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1.  Influence of HIV and HCV on T cell antigen presentation and challenges in the development of vaccines 
Some of the central challenges for developing effective vaccines against HIV and hepatitis C virus (HCV) are similar. Both infections are caused by small, highly mutable, rapidly replicating RNA viruses with the ability to establish long-term chronic pathogenic infection in human hosts. HIV has caused 60 million infections globally and HCV 180 million and both viruses may co-exist among certain populations by virtue of common blood-borne, sexual, or vertical transmission. Persistence of both pathogens is achieved by evasion of intrinsic, innate, and adaptive immune defenses but with some distinct mechanisms reflecting their differences in evolutionary history, replication characteristics, cell tropism, and visibility to mucosal versus systemic and hepatic immune responses. A potent and durable antibody and T cell response is a likely requirement of future HIV and HCV vaccines. Perhaps the single biggest difference between the two vaccine design challenges is that in HCV, a natural model of protective immunity can be found in those who resolve acute infection spontaneously. Such spontaneous resolvers exhibit durable and functional CD4+ and CD8+ T cell responses (Diepolder et al., 1995; Cooper et al., 1999; Thimme et al., 2001; Grakoui et al., 2003; Lauer et al., 2004; Schulze Zur Wiesch et al., 2012). However, frequent re-infection suggests partial or lack of protective immunity against heterologous HCV strains, possibly indicative of the degree of genetic diversity of circulating HCV genotypes and subtypes. There is no natural model of protective immunity in HIV, however, studies of “elite controllers,” or individuals who have durably suppressed levels of plasma HIV RNA without antiretroviral therapy, has provided the strongest evidence for CD8+ T cell responses in controlling viremia and limiting reservoir burden in established infection. Here we compare and contrast the specific mechanisms of immune evasion used by HIV and HCV, which subvert adaptive human leukocyte antigen (HLA)-restricted T cell immunity in natural infection, and the challenges these pose for designing effective preventative or therapeutic vaccines.
doi:10.3389/fmicb.2014.00514
PMCID: PMC4195390  PMID: 25352836
HIV; HCV; viral immune escape; preventative vaccine; anti-viral immune responses
2.  Kidney and liver organ transplantation in persons with human immunodeficiency virus 
Executive Summary
Objective
The objective of this analysis is to determine the effectiveness of solid organ transplantation in persons with end stage organ failure (ESOF) and human immunodeficiency virus (HIV+)
Clinical Need: Condition and Target Population
Patients with end stage organ failure who have been unresponsive to other forms of treatment eventually require solid organ transplantation. Similar to persons who are HIV negative (HIV−), persons living with HIV infection (HIV+) are at risk for ESOF from viral (e.g. hepatitis B and C) and non-viral aetiologies (e.g. coronary artery disease, diabetes, hepatocellular carcinoma). Additionally, HIV+ persons also incur risks of ESOF from HIV-associated nephropathy (HIVAN), accelerated liver damage from hepatitis C virus (HCV+), with which an estimated 30% of HIV positive (HIV+) persons are co-infected, and coronary artery disease secondary to antiretroviral therapy. Concerns that the need for post transplant immunosuppression and/or the interaction of immunosuppressive drugs with antiretroviral agents may accelerate the progression of HIV disease, as well as the risk of opportunistic infections post transplantation, have led to uncertainty regarding the overall benefit of transplantation among HIV+ patients. Moreover, the scarcity of donor organs and their use in a population where the clinical benefit of transplantation is uncertain has limited the availability of organ transplantation to persons living with ESOF and HIV.
With the development of highly active anti retroviral therapy (HAART), which has been available in Canada since 1997, there has been improved survival and health-related quality of life for persons living with HIV. HAART can suppress HIV replication, enhance immune function, and slow disease progression. HAART managed persons can now be expected to live longer than those in the pre-HAART era and as a result many will now experience ESOF well before they experience life-threatening conditions related to HIV infection. Given their improved prognosis and the burden of illness they may experience from ESOF, the benefit of solid organ transplantation for HIV+ patients needs to be reassessed.
Evidence-Based Analysis Methods
Research Questions
What are the effectiveness and cost effectiveness of solid organ transplantation in HIV+ persons with ESOF?
Literature Search
A literature search was performed on September 22, 2009 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, the Cumulative Index to Nursing & Allied Health Literature (CINAHL), the Cochrane Library, and the International Agency for Health Technology Assessment (INAHTA) for studies published from January 1, 1996 to September 22, 2009.
Inclusion Criteria
Systematic review with or without a Meta analysis, RCT, Non-RCT with controls
HIV+ population undergoing solid organ transplantation
HIV+ population managed with HAART therapy
Controls include persons undergoing solid organ transplantation who are i) HIV− ii) HCV+ mono-infected, and iii) HIV+ persons with ESOF not transplanted.
Studies that completed and reported results of a Kaplan-Meier Survival Curve analysis.
Studies with a minimum (mean or medium) follow up of 1-year.
English language citations
Exclusion Criteria
Case reports and case series were excluded form this review.
Outcomes of Interest
i) Risk of Death after transplantation
ii) Death censored graft survival (DCGS)
iii) HIV disease progression defined as the post transplant incidence of:
- opportunistic infections or neoplasms,
- CD4+ T-cell count < 200mm3, and
- any detectable level of plasma HIV viral load.
iv) Acute graft rejection,
v) Return to dialysis,
vi) Recurrence of HCV infection
Summary of Findings
No direct evidence comparing an HIV+ cohort undergoing transplantation with the same not undergoing transplantation (wait list) was found in the literature search.
The results of this review are reported for the following comparison cohorts undergoing transplantation:
i) Kidney Transplantation: HIV+ cohort compared with HIV− cohort
ii) Liver Transplantation: HIV+ cohort compared with HIV− negative cohort
iii) Liver Transplantation: HIV+ HCV+ (co-infected) cohort compared with HCV+ (mono-infected) cohort
Kidney Transplantation: HIV+ vs. HIV−
Based on a pooled HIV+ cohort sample size of 285 patients across four studies, the risk of death after kidney transplantation in an HIV+ cohort does not differ to that of an HIV− cohort [hazard ratio (HR): 0.90; 95% CI: 0.36, 2.23]. The quality of evidence supporting this outcome is very low.
Death censored graft survival was reported in one study with an HIV+ cohort sample size of 100, and was statistically significantly different (p=.03) to that in the HIV− cohort (n=36,492). However, the quality of evidence supporting this outcome was determined to be very low. There was also uncertainty in the rate of return to dialysis after kidney transplantation in both the HIV+ and HIV− groups and the effect, if any, this may have on patient survival. Because of the very low quality evidence rating, the effect of kidney transplantation on HIV-disease progression is uncertain.
The rate of acute graft rejection was determined using the data from one study. There was a nonsignificant difference between the HIV+ and HIV− cohorts (OR 0.13; 95% CI: 0.01, 2.64), although again, because of very low quality evidence there is uncertainty in this estimate of effect.
Liver Transplantation: HIV+ vs. HIV−
Based on a combined HIV+ cohort sample size of 198 patient across five studies, the risk of death after liver transplantation in an HIV+ cohort (with at least 50% of the cohort co-infected with HCV+) is statistically significantly 64% greater compared with an HIV− cohort (HR: 1.64; 95% CI: 1.32, 2.02). The quality of evidence supporting this outcome is very low.
Death censored graft survival was reported for an HIV+ cohort in one study (n=11) however the DCGS rate of the contemporaneous control HIV− cohort was not reported. Because of sparse data the quality of evidence supporting this outcome is very low indicating death censored graft survival is uncertain.
Both the CD4+ T-cell count and HIV viral load appear controlled post transplant with an incidence of opportunistic infection of 20.5%. However, the quality of this evidence for these outcomes is very low indicating uncertainty in these effects. Similarly, because of very low quality evidence there is uncertainty in the rate of acute graft rejection among both the HIV+ and HIV− groups
Liver Transplantation: HIV+/HCV+ vs. HCV+
Based on a combined HIV+/HCV+ cohort sample size of 156 from seven studies, the risk of death after liver transplantation is significantly greater (2.8 fold) in a co-infected cohort compared with an HCV+ mono-infected cohort (HR: 2.81; 95% CI: 1.47, 5.37). The quality of evidence supporting this outcome is very low. Death censored graft survival evidence was not available.
Regarding disease progression, based on a combined sample size of 71 persons in the co-infected cohort, the CD4+ T-cell count and HIV viral load appear controlled post transplant; however, again the quality of evidence supporting this outcome is very low. The rate of opportunistic infection in the co-infected cohort was 7.2%. The quality of evidence supporting this estimate is very low, indicating uncertainty in these estimates of effect.
Based on a combined HIV+/HCV+ cohort (n=57) the rate of acute graft rejection does not differ to that of an HCV+ mono-infected cohort (OR: 0.88; 95% CI: 0.44, 1.76). Also based on a combined HIV+/HCV+ cohort (n=83), the rate of HCV+ recurrence does not differ to that of an HCV+ mono-infected cohort (OR: 0.66; 95% CI: 0.27, 1.59). In both cases, the quality of the supporting evidence was very low.
Overall, because of very low quality evidence there is uncertainty in the effect of kidney or liver transplantation in HIV+ persons with end stage organ failure compared with those not infected with HIV. Examining the economics of this issue, the cost of kidney and liver transplants in an HIV+ patient population are, on average, 56K and 147K per case, based on both Canadian and American experiences.
PMCID: PMC3377507  PMID: 23074407
3.  Impaired Hepatitis C Virus-Specific T Cell Responses and Recurrent Hepatitis C Virus in HIV Coinfection 
PLoS Medicine  2006;3(12):e492.
Background
Hepatitis C virus (HCV)-specific T cell responses are critical for spontaneous resolution of HCV viremia. Here we examined the effect of a lymphotropic virus, HIV-1, on the ability of coinfected patients to maintain spontaneous control of HCV infection.
Methods and Findings
We measured T cell responsiveness by lymphoproliferation and interferon-γ ELISPOT in a large cohort of HCV-infected individuals with and without HIV infection. Among 47 HCV/HIV-1-coinfected individuals, spontaneous control of HCV was associated with more frequent HCV-specific lymphoproliferative (LP) responses (35%) compared to coinfected persons who exhibited chronic HCV viremia (7%, p = 0.016), but less frequent compared to HCV controllers who were not HIV infected (86%, p = 0.003). Preservation of HCV-specific LP responses in coinfected individuals was associated with a higher nadir CD4 count (r2 = 0.45, p < 0.001) and the presence and magnitude of the HCV-specific CD8+ T cell interferon-γ response (p = 0.0014). During long-term follow-up, recurrence of HCV viremia occurred in six of 25 coinfected individuals with prior control of HCV, but in 0 of 16 HIV-1-negative HCV controllers (p = 0.03, log rank test). In these six individuals with recurrent HCV viremia, the magnitude of HCV viremia following recurrence inversely correlated with the CD4 count at time of breakthrough (r = −0.94, p = 0.017).
Conclusions
These results indicate that HIV infection impairs the immune response to HCV—including in persons who have cleared HCV infection—and that HIV-1-infected individuals with spontaneous control of HCV remain at significant risk for a second episode of HCV viremia. These findings highlight the need for repeat viral RNA testing of apparent controllers of HCV infection in the setting of HIV-1 coinfection and provide a possible explanation for the higher rate of HCV persistence observed in this population.
HIV infection impairs the immune response to HCV. Even individuals who have cleared HCV infection remain at significant risk for a second episode of HCV viremia.
Editors' Summary
Background.
Because of shared transmission routes (contaminated needles, contaminated blood products, and, to a lesser extent, unprotected sex), a large proportion of HIV-infected individuals (estimates range between 25% and 33%) are also infected with the hepatitis C virus (HCV). In most but not all individuals infected with HCV, the virus infection is chronic and causes liver disease that can eventually lead to liver failure. Disease progress is slow; it often takes decades until infected individuals develop serious liver disease. In people infected with both HCV and HIV, however, liver disease caused by HCV often appears sooner and progresses faster. As highly active antiretroviral therapy (HAART) and prophylaxis of opportunistic infections increase the life span of persons living with HIV, HCV-related liver disease has become a major cause of hospital admissions and deaths among HIV-infected persons.
Why Was This Study Done?
A sizable minority of people who are infected with HCV manage to control the virus and never get liver disease, and scientists have found that these people somehow mounted a strong immune response against the hepatitis C virus. CD4+ T cells, the very immune cells that are infected and destroyed by HIV, play an important role in this immune response. The goal of the present study was to better understand how infection with HIV compromises the specific immune response to HCV and thereby the control of HCV disease progression.
What Did the Researchers Do and Find?
The researchers recruited four groups of patients, 94 in total, all of whom were infected with HCV. Two groups comprised patients who were infected with HIV as well as HCV, with either high or undetectable levels of HCV (30 patients in each group). The two other groups included patients not infected with HIV, either with high or undetectable levels of HCV (17 patients in each group). The researchers focused on the individuals who, despite coinfection with HIV, were able to control their HCV infection. They found that those individuals managed to maintain relatively high levels of CD4+ T cells that specifically recognize HCV. However, a quarter of these patients (six out of 25) failed to keep HCV levels down for the entire observation period of up to 2.5 years; their blood levels of HCV rose substantially, most likely due to recurrence of the previously suppressed virus (the researchers could not be certain that none of the patients had become infected again after a new exposure to HCV-contaminated blood, but there was no evidence that they had engaged in risky behavior). The rise of HCV levels in the blood of the relapsed patients coincided with a drop in overall CD4+ T cell numbers. Following relapse in these individuals, HCV did not return to undetectable levels during the study. During the same period none of the 16 HIV-uninfected people with controlled HCV infection experienced a recurrence of detectable HCV.
What Do These Findings Mean?
Despite the relatively small numbers of patients, these results suggest that recurrence of HCV after initial control of the virus is more likely in people who are coinfected with HIV, and that HCV control is lost when CD4+ T cell counts fall. This is one more reason to test all HIV-positive patients for HCV coinfection. Coinfected patients, even those who seem to be controlling HCV and would not automatically receive HCV treatment, should be regularly tested for a rise of HCV levels. In addition, maintaining CD4+ T cells at a high level might be particularly important for those patients, which means that doctors might consider starting HAART therapy earlier than is generally recommended for HIV-infected individuals. Additional studies are needed to support these recommendations, however, especially as this study did not follow the patients long enough to determine the consequences of the observed loss of control of HCV.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030492.
AIDS Treatment Data Network factsheet on HIV/HCV coinfection
US CDC factsheet on HIV/HCV coinfection
American Liver Foundation, information on HIV and HCV
MedlinePlus pages on HCV
doi:10.1371/journal.pmed.0030492
PMCID: PMC1705826  PMID: 17194190
4.  Host-Specific Response to HCV Infection in the Chimeric SCID-beige/Alb-uPA Mouse Model: Role of the Innate Antiviral Immune Response 
PLoS Pathogens  2006;2(6):e59.
The severe combined immunodeficiency disorder (SCID)-beige/albumin (Alb)-urokinase plasminogen activator (uPA) mouse containing a human-mouse chimeric liver is currently the only small animal model capable of supporting hepatitis C virus (HCV) infection. This model was utilized to characterize the host transcriptional response to HCV infection. The purpose of these studies was to investigate the genetic component of the host response to HCV infection and also to distinguish virus-induced gene expression changes from adaptive HCV-specific immune-mediated effects. Gene expression profiles from HCV-infected mice were also compared to those from HCV-infected patients. Analyses of the gene expression data demonstrate that host factors regulate the response to HCV infection, including the nature of the innate antiviral immune response. They also indicate that HCV mediates gene expression changes, including regulation of lipid metabolism genes, which have the potential to be directly cytopathic, indicating that liver pathology may not be exclusively mediated by HCV-specific adaptive immune responses. This effect appears to be inversely related to the activation of the innate antiviral immune response. In summary, the nature of the initial interferon response to HCV infection may determine the extent of viral-mediated effects on host gene expression.
Synopsis
The natural history of hepatitis C virus (HCV) infection is highly variable, and approximately 30% of chronically infected patients will develop progressive liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). This high variability in HCV-associated liver disease, ranging from mild inflammation to rapidly progressive fibrosis, suggests that host factors play an important role in both infection outcome and viral pathogenesis. In the current study, the severe combined immunodeficiency disorder-beige/albumin-urokinase plasminogen activator mouse model was used to investigate how host-specific factors influence the host response to HCV infection. Cohorts of mice transplanted with hepatocytes from different donors were inoculated with a single source of HCV. Gene expression profiling was performed to characterize the host response to infection. The results indicate that host factors do contribute to the variation in host response to HCV infection, including the activation of innate antiviral signaling pathways. They also suggest that the nature of the innate antiviral immune response during the acute phase of infection may determine the extent of viral-mediated effects on host gene expression, including regulation of lipid metabolism genes and induction of stress-response genes. In addition, the presence of apoptotic hepatocytes in HCV-infected mice suggests that liver injury can occur in the absence of an adaptive HCV-specific immune response.
doi:10.1371/journal.ppat.0020059
PMCID: PMC1480599  PMID: 16789836
5.  Immune Biomarker Differences and Changes Comparing HCV Mono-Infected, HIV/HCV Co-Infected, and HCV Spontaneously Cleared Patients 
PLoS ONE  2013;8(4):e60387.
Background
Immune biomarkers are implicated in HCV treatment response, fibrosis, and accelerated pathogenesis of comorbidities, though only D-dimer and C-reactive protein have been consistently studied. Few studies have evaluated HIV/HCV co-infection, and little longitudinal data exists describing a broader antiviral cytokine response
Methods
Fifty immune biomarkers were analyzed at baseline(BL) and HCV end of treatment follow-up(FU) time point using the Luminex 50-plex assay in plasma samples from 15 HCV-cleared, 24 HCV mono- and 49 HIV/HCV co-infected patients receiving antiretroviral treatment, who either did or did not receive pegylated-interferon/ribavirin HCV treatment. Biomarker levels were compared among spontaneous clearance patients, mono- and co-infected, untreated and HCV-treated, and sustained virologic responders (SVR) and non-responders (NR) at BL and FU using nonparametric analyses. A Bonferroni correction, adjusting for tests of 50 biomarkers, was used to reduce Type I error
Results
Compared to HCV patients at BL, HIV/HCV patients had 22 significantly higher and 4 significantly lower biomarker levels, following correction for multiple testing. There were no significantly different BL levels when comparing SVR and NR in mono- or co-infected patients; however, FU levels changed considerably in co-infected patients, with seven becoming significantly higher and eight becoming significantly lower in SVR patients. Longitudinally between BL and FU, 13 markers significantly changed in co-infected SVR patients, while none significantly changed in co-infected NR patients. There were also no significant changes in longitudinal analyses of mono-infected patients achieving SVR or mono-infected and co-infected groups deferring treatment
Conclusions
Clear differences exist in pattern and quantity of plasma immune biomarkers among HCV mono-infected, HIV/HCV co-infected, and HCV-cleared patients; and with SVR in co-infected patients treated for HCV. Though >90% of patients were male and co-infected had a larger percentage of African American patients, our findings may have implications for better understanding HCV pathogenesis, treatment outcomes, and future therapeutic targets
doi:10.1371/journal.pone.0060387
PMCID: PMC3617231  PMID: 23593207
6.  HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1 
PLoS Pathogens  2013;9(4):e1003248.
Upon recognition of viral components by pattern recognition receptors, such as the toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like helicases, cells are activated to produce type I interferon (IFN) and proinflammatory cytokines. These pathways are tightly regulated by the host to prevent an inappropriate cellular response, but viruses can modulate these pathways to proliferate and spread. In this study, we revealed a novel mechanism in which hepatitis C virus (HCV) evades the immune surveillance system to proliferate by activating microRNA-21 (miR-21). We demonstrated that HCV infection upregulates miR-21, which in turn suppresses HCV-triggered type I IFN production, thus promoting HCV replication. Furthermore, we demonstrated that miR-21 targets two important factors in the TLR signaling pathway, myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase 1 (IRAK1), which are involved in HCV-induced type I IFN production. HCV-mediated activation of miR-21 expression requires viral proteins and several signaling components. Moreover, we identified a transcription factor, activating protein-1 (AP-1), which is partly responsible for miR-21 induction in response to HCV infection through PKCε/JNK/c-Jun and PKCα/ERK/c-Fos cascades. Taken together, our results indicate that miR-21 is upregulated during HCV infection and negatively regulates IFN-α signaling through MyD88 and IRAK1 and may be a potential therapeutic target for antiviral intervention.
Author Summary
Hepatitis C virus (HCV), a major cause of chronic hepatitis, end-stage cirrhosis, and hepatocellular carcinoma, has chronically infected 200 million people worldwide and 3–4 million more each year. When triggered by viral infection, host cells produce type I interferon (IFN) and proinflammatory cytokines to antagonize the virus. Despite extensive research, the mechanism underlying HCV immune system evasion remains elusive. Our results provided the first direct evidence that microRNA-21 (miR-21) feedback inhibits type I IFN signaling when cells are challenged with HCV, thus promoting the infection. MicroRNA is a kind of endogenous non-coding small RNA that regulates a wide range of biological processes and participate in innate and adaptive immune responses through complementarily pairing with target mRNA, which can regulate its expression or translation. Currently, miRNAs have intrigued many scientists as potent targets or therapeutic agents for diseases. In our study, the targets of miR-21, myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase 1 (IRAK1), which are important for HCV-induced type I IFN production, have also been found. Moreover, we identified a transcription factor, AP-1, which is partly responsible for miR-21 induction in response to HCV infection. Taken together, our research has provided new insights into understanding the effects of miRNA on host-virus interactions, and revealed a potential therapeutic target for antiviral intervention.
doi:10.1371/journal.ppat.1003248
PMCID: PMC3635988  PMID: 23633945
7.  Innate Immune Responses in Hepatitis C Virus Infection1 
Seminars in immunopathology  2012;35(1):53-72.
Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma worldwide and thus poses a significant public health threat. A hallmark of HCV infection is the extraordinary ability of the virus to persist in a majority of infected people. Innate immune responses represent the front line of defense of the human body against HCV immediately after infection. They also play a crucial role in orchestrating subsequent HCV-specific adaptive immunity that is pivotal for viral clearance. Accumulating evidence suggests that the host has evolved multifaceted innate immune mechanisms to sense HCV infection and elicit defense responses, while HCV has developed elaborate strategies to circumvent many of these. Defining the interplay of HCV with host innate immunity reveals mechanistic insights into hepatitis C pathogenesis and informs approaches to therapy. In this review, we summarize recent advances in understanding innate immune responses to HCV infection, focusing on induction and effector mechanisms of the interferon antiviral response as well as the evasion strategies of HCV.
doi:10.1007/s00281-012-0332-x
PMCID: PMC3732459  PMID: 22868377
8.  Enhanced IL-10 production in response to hepatitis C virus proteins by peripheral blood mononuclear cells from human immunodeficiency virus-monoinfected individuals 
BMC Immunology  2008;9:28.
Background
Multiple immune evasion strategies by which HCV establishes chronic infection have been proposed, including manipulation of cytokine responses. Prior infection with HIV increases the likelihood of chronic HCV infection and accelerates development of HCV-related morbidity. Therefore, we investigated in vitro cytokine responses to HCV structural and non-structural proteins in peripheral blood mononuclear cells (PBMC) from uninfected, HIV-infected, HCV-infected and HIV/HCV-coinfected individuals.
Results
Intracellular flow cytometry was used to assess IL-2, IL-10, IL-12, and IFN-γ production by freshly isolated PBMC incubated for 16 hours with recombinant HCV core, non-structural protein 3 (NS3), and NS4 proteins. Anti-HCV cellular responses were assessed in HIV/HCV-coinfected individuals by 3H-thymidine proliferation assay. Exposure to HCV antigens increased IL-10 production by PBMC, especially in uninfected and HIV-monoinfected individuals. This IL-10 response was attenuated in chronic HCV infection even with HCV/HIV-coinfection. The cells producing IL-10 in response to HCV proteins in vitro matched a PBMC subset recently shown to constitutively produce IL-10 in vivo. This subset was found at similar frequencies in uninfected, HIV-infected, HCV-infected and HIV/HCV-coinfected individuals before exposure to HCV proteins. HCV-specific T cell proliferation was detectable in only one HIV/HCV-coinfected individual who demonstrated no HCV-induced IL-10 response.
Conclusion
This pattern suggests that selective induction of IL-10 in uninfected individuals and especially in HIV-monoinfected individuals plays a role in establishing chronic HCV infection and conversely, that attenuation of this response, once chronic infection is established, favours development of hepatic immunopathology.
doi:10.1186/1471-2172-9-28
PMCID: PMC2443791  PMID: 18554409
9.  Hepatitis C Virus Cell-Cell Transmission and Resistance to Direct-Acting Antiviral Agents 
PLoS Pathogens  2014;10(5):e1004128.
Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs.
Author Summary
In spite of the rapid development of antiviral agents, antiviral resistance remains a challenge for the treatment of viral infections including hepatitis B and C virus (HBV, HCV), human immunodeficiency virus (HIV) and influenza. Virus spreads from infected cells to surrounding uninfected host cells to develop infection through cell-free and cell-cell transmission routes. Understanding the spread of resistant virus is important for the development of novel antiviral strategies to prevent and treat antiviral resistance. Here, we characterize the spread of resistant viruses and its impact for emergence and prevention of resistance using HCV as a model system. Our results show that cell-cell transmission is the main transmission route for antiviral resistant HCV strains and is crucial for the maintenance of infection. Monoclonal antibodies or small molecules targeting HCV entry factors are effective in inhibiting the spread of resistant HCV in cell culture models and thus should be evaluated clinically for prevention and treatment of HCV resistance. Combination of inhibitors targeting viral entry and clinically used direct-acting antivirals (DAAs) prevents antiviral resistance and leads to viral eradication in cell culture models. Collectively, the investigation provides a new strategy for prevention of viral resistance to antiviral agents.
doi:10.1371/journal.ppat.1004128
PMCID: PMC4022730  PMID: 24830295
10.  Hepatitis C virus versus innate and adaptive immune responses: a tale of coevolution and coexistence 
The Journal of Clinical Investigation  2009;119(7):1745-1754.
Since the identification of the hepatitis C virus (HCV) 20 years ago, much progress has been made in our understanding of its life cycle and interaction with the host immune system. Much has been learned from HCV itself, which, via decades of coevolution, gained an intricate knowledge of host innate and adaptive immune responses and developed sophisticated ways to preempt, subvert, and antagonize them. This review discusses the clinical, virological, and immunological features of acute and chronic hepatitis C and the role of the immune response in spontaneous and treatment-induced HCV clearance.
doi:10.1172/JCI39133
PMCID: PMC2701885  PMID: 19587449
11.  Variation in Both IL28B and KIR2DS3 Genes Influence Pegylated Interferon and Ribavirin Hepatitis C Treatment Outcome in HIV-1 Co-Infection 
PLoS ONE  2013;8(6):e66831.
Pegylated-IFN and ribavirin remains the current treatment for chronic HCV infection in patients co-infected with HIV-1, but this regimen has low efficacy rates, particularly for HCV genotype 1/4 infection, has severe side effects and is extremely costly. Therefore, accurate prediction of treatment response is urgently required. We have recently shown that the NK cell gene, KIR2DS3 and a SNP associated with the IL28B gene synergise to increase the risk of chronic infection in primary HCV mono-infected patients. Identification of SNPs associated with the IL28B gene has also proven very powerful for predicting patient response to treatment. Patients co-infected with HIV-1 are of particular concern given they respond less well to HCV treatment, have more side effects and suffer a more rapid liver disease progression. In this study, we examined both IL28B and KIR2DS3 for their ability to predict treatment response in a cohort of HIV-1/HCV co-infected patients attending two treatment centres in Europe. We found that variation in both host genetic risk factors, IL28B and KIR2DS3, was strongly associated with sustained virological response (SVR) to treatment in our co-infected cohort (n = 149). The majority of patients who achieved a rapid virological response (RVR) achieved a SVR. However, it is currently impossible to predict treatment outcome in patients who fail to achieve an RVR. In our cohort, the presence of host genetic risk factors, IL28B-T and KIR2DS3 alleles, resulted in increased odds of treatment failure in these RVR negative patients (n = 88). Our data suggests that testing for host genetic factors will improve predicting treatment responsiveness in the clinical management of co-infected patients, and provides further evidence of the importance of the innate immune system in the immune response to HCV.
doi:10.1371/journal.pone.0066831
PMCID: PMC3691248  PMID: 23826153
12.  MicroRNAs, Hepatitis C Virus, and HCV/HIV-1 Co-Infection: New Insights in Pathogenesis and Therapy 
Viruses  2012;4(11):2485-2513.
MicroRNAs (miRNAs) can exert a profound effect on Hepatitis C virus (HCV) replication. The interaction of HCV with the highly liver-enriched miRNA, miR-122 represents one such unique example of viruses having evolved mechanism(s) to usurp the host miRNA machinery to support viral life cycle. Furthermore, HCV infection can also trigger changes in the cellular miRNA profile, which may ultimately contribute to the outcome of viral infection. Accumulating knowledge on HCV-host miRNA interactions has ultimately influenced the design of therapeutic interventions against chronic HCV infection. The importance of microRNA modulation in Human Immunodeficiency Virus (HIV-1) replication has been reported, albeit only in the context of HIV-1 mono-infection. The development of HCV infection is dramatically influenced during co-infection with HIV-1. Here, we review the current knowledge on miRNAs in HCV mono-infection. In addition, we discuss the potential role of some miRNAs, identified from the analyses of public data, in HCV/HIV-1 co-infection.
doi:10.3390/v4112485
PMCID: PMC3509660  PMID: 23202492
microRNA; miR-122, exosomes, HCV; hepatitis C virus; hepatitis; antiviral response; HIV-1; HIV-1/HCV co-infection; antagomir; therapeutics
13.  Complement Lysis Activity in Autologous Plasma Is Associated with Lower Viral Loads during the Acute Phase of HIV-1 Infection 
PLoS Medicine  2006;3(11):e441.
Background
To explore the possibility that antibody-mediated complement lysis contributes to viremia control in HIV-1 infection, we measured the activity of patient plasma in mediating complement lysis of autologous primary virus.
Methods and Findings
Sera from two groups of patients—25 with acute HIV-1 infection and 31 with chronic infection—were used in this study. We developed a novel real-time PCR-based assay strategy that allows reliable and sensitive quantification of virus lysis by complement. Plasma derived at the time of virus isolation induced complement lysis of the autologous virus isolate in the majority of patients. Overall lysis activity against the autologous virus and the heterologous primary virus strain JR-FL was higher at chronic disease stages than during the acute phase. Most strikingly, we found that plasma virus load levels during the acute but not the chronic infection phase correlated inversely with the autologous complement lysis activity. Antibody reactivity to the envelope (Env) proteins gp120 and gp41 were positively correlated with the lysis activity against JR-FL, indicating that anti-Env responses mediated complement lysis. Neutralization and complement lysis activity against autologous viruses were not associated, suggesting that complement lysis is predominantly caused by non-neutralizing antibodies.
Conclusions
Collectively our data provide evidence that antibody-mediated complement virion lysis develops rapidly and is effective early in the course of infection; thus it should be considered a parameter that, in concert with other immune functions, steers viremia control in vivo.
Antibody-mediated complement lysis of HIV virions develops rapidly and is effective already early in the course of HIV infection.
Editors' Summary
Background.
If untreated, most people who become infected with the human immunodeficiency virus (HIV) eventually develop acquired immunodeficiency syndrome (AIDS). Over time, HIV infects and kills their CD4 T lymphocytes—immune system cells that stimulate B lymphocytes to make antibodies (proteins that recognize and destroy infectious agents) and that help CD8 T lymphocytes to kill cells that contain viruses and bacteria. The loss of CD4 T lymphocytes—a central player in “adaptive immunity”—leaves patients very susceptible to infections. However, the immune system does not die quietly. It does its best to fight HIV infection by mounting a cell-mediated immune response in which T lymphocytes attack HIV-infected cells. It also mounts a “humoral” immune response in which antibodies that recognize HIV are made. Some of these are neutralizing antibodies, which prevent HIV entering its host cells and replicating. Other antibodies may limit viral spread by inducing destruction of the virus. One way they can do this is by activating another part of the immune system called the complement system, which can break open and kill viruses (this is known as antibody-mediated complement lysis). In addition, antibodies and complement can coat the HIV virus particles so that phagocytes (for instance macrophages—yet another type of immune system cell) engulf and destroy the virus.
Why Was This Study Done?
The role that humoral immunity plays in fighting HIV infection is complex and poorly understood. In particular, it is not clear whether the complement system helps to stop the spread of HIV or whether it inadvertently helps it to spread by facilitating its entry into host cells. It is important to understand as much as possible about the humoral immune response to HIV infection so that vaccines can be designed to provide maximum protection against HIV. In this study, the researchers have investigated whether antibody-mediated complement lysis controls the amount of virus in the blood of patients infected with HIV.
What Did the Researchers Do and Find?
The researchers collected plasma (the liquid part of blood that contains circulating antibodies) from patients recently infected with HIV (acute infection) and patients who had been infected for some time (chronically infected). They also isolated HIV from each of the patients—so-called autologous virus. They then used a sensitive molecular biology assay to test each plasma sample for its ability to lyse the autologous virus (and also a standard virus) when supplied with complement from a healthy donor. Most of the plasma samples were able to lyse HIV, although the samples taken from chronically infected patients generally caused more lysis than those from acutely infected patients. In the chronically infected patients, the level of lysis induced was not related to the amount of virus in the patients' blood (viremia). However, plasma taken from acutely infected patients with higher viral loads was less active in the lysis assay than plasma taken from patients with lower viral loads. Finally, the researchers showed that the levels of antibodies in the various plasma samples to the two envelope proteins of HIV correlated strongly with the ability of each sample to lyse the standard virus and that these antibodies were mainly non-neutralizing antibodies.
What Do These Findings Mean?
By showing that antibody-mediated complement lysis of HIV in the laboratory is inversely related to the patients' viral loads during acute infection, these findings suggest (but do not prove) that antibody-mediated complement lysis of HIV contributes to the control of viremia early in HIV infections. But, the importance of this form of humoral immunity in combating HIV infections remains uncertain, since complement has the potential to enhance as well as block viral spread. Further work is needed to unravel which of these effects is dominant in patients and to characterize fully the antibodies that activate complement. Nevertheless, the results of this study suggest that complement-activating antibodies should be considered in future attempts to design an effective HIV vaccine.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030441.
National Institute of Allergy and Infectious Diseases fact sheet on HIV infection and AIDS
US Department of Health and Human Services information on AIDS, including information on vaccines
US Centers for Disease Control and Prevention information on HIV/AIDS
Aidsmap information on HIV and the immune system provided by the charity NAM
Wikipedia pages on the complement system (note: Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030441
PMCID: PMC1637124  PMID: 17121450
14.  Viral Factors Associated with Cytokine Expression During HCV/HIV Co-Infection 
Co-infection with human immunodeficiency virus (HIV) is associated with reduced hepatitis C virus (HCV) treatment response and accelerated HCV disease. Cytokines, as mediators of immune responses, inflammation, and fibrogenesis, may underlie important differences in HCV pathogenesis during HIV co-infection. We previously found that serum interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) increased after HCV therapy with interferon (IFN) in HCV/HIV co-infected patients; however, cytokine levels were not predictive of HCV therapeutic response. Here, we examined viral factors associated with expression of IL-8, TNF-α, and transforming growth factor-β1 (TGF-β1) in uninfected, HCV mono-infected, HIV mono-infected, and HCV/HIV co-infected persons. HIV co-infection was associated with decreased IL-8 detection but not TNF-α detection. A significant interaction effect demonstrated that HIV infection was associated with elevated TGF-β1 in HCV-positive individuals but not in HCV-negative individuals. The induction of a sustained profibrotic signal, such as TGF-β1, by HIV may cause accelerated liver fibrosis during HCV/HIV co-infection and may hinder the host’s ability to mount an effective HCV-specific immune response. Further studies are warranted to identify noninvasive markers of liver disease for the clinical management of HCV disease, particularly when liver biopsies have not been performed or are contraindicated.
doi:10.1089/jir.2006.0147
PMCID: PMC4066618  PMID: 17477814
15.  Convergent Evolution of Escape from Hepaciviral Antagonism in Primates 
PLoS Biology  2012;10(3):e1001282.
Escape from antagonism by hepatitis C and related viruses has repeatedly evolved in antiviral factor MAVS via convergent evolution, revealing an ancient history of previous viral encounters in primates.
The ability to mount an interferon response on sensing viral infection is a critical component of mammalian innate immunity. Several viruses directly antagonize viral sensing pathways to block activation of the host immune response. Here, we show that recurrent viral antagonism has shaped the evolution of the host protein MAVS—a crucial component of the viral-sensing pathway in primates. From sequencing and phylogenetic analyses of MAVS from 21 simian primates, we found that MAVS has evolved under strong positive selection. We focused on how this positive selection has shaped MAVS' susceptibility to Hepatitis C virus (HCV). We functionally tested MAVS proteins from diverse primate species for their ability to resist antagonism by HCV, which uses its protease NS3/4A to cleave human MAVS. We found that MAVS from multiple primates are resistant to inhibition by the HCV protease. This resistance maps to single changes within the protease cleavage site in MAVS, which protect MAVS from getting cleaved by the HCV protease. Remarkably, most of these changes have been independently acquired at a single residue 506 that evolved under positive selection. We show that “escape” mutations lower affinity of the NS3 protease for MAVS and allow it to better restrict HCV replication. We further show that NS3 proteases from all other primate hepaciviruses, including the highly divergent GBV-A and GBV-C viruses, are functionally similar to HCV. We conclude that convergent evolution at residue 506 in multiple primates has resulted in escape from antagonism by hepaciviruses. Our study provides a model whereby insights into the ancient history of viral infections in primates can be gained using extant host and virus genes. Our analyses also provide a means by which primates might clear infections by extant hepaciviruses like HCV.
Author Summary
Hepatitis C virus (HCV) causes chronic liver disease and is estimated to infect 170 million people worldwide. HCV is able to establish a persistent infection in part by inhibiting the innate immune response. It does so by using its protease, NS3, to cleave the host's antiviral factor MAVS, which normally activates the interferon response. Using an assay that measures MAVS activity, we found that multiple primate species contain a version of MAVS that is resistant to HCV antagonism. Surprisingly, most of these primates have independently converged on changes in the same amino acid residue of MAVS to escape cleavage by the HCV protease. We found that the HCV protease has lower binding affinity for these resistant MAVS variants, which consequently are more effective at restricting HCV infection. Using a combination of phylogenetic and functional analyses of proteases from other HCV-related viruses, we infer that ancestral primates were likely exposed to and adapted to HCV-like viruses. One consequence of this adaptation is that changes that have given rise to extant MAVS variants may now provide protection from modern-day viruses.
doi:10.1371/journal.pbio.1001282
PMCID: PMC3302847  PMID: 22427742
16.  Adaptation to Human Populations Is Revealed by Within-Host Polymorphisms in HIV-1 and Hepatitis C Virus 
PLoS Pathogens  2007;3(3):e45.
CD8+ cytotoxic T-lymphocytes (CTLs) perform a critical role in the immune control of viral infections, including those caused by human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). As a result, genetic variation at CTL epitopes is strongly influenced by host-specific selection for either escape from the immune response, or reversion due to the replicative costs of escape mutations in the absence of CTL recognition. Under strong CTL-mediated selection, codon positions within epitopes may immediately “toggle” in response to each host, such that genetic variation in the circulating virus population is shaped by rapid adaptation to immune variation in the host population. However, this hypothesis neglects the substantial genetic variation that accumulates in virus populations within hosts. Here, we evaluate this quantity for a large number of HIV-1– (n ≥ 3,000) and HCV-infected patients (n ≥ 2,600) by screening bulk RT-PCR sequences for sequencing “mixtures” (i.e., ambiguous nucleotides), which act as site-specific markers of genetic variation within each host. We find that nonsynonymous mixtures are abundant and significantly associated with codon positions under host-specific CTL selection, which should deplete within-host variation by driving the fixation of the favored variant. Using a simple model, we demonstrate that this apparently contradictory outcome can be explained by the transmission of unfavorable variants to new hosts before they are removed by selection, which occurs more frequently when selection and transmission occur on similar time scales. Consequently, the circulating virus population is shaped by the transmission rate and the disparity in selection intensities for escape or reversion as much as it is shaped by the immune diversity of the host population, with potentially serious implications for vaccine design.
Author Summary
The rapid accumulation of genetic variation in human viruses, such as human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV), enables these pathogens to elude the immune system and forestalls the development of effective vaccines. This variation may be shaped by selection due to host-specific immune responses, such that the total virus population mirrors the immune diversity of the host population. However, the often-neglected viral genetic variation within hosts may also play an important role in shaping variation in the total virus population. We carry out an innovative analysis of bulk HIV-1 and HCV sequences isolated from over 4,000 human patients, exploiting “mixtures” (i.e., ambiguous nucleotides) as a site-specific marker of within-host genetic variation. We find that nonsynonymous mixtures are disproportionately abundant at codon positions under strong host-specific immune selection. Because existing models of virus evolution provide no explanation for this outcome, we have formulated a new model supplemented with stochastic simulations to demonstrate that the rapid transmission of viruses through diverse selective environments creates a positive correlation between nonsynonymous variation within and among hosts.
doi:10.1371/journal.ppat.0030045
PMCID: PMC1839164  PMID: 17397261
17.  Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells 
PLoS Pathogens  2013;9(4):e1003316.
Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell in the defense against viruses. Through pattern recognition receptors (PRRs), these cells detect viral pathogen associated molecular patterns (PAMPs) and initiate an Interferon (IFN) response. pDCs produce the antiviral IFNs including the well-studied Type I and the more recently described Type III. Recent genome wide association studies (GWAS) have implicated Type III IFNs in HCV clearance. We examined the IFN response induced in a pDC cell line and ex vivo human pDCs by a region of the HCV genome referred to as the HCV PAMP. This RNA has been shown previously to be immunogenic in hepatocytes, whereas the conserved X-region RNA is not. We show that in response to the HCV PAMP, pDC-GEN2.2 cells upregulate and secrete Type III (in addition to Type I) IFNs and upregulate PRR genes and proteins. We also demonstrate that the recognition of this RNA is dependent on RIG-I-like Receptors (RLRs) and Toll-like Receptors (TLRs), challenging the dogma that RLRs are dispensable in pDCs. The IFNs produced by these cells in response to the HCV PAMP also control HCV replication in vitro. These data are recapitulated in ex vivo pDCs isolated from healthy donors. Together, our data shows that pDCs respond robustly to HCV RNA to make Type III Interferons that control viral replication. This may represent a novel therapeutic strategy for the treatment of HCV.
Author Summary
Hepatitis C Virus (HCV) is the most common bloodborne pathogen for which no vaccine is available. Infection with the virus often leads to persistent (or chronic) infection. Patients with chronic HCV infection can develop progressive liver disease and liver failure, leading to the need for a transplant. It is not fully understood why some people clear the virus and others develop persistent infection. Understanding differences in how patients respond to the virus in the early phases of infection may lead to better treatment of HCV. Here, we use a highly conserved region of the HCV genome to examine innate immunological responses to HCV. We found that plasmacytoid dendritic cells, innate cells keyed to respond with anti-viral interferon proteins, recognize the virus. Additionally, we show that pDCs use RIG-I in the recognition of this virus, which was previously thought to be dispensable in pDCs. The proteins secreted by these cells can control viral replication in a cell-based laboratory system. In cells isolated from healthy donors, we found that fresh human cells can respond in the same manner to the virus as the laboratory strain of cells, and there was a correlation with genetic differences. Our study offers novel insight to how the body recognizes HCV during early infection and host-virus interactions that mediate viral control of this common infection.
doi:10.1371/journal.ppat.1003316
PMCID: PMC3630164  PMID: 23637605
18.  Differential regulation of cytotoxicity pathway discriminating between HIV, HCV mono- and co-infection identified by transcriptome profiling of PBMCs 
Virology Journal  2015;12:4.
Background
Despite the easy accessibility and diagnostic utility of PBMCs and their potential to show distinct expression patterns associated with the accelerated disease progression in HIV/HCV co-infection, there has not been a systematic study focusing on the global dysregulations of the biological pathways in PBMCs from HIV, HCV mono- and co-infected individuals. This study aimed at identifying the transcriptome distinctions of PBMCs between these patient groups.
Methods
Genome-wide transcriptomes of PBMCs from 10 HIV/HCV co-infected patients, 7 HIV+ patients, 5 HCV+ patients, and 5 HIV/HCV sero-negative healthy controls were analyzed using Illumina microarray. Pairwise comparisons were performed to identify differentially expressed genes (DEGs), followed by gene set enrichment analysis (GSEA) to detect the global dysregulations of the biological pathways between HIV, HCV mono- and co-infection.
Results
Forty-one, 262, and 44 DEGs with fold change > 1.5 and FDR (false discovery rate) <0.05 for the comparisons of HCV versus co-infection, HIV versus co-infection, and HIV versus HCV were identified, respectively. Significantly altered pathways (FDR < 0.05), featured by those involved in immune system, signaling transduction, and cell cycle, were detected. Notably, the differential regulation of cytotoxicity pathway discriminated between HIV, HCV mono- and co-infection (up-regulated in the former versus the latter group: co-infection versus HIV or HCV, HIV versus HCV; FDR <0.001 ~ 0.019). Conversely, the cytokine-cytokine receptor interaction pathway was down-regulated in co-infection versus either HCV (FDR = 0.003) or HIV (FDR = 0.028). For the comparison of HIV versus HCV, the cell cycle (FDR = 0.016) and WNT signaling (FDR = 0.006) pathways were up- and down-regulated in HIV, respectively.
Conclusions
Our study is the first to identify the differential regulation of cytotoxicity pathway discriminating between HIV, HCV mono- and co-infection, which may reflect the distinct patterns of virus-host cell interactions underlying disease progression. Further inspection of cytotoxicity pathway has pinned down to the expression of the KIR genes to be associated with specific patterns of particular virus-host interactions. Between HIV and HCV, the altered cell cycle and WNT signaling pathways may suggest the different impact of HIV and HCV on cell proliferation and differentiation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-014-0236-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12985-014-0236-6
PMCID: PMC4312599  PMID: 25623235
HIV; HCV; HIV/HCV co-infection; Transcriptome; Cytotoxicity pathway
19.  Constrained Pattern of Viral Evolution in Acute and Early HCV Infection Limits Viral Plasticity 
PLoS ONE  2011;6(2):e16797.
Cellular immune responses during acute Hepatitis C virus (HCV) and HIV infection are a known correlate of infection outcome. Viral adaptation to these responses via mutation(s) within CD8+ T-cell epitopes allows these viruses to subvert host immune control. This study examined HCV evolution in 21 HCV genotype 1-infected subjects to characterise the level of viral adaptation during acute and early HCV infection. Of the total mutations observed 25% were within described CD8+ T-cell epitopes or at viral adaptation sites. Most mutations were maintained into the chronic phase of HCV infection (75%). The lack of reversion of adaptations and high proportion of silent substitutions suggests that HCV has structural and functional limitations that constrain evolution. These results were compared to the pattern of viral evolution observed in 98 subjects during a similar phase in HIV infection from a previous study. In contrast to HCV, evolution during acute HIV infection is marked by high levels of amino acid change relative to silent substitutions, including a higher proportion of adaptations, likely reflecting strong and continued CD8+ T-cell pressure combined with greater plasticity of the virus. Understanding viral escape dynamics for these two viruses is important for effective T cell vaccine design.
doi:10.1371/journal.pone.0016797
PMCID: PMC3035653  PMID: 21347433
20.  Neutralizing Antibodies and Pathogenesis of Hepatitis C Virus Infection 
Viruses  2012;4(10):2016-2030.
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The interplay between the virus and host innate and adaptive immune responses determines the outcome of infection. There is increasing evidence that host neutralizing responses play a relevant role in the resulting pathogenesis. Furthermore, viral evasion from host neutralizing antibodies has been revealed to be an important contributor in leading both to viral persistence in acute liver graft infection following liver transplantation, and to chronic viral infection. The development of novel model systems to study HCV entry and neutralization has allowed a detailed understanding of the molecular mechanisms of virus-host interactions during antibody-mediated neutralization. The understanding of these mechanisms will ultimately contribute to the development of novel antiviral preventive strategies for liver graft infection and an urgently needed vaccine. This review summarizes recent concepts of the role of neutralizing antibodies in viral clearance and protection, and highlights consequences of viral escape from neutralizing antibodies in the pathogenesis of HCV infection.
doi:10.3390/v4102016
PMCID: PMC3497039  PMID: 23202451
antiviral; evasion; liver; transplantation; vaccine
21.  Elucidation of Hepatitis C Virus Transmission and Early Diversification by Single Genome Sequencing 
PLoS Pathogens  2012;8(8):e1002880.
A precise molecular identification of transmitted hepatitis C virus (HCV) genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of 2,922 half or quarter genomes from plasma viral RNA to identify transmitted/founder (T/F) viruses in 17 subjects with acute community-acquired HCV infection. Sequences from 13 of 17 acute subjects, but none of 14 chronic controls, exhibited one or more discrete low diversity viral lineages. Sequences within each lineage generally revealed a star-like phylogeny of mutations that coalesced to unambiguous T/F viral genomes. Numbers of transmitted viruses leading to productive clinical infection were estimated to range from 1 to 37 or more (median = 4). Four acutely infected subjects showed a distinctly different pattern of virus diversity that deviated from a star-like phylogeny. In these cases, empirical analysis and mathematical modeling suggested high multiplicity virus transmission from individuals who themselves were acutely infected or had experienced a virus population bottleneck due to antiviral drug therapy. These results provide new quantitative and qualitative insights into HCV transmission, revealing for the first time virus-host interactions that successful vaccines or treatment interventions will need to overcome. Our findings further suggest a novel experimental strategy for identifying full-length T/F genomes for proteome-wide analyses of HCV biology and adaptation to antiviral drug or immune pressures.
Author Summary
Hepatitis C virus infects as many as 170 million people worldwide. Globally, there are seven major genotypes of HCV that differ by approximately 30% in nucleotide sequence. Importantly, the natural history of HCV infection is variable, ranging from spontaneous resolution to persistent viremia and chronic disease. Factors responsible for this variability in clinical outcome are unknown but likely involve a combination of viral and host determinants. To this end, a precise molecular identification of transmitted HCV genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of plasma viral RNA to identify transmitted viral genomes and their progeny in 17 subjects with acute infection. Numbers of transmitted viruses leading to productive clinical infection ranged from 1 to 37 or more (median = 4). Surprisingly, we found evidence of high multiplicity acute-to-acute HCV transmission in 3 of 17 subjects, which suggests that clinical transmission of HCV, like that of HIV-1, may be enhanced in early infection when virus titers are highest and neutralizing antibodies are absent. These results provide novel insight into HCV transmission and early virus diversification key to our understanding of virus natural history and response to drug selection and immune pressure.
doi:10.1371/journal.ppat.1002880
PMCID: PMC3426529  PMID: 22927816
22.  Hepatitis C Virus p7 Protein Is Crucial for Assembly and Release of Infectious Virions 
PLoS Pathogens  2007;3(7):e103.
Hepatitis C virus (HCV) infection is associated with chronic liver disease and currently affects about 3% of the world population. Although much has been learned about the function of individual viral proteins, the role of the HCV p7 protein in virus replication is not known. Recent data, however, suggest that it forms ion channels that may be targeted by antiviral compounds. Moreover, this protein was shown to be essential for infectivity in chimpanzee. Employing the novel HCV infection system and using a genetic approach to investigate the function of p7 in the viral replication cycle, we find that this protein is essential for efficient assembly and release of infectious virions across divergent virus strains. We show that p7 promotes virus particle production in a genotype-specific manner most likely due to interactions with other viral factors. Virus entry, on the other hand, is largely independent of p7, as the specific infectivity of released virions with a defect in p7 was not affected. Together, these observations indicate that p7 is primarily involved in the late phase of the HCV replication cycle. Finally, we note that p7 variants from different isolates deviate substantially in their capacity to promote virus production, suggesting that p7 is an important virulence factor that may modulate fitness and in turn virus persistence and pathogenesis.
Author Summary
The hepatitis C virus (HCV), a major human pathogen associated with severe liver disease, encodes a small membrane protein designated p7. Although recent reports indicated that p7 forms channels conducting ions across membranes and is essential for HCV infection, its exact role in the viral life cycle remained elusive. In this study, we illustrate that HCV relies on p7 function for efficient assembly and release of infectious progeny virions from liver cells. Conversely, entry of HCV particles into new host cells is independent of p7. This new evidence supports the recent proposal to include p7 into the family of viroporins that comprises proteins from diverse viruses, for instance, HIV-1 and influenza A virus. Members of this group of functionally related proteins form membrane pores that promote virus release and in some cases also virus entry. Moreover, we identify several conserved p7 residues crucial for functioning of this protein. These amino acids possibly stabilize the structure of p7 or directly participate in channelling of ions. Interestingly, p7 variants from divergent patient isolates differ with regard to their ability to promote virus production, suggesting that p7 modulates viral fitness. Together these observations shed new light on fundamental aspects of the HCV replication strategy.
doi:10.1371/journal.ppat.0030103
PMCID: PMC1924870  PMID: 17658949
23.  German cohort of HCV mono-infected and HCV/HIV co-infected patients reveals relative under-treatment of co-infected patients 
Background
Current German and European HIV guidelines recommend early evaluation of HCV treatment in all HIV/HCV co-infected patients. However, there are still considerable barriers to initiate HCV therapy in everyday clinical practice. This study evaluates baseline characteristics, “intention-to-treat” pattern and outcome of therapy of HCV/HIV co-infected patients in direct comparison to HCV mono-infected patients in a “real-life” setting.
Methods
A large, single-center cohort of 172 unselected HCV patients seen at the Infectious Diseases Unit at the University Medical Center Hamburg-Eppendorf from 2000–2011, 88 of whom HCV/HIV co-infected, was retrospectively analyzed by chart review with special focus on demographic, clinical and virologic aspects as well as treatment outcome.
Results
Antiviral HCV combination therapy with PEG-interferon plus weight-adapted ribavirin was initiated in 88/172 (52%) patients of the entire cohort and in n = 36 (40%) of all HCV/HIV co-infected patients (group A) compared to n = 52 (61%) of the HCV mono-infected group (group B) (p = 0.006). There were no significant differences of the demographics or severity of the liver disease between the two groups with the exception of slightly higher baseline viral loads in group A. A sustained virologic response (SVR) was observed in 50% (n = 18) of all treated HIV/HCV co-infected patients versus 52% (n = 27) of all treated HCV mono-infected patients (p = 0.859). Genotype 1 was the most frequent genotype in both groups (group A: n = 37, group B: n = 49) and the SVR rates for these patients were only slightly lower in the group of co-infected patients (group A: n = 33%, group B: 40% p = 0.626). During the course of treatment HCV/HIV co-infected patients received less ribavirin than mono-infected patients.
Conclusion
Overall, treatment was only initiated in half of the patients of the entire cohort and in an even smaller proportion of HCV/HIV co-infected patients despite comparable outcome (SVR) and similar baseline characteristics. In the light of newer treatment options, greater efforts to remove the barriers to treatment that still exist for a great proportion of patients especially with HIV/HCV co-infection have to be undertaken.
doi:10.1186/1742-6405-11-16
PMCID: PMC4086688  PMID: 25006340
HCV/HIV co-infection; Hepatitis C; PEG interferon/ribavirin treatment
24.  Integrative Functional Genomics of Hepatitis C Virus Infection Identifies Host Dependencies in Complete Viral Replication Cycle 
PLoS Pathogens  2014;10(5):e1004163.
Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.
Author Summary
Hepatitis C virus (HCV) is a positive strand RNA virus that belongs to the Flaviridae family. Chronic HCV infection is a leading cause of end-stage liver disease, which is associated with significant morbidity and mortality in the world. Our recent genome-wide siRNA screen has revealed that HCV depends extensively on host factors for efficient infection and propagation. Here we systematically and functionally catalogued these host dependencies to various stages of the HCV replication cycle. Applying systems virology and functional genomics approaches with various in vitro HCV model systems, we further defined multiple previously unrecognized host factors or pathways that are involved in either HCV entry, IRES-mediated translation, RNA replication, or assembly/secretion. By bioinformatics meta-analyses and literature mining of existing publications and databases, we constructed an extensive roadmap of the cellular networks and pathways requisite for the complete HCV replication cycle. Our study yields novel insights into viral infection, pathogenesis and potential therapeutic targets. Furthermore, this study serves as a valuable reference source for subsequent work on host pathways and virus-host interactions in general.
doi:10.1371/journal.ppat.1004163
PMCID: PMC4095987  PMID: 24852294
25.  Immunity and Hepatitis C: A Review 
Current HIV/AIDS reports  2013;10(1):51-58.
Hepatitis C virus (HCV) is a major cause of chronic hepatitis and hepatocellular carcinoma worldwide. Due to shared transmission routes, the prevalence of HCV is especially high among individuals infected with HIV. HIV uninfected individuals spontaneously clear HCV approximately 30% of the time, while the rate of control in HIV infected individuals who subsequently acquire HCV is substantially lower. In addition, complications of HCV are more frequent in those with HIV infection, making liver disease the leading cause of non-AIDS-related death in HIV infected individuals. This review summarizes recent advances in understanding the role of the innate and adaptive immune responses to HCV in those with and without HIV. Further defining the interaction between hepatitis C and the host immune system will potentially reveal insights into HCV pathogenesis and the host’s ability to prevent persistent infection, as well as direct the development of vaccines.
doi:10.1007/s11904-012-0146-4
PMCID: PMC3567217  PMID: 23180007
Hepatitis C; HCV; viral hepatitis; hepatitis; Immunity; innate immune response; Interferons; IFN; cellular immunity; adaptive immune response; neutralizing antibodies; chronic infection; acute infection; liver disease; spontaneous clearance; treatment response; SVR; vaccination; HIV; HIV-HCV coinfection; coinfection; T cell response; B cell response; interleukin 18; interleukin 28B; virus

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