Preservation of memory B cell homeostasis in an individual producing broadly neutralising antibodies against HIV-1.

Immunological determinants favouring emergence of broadly neutralising antibodies are crucial to the development of HIV-1 vaccination strategies. Here, we combined RNAseq and B cell cloning approaches to isolate a broadly neutralising antibody (bnAb) ELC07 from an individual living with untreated HIV-1. Using single particle cryogenic electron microscopy (cryo-EM), we show that the antibody recognises a conformational epitope at the gp120-gp41 interface. ELC07 binds the closed state of the viral glycoprotein causing considerable perturbations to the gp41 trimer core structure. Phenotypic analysis of memory B cell subsets from the ELC07 bnAb donor revealed a lack of expected HIV-1-associated dysfunction, specifically no increase in CD21-/CD27- cells was observed whilst the resting memory (CD21+/CD27+) population appeared preserved despite uncontrolled HIV-1 viraemia. Moreover, single cell transcriptomes of memory B cells from this bnAb donor showed a resting memory phenotype irrespective of the epitope they targeted or their ability to neutralise diverse strains of HIV-1. Strikingly, single memory B cells from the ELC07 bnAb donor were transcriptionally similar to memory B cells from HIV-negative individuals. Our results demonstrate that potent bnAbs can arise without the HIV-1-induced dysregulation of the memory B cell compartment and suggest that sufficient levels of antigenic stimulation with a strategically designed immunogen could be effective in HIV-negative vaccine recipients.

Effect of a 2-week interruption in methotrexate treatment on COVID-19 vaccine response in people with immune-mediated inflammatory diseases (VROOM study): a randomised, open label, superiority trial.

BACKGROUND: Methotrexate is the first-line treatment for immune-mediated inflammatory diseases and reduces vaccine-induced immunity. We evaluated if a 2-week interruption of methotrexate treatment immediately after COVID-19 booster vaccination improved antibody response against the S1 receptor binding domain (S1-RBD) of the SARS-CoV-2 spike protein and live SARS-CoV-2 neutralisation compared with uninterrupted treatment in patients with immune-mediated inflammatory diseases. METHOD: We did a multicentre, open-label, parallel-group, randomised, superiority trial in secondary-care rheumatology and dermatology clinics in 26 hospitals in the UK. Adults (aged ≥18 years) with immune-mediated inflammatory diseases taking methotrexate (≤25 mg per week) for at least 3 months, who had received two primary vaccine doses from the UK COVID-19 vaccination programme were eligible. Participants were randomly assigned (1:1) using a centralised validated computer program, to temporarily suspend methotrexate treatment for 2 weeks immediately after COVID-19 booster vaccination or continue treatment as usual. The primary outcome was S1-RBD antibody titres 4 weeks after COVID-19 booster vaccination and was assessed masked to group assignment. All randomly assigned patients were included in primary and safety analyses. This trial is registered with ISRCTN, ISRCTN11442263; following a pre-planned interim analysis, recruitment was stopped early. FINDING: Between Sept 30, 2021, and March 7, 2022, we screened 685 individuals, of whom 383 were randomly assigned: to either suspend methotrexate (n=191; mean age 58·8 years [SD 12·5], 118 [62%] women and 73 [38%] men) or to continue methotrexate (n=192; mean age 59·3 years [11·9], 117 [61%] women and 75 [39%] men). At 4 weeks, the geometric mean S1-RBD antibody titre was 25 413 U/mL (95% CI 22 227-29 056) in the suspend methotrexate group and 12 326 U/mL (10 538-14 418) in the continue methotrexate group with a geometric mean ratio (GMR) of 2·08 (95% CI 1·59-2·70; p<0·0001). No intervention-related serious adverse events occurred. INTERPRETATION: 2-week interruption of methotrexate treatment in people with immune-mediated inflammatory diseases enhanced antibody responses after COVID-19 booster vaccination that were sustained at 12 weeks and 26 weeks. There was a temporary increase in inflammatory disease flares, mostly self-managed. The choice to suspend methotrexate should be individualised based on disease status and vulnerability to severe outcomes from COVID-19. FUNDING: National Institute for Health and Care Research.

Curbside particulate matter and susceptibility to SARS-CoV-2 infection.

Background: Biologic plausibility for the association between exposure to particulate matter (PM) less than 10 µm in aerodynamic diameter (PM10) and coronavirus disease 2019 (COVID-19) morbidity in epidemiologic studies has not been determined. The upregulation of angiotensin-converting enzyme 2 (ACE2), the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) entry receptor on host cells, by PM10 is a putative mechanism. Objective: We sought to assess the effect of PM10 on SARS-CoV-2 infection of cells in vitro. Methods: PM10 from the curbside of London's Marylebone Road and from exhaust emissions was collected by cyclone. A549 cells, human primary nasal epithelial cells (HPNEpCs), SARS-CoV-2-susceptible Vero-E6 and Calu3 cells were cultured with PM10. ACE2 expression (as determined by median fluorescent intensity) was assessed by flow cytometry, and ACE2 mRNA transcript level was assessed by PCR. The role of oxidative stress was determined by N-acetyl cysteine. The cytopathic effect of SARS-CoV-2 (percentage of infection enhancement) and expression of SARS-CoV-2 genes' open reading frame (ORF) 1ab, S protein, and N protein (focus-forming units/mL) were assessed in Vero-E6 cells. Data were analyzed by either the Mann-Whitney U test or Kruskal-Wallis test with the Dunn multiple comparisons test. Results: Curbside PM10 at concentrations of 10 µg/mL or more increased ACE2 expression in A549 cells (P = .0021). Both diesel PM10 and curbside PM10 in a concentration of 10 µg/mL increased ACE2 expression in HPNEpCs (P = .0022 and P = .0072, respectively). ACE2 expression simulated by curbside PM10 was attenuated by N-acetyl cysteine in HPNEpCs (P = .0464). Curbside PM10 increased ACE2 expression in Calu3 cells (P = .0256). In Vero-E6 cells, curbside PM10 increased ACE2 expression (P = .0079), ACE2 transcript level (P = .0079), SARS-CoV-2 cytopathic effect (P = .0002), and expression of the SARS-CoV-2 genes' ORF1ab, S protein, and N protein (P = .0079). Conclusions: Curbside PM10 increases susceptibility to SARS-COV-2 infection in vitro.

Persistent symptoms after COVID-19 are not associated with differential SARS-CoV-2 antibody or T cell immunity.

Among the unknowns in decoding the pathogenesis of SARS-CoV-2 persistent symptoms in Long Covid is whether there is a contributory role of abnormal immunity during acute infection. It has been proposed that Long Covid is a consequence of either an excessive or inadequate initial immune response. Here, we analyze SARS-CoV-2 humoral and cellular immunity in 86 healthcare workers with laboratory confirmed mild or asymptomatic SARS-CoV-2 infection during the first wave. Symptom questionnaires allow stratification into those with persistent symptoms and those without for comparison. During the period up to 18-weeks post-infection, we observe no difference in antibody responses to spike RBD or nucleoprotein, virus neutralization, or T cell responses. Also, there is no difference in the profile of antibody waning. Analysis at 1-year, after two vaccine doses, comparing those with persistent symptoms to those without, again shows similar SARS-CoV-2 immunity. Thus, quantitative differences in these measured parameters of SARS-CoV-2 adaptive immunity following mild or asymptomatic acute infection are unlikely to have contributed to Long Covid causality. ClinicalTrials.gov (NCT04318314).

Behçet's patients' response to COVID-19 vaccination.

Immune hyperstimulation by SARS-CoV2 results in multi-system involvement with consequent organ damage not dissimilar to Behçet's Disease (BD). Management of BD includes immunosuppressive medication, which led to concerns that; firstly, SARS-CoV-2 would stimulate BD activity, thrombin, clotting times, TPO antibodies, and the effectiveness and duration of the COVID-19 vaccines' response in this potentially vulnerable group. The main objectives of this study were: to assess BD patients' immune response to the COVID-19 vaccines based on age, gender, disease activity, BD phenotype, and immunomodulatory medication compared to healthy control participants by measuring anti-spike IgG levels. Further to evaluate the effect of the COVID-19 vaccines on T and B cells, immunoglobulins, thrombophilia, thyroid function and COVID-19 antibody production. Patients on immunosuppressive medication had a reduced immune response to COVID-19 vaccines. -Also, patients over 40 years and with the neurologic BD phenotype had lower responses. mRNA COVID-19 vaccines were more effective and had fewer side effects compared to conventional COVID-19 vaccines.

Large clones of pre-existing T cells drive early immunity against SARS-COV-2 and LCMV infection.

T cell responses precede antibody and may provide early control of infection. We analyzed the clonal basis of this rapid response following SARS-COV-2 infection. We applied T cell receptor (TCR) sequencing to define the trajectories of individual T cell clones immediately. In SARS-COV-2 PCR+ individuals, a wave of TCRs strongly but transiently expand, frequently peaking the same week as the first positive PCR test. These expanding TCR CDR3s were enriched for sequences functionally annotated as SARS-COV-2 specific. Epitopes recognized by the expanding TCRs were highly conserved between SARS-COV-2 strains but not with circulating human coronaviruses. Many expanding CDR3s were present at high frequency in pre-pandemic repertoires. Early response TCRs specific for lymphocytic choriomeningitis virus epitopes were also found at high frequency in the preinfection naive repertoire. High-frequency naive precursors may allow the T cell response to respond rapidly during the crucial early phases of acute viral infection.

Vitamin D Supplementation Does Not Influence SARS-CoV-2 Vaccine Efficacy or Immunogenicity: Sub-Studies Nested within the CORONAVIT Randomised Controlled Trial.

Vitamin D deficiency has been reported to associate with the impaired development of antigen-specific responses following vaccination. We aimed to determine whether vitamin D supplements might boost the immunogenicity and efficacy of SARS-CoV-2 vaccination by conducting three sub-studies nested within the CORONAVIT randomised controlled trial, which investigated the effects of offering vitamin D supplements at a dose of 800 IU/day or 3200 IU/day vs. no offer on risk of acute respiratory infections in UK adults with circulating 25-hydroxyvitamin D concentrations <75 nmol/L. Sub-study 1 (n = 2808) investigated the effects of vitamin D supplementation on the risk of breakthrough SARS-CoV-2 infection following two doses of SARS-CoV-2 vaccine. Sub-study 2 (n = 1853) investigated the effects of vitamin D supplementation on titres of combined IgG, IgA and IgM (IgGAM) anti-Spike antibodies in eluates of dried blood spots collected after SARS-CoV-2 vaccination. Sub-study 3 (n = 100) investigated the effects of vitamin D supplementation on neutralising antibody and cellular responses in venous blood samples collected after SARS-CoV-2 vaccination. In total, 1945/2808 (69.3%) sub-study 1 participants received two doses of ChAdOx1 nCoV-19 (Oxford−AstraZeneca); the remainder received two doses of BNT162b2 (Pfizer). Mean follow-up 25(OH)D concentrations were significantly elevated in the 800 IU/day vs. no-offer group (82.5 vs. 53.6 nmol/L; mean difference 28.8 nmol/L, 95% CI 22.8−34.8) and in the 3200 IU/day vs. no offer group (105.4 vs. 53.6 nmol/L; mean difference 51.7 nmol/L, 45.1−58.4). Vitamin D supplementation did not influence the risk of breakthrough SARS-CoV-2 infection in vaccinated participants (800 IU/day vs. no offer: adjusted hazard ratio 1.28, 95% CI 0.89 to 1.84; 3200 IU/day vs. no offer: 1.17, 0.81 to 1.70). Neither did it influence IgGAM anti-Spike titres, neutralising antibody titres or IFN-γ concentrations in the supernatants of S peptide-stimulated whole blood. In conclusion, vitamin D replacement at a dose of 800 or 3200 IU/day effectively elevated 25(OH)D concentrations, but it did not influence the protective efficacy or immunogenicity of SARS-CoV-2 vaccination when given to adults who had a sub-optimal vitamin D status at baseline.

Quantitative, multiplexed, targeted proteomics for ascertaining variant specific SARS-CoV-2 antibody response.

Determining the protection an individual has to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VoCs) is crucial for future immune surveillance, vaccine development, and understanding of the changing immune response. We devised an informative assay to current ELISA-based serology using multiplexed, baited, targeted proteomics for direct detection of multiple proteins in the SARS-CoV-2 anti-spike antibody immunocomplex. Serum from individuals collected after infection or first- and second-dose vaccination demonstrates this approach and shows concordance with existing serology and neutralization. Our assays show altered responses of both immunoglobulins and complement to the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.1) VoCs and a reduced response to Omicron (B1.1.1529). We were able to identify individuals who had prior infection, and observed that C1q is closely associated with IgG1 (r > 0.82) and may better reflect neutralization to VoCs. Analyzing additional immunoproteins beyond immunoglobulin (Ig) G, provides important information about our understanding of the response to infection and vaccination.

Correlation Between Postvaccination Anti-Spike Antibody Titers and Protection Against Breakthrough Severe Acute Respiratory Syndrome Coronavirus 2 Infection: A Population-Based Longitudinal Study.

In this population-based cohort of 7538 adults, combined immunoglobulin (Ig) G, IgA, and IgM (IgG/A/M) anti-spike titers measured after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination were predictive of protection against breakthrough SARS-CoV-2 infection. Discrimination was significantly improved by adjustment for factors influencing risk of SARS-CoV-2 exposure, including household overcrowding, public transport use, and visits to indoor public places. Anti-spike IgG/A/M titers showed positive correlation with neutralizing antibody titers (rs = 0.80 [95% confidence interval, .72-.86]; P < .001) and S peptide-stimulated interferon-γ concentrations (rs = 0.31 [.13-.47]; P < .001).

Immune boosting by B.1.1.529 (Omicron) depends on previous SARS-CoV-2 exposure.

The Omicron, or Pango lineage B.1.1.529, variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carries multiple spike mutations with high transmissibility and partial neutralizing antibody (nAb) escape. Vaccinated individuals show protection against severe disease, often attributed to primed cellular immunity. We investigated T and B cell immunity against B.1.1.529 in triple BioNTech BNT162b2 messenger RNA-vaccinated health care workers (HCWs) with different SARS-CoV-2 infection histories. B and T cell immunity against previous variants of concern was enhanced in triple-vaccinated individuals, but the magnitude of T and B cell responses against B.1.1.529 spike protein was reduced. Immune imprinting by infection with the earlier B.1.1.7 (Alpha) variant resulted in less durable binding antibody against B.1.1.529. Previously infection-naïve HCWs who became infected during the B.1.1.529 wave showed enhanced immunity against earlier variants but reduced nAb potency and T cell responses against B.1.1.529 itself. Previous Wuhan Hu-1 infection abrogated T cell recognition and any enhanced cross-reactive neutralizing immunity on infection with B.1.1.529.

Generation of Novel Severe Acute Respiratory Syndrome Coronavirus 2 Variants on the B.1.1.7 Lineage in 3 Patients With Advanced Human Immunodeficiency Virus-1 Disease.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is of public health concern in case of vaccine escape. Described are 3 patients with advanced human immunodeficiency virus (HIV)-1 and chronic SARS-CoV-2 infection in whom there is evidence of selection and persistence of novel mutations that are associated with increased transmissibility and immune escape.

Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections.

Effective control of SARS-CoV-2 infection on primary exposure may reveal correlates of protective immunity to future variants, but we lack insights into immune responses before or at the time virus is first detected. We use blood transcriptomics, multiparameter flow cytometry, and T cell receptor (TCR) sequencing spanning the time of incident non-severe infection in unvaccinated virus-naive individuals to identify rapid type 1 interferon (IFN) responses common to other acute respiratory viruses and cell proliferation responses that discriminate SARS-CoV-2 from other viruses. These peak by the time the virus is first detected and sometimes precede virus detection. Cell proliferation is most evident in CD8 T cells and associated with specific expansion of SARS-CoV-2-reactive TCRs, in contrast to virus-specific antibodies, which lag by 1-2 weeks. Our data support a protective role for early type 1 IFN and CD8 T cell responses, with implications for development of universal T cell vaccines.

HLA-DR polymorphism in SARS-CoV-2 infection and susceptibility to symptomatic COVID-19.

SARS-CoV-2 infection results in different outcomes ranging from asymptomatic infection to mild or severe disease and death. Reasons for this diversity of outcome include differences in challenge dose, age, gender, comorbidity and host genomic variation. Human leukocyte antigen (HLA) polymorphisms may influence immune response and disease outcome. We investigated the association of HLAII alleles with case definition symptomatic COVID-19, virus-specific antibody and T-cell immunity. A total of 1364 UK healthcare workers (HCWs) were recruited during the first UK SARS-CoV-2 wave and analysed longitudinally, encompassing regular PCR screening for infection, symptom reporting, imputation of HLAII genotype and analysis for antibody and T-cell responses to nucleoprotein (N) and spike (S). Of 272 (20%) HCW who seroconverted, the presence of HLA-DRB1*13:02 was associated with a 6·7-fold increased risk of case definition symptomatic COVID-19. In terms of immune responsiveness, HLA-DRB1*15:02 was associated with lower nucleocapsid T-cell responses. There was no association between DRB1 alleles and anti-spike antibody titres after two COVID vaccine doses. However, HLA DRB1*15:01 was associated with increased spike T-cell responses following both first and second dose vaccination. Trial registration: NCT04318314 and ISRCTN15677965.

Pre-existing polymerase-specific T cells expand in abortive seronegative SARS-CoV-2.

Individuals with potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) do not necessarily develop PCR or antibody positivity, suggesting that some individuals may clear subclinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections1-3. Here we hypothesize that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-2 (refs. 4-11), would expand in vivo to support rapid viral control, aborting infection. We measured SARS-CoV-2-reactive T cells, including those against the early transcribed replication-transcription complex (RTC)12,13, in intensively monitored healthcare workers (HCWs) who tested repeatedly negative according to PCR, antibody binding and neutralization assays (seronegative HCWs (SN-HCWs)). SN-HCWs had stronger, more multispecific memory T cells compared with a cohort of unexposed individuals from before the pandemic (prepandemic cohort), and these cells were more frequently directed against the RTC than the structural-protein-dominated responses observed after detectable infection (matched concurrent cohort). SN-HCWs with the strongest RTC-specific T cells had an increase in IFI27, a robust early innate signature of SARS-CoV-2 (ref. 14), suggesting abortive infection. RNA polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and SARS-CoV-2 clades. RNA polymerase was preferentially targeted (among the regions tested) by T cells from prepandemic cohorts and SN-HCWs. RTC-epitope-specific T cells that cross-recognized HCoV variants were identified in SN-HCWs. Enriched pre-existing RNA-polymerase-specific T cells expanded in vivo to preferentially accumulate in the memory response after putative abortive compared to overt SARS-CoV-2 infection. Our data highlight RTC-specific T cells as targets for vaccines against endemic and emerging Coronaviridae.

Heterologous infection and vaccination shapes immunity against SARS-CoV-2 variants.

The impact of the initial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infecting strain on downstream immunity to heterologous variants of concern (VOCs) is unknown. Studying a longitudinal healthcare worker cohort, we found that after three antigen exposures (infection plus two vaccine doses), S1 antibody, memory B cells, and heterologous neutralization of B.1.351, P.1, and B.1.617.2 plateaued, whereas B.1.1.7 neutralization and spike T cell responses increased. Serology using the Wuhan Hu-1 spike receptor binding domain poorly predicted neutralizing immunity against VOCs. Neutralization potency against VOCs changed with heterologous virus encounter and number of antigen exposures. Neutralization potency fell differentially depending on targeted VOCs over the 5 months from the second vaccine dose. Heterologous combinations of spike encountered during infection and vaccination shape subsequent cross-protection against VOC, with implications for future-proof next-generation vaccines.

Prior SARS-CoV-2 infection rescues B and T cell responses to variants after first vaccine dose.

SARS-CoV-2 vaccine rollout has coincided with the spread of variants of concern. We investigated if single dose vaccination, with or without prior infection, confers cross protective immunity to variants. We analyzed T and B cell responses after first dose vaccination with the Pfizer/BioNTech mRNA vaccine BNT162b2 in healthcare workers (HCW) followed longitudinally, with or without prior Wuhan-Hu-1 SARS-CoV-2 infection. After one dose, individuals with prior infection showed enhanced T cell immunity, antibody secreting memory B cell response to spike and neutralizing antibodies effective against B.1.1.7 and B.1.351. By comparison, HCW receiving one vaccine dose without prior infection showed reduced immunity against variants. B.1.1.7 and B.1.351 spike mutations resulted in increased, abrogated or unchanged T cell responses depending on human leukocyte antigen (HLA) polymorphisms. Single dose vaccination with BNT162b2 in the context of prior infection with a heterologous variant substantially enhances neutralizing antibody responses against variants.

The ß-NGF/TrkA Signalling Pathway Is Associated With the Production of Anti-Nucleoprotein IgG in Convalescent COVID-19.

Background: The presentation of SARS-CoV-2 infection varies from asymptomatic to severe COVID-19. Similarly, high variability in the presence, titre and duration of specific antibodies has been reported. While some host factors determining these differences, such as age and ethnicity have been identified, the underlying molecular mechanisms underpinning these differences remain poorly defined. Methods: We analysed serum and PBMC from 17 subjects with a previous PCR-confirmed SARS-CoV-2 infection and 10 unexposed volunteers following the first wave of the pandemic, in the UK. Anti-NP IgG and neutralising antibodies were measured, as well as a panel of infection and inflammation related cytokines. The virus-specific T cell response was determined by IFN-γ ELISPOT and flow cytometry after overnight incubation of PBMCs with pools of selected SARS-CoV-2 specific peptides. Results: Seven of 17 convalescent subjects had undetectable levels of anti-NP IgG, and a positive correlation was shown between anti-NP IgG levels and the titre of neutralising antibodies (IC50). In contrast, a discrepancy was noted between antibody levels and T cell IFN-γ production by ELISpot following stimulation with specific peptides. Among the analysed cytokines, ß-NGF and IL-1α levels were significantly different between anti-NP positive and negative subjects, and only ß-NGF significantly correlated with anti-NP positivity. Interestingly, CD4+ T cells of anti-NP negative subjects expressed lower amounts of the ß-NGF-specific receptor TrkA. Conclusions: Our results suggest that the ß-NGF/TrkA signalling pathway is associated with the production of anti-NP specific antibody in mild SARS-CoV-2 infection and the mechanistic regulation of this pathway in COVID-19 requires further investigation.

Discordant neutralizing antibody and T cell responses in asymptomatic and mild SARS-CoV-2 infection.

Understanding the nature of immunity following mild/asymptomatic infection with SARS-CoV-2 is crucial to controlling the pandemic. We analyzed T cell and neutralizing antibody responses in 136 healthcare workers (HCW) 16-18 weeks after United Kingdom lockdown, 76 of whom had mild/asymptomatic SARS-CoV-2 infection captured by serial sampling. Neutralizing antibodies (nAb) were present in 89% of previously infected HCW. T cell responses tended to be lower following asymptomatic infection than in those reporting case-definition symptoms of COVID-19, while nAb titers were maintained irrespective of symptoms. T cell and antibody responses were sometimes discordant. Eleven percent lacked nAb and had undetectable T cell responses to spike protein but had T cells reactive with other SARS-CoV-2 antigens. Our findings suggest that the majority of individuals with mild or asymptomatic SARS-CoV-2 infection carry nAb complemented by multispecific T cell responses at 16-18 weeks after mild or asymptomatic SARS-CoV-2 infection.