Longevity and neutralisation activity of secretory IgA following SARS-CoV-2 infection

The mucosal barrier is a primary defence against inhaled pathogens, comprising secretory antibodies which have the potential to block viral entry and neutralise infection. There is an ongoing need for greater understanding of the mucosal immunity to SARS-CoV-2 infection. In this study, we investigated mucosal IgA through non-invasive saliva sampling of healthcare workers. A total of 551 saliva samples were collected from staff at Great Ormond Street Children’s Hospital who previously tested positive for COVID-19. Participant metadata included age, gender, ethnicity and symptoms. IgA titres were measured by ELISA against viral antigens spike protein, nucleocapsid protein, and spike receptor-binding domain. SARS-CoV-2 neutralisation was measured using a VERO E6 cell culture infection assay. We found that approximately 30% of saliva samples contained detectable IgA specific for at least one of the SARS-CoV-2 antigens. IgA levels in saliva decreased with the time post-infection, and were largely undetectable after six months. IgA titres specific to SARS-CoV-2 were lowest in participants over 60 years old. Specific saliva samples were identified which effectively neutralised SARS-CoV-2 virus infection of epithelial cells. Our results suggest secretory IgA specific to SARS-CoV-2 can be detected in saliva following infection, an accessible sample type for testing, although titres decreased over time. Some saliva samples were able to neutralise SARS-CoV-2 infectivity against cultured epithelial cells. This data could be used to assess the risk of re-infection with SARS-CoV-2, as well as accelerate efforts to develop effective mucosal vaccination with longer lasting protection

Genetic testing of Behçet’s disease using next-generation sequencing to identify monogenic mimics and HLA-B*51

Objective: Several monogenic autoinflammatory disorders and primary immunodeficiencies can present early in life with features that may be mistaken for Behçet's disease (BD). We aimed to develop a genetic analysis workflow to identify rare monogenic BD-like diseases and establish the contribution of HLA haplotype in a cohort of patients from the UK. // Methods: Patients with clinically suspected BD were recruited from four BD specialist care centres in the UK. All participants underwent whole exome sequencing (WES), and genetic analysis thereafter by 1. examining genes known to cause monogenic immunodeficiency, autoinflammation or vasculitis by virtual panel application; 2. scrutiny of variants prioritised by Exomiser using Human Phenotype Ontology (HPO); 3. identification of copy number variants using ExomeDepth; and 4. HLA-typing using OptiType. // Results: Thirty-one patients were recruited: median age 15 (4-52), and median disease onset age 5 (0-20). Nine/31 (29%) patients had monogenic disease mimicking BD: 5 cases of Haploinsufficiency of A20 with novel TNFAIP3 variants (p.T76I, p.M112Tfs*8, p.S548Dfs*128, p.C657Vfs*14, p.E661Nfs*36); 1 case of ISG15 deficiency with a novel nonsense variant (ISG15:p.Q16X) and 1p36.33 microdeletion; 1 case of Common variable immune deficiency (TNFRSF13B:p.A181E); and 2 cases of TNF receptor associated periodic syndrome (TNFRSF1A:p.R92Q). Of the remaining 22 patients, 8 (36%) were HLA-B*51 positive. // Conclusion: We describe a novel genetic workflow for BD, which can efficiently detect known and potentially novel monogenic forms of BD, whilst additionally providing HLA-typing. Our results highlight the importance of genetic testing before BD diagnosis, since this has impact on choice of therapy, prognosis, and genetic counselling

Salivary IgA and vimentin differentiate in vitro SARS-CoV-2 infection: a study of 290 convalescent COVID-19 patients

SARS-CoV-2 initially infects cells in the nasopharynx and oral cavity. The immune system at these mucosal sites plays a crucial role in minimizing viral transmission and infection. To develop new strategies for preventing SARS-CoV-2 infection, this study aimed to identify proteins that protect against viral infection in saliva. We collected 551 saliva samples from 290 healthcare workers who had tested positive for COVID-19, before vaccination, between June and December 2020. The samples were categorized based on their ability to block or enhance infection using in vitro assays. Mass spectrometry and ELISA experiments were used to identify and measure the abundance of proteins that specifically bind to SARS-CoV-2 antigens. IgA specific to SARS-CoV-2 antigens was detectable in over 83% of the convalescent saliva samples. We found that concentrations of anti-RBD IgA >500 pg/µg total protein in saliva correlates with reduced viral infectivity in vitro. However, there is a dissociation between the salivary IgA response to SARS-CoV-2, and systemic IgG titres in convalescent COVID19 patients. Then, using an innovative technique known as spike-baited mass spectrometry, we identified novel spike-binding proteins in saliva, most notably vimentin, which correlated with increased viral infectivity in vitro, could serve as a therapeutic target against COVID-19

Neuroinflammation, autoinflammation, splenomegaly and anemia caused by bi-allelic mutations in IRAK4

We describe a novel, severe autoinflammatory syndrome characterized by neuroinflammation, systemic autoinflammation, splenomegaly, and anemia (NASA) caused by bi-allelic mutations in IRAK4. IRAK-4 is a serine/threonine kinase with a pivotal role in innate immune signaling from toll-like receptors and production of pro-inflammatory cytokines. In humans, bi-allelic mutations in IRAK4 result in IRAK-4 deficiency and increased susceptibility to pyogenic bacterial infections, but autoinflammation has never been described. We describe 5 affected patients from 2 unrelated families with compound heterozygous mutations in IRAK4 (c.C877T (p.Q293*)/c.G958T (p.D320Y); and c.A86C (p.Q29P)/c.161 + 1G>A) resulting in severe systemic autoinflammation, massive splenomegaly and severe transfusion dependent anemia and, in 3/5 cases, severe neuroinflammation and seizures. IRAK-4 protein expression was reduced in peripheral blood mononuclear cells (PBMC) in affected patients. Immunological analysis demonstrated elevated serum tumor necrosis factor (TNF), interleukin (IL) 1 beta (IL-1β), IL-6, IL-8, interferon α2a (IFN-α2a), and interferon β (IFN-β); and elevated cerebrospinal fluid (CSF) IL-6 without elevation of CSF IFN-α despite perturbed interferon gene signature. Mutations were located within the death domain (DD; p.Q29P and splice site mutation c.161 + 1G>A) and kinase domain (p.Q293*/p.D320Y) of IRAK-4. Structure-based modeling of the DD mutation p.Q29P showed alteration in the alignment of a loop within the DD with loss of contact distance and hydrogen bond interactions with IRAK-1/2 within the myddosome complex. The kinase domain mutation p.D320Y was predicted to stabilize interactions within the kinase active site. While precise mechanisms of autoinflammation in NASA remain uncertain, we speculate that loss of negative regulation of IRAK-4 and IRAK-1; dysregulation of myddosome assembly and disassembly; or kinase active site instability may drive dysregulated IL-6 and TNF production. Blockade of IL-6 resulted in immediate and complete amelioration of systemic autoinflammation and anemia in all 5 patients treated; however, neuroinflammation has, so far proven recalcitrant to IL-6 blockade and the janus kinase (JAK) inhibitor baricitinib, likely due to lack of central nervous system penetration of both drugs. We therefore highlight that bi-allelic mutation in IRAK4 may be associated with a severe and complex autoinflammatory and neuroinflammatory phenotype that we have called NASA (neuroinflammation, autoinflammation, splenomegaly and anemia), in addition to immunodeficiency in humans

Monogenic mimics of Behçet’s Disease

Behçet’s Disease (BD) is a rare multisystemic variable vessel vasculitis with a chronic and relapsing disease course. BD is polygenic, and genome wide association studies in cohorts around the world have defined risk alleles across various loci which predispose individuals to the disease. The strongest associated genotype is HLA-B*51. Several monogenic autoinflammatory disorders and primary immunodeficiencies can present with features that may be mistaken for BD, and it is important that they are diagnosed as the treatments and prognoses can differ significantly. In this thesis, patients with clinically suspected BD were recruited from four specialist BD care centres in the UK. I designed a novel genetic workflow specifically to identify variants causing monogenic disease and perform HLA-typing from the same dataset. All participants underwent whole exome sequencing, and genetic analysis was undertaken thereafter by 1. examining germline and somatic variants in genes known to cause monogenic immunodeficiency, autoinflammation or vasculitis by virtual gene panel application; 2. scrutiny of data prioritised by human phenotype ontology using Exomiser; 3. identification of copy number variants using ExomeDepth; 4. HLA and ERAP1 genotyping using OptiType and ERAPlotype. Using this approach, I identified a series of patients with monogenic diseases mimicking BD, including some with novel variants in TNFAIP3 and ISG15. HLA-B*51 positivity was also confirmed in some cases. I characterised the novel variants through functional investigation of these patients, which led to the first case of successful targeted treatment of ISG15 deficiency with Janus kinase inhibition. In summary, I have described a novel genetic workflow for BD facilitating precise genetic diagnoses, and more accurate patient classification based on characterisation of the genetic architecture of BD. This approach can efficiently detect known and potentially novel monogenic forms of BD, whilst additionally providing HLA-typing which can be helpful for the classification of typical polygenic BD

Table_1_A rapid turnaround gene panel for severe autoinflammation: Genetic results within 48 hours.docx

There is an important unmet clinical need for fast turnaround next generation sequencing (NGS) to aid genetic diagnosis of patients with acute and sometimes catastrophic inflammatory presentations. This is imperative for patients who require precise and targeted treatment to prevent irreparable organ damage or even death. Acute and severe hyper- inflammation may be caused by primary immunodeficiency (PID) with immune dysregulation, or more typical autoinflammatory diseases in the absence of obvious immunodeficiency. Infectious triggers may be present in either immunodeficiency or autoinflammation. We compiled a list of 25 genes causing monogenetic immunological diseases that are notorious for their acute first presentation with fulminant inflammation and which may be amenable to specific treatment, including hemophagocytic lymphohistiocytosis (HLH); and autoinflammatory diseases that can present with early-onset stroke or other irreversible neurological inflammatory complications. We designed and validated a pipeline that enabled return of clinically actionable results in hours rather than weeks: the Rapid Autoinflammation Panel (RAP). We demonstrated accuracy of this new pipeline, with 100% sensitivity and 100% specificity. Return of results to clinicians was achieved within 48-hours from receiving the patient’s blood or saliva sample. This approach demonstrates the potential significant diagnostic impact of NGS in acute medicine to facilitate precision medicine and save “life or limb” in these critical situations.</p