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Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein ␣olf and adenylyl cyclase type-III to the Ca 2؉ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of G␣olf in odorant-specific signaling out of OR. The employment of the non-typical G protein ␣15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescenceimaging plate reader experiments, resulting in the deorphaning of two new OR for the odorant (؊)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (؊)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of G␣15 or G␣olf. We finally established an EC 50 -ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.

Anti-SARS-CoV-2 mRNA vaccine in patients with rheumatoid arthritisLong-term vaccine-induced immunity is crucial for controlling the COVID-19 pandemic. Vaccination against COVID-19 is recommended for patients with rheumatic diseases, but a paucity of data are available regarding COVID-19 vaccines in patients with rheumatoid arthritis. Because patients receiving immunosuppressive treatment were excluded from the phase 3 clinical trials, 1,2 it is not clear whether disease-modifying anti-rheumatic drug (DMARD) treatment should be continued before and after vaccination. In addition, some published reports are limited to follow-up after a single vaccine dose. [3][4][5] Here we report 53 consecutive patients with rheumatoid arthritis on DMARDs and 20 healthy controls (appendix p 1) who were eligible for vaccination according to the Swiss federal regulations and were enrolled in the RECOVER study, a non-randomised, prospective, observational trial. The RECOVER study was approved by the Ethical Committee of St Gallen, Switzerland, and written consent was obtained from all patients before inclusion. The vaccination itself was not part of the study. Nine patients received two doses of the mRNA-1273 vaccine (Moderna), all others received two doses of the BNT162b2 vaccine (Pfizer-BioNTech). Serum samples were collected at baseline, 3 weeks after the first vaccination, and 2 weeks after the second vaccination. Quantitative antibody testing was done using the Roche Elecsys Anti-SARS-CoV-2 spike subunit 1 (S1) assay that measures antibodies to SARS-CoV-2 spike protein 1 (range 0•4-2500 U/mL) and to SARS-CoV-2 nucleoprotein. This assay was used because it can distinguish between people who develop an anti-S1 response after vaccination or after natural infection, when typically antibodies to both S1 and nucleoprotein are generated. The threshold for this anti-SARS-CoV-2 S1 assay that might correspond to neutralisation of viral infectivity is still being discussed, but a cutoff level of 133 U/mL has been proposed. 6 A lower cutoff level of >15 U/mL has been suggested, 7 emphasising the need to establish formal cutoff levels of anti-SARS-CoV-2 antibody titres associated with protection against SARS-CoV-2 infection and severe disease.Intervals between the first and second vaccine dose and the intervals between vaccination and serum

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CD4 T cells in human infants and adults differ in the initiation and strength of their responses. The molecular basis for these differences is not yet understood. To address this the principle key molecular events of TCR- and CD28-induced signaling in naive CD4 T cells, such as Ca2+ influx, NFAT expression, phosphorylation and translocation into the nucleus, ERK activation and IL-2 response, were analyzed over at least the first 3 years of life. We report dramatically reduced IL-2 and TNFα responses in naive CD31+ T cells during infancy. Looking at the obligatory Ca2+ influx required to induce T cell activation and proliferation, we demonstrate characteristic patterns of impairment for each stage of infancy that are partly due to the differential usage of Ca2+ stores. Consistent with those findings, translocation of NFATc2 is limited, but still dependent on Ca2+ influx as demonstrated by sensitivity to cyclosporin A (CsA) treatment. Thus weak Ca2+ influx functions as a catalyst for the implementation of restricted IL-2 response in T cells during infancy. Our studies also define limited mobilization of Ca2+ ions as a characteristic property of T cells during infancy. This work adds to our understanding of infants’ poor T cell responsiveness against pathogens.

Background COVID‐19 and some anti‐SARS‐CoV‐2 vaccines trigger a humoral autoimmune response against a broad range of endogenous components, which may affect recipients’ prognosis in predisposed individuals. Autoantibodies directed against apolipoprotein A‐1 (AAA1 IgG) the major protein fraction of High Density Lipoprotein have been shown to be raised in COVID‐19 and in rheumatoid arthritis (RA) patients and other populations where they have been associated with poorer outcomes. We wanted to assess the impact of anti‐SARS‐CoV‐2 mRNA‐based vaccination on AAA1 autoimmune biomarkers in RA patients. Methods 20 healthy controls and 77 RA mRNA‐based vaccinated patients were collected at baseline, 3 weeks after the first vaccination, 2 and 8 weeks after the second vaccination. AAA1 and SARS‐CoV‐2 serologies were measured by immunoassays. Systemic and local symptoms occurring during the vaccination protocol were recorded. Results mRNA‐based vaccination induced a significant increase in median AAA1 IgG levels in both healthy controls and RA patients overtime. However, in both populations, these medians trend did not translate into significant increase in AAA1 IgG seropositivity rates despite evolving from 5 to 10% in healthy controls, and from 9 to 12.9% in RA patients. No associations were retrieved between AAA1 IgG and symptoms of any kind during the vaccination protocol. Conclusions mRNA‐based vaccination seems to induce a light AAA1 IgG response in immunocompetent individuals within 2 months after the last injection. Although we did not observe any warning signs, the formal demonstration of the harmlessness of such biological warrants further studies.

ObjectivesTo correlate immune responses following a two-dose regimen of mRNA anti-SARS-CoV-2 vaccines in patients with rheumatoid arthritis (RA) to the development of a potent neutralising antiviral activity.MethodsThe RECOVER study was a prospective, monocentric study including patients with RA and healthy controls (HCs). Assessments were performed before, and 3, 6, 12 and 24 weeks, after the first vaccine dose, respectively, and included IgG, IgA and IgM responses (against receptor binding domain, S1, S2, N), IFN-γ ELISpots as well as neutralisation assays.ResultsIn patients with RA, IgG responses developed slower with lower peak titres compared with HC. Potent neutralising activity assessed by a SARS-CoV-2 pseudovirus neutralisation assay after 12 weeks was observed in all 21 HCs, and in 60.3% of 73 patients with RA. A significant correlation between peak anti-S IgG levels 2 weeks after the second vaccine dose and potent neutralising activity against SARS-CoV-2 was observed at weeks 12 and 24. The analysis of IgG, IgA and IgM isotype responses to different viral proteins demonstrated a delay in IgG but not in IgA and IgM responses. T cell responses were comparable in HC and patients with RA but declined earlier in patients with RA.ConclusionIn patients with RA, vaccine-induced IgG antibody levels were diminished, while IgA and IgM responses persisted, indicating a delayed isotype switch. Anti-S IgG levels 2 weeks after the second vaccine dose correlate with the development of a potent neutralising activity after 12 and 24 weeks and may allow to identify patients who might benefit from additional vaccine doses or prophylactic regimen.

BackgroundAcetaminophen (APAP = paracetamol) may potentially impact vaccine-associated immune responses as the intake of APAP has been associated with a worse outcome in tumor patients receiving checkpoint inhibitors.[1]Different DMARD regimen have been shown to impair the humoral immune response to mRNA SARS-CoV-2 vaccines in patients with rheumatoid arthritis but the effect of paracetamol has not been explored thus far.ObjectivesTo analyse whether the intake of APAP may interfere with antiviral humoral immune responses following two doses of an anti-SARS-CoV-2 mRNA based vaccine in patients with rheumatoid arthritis (RA) on DMARD therapy.MethodsThe RECOVER trial (RheumatoidCovid-19VaccineImmuneResponse) was a non-randomised, prospective observational control group trial and enrolled 77 RA patients on DMARD therapy and 21 healthy controls (HC). We performed a posthoc analysis of blood samples taken before the first vaccine dose (T0), two (T1) and three (T2) weeks after the first and second vaccine dose, and at 12 (T3) weeks. APAP intake was measured using ELISA. The antibody response (anti-S) to the receptor binding domain (RBD) within the SARS-CoV-2 S1 protein was measured with the Elecsys Anti-SARS-CoV-2-S (Roche Diagnostics GmbH) test. The neutralizing activity NT50 at week 12 was assessed using an HIV-based pseudovirus neutralization assay against Wuhan-Hu-1.ResultsBaseline characteristics of participants are detailed in Table 1. The immunogenicity analyses were based on 73 RA patients after exclusion of 4 patients with previously unnoticed SARS-CoV-2 infection (positive for anti-nucleoprotein at baseline). APAP was detected in serum samples from 34/73 (25%) RA patients and in 7/21 (33%) HC (least at one timepoint T0, T1 and/or T2). APAP intake in HC did not affect levels of anti-S at any timepoint and all HC developed potent neutralizing activity (NT50 ≥ 250) at week 12. RA patients, who tested positive for APAP at T1, showed comparable anti-S levels at T1, T2 and T3 compared to RA patients not exposed to APAP. The detection of APAP at T2 corresponded to lower anti-S levels at T2 (Figure 1 A, B). The detection of APAP at T2 was associated with a significantly lower SARS-CoV-2 neutralizing activity at week 12 compared to patients without perivaccination APAP exposure (p =0.04) (Figure 1 C).ConclusionA decrease of antiviral humoral immune responses was observed in RA patients (but not in HC) who were exposed to APAP at the time of the second mRNA vaccine dose compared to patients in whom APAP was not detected. Our data suggest that the use of paracetamol within the time period around vaccination may impair vaccine-induced immune responses in patients with an already higher risk for blunted immune responses.Reference[1]Bessede A et al. Ann Oncol 2022; 33: 909-915Table 1.Baseline characteristics: RA patients and HC with/without APAP exposureRAAPAP –n = 37RAAPAP +n = 36p-valueHCAPAP –n = 8HCAPAP +n = 13p-valueAge (yrs), mean (± SD)62 (13)67 (10)0.07(NS)45 (12)44 (14)0.90(NS)Female sex, n (%)24 (65)19 (53)0.29(NS)2 (25)5 (38)0.53(NS)Vaccination type/schedulemRNA-1273, n (%)4 (11)8 (22.2)0.19(NS)0 (0)0 (0)BNT162b2, n (%)33 (89)28 (77.8)0.19(NS)8 (100)13 (100)RA disease characteristicsACPA ± RF, n (%)17/37 (46)19/36 (53)0.56(NS)NANANARA disease duration (yrs ± SD)9.2 (9.8)10.2 (8.1)0.67(NS)NANANADMARD therapycsDMARD-mono, n (%)13/37(35)9/36(25)0.35(NS)NANANAbDMARD-mono/combo, n (%)16/37(43)16/36(44)0.92(NS)NANANAtsDMARDs-mono/combo, n (%)8/37(22)11/36(31)0.38(NS)NANANAPrednisone, n (%)15/37 (41)12/36 (33.3)0.52(NS)NANANAMean daily dose prednisone (mg ± SD)4.6 ± 1.13.9 ± 2.30.39(NS)NANANA* APAP = acetaminophenFigure 1.Acknowledgements:NIL.Disclosure of InterestsNone Declared.