Daire O'Shea

IL-28B is a Key Regulator of B- and T-Cell Vaccine Responses against Influenza

Influenza is a major cause of morbidity and mortality in immunosuppressed persons, and vaccination often confers insufficient protection. IL-28B, a member of the interferon (IFN)-λ family, has variable expression due to single nucleotide polymorphisms (SNPs). While type-I IFNs are well known to modulate adaptive immunity, the impact of IL-28B on B- and T-cell vaccine responses is unclear. Here we demonstrate that the presence of the IL-28B TG/GG genotype (rs8099917, minor-allele) was associated with increased seroconversion following influenza vaccination (OR 1.99 p = 0.038). Also, influenza A (H1N1)-stimulated T- and B-cells from minor-allele carriers showed increased IL-4 production (4-fold) and HLA-DR expression, respectively. In vitro, recombinant IL-28B increased Th1-cytokines (e.g. IFN-γ), and suppressed Th2-cytokines (e.g. IL-4, IL-5, and IL-13), H1N1-stimulated B-cell proliferation (reduced 70%), and IgG-production (reduced>70%). Since IL-28B inhibited B-cell responses, we designed antagonistic peptides to block the IL-28 receptor α-subunit (IL28RA). In vitro, these peptides significantly suppressed binding of IFN-λs to IL28RA, increased H1N1-stimulated B-cell activation and IgG-production in samples from healthy volunteers (2-fold) and from transplant patients previously unresponsive to vaccination (1.4-fold). Together, these findings identify IL-28B as a key regulator of the Th1/Th2 balance during influenza vaccination. Blockade of IL28RA offers a novel strategy to augment vaccine responses.

An Analysis of Regulatory T-Cell and Th-17 Cell Dynamics during Cytomegalovirus Replication in Solid Organ Transplant Recipients

Background CMV-specific T-cells are crucial to control CMV-replication post-transplant. Regulatory T-cells (T-regs) are associated with a tolerant immune state and may contribute to CMV-replication. However, T-cell subsets such as T-regs and IL-17 producing T-cells (Th-17) are not well studied in this context. We explored T-regs and Th-17 frequencies during CMV-replication after transplantation. Methods We prospectively evaluated 30 transplant patients with CMV-viremia. We quantified CMV-specific CD4+ and CD8+ T-cells, T-regs (CD4+CD25+FoxP3+) and Th-17 frequencies using flow-cytometry and followed patients requiring anti-viral treatment. Two subsets were compared: anti-viral treatment requirement (n = 20) vs. spontaneous clearance of viremia (n = 10). Results Higher initial CMV-specific CD4+ T-cells and lower T-regs were observed in patients with spontaneous clearance (p = 0.043; p = 0.021 respectively). Using a ratio of CMV-specific CD4+ T-cells to T-regs allowed prediction of viral clearance with 80% sensitivity and 90% specificity (p = 0.001). One month after stop of treatment, the same correlation was observed in patients protected from CMV-relapse. The ratio of CMV-specific CD4+ T-cells to T-regs allowed prediction of relapse with 85% sensitivity and 86% specificity (p = 0.004). Th-17 responses were not correlated with virologic outcomes. Conclusions This study provides novel insights into T-regs and Th-17 subpopulations during CMV-replication after transplantation. These preliminary data suggest that measurement of CMV-specific CD4+ T-cells together with T-regs has value in predicting spontaneous clearance of viremia and relapse.

Hcmv‐miR‐UL22A‐5p: A Biomarker in Transplantation With Broad Impact on Host Gene Expression and Potential Immunological Implications

Cytomegalovirus (CMV) encodes multiple microRNAs. While these have been partially characterized in vitro, their relevance to clinical CMV infection has not been evaluated. We analyzed samples from a cohort of solid organ transplant patients with CMV disease (n = 245) for viral microRNA expression. Several CMV microRNAs were readily detectable in patients with CMV disease in variable relative abundance. Expression level generally correlated with DNA viral load and the absence of viral microRNA was associated with faster viral clearance. Detection of hcmv‐miR‐UL22A‐5p at baseline independently predicted the recurrence of CMV viremia upon discontinuation of antiviral therapy (OR 3.024, 95% CI: 1.35–6.8; p = 0.007). A combination of direct mRNA targeting by the microRNA and indirect modulation of gene expression involving isoforms of the transcriptional regulator C‐MYC may be responsible for the broad effects seen in the association of gene transcripts with the RNA‐induced silencing complex and in global protein expression upon hcmv‐miR‐UL22A‐5p transfection. This novel study of in vivo viral microRNA expression profiles provides unique insight into the complexity of clinical CMV infection following transplantation. We provide evidence that viral microRNAs may have complex effects on gene expression and be associated with specific virologic and clinical outcomes, and thus could be further evaluated as biomarkers.

Effect of Immunosuppression on T-Helper 2 and B-Cell Responses to Influenza Vaccination.

BACKGROUND Influenza vaccine immunogenicity is suboptimal in immunocompromised patients. However, there are limited data on the interplay of T- and B- cell responses to vaccination with simultaneous immunosuppression. METHODS We collected peripheral blood mononuclear cells from transplant recipients before and 1 month after seasonal influenza vaccination. Before and after vaccination, H1N1-specific T- and B-cell activation were quantified with flow cytometry. We also developed a mathematical model using T- and B-cell markers and mycophenolate mofetil (MMF) dosage. RESULTS In the 47 patients analyzed, seroconversion to H1N1 antigen was demonstrated in 34%. H1N1-specific interleukin 4 (IL-4)-producing CD4(+) T-cell frequencies increased significantly after vaccination in 53% of patients. Prevaccine expression of H1N1-induced HLA-DR and CD86 on B cells was high in patients who seroconverted. Seroconversion against H1N1 was strongly associated with HLA-DR expression on B cells, which was dependent on the increase between prevaccine and postvaccine H1N1-specific IL-4(+)CD4(+) T cells (R(2) = 0.35). High doses of MMF (≥ 2 g/d) led to lower seroconversion rates, smaller increase in H1N1-specific IL-4(+)CD4(+) T cells, and reduced HLA-DR expression on B cells. The mathematical model incorporating a MMF-inhibited positive feedback loop between H1N1-specific IL-4(+)CD4(+) T cells and HLA-DR expression on B cells captured seroconversion with high specificity. CONCLUSIONS Seroconversion is associated with influenza-specific T-helper 2 and B-cell activation and seems to be modulated by MMF.

Prevention of hepatitis C virus infection using a broad cross‐neutralizing monoclonal antibody (AR4A) and epigallocatechin gallate

The anti–hepatitis C virus (HCV) activity of a novel monoclonal antibody (mAb; AR4A) and epigallocatechin gallate (EGCG) were studied in vitro using a HCV cell culture system and in vivo using a humanized liver mouse model capable of supporting HCV replication. Alone, both exhibit reliable cross‐genotype HCV inhibition in vitro, and combination therapy completely prevented HCV infection. In vitro AR4A mAb (alone and combined with EGCG) robustly protects against the establishment of HCV genotype 1a infection. EGCG alone fails to reliably protect against an HCV challenge. In conclusion, AR4A mAb represents a safe and efficacious broadly neutralizing antibody against HCV applicable to strategies to safely prevent HCV reinfection following liver transplantation, and it lends further support to the concept of HCV vaccine development. The poor bioavailability of EGCG limits HCV antiviral activity in vitro. Liver Transpl 22:324–332, 2016.

Complexity of Host Micro-RNA Response to Cytomegalovirus Reactivation After Organ Transplantation

Human (Homo sapiens) micro‐RNAs (hsa‐miRNAs) regulate virus and host‐gene translation, but the biological impact in patients with human cytomegalovirus (hCMV) infection is not well defined in a clinically relevant model. First, we compared hsa‐miRNA expression profiles in peripheral blood mononuclear cells from 35 transplant recipients with and without CMV viremia by using a microarray chip covering 847 hsa‐miRNAs. This approach demonstrated a set of 142 differentially expressed hsa‐miRNAs. Next, we examined the effect of each of these miRNAs on viral growth by using human fibroblasts (human foreskin fibroblast‐1) infected with the hCMV Towne strain, identifying a subset of proviral and antiviral hsa‐miRNAs. miRNA‐target prediction software indicated potential binding sites within the hCMV genome (e.g., hCMV‐UL52 and ‐UL100 [UL = unique long]) and host‐genes (e.g., interleukin‐1 receptor, IRF1). Luciferase‐expressing plasmid constructs and immunoblotting confirmed several predicted miRNA targets. Finally, we determined the expression of selected proviral and antiviral hsa‐miRNAs in 242 transplant recipients with hCMV‐viremia. We measured hsa‐miRNAs before and after antiviral therapy and correlated hsa‐miRNA expression levels to hCMV‐replication dynamics. One of six antiviral hsa‐miRNAs showed a significant increase during treatment, concurrent with viral decline. In contrast, six of eight proviral hsa‐miRNAs showed a decrease during viral decline. Our results indicate that a complex and multitargeted hsa‐miRNA response occurs during CMV replication in immunosuppressed patients. This study provides mechanistic insight and potential novel biomarkers for CMV replication.

Prevention of HCV infection using a broad cross‐neutralizing monoclonal antibody (AR4A) and Epigallocatechin‐Gallate

The anti–hepatitis C virus (HCV) activity of a novel monoclonal antibody (mAb; AR4A) and epigallocatechin gallate (EGCG) were studied in vitro using a HCV cell culture system and in vivo using a humanized liver mouse model capable of supporting HCV replication. Alone, both exhibit reliable cross‐genotype HCV inhibition in vitro, and combination therapy completely prevented HCV infection. In vitro AR4A mAb (alone and combined with EGCG) robustly protects against the establishment of HCV genotype 1a infection. EGCG alone fails to reliably protect against an HCV challenge. In conclusion, AR4A mAb represents a safe and efficacious broadly neutralizing antibody against HCV applicable to strategies to safely prevent HCV reinfection following liver transplantation, and it lends further support to the concept of HCV vaccine development. The poor bioavailability of EGCG limits HCV antiviral activity in vitro. Liver Transpl 22:324–332, 2016.

CCL8 and the Immune Control of Cytomegalovirus in Organ Transplant Recipients

Monitoring of cytomegalovirus cell‐mediated immunity is a promising tool for the refinement of preventative and therapeutic strategies posttransplantation. Typically, the interferon‐γ response to T cell stimulation is measured. We evaluated a broad range of cytokine and chemokines to better characterize the ex vivo host‐response to CMV peptide stimulation. In a cohort of CMV viremic organ transplant recipients, chemokine expression—specifically CCL8 (AUC 0.849 95% CI 0.721–0.978; p = 0.003) and CXCL10 (AUC 0.841, 95% CI 0.707–0.974; p = 0.004)—was associated with control of viral replication. In a second cohort of transplant recipients at high‐risk for CMV, the presence of a polymorphism in the CCL8 promoter conferred an increased risk of viral replication after discontinuation of antiviral prophylaxis (logrank hazard ratio 3.6; 95% CI 2.077–51.88). Using cell‐sorting experiments, we determined that the primary cell type producing CCL8 in response to CMV peptide stimulation was the monocyte fraction. Finally, in vitro experiments using standard immunosuppressive agents demonstrated a dose‐dependent reduction in CCL8 production. Chemokines appear to be important elements of the cell‐mediated response to CMV infection posttransplant, as here suggested for CCL8, and translation of this knowledge may allow for the tailoring and improvement of preventative strategies.

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions.

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.