately high values (for a chimpanzee) observed during primary infection suggest that liver disease was attenuated as

The chimpanzee animal model has been valuable in exploring factors involved in HCV infection such as the clinical outcome, genetic drift, and immune response. Chimpanzees are valuable for studying the early events in the immune response to HCV, because blood and tissue samples are readily available from the time of infection and throughout the acute phase of infection. Since most humans experience asymptomatic infections and the time of infection is rarely known, blood and tissue samples cannot be collected during the early acute phase of infection. Analysis of the immune response during this time period may reveal critical factors involved in viral clearance. Additionally, chimpanzees may be rechallenged with HCV to augment and explore immune responses that may be important in viral clearance but are too weak to detect during the initial acute infection. Although the antibody and


FIG. 6. (A) HCV RNA, ALT, and anti-HCV antibody profiles and (B) HCV-specific proliferative responses of x198, a chimpanzee that cleared a previous infection with the H77 genotype 1a strain of HCV and was rechallenged with a genotype 1b strain of HCV. The PBMC proliferation response to HCV antigens was analyzed at various time points after inoculation and is represented as the stimulation index. For details, see the legend to Fig. 1.

CTL responses have been studied in HCV-inoculated chimpanzees, nothing is currently known regarding HCV-specific in vitro recall proliferative responses of PBMC during infection in chimpanzees. We have previously determined that up to 60% of chimpanzees rapidly clear HCV infections, making the chimpanzee an excellent model for determining the mechanism of viral clearance.34 As an extension of these studies, we have examined the outcome of rechallenge experiments in which animals that had previously cleared infection were rechallenged with homologous or heterologous strains of HCV. The viral RNA levels in both the serum and liver were monitored by quantitative RT-PCR, and T-cell proliferative responses to HCV proteins were monitored to examine the relationship between the cellular immune response and disease outcome. Rechallenge was accompanied by rapid viral clearance, in the absence of detectable viremia in one case, despite either a long duration of

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time from the primary infection (16 years) or rechallenged with a different subtype. T-cell proliferation studies indicated that viral clearance was associated with an in vitro recall response to HCV nonstructural proteins. The rapid appearance of a proliferative response suggests that a memory T-cell response specific for HCV NS3-NS4 may contribute to a long-lasting, protective immune response in chimpanzees. This protective response extended to subgenotypes quite divergent (15%) from the strain of the original inoculum. Although clearance after rechallenge was rapid, it was not associated with a significant elevation in serum ALT levels. Two of the rechallenged animals had no increase in ALT levels, whereas minimal increases of 1 week in duration were observed in the other 2 chimpanzees. This suggested that extensive hepatocellular necroinflammatory disease was not involved in viral clearance in these studies. This may be due to the limited degree of spread of the infection in the liver, rather than the nature of the immune response involved in clearance in these animals. HCV-specific proliferative responses were not detected in PBMC from 4 chimpanzees that were persistently infected with HCV. The absence of a detectable HCV-specific recall response may be caused by T-cell anergy. Because recall responses were observed in chimpanzees that were rechallenged with heterologous inocula, it is unlikely that the lack of detectable response was caused by amino acid differences in the antigens. Similar to our findings in persistently infected chimpanzees, PBMC from a low percentage of humans with chronic HCV infection proliferate in response to stimulation with recombinant HCV proteins. Takaki et al. and Gerlach et al. recently reported that in vitro recall responses could be detected from only 25% to 44% of the chronically infected individuals in contrast to 79% to 100% of patients that cleared an HCV infection.25,32 HCV-specific proliferative responses were also examined in 2 chimpanzees with acutely resolving HCV infections. Proliferative responses to c200 and c-25 were detected in PBMC from chimpanzee x363. In contrast, HCV-specific proliferative responses for PBMC from x329 were below the SI cutoff used in our studies. Use of an alternative method of calculating the SI yielded results slightly above the cutoff, suggesting that a weak proliferative response probably occurred in this animal. Compartmentalization of T cells to the liver could have resulted in failure to detect proliferation in PBMC,39 although in humans PBMC proliferative responses are detected even when compartmentalization occurs. Another possibility is that the dominant T-cell response was elicited to peptides not used in our assays. Several reports suggest that viral clearance in acutely infected humans is associated with a strong proliferative response to HCV proteins. However, it must be emphasized that acutely infected humans are selected based on overt, symptomatic hepatitis. This certainly represents a subset of infected humans, because most humans experience an asymptomatic acute infection. Acutely infected chimpanzees generally experience a mild disease with low ALT elevations, as well. The strength of proliferative responses in acutely infected, asymptomatic humans cannot be examined, but may be more representative of the responses observed in chimpanzees. Viral clearance was also observed in 4 animals that resolved a previous HCV infection and were rechallenged. Proliferative responses to c200 and c-25 were observed in all 4 animals.

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Additionally, 2 chimpanzees (x187 and x198) responded to c100-3 and 2 chimpanzees (x187 and x361) responded to NS5. The HCV proteins used in these studies were derived from the HCV-1 (1a) strain. Although the sequence homology between HCV-1 and H77 is quite high, considerable divergence (approximately 15%) at the amino acid level exists between HCV-1 and genotype 1b strains. Some proliferative responses to HCV may have been undetected if differences in the amino acid sequences prevented epitope presentation or recognition. Proliferative responses were observed primarily in the NS3NS4 domain in the rechallenged chimpanzees even though they were inoculated with different strains and subtypes of HCV, supporting the hypothesis that conserved T-cell epitopes within NS3 may be important in viral clearance. Diepolder et al. suggested that proliferative responses specific for an immunodominant epitope within NS3 (aa 1251-1259) may be associated with viral clearance in humans.28-30 The epitope appears to be highly conserved among different HCV genotypes and is recognized in the context of at least 5 different HLA class II alleles. Although the T-cell responses observed during rechallenge in these studies were associated with rapid viral clearance, no data at this time shows that the proliferative T-cell response to NS3 was responsible for viral clearance. HCV-specific proliferative responses were detected very early after inoculation in the rechallenged chimpanzees. Proliferative responses were detected within 2 weeks after rechallenge in all animals. In contrast, the HCV-specific proliferative responses that were observed in the acutely infected chimpanzee were not detected until 10 weeks postinoculation. The rapid response observed in the rechallenged animals probably represents the presence of memory T cells, which probably played an important role in suppressing the infection. The proliferative response to the HCV proteins was short lived in most of the animals we analyzed. In humans, HCVspecific recall responses have been observed for 18 to 20 years after viral clearance.25 Gerlach et al. observed that transient HCV-specific T-cell responses were observed during the acute phase of infection in individuals that eventually progressed to chronic HCV infection.32 The T-cell responses were lost around 5 to 10 months during the acute phase of infection and the loss of T-cell responsiveness was associated with recurrence of HCV RNA. The transient proliferative response in the chimpanzees did not appear to be associated with progression to chronic infection and may reflect differences in HCV infection in chimpanzees versus humans. However, we have not had the opportunity to evaluate proliferative responses in a chimpanzee progressing to chronicity. HCV-specific antibodies may have contributed to viral clearance in the rechallenged chimpanzees, because the HCVspecific antibody titer increased rapidly after rechallenge. The antibody titer increased by at least 1,000-fold in all animals. In contrast, Cooper et al. did not observe a strong antibody response in a chimpanzee that previously cleared an HCV infection and was rechallenged.14 The differences in the humoral immune responses observed in these 2 studies may be caused by differences in the antigens and ELISAs used. The most striking observation in this study was that chimpanzees that were rechallenged with HCV cleared the virus very rapidly in comparison with acutely infected animals.


HCV RNA was detected in serum samples collected from x329, x363, x187, and x361 for an average of 14 weeks postinoculation during the acute phase of the initial HCV infection. In contrast, HCV viremia was detected only for an average of 5 weeks in the rechallenged animals, and the level of viremia was approximately 2 logs lower in rechallenged animals in comparison with the levels observed during primary infection. Chimpanzee x361 exhibited sterilizing immunity, because no viremia was detected after rechallenge; however, a low level of viral RNA was present in the liver at week 4 postinoculation. Thus, a strong protective immune response to HCV infection persists for at least 16 years, although the key immune mediators are not clear at this time. The chimpanzee animal model will continue to be valuable in elucidating the role of the immune response in viral clearance during the acute phase of HCV infection, and a better understanding of the protective cellular immune response to HCV proteins may lead to improved therapies and vaccines. The protective immunity observed in these animals support the concept that an efficacious vaccine to heterologous strains should be possible. REFERENCES 1. Anonymous. National Institutes of Health Consensus development conference panel statement: management of hepatitis C. HEPATOLOGY 1997; 26:2S-10S. 2. Robertson B, Myers G, Howard C, Brettin T, Bukh J, Gaschen B, Gojobori T, et al. Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. International Committee on Virus Taxonomy. Arch Virol 1998;143: 2493-2503. 3. Houghton M, Selby M, Weiner A, Choo QL. Hepatitis C virus: structure, protein, products and processing of the polyprotein precursor. Curr Stud Hematol Blood Transf 1994;61:1-11. 4. Reed KE, Rice CM. Molecular characterization of hepatitis C virus. Curr Stud Hematol Blood Transf 1998;62:1-37. 5. Walker CM. Comparative features of hepatitis C virus infection in humans and chimpanzees. Springer Semin Immunopathol 1997;19:85-98. 6. Abe K, Inchauspe G, Shikata T, Prince AM. Three different patterns of hepatitis C virus infection in chimpanzees. HEPATOLOGY 1992;15:690695. 7. Bassett SE, Thomas DL, Brasky KM, Lanford, RE. Viral persistence, antibody to E1 and E2, and hypervariable region 1 sequence stability in hepatitis C virus-inoculated chimpanzees. J Virol 1999;73:1118-1126. 8. Choo Q-L, Kuo G, Ralston R, Weiner A, Chien D, Van Nest G, Han J, et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci U S A 1994;91:1294-1298. 9. Hilfenhaus J, Krupka U, Nowak T, Cummins LB, Fuchs K, Roggendorf M. Follow-up of hepatitis C virus infection in chimpanzees: determination of viraemia and specific humoral immune response. J Gen Virol 1992;73: 1015-1019. 10. Lanford RE, Notvall L, Barbosa LH, Eichberg JW. Evaluation of a chimpanzee colony for antibodies to hepatitis C virus. J Med Virol 1991;34: 148-153. 11. Wang YF, Brotman B, Andrus L, Prince AM. Immune response to epitopes of hepatitis C virus (HCV) structural proteins in HCV-infected humans and chimpanzees. J Infect Dis 1996;173:808-821. 12. Farci P, Alter HJ, Wong DC, Miller RH, Govindarajan S, Engle R, Shapiro M, et al. Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization. Proc Natl Acad Sci U S A 1994; 91:7792-7796. 13. Krawczynski K, Alter MJ, Tankersley DL, Beach M, Robertson BH, Lambert S, Kuo G, et al. Effect of immune globulin on the prevention of experimental hepatitis C virus infection. J Infect Dis 1996;173:822-828. 14. Cooper S, Erickson AL, Adams EJ, Kansopon J, Weiner AJ, Chien DY, Houghton M, et al. Analysis of a successful immune response against hepatitis C virus. Immunity 1999;10:439-449. 15. Farci P, Alter HJ, Govindarajan S, Wong DC, Engle R, Lesniewski RR, Mushahwar IK, et al. Lack of protective immunity against reinfection with hepatitis C virus. Science 1992;258:135-140.

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