Marian E. Major

Kinetics of CD4+ and CD8+ memory T-cell responses during hepatitis C virus rechallenge of previously recovered chimpanzees.

ABSTRACT The immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood. Antibodies to HCV structural proteins do not appear to play a key role in clearance of the virus and do not persist after recovery. Here, we studied the kinetics of the cellular immune responses of three HCV-recovered chimpanzees during rechallenge with increasing doses of homologous HCV. Although HCV envelope antibodies remained undetectable throughout the rechallenge, all animals mounted rapid HCV-specific T-cell responses. The pattern of the cellular immune response in blood and liver correlated with the virological outcome. The animal that most rapidly cleared circulating HCV as determined by nested reverse transcription-PCR (RT-PCR) displayed the most vigorous and sustained response of gamma interferon (IFN-γ)-producing and proliferating CD4+ T cells in the blood. Vigorous CD4+ T-cell proliferation during viremia was followed by an increased frequency and a phenotypic and functional change of the tetramer+ CD8+ T-cell population. The second animal cleared HCV initially with strong peripheral and intrahepatic CD4+ T-cell responses but experienced low-level HCV recrudescence 12 weeks later, when HCV-specific T cells became undetectable. The third animal maintained minute amounts of circulating HCV, detectable only by nested RT-PCR, in the face of a weak IFN-γ+ T-cell response. Collectively, the results suggest protective rather than sterilizing immunity after recovery from hepatitis C. The rate of HCV clearance following reexposure depends on the cellular immune response, the quality and quantity of which may vary among chimpanzees that recovered from HCV infection.

Previously Infected and Recovered Chimpanzees Exhibit Rapid Responses That Control Hepatitis C Virus Replication upon Rechallenge

ABSTRACT Responses in three chimpanzees were compared following challenge with a clonal hepatitis C virus (HCV) contained in plasma from an animal that had received infectious RNA transcripts. Two of the chimpanzees (Ch1552 and ChX0186) had recovered from a previous infection with HCV, while the third (Ch1605) was a naïve animal. All animals were challenged by reverse titration with decreasing dilutions of plasma and became serum RNA positive following challenge. Ch1605 displayed a typical disease profile for a chimpanzee. We observed increasing levels of serum RNA from week 1 postinoculation (p.i.), reaching a peak of 106 copies/ml at week 9 p.i., and alanine aminotransferase (ALT) elevations and seroconversion to HCV antibodies at week 10 p.i. In contrast, both Ch1552 and ChX0186 exhibited much shorter periods of viremia (4 weeks), low serum RNA levels (peak, 103 copies/ml), and minimal ALT elevations. A comparison of intrahepatic cytokine levels in Ch1552 and Ch1605 showed greater and earlier gamma interferon (IFN-γ) and tumor necrosis factor alpha responses in the previously infected animal, responses that were 30-fold greater than baseline responses at week 4 p.i. for IFN-γ in Ch1552 compared to 12-fold in Ch1605 at week 10 p.i. These data indicate (i) that clonal HCV generated from an infectious RNA transcript will lead to a typical HCV infection in naïve chimpanzees, (ii) that there are memory immune responses in recovered chimpanzees that control HCV infection upon rechallenge, and (iii) that these responses seem to be T-cell mediated, as none of the animals had detectable antibody against the HCV envelope glycoproteins. These observations have encouraging implications for the development of a vaccine for HCV.

Hepatitis C virus kinetics and host responses associated with disease and outcome of infection in chimpanzees

To study determinants of clinical outcome following HCV infection, viral kinetics, immune events, and intrahepatic cytokine markers were compared in 10 naive chimpanzees. Four of the animals cleared HCV; 6 developed persistent infections. All animals developed similar acute infections with increasing viremia from 1 to 2 weeks, followed by alanine aminotransferase (ALT) elevations and seroconversion. This viremia pattern consisted of a biphasic increase, a rapid slope (mean doubling time [t2] = 0.5 days) followed by a slower slope after the second week (t2 = 7.5 days). This slowing of virus replication correlated in all animals with increased intrahepatic 2′5′ oligoadenylate synthetase 1 (2OAS‐1) messenger RNA (mRNA) levels and was independent of disease outcome. An effective control of virus replication was observed following increases in intrahepatic interferon γ (IFN‐γ) mRNA and ALT levels. Although this control was associated in all animals with a 2‐log decrease in virus titer, the timing occurred approximately 2 weeks later in the chronic group (P < .05). Additionally, while cleared infections were characterized by a continual decrease in virus titer, the titers in the persistent infections reached a steady state level of 104 to 105 RNA copies/mL. This inability of the immune response to sustain viral clearance in the persistent infections was associated with a reduced intrahepatic CD3e and monocyte‐induced protein 1α (MIP‐1α) mRNA induction. In conclusion, these data indicate that, regardless of outcome, chimpanzees generate responses that control HCV replication during the early and late acute phase. However, the pathogenesis of HCV may be determined by a more rapid onset of the induced response and the cell population that migrates to the liver. (HEPATOLOGY 2004;39:1709–1720.)

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver.

We established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum‐free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP+ cells expressing ALB were detected in close proximity to “beating” myocytes after 7 days of EB cultures. GFP+ cells increased in number, acquired hepatocyte‐like morphology and hepatocyte‐specific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS‐purified GFP+ cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP‐uPA/SCID mice. The ES cell‐derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl4‐induced liver injury. The transplanted GFP+ cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy. (HEPATOLOGY 2006;44:1478–1486.)

Depletion of interfering antibodies in chronic hepatitis C patients and vaccinated chimpanzees reveals broad cross-genotype neutralizing activity

Using human immune globulins made from antihepatitis C virus (HCV)-positive plasma, we recently identified two antibody epitopes in the E2 protein at residues 412–426 (epitope I) and 434–446 (epitope II). Whereas epitope I is highly conserved among genotypes, epitope II varies. We discovered that epitope I was implicated in HCV neutralization whereas the binding of non-neutralizing antibody to epitope II disrupted virus neutralization mediated by antibody binding at epitope I. These findings suggested that, if this interfering mechanism operates in vivo during HCV infection, a neutralizing antibody against epitope I can be restrained by an interfering antibody, which may account for the persistence of HCV even in the presence of an abundance of neutralizing antibodies. We tested this hypothesis by affinity depletion and peptide-blocking of epitope-II-specific antibodies in plasma of a chronically HCV-infected patient and recombinant E1E2 vaccinated chimpanzees. We demonstrate that, by removing the restraints imposed by the interfering antibodies to epitope-II, neutralizing activity can be revealed in plasma that previously failed to neutralize viral stock in cell culture. Further, cross-genotype neutralization could be generated from monospecific plasma. Our studies contribute to understanding the mechanisms of antibody-mediated neutralization and interference and provide a practical approach to the development of more potent and broadly reactive hepatitis C immune globulins.

Meta-Analysis of Hepatitis C Virus Vaccine Efficacy in Chimpanzees Indicates an Importance for Structural Proteins

BACKGROUND & AIMS Studies in patients and chimpanzees that spontaneously cleared hepatitis C virus (HCV) infections demonstrated that natural immunity to the virus is induced during primary infections and that this immunity can be cross protective. These discoveries led to optimism about prophylactic HCV vaccines, and several studies were performed in chimpanzees, although most included fewer than 6 animals. To draw meaningful conclusions about the efficacy of HCV vaccines in chimpanzees, we performed statistical analyses of data from previously published studies from different groups. METHODS We performed a meta-analysis that compared parameters among naïve (n = 63), vaccinated (n = 53), and rechallenged (n = 36) animals, including peak RNA titer postchallenge, time points of peak RNA titer, duration of viremia, and proportion of persistent infections. RESULTS Each vaccination study induced immune responses that were effective in rapidly controlling HCV replication. Levels of induced T-cell responses did not indicate vaccine success. There was no reduction in the rate of HCV persistence in vaccinated animals, compared with naïve animals, when nonstructural proteins were included in the vaccine. Vaccines that contained only structural proteins had clearance rates that were significantly higher than vaccines that contained nonstructural components (P = .015). CONCLUSIONS The inclusion of nonstructural proteins in HCV vaccines might be detrimental to protective immune responses, and/or structural proteins might activate T-cell responses that mediate viral clearance.

CD4+ immune escape and subsequent T-cell failure following chimpanzee immunization against hepatitis C virus†

Hepatitis C is a major cause of chronic liver disease, with 170 million individuals infected worldwide and no available vaccine. We analyzed the effects of an induced T‐cell response in 3 chimpanzees, targeting nonstructural proteins in the absence of neutralizing antibodies. In all animals the specific T‐cell response modified the outcome of infection, producing a 10‐ to 1,000‐fold reduction in peak virus titers. The challenge of 2 immunized animals that had been previously exposed to hepatitis C virus resulted in subclinical infections. Immune responses in the third animal, naive prior to immunization, limited viral replication immediately, evidenced by a 30‐fold reduction in virus titer by week 2, declining to a nonquantifiable level by week 6. After 10 weeks of immunological control, we observed a resurgence of virus, followed by progression to a persistent infection. Comparing virus evolution with T‐cell recognition, we demonstrated that: (i) resurgence was concomitant with the emergence of new dominant viral populations bearing single amino acid changes in the NS3 and NS5A regions, (ii) these mutations resulted in a loss of CD4+ T‐cell recognition, and (iii) subsequent to viral resurgence and immune escape a large fraction of NS3‐specific T cells became impaired in their ability to secrete IFN‐γ and proliferate. In contrast, NS3‐specific responses were sustained in the recovered/immunized animals presenting with subclinical infections. In conclusion, viral escape from CD4+ T cells can result in the eventual failure of an induced T‐cell response that initially controls infection. Vaccines that can induce strong T‐cell responses prior to challenge will not necessarily prevent persistent HCV infection. (HEPATOLOGY 2006;44:736–745.)

Delayed Induction, Not Impaired Recruitment, of Specific CD8+ T Cells Causes the Late Onset of Acute Hepatitis C

BACKGROUND & AIMS Hepatitis C virus (HCV) infection is characterized by lack of immune-mediated liver injury despite a high level of HCV replication during the incubation phase, which lasts about 8 weeks. We investigated whether this results from delayed recruitment of HCV-specific T cells and whether it facilitates HCV persistence. METHODS Six chimpanzees were infected with HCV; blood and liver samples were collected for 28 weeks and analyzed for immune cells and chemokines. RESULTS Two chimpanzees developed self-limited infections, whereas the remaining 4 developed chronic infections. Levels of the chemokines CXCL10, CXCL11, CCL4, and CCL5 increased in blood and liver samples from all chimpanzees within 1 month of HCV infection. Chemokine induction correlated with intrahepatic type I interferon (IFN) responses in vivo and was blocked by neutralizing antibodies against IFN-β in vitro. Despite the early-stage induction of chemokines, the intrahepatic lymphocytic infiltrate started to increase no earlier than 8 weeks after HCV infection, when HCV-specific, tetramer-positive CD8(+) T cells appeared in the circulation. The HCV-specific CD8(+) T cells expressed chemokine receptors when they were initially detected in blood samples, so they could be recruited to the liver as soon as they entered the circulation. CONCLUSIONS Chemokines are induced during early stages of HCV infection, which requires a type I IFN-mediated response. The delayed onset of acute hepatitis does not result from delayed recruitment of HCV-specific T cells, but could instead be related to a primary delay in the induction of HCV-specific T cells. Divergent outcomes occur without evident differences in chemokine induction and T-cell recruitment.

Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay

The availability of molecular protocols for the detection and quantitation of very low numbers of hepatitis C virus (HCV) particles in biological samples is an issue of interest in both clinical and analytical fields of HCV research. A sensitive and reproducible assay is described for HCV RNA quantitation using the TaqMan PCR fluorogenic real-time detection system to establish the levels of HCV RNA in chimpanzee plasma. Our TaqMan PCR protocol and synthetic full length HCV RNA template show that the threshold of sensitivity for our TaqMan PCR is two copies per reaction. As few as 10 genome copies per reaction could be quantitated maintaining a linear range. The accuracy of the TaqMan PCR test was comparable to commercial bDNA and Amplicor tests. The RNA standards of the laboratory were tested in parallel with a World Health Organization (WHO) International Standard for HCV RNA obtaining ratios of 2.7+/-0.7 RNA copies per HCV international unit (IU). Our method using RNA extracted from chimpanzee samples had an estimated sensitivity of 200 RNA copies/ml of plasma (approximately eight copies/reaction or 74 WHO IU/ml). Serial plasma samples from HCV-infected chimpanzees were analyzed using this methodology to evaluate its applicability, and RNA profiles were observed consistent with the evolution of the pathology in each animal. The present study therefore illustrates the high reproducibility, sensitivity and reliability of our TaqMan methodology, providing a useful method for HCV research to consistently detect and quantify viral RNA throughout a range of concentrations.

Prospects for Prophylactic and Therapeutic Vaccines Against Hepatitis C Virus

Natural cross-protective immunity is induced after spontaneous clearance of primary hepatitis C virus (HCV) infection. Although this suggests that effective prophylactic vaccines against HCV are possible, there are still several areas that require further study. Current data indicate that, at best, vaccine-induced immunity may not completely prevent HCV infection but rather prevent persistence of the virus. However, this may be an acceptable goal, because chronic persistence of the virus is the main cause of pathogenesis and the development of serious liver conditions. Therapeutic vaccine development is also highly challenging; however, strategies have been pursued in combination with current or new treatments in an effort to reduce the costs and adverse effects associated with antiviral therapy. This review summarizes the current state of HCV vaccines and the challenges faced for future development and clinical trial design.