Inhibition of Phosphoinositide 3-Kinase p110delta Does Not Affect T Cell Driven Development of Type 1 Diabetes Despite Significant Effects on Cytokine Production.

Type 1 diabetes is caused by the destruction of insulin producing beta cells by the immune system. The p110δ isoform of PI3K is expressed primarily in cells of haematopoietic origin and the catalytic activity of p110δ is important for the activation of these cells. Targeting of this pathway offers an opportunity to reduce immune cell activity without unwanted side effects. We have explored the effects of a specific p110δ isoform inhibitor, IC87114, on diabetogenic T cells both in vitro and in vivo, and find that although pharmacological inhibition of p110δ has a considerable impact on the production of pro-inflammatory cytokines, it does not delay the onset of diabetes after adoptive transfer of diabetogenic cells. Further, we demonstrate that combination treatment with CTLA4-Ig does not improve the efficacy of treatment, but instead attenuates the protective effects seen with CTLA4-Ig treatment alone. Our results suggest that decreased IL-10 production by Foxp3+ CD4+ T cells in the presence of IC87114 negates individual anti-inflammatory effects of IC8114 and CTLA4-Ig.This work was supported by Grant number 09/0003840, Diabetes UK https://www.diabetes.org.uk/ (MW); Grant number 5-2006-229, Juvenile Diabetes Research Foundation https://www.jdrf.org.uk/ (KO); Grant number BBS/E/B/0000C236 Biotechnology and Biological Sciences Research Council (KO); Grant number health-f5-2009-241883 European Research Council 7th Frame Programme http://ec.europa.eu/research/fp7/index_en.cfm (AC); and Grant number 02BX12ACYD, the Britain Israel Research and Academic Exchange Partnership (BIRAX) http://www.britishcouncil.org.il/en/programmes/science/birax.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.014651

Combined point of care nucleic acid and antibody testing for SARS-CoV-2 following emergence of D614G Spike Variant

Rapid COVID-19 diagnosis in hospital is essential, though complicated by 30-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant now dominates the pandemic and it is unclear how serological tests designed to detect anti-Spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95CI 57.8-92.9%) by rapid NAAT alone. Combined point of care antibody test and rapid NAAT is not impacted by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission

Funder: Addenbrooke's Charitable Trust, Cambridge University Hospitals; FundRef: http://dx.doi.org/10.13039/501100002927Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3 week period (April 2020), 1032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in the asymptomatic screening group tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (COVID-19)>7 days prior to testing, most self-isolating, returning well. Clusters of HCW infection were discovered on two independent wards. Viral genome sequencing showed that the majority of HCWs had the dominant lineage B∙1. Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms. This approach will be critical for protecting patients and hospital staff

Age-related immune response heterogeneity to SARS-CoV-2 vaccine BNT162b2

Abstract: Although two-dose mRNA vaccination provides excellent protection against SARS-CoV-2, there is little information about vaccine efficacy against variants of concern (VOC) in individuals above eighty years of age1. Here we analysed immune responses following vaccination with the BNT162b2 mRNA vaccine2 in elderly participants and younger healthcare workers. Serum neutralization and levels of binding IgG or IgA after the first vaccine dose were lower in older individuals, with a marked drop in participants over eighty years old. Sera from participants above eighty showed lower neutralization potency against the B.1.1.7 (Alpha), B.1.351 (Beta) and P.1. (Gamma) VOC than against the wild-type virus and were more likely to lack any neutralization against VOC following the first dose. However, following the second dose, neutralization against VOC was detectable regardless of age. The frequency of SARS-CoV-2 spike-specific memory B cells was higher in elderly responders (whose serum showed neutralization activity) than in non-responders after the first dose. Elderly participants showed a clear reduction in somatic hypermutation of class-switched cells. The production of interferon-γ and interleukin-2 by SARS-CoV-2 spike-specific T cells was lower in older participants, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high-risk population and that specific measures to boost vaccine responses in this population are warranted, particularly where variants of concern are circulating

Complement lectin pathway activation is associated with COVID-19 disease severity, independent of MBL2 genotype subgroups

IntroductionWhile complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood.MethodsWe therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome.ResultsWe show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p<0.0001) and COVID-19 severity. Despite this, genetic variations in MBL2 are not associated with susceptibility to SARS-CoV-2 infection or disease outcomes such as mortality and the development of Long COVID.ConclusionIn conclusion, activation of the MBL-LP only plays a minor role in COVID-19 pathogenesis, since no clinically meaningful, consistent associations with disease outcomes were noted

Single-cell multi-omics analysis of the immune response in COVID-19

Funder: Lister Institute of Preventive Medicine; doi: https://doi.org/10.13039/501100001255Funder: University College London, Birkbeck MRC Doctoral Training ProgrammeFunder: The Jikei University School of MedicineFunder: Action Medical Research (GN2779)Funder: NIHR Clinical Lectureship (CL-2017-01-004)Funder: NIHR (ACF-2018-01-004) and the BMA FoundationFunder: Chan Zuckerberg Initiative (grant 2017-174169) and from Wellcome (WT211276/Z/18/Z and Sanger core grant WT206194)Funder: UKRI Innovation/Rutherford Fund Fellowship allocated by the MRC and the UK Regenerative Medicine Platform (MR/5005579/1 to M.Z.N.). M.Z.N. and K.B.M. have been funded by the Rosetrees Trust (M944)Funder: Barbour FoundationFunder: ERC Consolidator and EU MRG-Grammar awardsFunder: Versus Arthritis Cure Challenge Research Grant (21777), and an NIHR Research Professorship (RP-2017-08-ST2-002)Funder: European Molecular Biology Laboratory (EMBL)Abstract: Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

Abstract: The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era

T cell targeted interventions for experimental autoimmunity

Autoimmune diseases are chronic conditions caused by a loss of tolerance to self-antigens due to inappropriate activation of the immune system. Rheumatoid arthritis (RA) is a chronic inflammatory disease where the inadequate activation of T cells with pro-inflammatory characteristics leads to synovial hyperplasia and joint inflammation. In spite of excellent results in patients responding to biological therapies approved for the treatment of these patients, only partial responses have been achieved and a continuous treatment is required. In the case of type 1 diabetes (T1D), no drugs have been approved to halt the autoimmune process that causes the destruction of insulin producing β cells. There is an increasing understanding that pro-inflammatory responses directed to self-antigens become chronic in autoimmune diseases because regulatory mechanisms fail to control them. As autoimmune disorders are T-cell driven, therapies that initiate the deletion of effector T (Teff) cells, skew their pro-inflammatory profile or induce a regulatory phenotype in auto-antigen specific T cells, seem promising. The research described in this thesis focuses on two approaches aimed to restore a proper balance between pathogenic and regulatory T cells in the context of two autoimmune diseases. The first part describes the regulatory properties of a peptide from heat shock protein of 60 kDa (HSP-60) in the context of RA. The second part focuses on the effect of the pharmacological inhibition of p110δ alone or in combination with abatacept to restore the balance between diabetogenic and regulatory T cells in the context of T1D. P110δ is the main phosphoinositide 3-kinases (PI3K) isoform expressed in leukocytes that triggers antigen-specific immune responses. These enzymes have the ability to control essential functions for cells like: proliferation, differentiation, growth, survival and intracellular trafficking

T Cell-Mediated Chronic Inflammatory Diseases Are Candidates for Therapeutic Tolerance Induction with Heat Shock Proteins

Failing immunological tolerance for critical self-antigens is the problem underlying most chronic inflammatory diseases of humans. Despite the success of novel immunosuppressive biological drugs, the so-called biologics, in the treatment of diseases such rheumatoid arthritis (RA) and type 1 diabetes, none of these approaches does lead to a permanent state of medicine free disease remission. Therefore, there is a need for therapies that restore physiological mechanisms of self-tolerance. Heat shock proteins (HSPs) have shown disease suppressive activities in many models of experimental autoimmune diseases through the induction of regulatory T cells (Tregs). Also in first clinical trials with HSP-based peptides in RA and diabetes, the induction of Tregs was noted. Due to their exceptionally high degree of evolutionary conservation, HSP protein sequences (peptides) are shared between the microbiota-associated bacterial species and the self-HSP in the tissues. Therefore, Treg mechanisms, such as those induced and maintained by gut mucosal tolerance for the microbiota, can play a role by targeting the more conserved HSP peptide sequences in the inflamed tissues. In addition, the stress upregulated presence of HSP in these tissues may well assist the targeting of the HSP induced Treg specifically to the sites of inflammation

miR-374a-5p regulates inflammatory genes and monocyte function in patients with inflammatory bowel disease.

MicroRNAs are critical regulators of gene expression controlling cellular processes including inflammation. We explored their role in the pathogenesis of inflammatory bowel disease (IBD) and identified reduced expression of miR-374a-5p in IBD monocytes that correlated with a module of up-regulated genes related to the inflammatory response. Key proinflammatory module genes, including for example TNFα, IL1A, IL6, and OSM, were inversely correlated with miR-374a-5p and were validated in vitro. In colonic biopsies, miR-374a-5p was again reduced in expression and inversely correlated with the same inflammatory module, and its levels predicted subsequent response to anti-TNF therapy. Increased miR-374a-5p expression was shown to control macrophage-driven inflammation by suppressing proinflammatory mediators and to reduce the capacity of monocytes to migrate and activate T cells. Our findings suggest that miR-374a-5p reduction is a central driver of inflammation in IBD, and its therapeutic supplementation could reduce monocyte-driven inflammation in IBD or other immune-mediated diseases