Innate resistance to hepatitis C virus infection in the Irish anti-D cohort
Citation:
Sugrue, Jamie Alan, Innate resistance to hepatitis C virus infection in the Irish anti-D cohort, Trinity College Dublin.School of Biochemistry & Immunology, 2022Download Item:
Abstract:
Even within homogenous populations, outcome following exposure to a virus can vary substantially. This continuum of infection outcome spans viral resistance, spontaneous resolution and chronic infection, with further immunological and pathological heterogeneity observed within each of these subgroups. This heterogeneity is particularly evident in hepatitis C virus (HCV) outbreaks. HCV candidate viral resistors, also known as exposed seronegative (ESN), remain antibody (Ab) and PCR negative following viral exposure, and are identifiable based on risk or documented exposure. Spontaneous resolvers (SR) are a distinct group that clear infection via an adaptive immune response, and are identifiable as Ab-positive but PCR-negative. The final group, those with chronic infection (CI), are identifiable as Ab-positive and PCR-positive. Study of the full spectrum of outcome in a single group has been stymied by the lack of suitable cohorts exposed to a virus in a controlled manner.
Between 1977-79 in Ireland, over 2000 Rhesus (RhD)-negative females were exposed to HCV following receipt of HCV-contaminated anti-D immunoglobulin. Analysis of records from the Irish Blood Transfusion Service (IBTS)indicated that there were three infection groups: ESNs, SRs and CI. Work over the past 40 years has focused on SRs and CIs as they were easily identifiable based on clinical assays. However, up to 611 (47%) women tested Ab and PCR-negative following exposure to HCV out of 1293 women who received highly infectious batches of anti-D. Through a nationwide media campaign we sought recruit women from each of these groups to study correlates of protective immunity. We successfully recruited and record-matched 34 ESN women, 98 seropositive women (SP; 48 SR and 50 CI [All the women with CI were treated successfully and achieved a sustained virological response; SVR]). We hypothesised that ESN women from the anti-D cohort have an enhanced innate immune response that protected them from infection with HCV.
To test for anti-HCV antibodies in our ESNs, and to test the hypothesis that ESN donors may also be protected against other common viral pathogens, we used VirScan, a novel technology to explore the history of viral infection. We compared antibody levels against 206 human pathogens in our subgroups. We observed no significant differences in antibody levels to any virus other than HCV. HCV Abs were detectable in 84% (16/17) of the SVR donors. Raised antibodies to some HCV epitopes were detected in 16% (3/19) of SR women. No HCV Ab positivity was detected in the ESN donor group. To further confirm the absence of a HCV adaptive immune response in our ESNs, we used IFNg ELISpots to assess the HCV-specific T memory response. We did not find any T cell activity to HCV peptides in our ESNs or controls but observed some positivity in the SRs and SVRs.
Assessing the immune response in humans is often confounded by labour intensive technologies with handling steps that can compromise reproducibility. We therefore opted to use standardised whole blood stimulations using the TruCulture system previously used by the Milieu Interieur Cohort (MIC). We invited n=18 ESN women, n=19 SR and n=17 SVR women from our cohort to donate a blood sample for stimulation with a panel of viral ligands including polyIC (TLR3), R848 (TLR7), ODN (TLR9) and IFNa2 (IFNAR1/2). Analysis of stimulated whole blood was carried out using Luminex and Simoa proteomics and NanoString transcriptomics. We found that ESNs had an increased polyIC induced type I interferon (IFN-I) signature compared with SP donors as well as increased production of several pro-inflammatory cytokines including CCL8, CXCL11 and IL-6. ESN, SRs and CI all had similar responses to stimulation with R848 or IFNa2.
To assess the potential genetic differences in innate immune response genes associated with resistance to HCV infection we genotyped our full cohort, alongside additional age and sex matched donors from previous studies, for tagSNPs in TLR3 (rs3775291), IRF3 (rs7251) and IFNAR1 (rs2257167; n ESN = 38, n SR = 63, n SVR = 77 and n unexposed control [UC] = 119). Using this approach, we identified an association between rs2257167 and resistance to HCV in our cohort. Subsequent analysis of transcriptomic data from UC donors in our cohort and the MIC showed that rs2257167 is a baseline expression quantitative trait locus (eQTL) for IFNAR1. Analysis of NanoString data from the MIC showed that rs2257167 is associated with increased interferon stimulated gene (IRG) responses following stimulation influenza virus (IAV), polyIC and LPS compared with the wild-type IFNAR1 receptor. rs2257167 is associated with decreased non-IRG pro-inflammatory responses to these stimuli.
Differences in the innate immune response may also impact the ability to spontaneously resolve infection. Seropositive women include those who cleared the virus themselves (SRs) and those who required therapeutic intervention to clear infection (SVR). SP women responded similarly to stimulation with R848, polyIC or IFNa2. However, in response to ODN, we observed differential expression of over 50 genes between SR and SVRs. Further interrogation of the differences between groups showed enrichment for IFN-I signalling in ESNs and SVRs compared to SRs. Using gene signature scores, the SRs had a reduced IFN-I signature. This was also reflected at the protein level, with SRs having decreased IFNg production compared to SVRs.
Association studies during the COVID-19 pandemic uncovered interesting associations between blood groups and risk of hospitalisation and severe disease. As our cohort is comprised of RhD-negative females, and appeared to have increased viral resistance compared with other cohorts, we sought to determine whether RhD status impacts on the whole blood immune response. We analysed genetic, proteomic and transcriptomic data previously generated on the 1000 person MIC in response to stimulation with LPS, polyIC and IAV. No study had yet explored the impact of RhD on the immune response. Donors were classified as RhD-negative or -positive using the rs590787 SNP in RHD and split into males and females. We found that RhD-negative males have increased responsiveness to IAV stimulation compared with RhD-positive males. Analysis of this response, using gene set enrichment analysis, showed increased IFNg signalling in the RhD-negative males but not in RhD-negative females. This increased IFNg response may explain, at least in part, the protective association of RhD-negativity with viral infection in males.
Collectively, the work here identifies for the first time, a unique cohort of HCV resistant individuals from the Irish anti-D cohort. Based on data from the IBTS, we show that viral resistance may be more common than originally thought- with up to 50% of individuals displaying natural resistance to HCV infection. We identify a TLR3-induced IFN-I signature as being associated with viral resistance, and uncover novel genetic associations with viral protection. We also show that resolution of acute HCV is associated with an reduced IFN-I response to ODN. Our work here underscores the power of whole blood analysis when coupled with robust multiplex assays to define the signatures of induced immune responses in small well-defined cohorts of volunteers.
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Grant Number
Science Foundation Ireland (SFI)
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APPROVED
Author: Sugrue, Jamie Alan
Advisor:
O'Farrelly, ClionaPublisher:
Trinity College Dublin. School of Biochemistry & Immunology. Discipline of BiochemistryType of material:
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