Hepatitis C virus (HCV), a single-stranded positive RNA virus belonging to the Flavirvirdae family, causes chronic disease in the vast majority of individuals infected. Due to shared routes of transmission, HIV/HCV coinfection is common; of the 33 million known HIV-infected individuals worldwide, 4–5 million are coinfected with HCV . Since the introduction of HAART and the reduction in morbidity and mortality, HCV has emerged as a major pathogen within the HIV-infected population .
HCV is a hepatotropic virus known to be associated with a number of extrahepatic manifestations. It replicates in extrahepatic sites, including peripheral blood mononuclear cells (PBMCs), during HCV infection [3,4]. In some patients, HCV infection is self-limiting and spontaneously resolves before proceeding beyond the acute phase. Spontaneous clearance occurs in up to as many as 42% of monoinfected individuals . However, robust and multispecific CD4 and CD8 T-cell responses are required for spontaneous clearance, and therefore, it is unsurprising that in HIV coinfection, spontaneous clearance is less common, ranging from 4 to 25% [6–8]. Treatment for HCV consists of administration of pegylated interferon in combination with ribavirin. The duration of treatment depends on the chronicity of the infection, the genotype and the response to initial therapy. Treatment is deemed successful if individuals achieve a sustained virological response (SVR), an undetectable serum HCV RNA at 6 months after the end of treatment. Success rates, especially for those with chronic genotype 1 or 4 virus, are unsatisfactory at approximately 50%  in the monoinfected population and 30%  in those coinfected with HIV. Until recently, it has been assumed that eradication of HCV RNA from the serum by spontaneous or therapeutic clearance reflects viral eradication from the liver and extrahepatic sites. However, it has been suggested that HCV may persist and replicate in these sites, even after serological clearance. Recent reports have provided conflicting data about the persistence of HCV RNA in PBMCs and hepatocytes of individuals who have achieved viral clearance from the serum (see Table 1) [11–17]. Not all of these studies have determined active viral replication. PBMCs may provide a privileged site for HCV, allowing persistent replication even after spontaneous or therapeutic clearance from the serum. Silent persistence of replicating HCV may have significant consequences by permitting late relapse in favourable conditions such as HIV or postliver transplant, perpetuation of subclinical liver disease and theoretically transmission through blood and organ donations.
The mechanism of viral replication involves the synthesis of a negative-strand RNA molecule that acts as a template for the production of positive or genomic HCV RNA. Thus, detection of the HCV RNA negative strand is indicative of viral replication. The aim of this prospective cross-sectional study was to determine whether HCV RNA persists in PBMCs after viral clearance from the serum, either spontaneously or after successful therapeutic intervention for acute or chronic HCV. Our objectives were to determine the frequency of HCV persistence in PBMCs of HIV-positive patients with clearance of the virus from serum and the frequency of negative-strand RNA in those found to have HCV persistence in PBMCs.
HIV antibody-positive individuals with previous exposure to HCV (HCV antibody positive), but not current infection with HCV (serum HCV RNA negative for at least 6 months) were eligible. We recruited to three cohorts: spontaneous HCV RNA clearance from the serum without therapeutic intervention following a clinical episode of acute hepatitis C (AHC), HCV RNA clearance from the serum following successful treatment of AHC and chronic hepatitis C (CHC). Participants fulfilling the criteria were identified in the combined HIV/Hepatitis clinic at Chelsea and Westminster Hospital. Written informed consent was obtained from all participants recruited to the study. Ethical approval was obtained from the Ealing & West London Mental Health Trust Research Ethics Committee.
On the day of recruitment, blood sampling was utilized to identify HCV RNA in both serum and PBMCs. PBMCs were isolated by density gradient sedimentation (lymphoprep) and washed three times in PBS to remove any serum-associated RNA. The cells were reconstituted in PBS to a concentration of 1 × 107 cells per vial. When samples were not tested immediately for RNA, they were stored at −80°C in lysis buffer after disruption and homogenization. Frozen samples were stable for up to 6 months. Intracellular HCV RNA was extracted using the QIAamp RNA Blood MiniKit (Qiagen). Reverse transcriptase-PCR was performed using a modification of the COBAS TaqMan HCV Test for use with the high pure system (Roche Diagnostics). The performance of the assay was determined using washed cell preparations, and the minimum detection level of HCV RNA in the presence of PBMC RNA was determind by the addition of exogenous HCV. HCV RNA could be detected to at least 600 IU/1 × 107 cells.
Twenty-six HIV-positive individuals were recruited to the study (see Table 2). All had previously been infected with HCV. Six individuals had spontaneously cleared HCV RNA from the serum, without therapeutic intervention, following a clinical episode of AHC (group 1), 10 had achieved a SVR following 24 weeks of pegylated interferon and ribavirin from AHC (group 2), and 10 had achieved a SVR following standard pegylated interferon and ribavirin therapy (24 weeks for genotype 2 or 3 and 48 weeks for genotype 1 or 4) for chronic HCV (group 3). Of the six individuals recruited to group 1, five were men, and all had previously been infected with genotype 1 HCV. All were on HAART with undetectable HIV viral loads (<50 copies/ml) at the time of recruitment. The median CD4 cell count was 385 cells/μl (range 275–587 cells/μl). The median duration from HCV spontaneous clearance to recruitment was 42 months (range 24–74 months). None demonstrated HCV RNA persistence in either serum or PBMCs.
Individuals recruited to group 2 were all men. All had previously been infected with genotype 1 HCV. Three individuals were HAART naive, with CD4 cell counts ranging from 225 to 575 cells/μl. All but one of those on HAART had an undetectable HIV viral load. The median CD4 cell count of those on treatment was 377 cells/μl (range 253–610 cells/μl). The median duration from achieving an SVR to recruitment was 15 months (range 5–29 months). None of the participants demonstrated HCV RNA persistence in either serum or PBMCs.
Nine individuals recruited to group 3 were men. Five individuals had previously been infected with genotype 1 HCV, one with genotype 2 HCV, two with genotype 3 HCV and two with genotype 4 HCV. Two were HAART naive at the time of recruitment, with CD4 cell counts of 379 and 1508 cells/μl, respectively. All of the eight individuals on HAART had undetectable HIV viral loads with a median CD4 cell count of 441.5 cells/μl (range 117–844 cells/μl). The median duration from achieving an SVR to recruitment was 28 months (range 8–73 months). None of the participants demonstrated HCV RNA persistence in either serum or PBMCs.
Several studies have examined the persistence and replication of HCV RNA in hepatocytes and PBMCs after spontaneous or therapeutic clearance from the sera in HCV-monoinfected individuals but with varying results (see Table 1). Early studies [11–14] from USA and Spain were relatively small. All showed evidence of HCV RNA in PBMCs, and the three studies that looked for HCV RNA persistence in hepatocytes found it in a significant proportion. When assessed for, evidence of viral replication via the presence of antigenomic HCV RNA was found in both PBMCs and liver tissue. More recently, Cavalherio Nde et al.  identified 54 individuals who had previously completed treatment for HCV. Samples were collected from 7 to 2730 days after the end of treatment. Thirty-five of these individuals had a positive serum HCV RNA. Of the remaining 19 individuals with negative serum HCV RNA after the end of treatment, three had HCV RNA identified in PBMCs. The presence of active viral replication was not determined. However, two studies [16,17] published in 2008 recruiting 413 individuals between them, who had previously spontaneously or therapeutically cleared HCV, detected no HCV RNA in PBMCs of any participants. Thus, the question of whether PBMCs act as sanctuary sites for HCV after clearance from the serum, in monoinfected individuals is, as yet, unresolved.
Theoretically, in HIV/HCV coinfection, the possible existence of HCV reservoirs is quite plausible due to the presence of a chronically impaired immune system. A recent study  analysed the association between the presence of positive/negative-strand HCV RNA in different isolated cell subsets at the end of treatment and treatment response. However, to our knowledge, this is the first study to investigate the presence of HCV RNA in PBMCs of HIV-infected individuals at least 6 months after clearing HCV from the serum.
Our study was limited by the small population and the lack of analysis of liver tissue that, although beyond the scope of this research, may be an important reservoir site for HCV and thus warrants further investigation. Our results are consistent with the findings from two recent large studies from Maylin et al.  and Bernardin et al. . Why other studies have detected HCV RNA in PBMCs when we have not is unclear. The Roche TaqMan assay used in this study has been calibrated against international standards to amplify HCV RNA at levels above 15 IU/ml. It can detect but not quantify HCV RNA below this value. Positive and negative controls were included on each run. The measured HCV RNA concentration for the positive control samples fell within the desired target range; inter-run variability was less than 10%. None of the negative control samples quantified. Some of the authors who did detect HCV RNA in PBMCs used an older and less sensitive version of the same assay that we used [12,15]. One other possible reason for the observed difference is that in some of the earlier studies, PBMCs were cultured  and mitogen stimulated  before PCR was performed. The effect of this manipulation on the detection of HCV RNA is unknown and requires further study.
Our findings lend support to the view that, clearance of HCV RNA from serum in HIV/HCV coinfection indicates eradication from PBMCs. The absence of HCV RNA in PBMCs of 26 previously HIV/HCV coinfected individuals is reassuring and confirms the use of SVR as a marker of treatment success in interferon-based therapies in coinfected individuals. However, the clinical endpoint of SVR may only be applicable to interferon-based therapies, and the advent of small molecule HCV inhibitors may require us to rethink our definitions of response and cure. For HIV-infected individuals, this issue may be particularly important, given the overall slower decay of HCV RNA under interferon-based therapies and the associated lower rates of SVR. For these reasons, further basic research into the biology of persisting HCV RNA in both PBMCs and hepatocytes is needed.
E.E.P. was involved in writing THE study proposal and protocol, patient recruitment and writing the article.
A.C. was involved in laboratory investigations and writing the article.
M.A. was involved in study concept, writing study proposal and protocol and writing the article.
M.R.N. was involved in study concept, writing study proposal and protocol, patient recruitment and writing the article.
Funding for this study was obtained from Roche Pharmaceuticals Limited and The British HIV Association.
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