Transmission of hepatitis C virus following antiviral treatment
July 8, 2014
Millions of people throughout the world are infected with hepatitis C virus (HCV). Left untreated, infection results in serious complications such as cirrhosis of the liver and cancer. Many HCV-infected patients respond well to anti-viral therapy and remain virus free. However, trace amounts of HCV RNA are sporadically detected in patients thought to have successfully responded to HCV treatment.
A recent study in the Journal of Clinical Investigation tested if this reappearing HCV RNA is infectious. Using an animal model, Barbara Rehermann and colleagues at the NIH found that blood from patients with trace amounts of HCV RNA is able to cause HCV infection, though the animals did not immediately show signs of HCV infection.
This study demonstrates that small amounts of HCV RNA in successfully treated patients can be infectious, but these transmission events may be hard to detect due to delayed onset of disease.
Source: Journal of Clinical Investigation
Trace amounts of sporadically reappearing HCV RNA can cause infection
Naga Suresh Veerapu1, Su-Hyung Park1, Damien C. Tully2, Todd M. Allen2 and Barbara Rehermann1
1Immunology Section, Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Department of Health and Human Services (DHHS), Bethesda, Maryland, USA.
2Ragon Institute of MGH, MIT, and Harvard, Boston, Massachusetts, USA.
Address correspondence to: Barbara Rehermann, Immunology Section, Liver Diseases Branch, NIDDK, National Institutes of Health, 10 Center Drive, Bldg. 10, Room 9B16C, Bethesda, Maryland 20892, USA. Phone: 301.402.7144; Fax: 301.402.0491; E-mail: Rehermann@nih.gov.
Authorship note: Naga Suresh Veerapu and Su-Hyung Park contributed equally to this work.
Published July 8, 2014
Received for publication September 6, 2013, and accepted in revised form May 29, 2014.
Successful hepatitis C virus (HCV) treatment is defined as the absence of viremia 6 months after therapy cessation. We previously reported that trace amounts of HCV RNA, below the sensitivity of the standard clinical assay, can reappear sporadically in treatment responders. Here, we assessed the infectivity of this RNA and infused 3 chimpanzees sequentially at 9-week intervals with plasma or PBMCs from patients who tested positive for trace amounts of HCV RNA more than 6 months after completing pegylated IFN-α/ribavirin therapy. A fourth chimpanzee received HCV RNA–negative plasma and PBMCs from healthy blood donors. The 3 experimental chimpanzees, but not the control chimpanzee, generated HCV-specific T cell responses against nonstructural and structural HCV sequences 6–10 weeks after the first infusion of patient plasma and during subsequent infusions. In 1 chimpanzee, T cell responses declined, and this animal developed high-level viremia at week 27. Deep sequencing of HCV demonstrated transmission of a minor HCV variant from the first infusion donor that persisted in the chimpanzee for more than 6 months despite undetectable systemic viremia. Collectively, these results demonstrate that trace amounts of HCV RNA, which appear sporadically in successfully treated patients, can be infectious; furthermore, transmission can be masked in the recipient by an extended eclipse phase prior to establishing high-level viremia.
At least 170 million people worldwide are persistently infected with hepatitis C virus (HCV), a leading cause of chronic inflammatory liver disease, cirrhosis, and cancer. The vast majority of patients who have been treated for chronic HCV infection received IFN-based treatment regimens. Pegylated interferon (PegIFN) in combination with ribavirin (RBV) has been the standard of care until the recent addition of direct-acting antivirals (1). A sustained virologic response (SVR) is defined as undetectable HCV RNA 6 months after the cessation of treatment. SVRs are considered cured because a virological relapse is exceedingly rare, and the risk of developing liver fibrosis and hepatocellular carcinoma decreases (2).
Considering the clinical experience of a long-term cure, it appears paradoxical that trace amounts of HCV RNA are sporadically detectable in the circulation (3) and in liver biopsies (2, 4–7) of some patients who experienced an SVR. Consistent with this, we recently reported that trace amounts of HCV RNA of pretreatment sequences, below the detection limit of the standard clinical assay at the NIH, reappeared sporadically in the blood of 15 of 98 (15%) patients in the first 8 years after an SVR. The sporadic reappearance of HCV RNA was sufficient to recall HCV-specific T cell responses and did not result in high-level viremia (8).
At present, it is not clear whether this RNA represents replication-competent HCV, whether it is associated with intact virions, and whether it can transmit infection. These questions are of interest not only from epidemiological and infectious disease standpoints, but also from a virological standpoint. Based on our current virological knowledge, HCV should not be able to achieve low-level persistence over extended periods of time, because it is an RNA virus with a short 40-minute plasma half-life (9) and without the ability to integrate into the host genome. We therefore asked whether cryopreserved plasma and PBMCs from patients with an SVR to IFN-based therapy, in whom we had previously described sporadic recurrence of trace amounts of HCV, transmit HCV infection to chimpanzees and establish persistent infection. The results demonstrate that (a) such plasma can be infectious and establish high-level viremia and chronic hepatitis in the recipient, (b) the course of viremia in the recipient can differ from the typical course of acute hepatitis, in that HCV persists for more than 6 months in the absence of viremia prior to establishing high-level systemic viremia, and (c) T cell responses correlate with temporary control of the low-level HCV infection.
Trace amounts of HCV RNA that sporadically reappear in patients after successful antiviral therapy can transmit HCV infection.
Three HCV-naive chimpanzees, A3A013, A3A015, and A3A017, were intravenously infused at 9-week intervals with human plasma or PBMCs (Table 1 and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI73104DS1). These samples were derived from previously described anti-HCV–positive patients who had experienced an SVR to IFN-based therapy by qualitative COBAS Amplicor HCV Test 2.0 (the standard clinical test used at the NIH) but sporadically tested positive for trace amounts of HCV in plasma or PBMCs using a nested RT-PCR specific for the 5′-UTR of the virus (Table 1 and ref. 8). A fourth HCV-naive chimpanzee, A3A025, served as a control and was infused at the same time intervals with HCV RNA–negative plasma and PBMCs from blood donors without any history of HCV infection.
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We previously described a window of time toward the end of HCV infection in which most of the HCV has been cleared, but traces of virus sporadically reappear in the circulation (8). These findings were made in a cohort of patients who sporadically tested positive for HCV RNA by nested RT-PCR within the first years after an SVR to IFN-based therapy (8). The results are consistent with reports showing residual virus in approximately 6% of SVRs by highly sensitive transcription-mediated amplification assays or nested RT-PCR (2, 10, 11), even though a relapse with high levels of viremia is exceedingly rare (12–15).
Our study now provides proof-of-principle that these PCR signals do indeed reflect replication-competent infectious virus. Indeed, our study may have underestimated the transmission rate, because we had to re-use each chimpanzee for 4 sequential infusions. Thus, HCV-specific T cell responses that were induced by the first infusion may have conferred protective immunity against subsequent infusions with human plasma and PBMCs with low levels of HCV. Consistent with this notion, chimpanzee A3A013 developed systemic viremia only when HCV-specific T cell responses declined.
Although this study documents that sporadically detected HCV RNA can represent infectious virus, it should not be used to justify virological testing beyond 24 weeks after treatment in patients with an SVR. However, the observed delayed viremia after low-level HCV infection may warrant a longer follow-up of health care workers after HCV exposure. Consistent with the results of our study, a case report described 2 health care workers who developed viremia 5 months and 9 months after needlestick injuries, respectively (16). HCV-specific T cell responses were analyzed in 1 of these individuals and, as in our study, were found to decline just prior to the emergence of systemic viremia (16). Thus, the final testing for HCV RNA and HCV antibodies should occur more than 7 months after exposure, which is a longer follow-up than currently recommended by the Centers for Disease Control and Prevention (CDC) (17).
The delayed appearance of systemic viremia may be due to several factors. First, the fact that HCV persisted in the absence of systemic viremia in both the source patient (8) and the infused chimpanzee in this study suggests that the unusual course of infection is due to the specific viral isolate rather than to host factors. While we excluded IFN resistance as described in a Japanese population due to mutations in the N-terminal NS5A region (18), it is possible that HCV persists in hepatocytes that become refractory to IFN signaling (19, 20). Notably, the source patient experienced a very slow second-phase virological response to IFN-based therapy (not shown). Second, the low viral titer in the injected plasma may have delayed viremia. Recently, Asabe et al. showed delayed systemic viremia in chimpanzees that had been inoculated with 101 genomic equivalents (GE) of HBV per milliliter as compared with those inoculated with 107 and 104 HBV GE per milliliter (21). As in our study, injection of the low-dose inoculum resulted in chronic infection. Finally, our results support the interesting hypothesis that HCV persists in a form that is refractory to eradication by IFN-based — and possibly even direct-acting antiviral — therapy, as recently proposed by Ralston et al. (22). Consistent with this notion, Bauhofer et al. demonstrated in an in vitro study that long-term exposure of differentiated quiescent hepatoma cells to IFN-α reduced HCV replication 1,000-fold, but did not eliminate HCV, and that viral replication rebounded after IFN-α withdrawal (23).
A final interesting aspect of this study is the immune response that the infected chimpanzees mounted. The passively transferred antibodies likely did not contribute much to the control of HCV infection, because they were unable to eliminate the autologous virus in the source patients, and because their titer decreased rapidly after each infusion in the chimpanzees. While the presence of strain-specific neutralizing antibodies cannot be formally excluded, the current data point to a role of the cellular immune response in the control of low-level HCV infection. Specifically, the induction of T cell responses against nonstructural HCV antigens that are not part of the HCV particle suggests that translation of HCV RNA occurred in infected cells. This was consistent with the detection of intrahepatic HCV RNA and ISG induction in chimpanzee A3A013. Chimpanzees A3A015 and A3A017, which also mounted T cell responses, either may have rapidly controlled intrahepatic HCV RNA, or the biopsies may have sampled uninfected rather than infected hepatocytes.
The presence of T cell responses in the absence of viremia and seroconversion in the 3 experimental chimpanzees is reminiscent of the immune status of subgroups of injection drug users, health care workers, and family members of chronic HCV patients who are frequently exposed to low-level HCV but test negative for HCV RNA and antibodies (24–30). Of note, however, there was no substantial increase in the breadth or strength of the immune response after each exposure, which is consistent with attrition of memory T cell subpopulations after heterologous exposures (31, 32) and with the observation that the T cell responses of the low-dose HCV–exposed chimpanzees did not protect against high-dose HCV challenge (33).
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