Anti-SARS-CoV-2 antibodies following vaccination are associated with lymphocyte count and serum immunoglobulins in SLE.

OBJECTIVES: Patients with Systemic Lupus Erythematosus are known to have dysregulated immune responses and may have reduced response to vaccination against COVID-19 while being at risk of severe COVID-19 disease. The aim of this study was to identify whether vaccine responses were attenuated in SLE and to assess disease- and treatment-specific associations. METHODS: Patients with SLE were matched by age, sex and ethnic background to healthcare worker healthy controls (HC). Anti-SARS-CoV-2 spike glycoprotein antibodies were measured at 4-8 weeks following the second COVID-19 vaccine dose (either BNT162b2 or ChAdOx1 nCoV-19) using a CE-marked combined ELISA detecting IgG, IgA and IgM (IgGAM). Antibody levels were considered as a continuous variable and in tertiles and compared between SLE patients and HC and associations with medication, disease activity and serological parameters were determined. RESULTS: Antibody levels were lower in 43 SLE patients compared to 40 HC (p < 0.001). There was no association between antibody levels and medication, lupus disease activity, vaccine type or prior COVID infection. Higher serum IgA, but not IgG or IgM, was associated with being in a higher anti-SARS-CoV-2 antibody level tertile (OR [95% CI] 1.820 [1.050, 3.156] p = 0.033). Similarly, higher lymphocyte count was also associated with being in a higher tertile of anti-SARS-CoV-2 (OR 3.330 [1.505, 7.366] p = 0.003). CONCLUSION: Patients with SLE have lower antibody levels following 2 doses of COVID-19 vaccines compared to HC. In SLE lower lymphocyte counts and serum IgA levels are associated with lower antibody levels post vaccination, potentially identifying a subgroup of patients who may therefore be at increased risk of infection.

SARS-CoV-2 Testing in the Community: Testing Positive Samples with the TaqMan SARS-CoV-2 Mutation Panel To Find Variants in Real Time.

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages; however, there can be limitations due to sequence dropout when used to identify specific key mutations. Recently, ThermoFisher Scientific has developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the ThermoFisher TaqMan mutation panel genotyping assay, initially for three mutations of concern and then for an additional two mutations of concern, against SARS-CoV-2-positive clinical samples and the corresponding COVID-19 Genomics UK Consortium (COG-UK) sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, is an excellent complement to genome sequencing, and has real clinical health value potential, allowing laboratories to report and take action on variants of concern much more quickly.

Development of a high-sensitivity ELISA detecting IgG, IgA and IgM antibodies to the SARS-CoV-2 spike glycoprotein in serum and saliva.

Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven relatively straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. We systematically developed an ELISA, optimizing different antigens and amplification steps, in serum and saliva from non-hospitalized SARS-CoV-2-infected subjects. Using trimeric spike glycoprotein, rather than nucleocapsid, enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike IgG, IgA and IgM antibody responses were readily detectable in saliva from a minority of RT-PCR confirmed, non-hospitalized symptomatic individuals, and these were mostly subjects who had the highest levels of anti-spike serum antibodies. Therefore, detecting antibody responses in both saliva and serum can contribute to determining virus exposure and understanding immune responses after SARS-CoV-2 infection.

Validation testing to determine the sensitivity of lateral flow testing for asymptomatic SARS-CoV-2 detection in low prevalence settings: Testing frequency and public health messaging is key.

Lateral flow devices (LFDs) are quickly being implemented for use in large-scale population surveillance programs for SARS-CoV-2 infection in the United Kingdom. These programs have been piloted in city-wide screening in the city of Liverpool and are now being rolled out to support care home visits and the return home of University students for the Christmas break. Here, we present data on the performance of LFDs to test almost 8,000 students at the University of Birmingham between December 2 and December 9, 2020. The performance is validated against almost 800 samples using PCR performed in the University Pillar 2 testing lab and theoretically validated on thousands of Pillar 2 PCR testing results performed on low-prevalence care home testing samples. Our data show that LFDs do not detect infections presenting with PCR Ct values over 29 to 30 as determined using the Thermo Fisher TaqPath asssay. This may be of particular importance in detecting individuals that are either at the early, or late stages of infection, and reinforces the need for frequent, recurrent testing.

S-Variant SARS-CoV-2 Lineage B1.1.7 Is Associated With Significantly Higher Viral Load in Samples Tested by TaqPath Polymerase Chain Reaction.

A SARS-CoV-2 variant B1.1.7 containing mutation Δ69/70 has spread rapidly in the United Kingdom and shows an identifiable profile in ThermoFisher TaqPath RT-qPCR, S gene target failure (SGTF). We analyzed recent test data for trends and significance. Linked cycle threshold (Ct) values for respiratory samples showed that a low Ct for ORF1ab and N were clearly associated with SGTF. Significantly more SGTF samples had higher inferred viral loads between 1×107 and 1×108. Our conclusion is that patients whose samples exhibit the SGTF profile are more likely to have high viral loads, which may explain higher infectivity and rapidity of spread.

Antibodies to gp210 and understanding risk in patients with primary biliary cholangitis.

BACKGROUND AND AIMS: A variety of auto-antibody assays are available as part of the clinical care of patients with liver disease. We sought to better understand the clinical utility of immune serological testing in patients with primary biliary cholangitis (PBC). METHODS: We retrospectively analysed data from 2846 patients investigated for liver disease at a UK liver centre between 2001 and 2017. A total of 499 patients with PBC were identified. Immune serology results were examined for their diagnostic utility and prognostic significance to predict transplant-free survival. RESULTS: Antimitochondrial antibodies (AMAs) were specific (94.5%) and sensitive (85.6%) for PBC; antinuclear antibodies (ANAs) against glycoprotein 210 (gp210) and sp100 were specific (>98%) but not sensitive (<25%). The disease-specific ANAs were detectable in 29.6% of AMA-negative patients. Anti-gp210 auto-antibodies were significantly associated with elevated serum aminotransferase activity, bilirubin and liver stiffness at presentation (P < .010). Anti-gp210 auto-antibodies predicted non-response to ursodeoxycholic acid (UDCA) by GLOBE criteria (39.3% vs 16.7%, P = .005). Moreover, anti-gp210 was independently associated with death or liver transplantation (HR 3.22, 95% CI 1.49-6.96; P = .003), after accounting for other significant baseline determinants of outcome. Serologic finding of anti-gp210 antibodies conferred an independent risk of death or transplantation (HR 4.13, 95% CI 1.85-9.22; P = .001) after accounting for treatment response. CONCLUSION: In our single-centre cohort of patients with PBC, the presence of anti-gp210 was associated with an adverse presenting phenotype, predicted treatment non-response and independently predicted reduced transplant-free survival.

Rapid implementation and validation of a cold-chain free SARS-CoV-2 diagnostic testing workflow to support surge capacity.

BACKGROUND: In January 2020 reports of unidentified severe respiratory illness were described in Wuhan, China. A rapid expansion in cases affecting most countries around the globe led to major changes in the way people live their daily lives. In the United Kingdom, the Department of Health and Social Care directed healthcare providers to establish additional resources to manage the anticipated surge in cases that could overwhelm the health services. A priority area was testing for SARS-CoV-2 RNA and its detection by qualitative RT-PCR. DESIGN: A laboratory workflow twinning research environment with clinical laboratory capabilities was implemented and validated in the University of Birmingham within 4 days of the project initiation. The diagnostic capability was centred on an IVD CE-marked RT-PCR kit and designed to provide surge capacity to the nearby Queen Elizabeth Hospital. The service was initially tasked with testing healthcare workers (HCW) using throat swabs, and subsequently the process investigated the utility of using saliva as an alternative sample type. RESULTS: Between the 8th April 2020 and the 30th April 2020, the laboratory tested a total of 1282 HCW for SARS-CoV-2 RNA in throat swabs. RNA was detected in 54 % of those who reported symptoms compatible with COVID-19, but in only 4% who were asymptomatic. CONCLUSION: This capability was established rapidly and utilised a cold-chain free methodology, applicable to a wide range of settings, and which can provide surge capacity and support to clinical laboratories facing increasing pressure during periods of national crisis.

Detection of antibodies to the SARS-CoV-2 spike glycoprotein in both serum and saliva enhances detection of infection.

BACKGROUND: Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. METHODS: We systemically developed an ELISA assay, optimising different antigens and amplification steps, in serum and saliva from symptomatic and asymptomatic SARS-CoV-2-infected subjects. RESULTS: Using trimeric spike glycoprotein, rather than nucleocapsid enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike, but not nucleocapsid, IgG, IgA and IgM antibody responses were readily detectable in saliva from non-hospitalized symptomatic and asymptomatic individuals. Antibody responses in saliva and serum were largely independent of each other and symptom reporting. CONCLUSIONS: Detecting antibody responses in both saliva and serum is optimal for determining virus exposure and understanding immune responses after SARS-CoV-2 infection. FUNDING: This work was funded by the University of Birmingham, the National Institute for Health Research (UK), the NIH National Institute for Allergy and Infectious Diseases, the Bill and Melinda Gates Foundation and the University of Southampton.

TRPC1 Regulates the Activity of a Voltage-Dependent Nonselective Cation Current in Hippocampal CA1 Neurons.

The cation channel subunit TRPC1 is strongly expressed in central neurons including neurons in the CA1 region of the hippocampus where it forms complexes with TRPC4 and TRPC5. To investigate the functional role of TRPC1 in these neurons and in channel function, we compared current responses to group I metabotropic glutamate receptor (mGluR I) activation and looked for major differences in dendritic morphology in neurons from TRPC1+/+ and TRPC1-/- mice. mGluR I stimulation resulted in the activation of a voltage-dependent nonselective cation current in both genotypes. Deletion of TRPC1 resulted in a modification of the shape of the current-voltage relationship, leading to an inward current increase. In current clamp recordings, the percentage of neurons that responded to depolarization in the presence of an mGluR I agonist with a plateau potential was increased in TRPC1-/- mice. There was also a small increase in the minor population of CA1 neurons that have more than one apical dendrite in TRPC1-/- mice. We conclude that TRPC1 has an inhibitory effect on receptor-operated nonselective cation channels in hippocampal CA1 neurons probably as a result of heterotetramer formation with other TRPC isoforms, and that TRPC1 deletion has only minor effects on dendritic morphology.

Analytical validation of new ELISAs for the quantitation of polyclonal free light chains and comparison to existing assays for healthy and patient samples.

BACKGROUND: Polyclonal FLCs can be used as a biomarker of inflammation and immune activation in a range of diseases. This study evaluated the performance of new FLC ELISAs (Seralite FLC ELISA) for the quantitation of polyclonal κ and λ FLC, including comparisons to existing assays. METHODS: Technical performance was assessed for the ELISA and reference ranges were generated using healthy donor serum (N = 91). Patients with a range of conditions associated with polyclonal FLC dysregulation (N = 164) were measured across platforms. RESULTS: The ELISAs generated references ranges of: 8.72-23.0 mg/L κ FLC, and 8.52-25.24 mg/L for λ FLC. ELISAs demonstrated linearity across the calibration range and intra-assay (≤ 8.7%) and inter-assay (≤ 12.3%) imprecision was low. The limit of detection was 0.63 mg/L for κ and 0.57 mg/L for λ FLC. Minimal cross-reactivity was observed for interference agents, alternate FLC and whole immunoglobulin (median change ≤3.6 mg/L). Assays showed good batch-to-batch consistency. For patient samples, methods generated different κ and λ FLC concentrations and differences were seen between methods for the number of patients classified as below, with and above references ranges for κ and λ FLC. There was no significant difference in the FLC sum between the different techniques. CONCLUSIONS: The ELISAs displayed good analytical and technical performance. The quantification of individual κ and λ FLC appears inherently different between platforms. These differences are attenuated if using the FLC sum, which was similar between methods and provided agreement in relation to patients having normal or elevated FLCs.

What does it mean if a patient is positive for anti-Jo-1 in routine hospital practice? A retrospective nested case-control study.

Background:  It is widely believed that patients bearing auto-antibodies to histidyl tRNA synthetase (anti-Jo-1) very likely have a connective tissue disease including myositis and interstitial lung disease.  The value of positive tests in low disease prevalence settings such as those tested in routine care is unknown.  We sought to determine the value of anti-Jo-1 auto-antibodies in routine practice. Methods: Our study was a nested case control study within a retrospective cohort of all patients tested for anti-ENA our hospital, from any hospital department, between January 2013 and December 2014.  Data was extracted from electronic records of anti-Jo-1 positive patients and randomly selected ENA negative patients (ratio of 1:2), allowing for a minimum follow up of at least 12 months after first testing. Results: 4009 samples (3581 patients) were tested.  Anti-ENA was positive in 616 (17.2%) patients, 40 (1.1%) were anti-Jo-1 positive. Repeat ENA testing was done for 350/3581 (9.8%) patients (428 of 4009 (10.7%) samples) and in 7/40 (17.5%) of anti-Jo-1 positive patients. The median interval between the first and second request was 124 days (inter-quartile range 233 days).  The frequencies of interstitial lung disease (ILD), myositis and Raynaud's were comparable for anti-Jo-1 positive patients (n=40) and 80 randomly selected ENA negative controls.  Positive tests led to additional diagnostic testing in the absence of clinical disease.  Sensitivity of Jo-1 for ILD was 50% (CI 19-81%), specificity 68% (CI 59-77%), positive predictive value 12.5% (CI 4 to 27%) and negative predictive value 93.8% (CI 86-98%). Of 10 (25%) patients with high anti-Jo1 levels, 3 had ILD, one myositis and two a malignancy (disseminated melanoma and CML).  Conclusion: Anti-Jo-1 is uncommon in a heterogenous hospital population and is only weakly predictive for ILD.  Repeated test requests were common and potentially unnecessary indicating that controls over repeat requests could yield significant cost savings.

Development of a rapid and quantitative lateral flow assay for the simultaneous measurement of serum κ and λ immunoglobulin free light chains (FLC): inception of a new near-patient FLC screening tool.

BACKGROUND: Serum free light chains (FLC) are sensitive biomarkers used for the diagnosis and management of plasma cell dyscrasias, such as multiple myeloma (MM), and are central to clinical screening algorithms and therapy response criteria. We have developed a portable, near-patient, lateral-flow test (Seralite®) that quantitates serum FLC in 10 min, and is designed to eliminate sample processing delays and accelerate decision-making in the clinic. METHODS: Assay interference, imprecision, lot-to-lot variability, linearity, and the utility of a competitive-inhibition design for the elimination of antigen-excess ('hook effect') were assessed. Reference ranges were calculated from 91 healthy donor sera. Preliminary clinical validation was conducted by retrospective analysis of sera from 329 patients. Quantitative and diagnostic results were compared to Freelite®. RESULTS: Seralite® gave a broad competitive-inhibition calibration curve from below 2.5 mg/L to above 200 mg/L, provided good assay linearity (between 1.6 and 208.7 mg/L for κ FLC and between 3.5 and 249.7 mg/L for λ FLC) and sensitivity (1.4 mg/L for κ FLC and 1.7 mg/L for λ FLC), and eliminated anomalous results from antigen-excess. Seralite® gave good diagnostic concordance with Freelite® (Roche Hitachi Cobas C501) identifying an abnormal FLC ratio and FLC difference in 209 patients with newly diagnosed MM and differentiating these patients from normal healthy donors with polyclonal FLC. CONCLUSIONS: Seralite® sensitively quantitates FLC and rapidly identifies clinical conditions where FLC are abnormal, including MM.

Brief Report: Immunization of HIV-Infected Adults in the UK With Haemophilus influenzae b/Meningococcal C Glycoconjugate and Pneumococcal Polysaccharide Vaccines.

U.K. guidelines for vaccinating HIV-infected adults against bacteria are based on limited data. We compared antibody responses between 211 HIV-infected and 73 HIV-uninfected adults vaccinated with pneumococcal polysaccharide vaccine (PPV) and Haemophilus influenzae b/meningococcal C polysaccharide-tetanus toxoid glycoconjugate vaccine (Hib/MenC-TT). IgG responses to Hib/MenC-TT were not significantly different. PPV induced median IgGs >1.3 µg/mL for 10/12 serotypes among HIV-uninfected participants and 5/12 in HIV-infected participants. HIV-uninfected adults had higher post-vaccination IgGs than HIV-infected adults for 4/12 serotypes (P < 0.001). Responses did not associate with CD4 count or viral suppression. In a U.K. HIV-infected population, Hib/MenC-TT induced similar responses to HIV-uninfected adults, whereas PPV induced poor responses.

Serum tryptase concentration and progression to end-stage renal disease.

BACKGROUND: Mast cell activation can lead to nonclassical activation of the Renin-Angiotensin-Aldosterone System. However, the relevance of this to human chronic kidney disease is unknown. We assessed the association between serum tryptase, a product of mast cell activation, and progression to end-stage renal disease or mortality in patients with advanced chronic kidney disease. We stratified patients by use of angiotensin-converting enzyme inhibitors/angiotensin receptor II blockers (ACEi/ARB). MATERIALS AND METHODS: This was a prospective cohort study of 446 participants recruited into the Renal Impairment in Secondary Care study. Serum tryptase was measured at recruitment by sandwich immunoassay. Cox regression analysis was undertaken to determine variables associated with progression to end-stage renal disease or death. RESULTS: Serum tryptase concentration was independently associated with progression to end-stage renal disease but not with death. In patients treated with ACEi or ARB, there was a strong independent association between higher tryptase concentrations and progression to end-stage renal disease; when compared to the lowest tertile, tryptase concentrations in the middle and highest tertiles had hazard ratios [HR] of 5·78 (95% confidence interval [CI] 1·19-28·03, P = 0·029) and 6·19 (95% CI 1·49-25·69, P = 0·012), respectively. The other independent risk factors for progression to end-stage renal disease were lower age, male gender, lower estimated glomerular filtration rate and higher urinary albumin creatinine ratio. CONCLUSION: Elevated serum tryptase concentration is an independent prognostic factor for progression to end-stage renal disease in patients with chronic kidney disease who are receiving treatment with an ACEi or ARB.

TRPs in mechanosensing and volume regulation.

Mechanosensitive channels allow cells to respond to changes in membrane stretch that occur due to external stimuli like pressure or flow or that occur because of osmotically induced cell swelling or shrinkage. Ion fluxes through the channels change the membrane potential and ion concentrations and link the stretch to cellular signalling. Changes in cellular activity evoked by mechanical stimuli can be used to elicit local tissue responses or can be transmitted further to generate more widespread responses. Channels can respond directly to membrane stress, can be conferred mechanosensitive by interaction with structural proteins, or can be activated by mechanosensitive signalling pathways. Because mechanosensitive channels are often nonselective cation channels, and invertebrate TRP isoforms are involved in mechanosensation, many of the mammalian TRP isoforms have been investigated with regard to their mechanosensitivity. There is evidence that members of the TRPC, TRPV, TRPM, TRPA and TRPP subfamilies could be in some way mechanosensitive, and each of the activation mechanisms described above is used by a TRP channel. TRP channels may be involved in mechanosensitive processes ranging from flow and pressure sensing in the vasculature and other organs to mechanosensation in sensory neurones and sensory organs. There is also evidence for a role of mechano- or osmosensitive TRP isoforms in osmosensing and the regulation of cell volume. Often, a number of different TRP isoforms have been implicated in a single type of mechanosensitive response. In many cases, the involvement of the isoforms needs to be confirmed, and their exact role in the signalling process determined.

Measurement of antibodies to pneumococcal, meningococcal and haemophilus polysaccharides, and tetanus and diphtheria toxoids using a 19-plexed assay.

The measurement of antibody responses to vaccination is useful in the assessment of immune status in suspected immune deficiency. Previous reliance on enzyme-linked immunoabsorbent assays (ELISA) has been cumbersome, time-consuming and expensive. The availability of flow cytometry systems has led to the development of multiplexed assays enabling simultaneous measurement of antibodies to several antigens. We optimized a flow cytometric bead-based assay to measure IgG and IgM concentrations in serum to 19 antigens contained in groups of bacterial subunit vaccines: pneumococcal vaccines, meningococcal vaccines, Haemophilus influenzae b (Hib), and tetanus and diphtheria toxoid vaccines. 89-SF was employed as the standard serum. The assay was used to determine specific antibody levels in serum from 193 healthy adult donors. IgG and pneumococcal IgM antibody concentrations were measurable across 3 log10 ranges encompassing the threshold protective IgG antibody levels for each antigen. There was little interference between antibody measurements by the 19-plexed assay compared with monoplexed assays, and a lack of cross-reactive IgG antibody, but evidence for cross-reacting IgM antibody for 3/19 pneumococcal antigens. 90th centile values for 15/19 IgG concentrations and 12/12 IgM concentrations of the 193 adult sera were within these ranges and percentages of sera containing protective IgG antibody levels varied from 4% to 95% depending on antigen. This multiplexed assay can simultaneously measure antibody levels to 19 bacterial vaccine antigens. It is suitable for use in standard clinical practice to assess the in vivo immune response to test vaccinations and measure absolute antibody levels to these antigens.

Dopamine-regulated microRNA MiR-181a controls GluA2 surface expression in hippocampal neurons.

The dynamic expression of AMPA-type glutamate receptors (AMPA-R) at synapses is a key determinant of synaptic plasticity, including neuroadaptations to drugs of abuse. Recently, microRNAs (miRNAs) have emerged as important posttranscriptional regulators of synaptic plasticity, but whether they target glutamate receptors to mediate this effect is not known. Here we used microarray screening to identify miRNAs that regulate synaptic plasticity within the nucleus accumbens, a brain region critical to forming drug-seeking habits. One of the miRNAs that showed a robust enrichment at medium spiny neuron synapses was miR-181a. Using bioinformatics tools, we detected a highly conserved miR-181a binding site within the mRNA encoding the GluA2 subunit of AMPA-Rs. Overexpression and knockdown of miR-181a in primary neurons demonstrated that this miRNA is a negative posttranscriptional regulator of GluA2 expression. Additionally, miR-181a overexpression reduced GluA2 surface expression, spine formation, and miniature excitatory postsynaptic current (mEPSC) frequency in hippocampal neurons, suggesting that miR-181a could regulate synaptic function. Moreover, miR-181a expression was induced by dopamine signaling in primary neurons, as well as by cocaine and amphetamines, in a mouse model of chronic drug treatment. Taken together, our results identify miR-181a as a key regulator of mammalian AMPA-type glutamate receptors, with potential implications for the regulation of drug-induced synaptic plasticity.