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BackgroundLimited data is available regarding long-term effectiveness of SARS-CoV-2 vaccines in patients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressive therapy. Whether the persistence of vaccine-induced humoral immunity against SARS-CoV-2 differs between this patient population and the general public is currently unknown.ObjectivesTo compare the persistence of anti-Spike antibodies following two SARS-CoV-2 vaccine doses between IMID patients using immunosuppressive medication and healthy controls and identify predictors of antibody decline.MethodsWe included patients with inflammatory joint- and bowel diseases on immunosuppressive medication and healthy controls enrolled in the prospective observational Nor-vaC study. Serum samples were collected at two time points following two dose SARS-CoV-2 vaccination (first assessment within 6–48 days and second within 49–123 days). Sera were analysed for antibodies binding the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. Anti-RBD ResultsA total of 1097 patients (400 rheumatoid arthritis, 189 psoriatic arthritis, 189 spondyloarthritis, 129 ulcerative colitis, 190 Crohn´s disease) (median age 54 years [IQR 43–64]; 56% women) and 133 controls (median age 45 years [IQR 35–56]; 83% women) provided blood samples within the defined intervals (median 19 days [IQR 15–24] and 97 days [86–105] after second vaccine dose). Antibody levels were significantly lower in patients compared to controls at both assessments, with median anti-RBD 1468 BAU/ml [IQR 500–5062] in patients and 5514 BAU/ml [2528–9580] in controls (pTable 1.Serological response in patients and controlsControls (n=133)Patients (n=1097)Anti-RBD antibodies (BAU/ml)1stassessment2ndassessment1stassessment2ndassessment0018 (1.6)54 (5)5-19, n (%)004 (0.4)60 (5)20-199, n (%)01 (1)40 (4)338 (31)200-1999, n (%)25 (19)89 (67)548 (50)558 (51)2000-8999, n (%)71 (53)40 (30)398 (36)82 (7.5)≥ 9000, n (%)37 (28)3 (2)89 (8)5 (0.5)1st assessment 6 - 48 days and 2nd assessment 49 -123 days after second vaccine dose. BAU= Binding antibody UnitsConclusionWithin four months after the second vaccine dose, anti-Spike antibody levels declined considerably in both IMID patients and controls. Patients had lower antibody levels at the first assessment and a more pronounced decline compared to controls, and were consequently more likely to have low antibody levels four months after the second vaccine dose. Our results support that IMID patients lose humoral protection and need additional vaccine doses sooner than healthy individuals.Disclosure of InterestsIngrid Egeland Christensen: None declared, Ingrid Jyssum: None declared, Anne Therese Tveter: None declared, Joe Sexton: None declared, Trung T. Tran: None declared, Siri Mjaaland: None declared, Grete B. Kro: None declared, Tore K. Kvien Speakers bureau: Amgen, Celltrion, Egis, Evapharma, Ewopharma, Hikma, Oktal, Sandoz, Sanofi, Consultant of: Abbvie, Amgen, Biogen, Celltrion, Eli Lilly, Gilead, Mylan, Novartis, Pfizer, Sandoz, Sanofi, Grant/research support from: Grants to institution (Diakonhjemmet Hospital): Abbvie, Amgen, BMS, MSD, Novartis, Pfizer, UCB, David Worren: None declared, Jørgen Jahnsen Speakers bureau: AbbVie, Astro Pharma, Boerhinger Ingelheim, BMS, Celltrion, Ferring, Gilead, Hikma, Janssen Cilag, Meda, MSD, Napp Pharma, Novartis, Orion Pharma Pfizer, Pharmacosmos, Roche, Takeda, Sandoz, Consultant of: AbbVie, Boerhinger Ingelheim, BMS, Celltrion, Ferring, Gilead, Janssen Cilag MSD, Napp Pharma, Novartis, Orion Pharma, Pfizer, Pharmacosmos, Takeda, Sandoz, Unimedic Pharma, Grant/research support from: Abbvie, Pharmacosmos, Ferring, Ludvig A. Munthe Speakers bureau: Novartis, Cellgene, Espen Haavardsholm: None declared, John Torgils Vaage: None declared, Gunnveig Grodeland Speakers bureau: Bayer, Sanofi Pasteur, Thermo Fisher, Consultant of: Consulting fees from the Norwegian System of Compensation to Patients and AstraZeneca, Fridtjof Lund-Johansen: None declared, Kristin Kaasen Jørgensen Speakers bureau: Roche, BMS, Consultant of: Celltrion, Norgine, Silje Watterdal Syversen: None declared, Guro Løvik Goll Speakers bureau: AbbVie, Pfizer, UCB, Sandoz, Orion Pharma, Novartis, Consultant of: Pfizer, AbbVie, Sella Aarrestad Provan: None declared

BackgroundPatients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressive therapy have an inadequate serologic response following two-dose SARS-CoV-2 vaccination, and a standard vaccination strategy of three doses for this patient group is currently under implementation in several countries. However, the serological response and safety of this strategy has not been evaluated.ObjectivesTo assess serological response and safety of a three-dose vaccination strategy in IMID patients on immunosuppressive therapy as compared to standard two-dose vaccination of healthy controls.MethodsThe prospective observational Nor-vaC study (NCT04798625) enrolled adult patients on immunosuppressive therapy for inflammatory joint- and bowel diseases. Healthy controls were health care workers from participating hospitals. All participants received standard vaccines according to the national vaccination program with three doses in patients and two doses in controls. The third dose was offered to IMID patients >4 weeks after the second dose. Analyses of antibodies binding the receptor-binding domain of the SARS-CoV-2 Spike protein were performed prior to, and 2-4 weeks after the second and third vaccine doses. Levels were compared across groups by Mann-Whitney U tests and multivariate linear regression was used to identify predictors of response.ResultsOverall, 961 patients (315 rheumatoid arthritis, 156 spondyloarthritis, 171 psoriatic arthritis, 132 ulcerative colitis and182 Crohn’s disease) (median age 54 years [IQR 43-64]; 56 % women) and 227 controls (median age 44 years [IQR 32-55]; 83 % women) were included in the present analyses. TNFi monotherapy was used by 399 patients, 229 used TNFi in combination with other immunomodulators, 189 methotrexate monotherapy, 39 vedolizumab, 32 JAKi and 73 patients used other drugs. Patients on rituximab were not included. Patients were vaccinated with Pfizer BNT162b2 (54% patients, 14% controls), Moderna mRNA-1273 (16% patients, 40% controls) or a combination of vaccines (30% patients, 46% controls). Patients received the third vaccine dose a median of 120 (IQR 102-143) days after the second dose. After two doses, median anti-Spike antibody levels were significantly lower in patients (861 BAU/ml (IQR 418-4275) than controls (6318 BAU/ml (IQR 2468-9857)), pAdverse events were reported by 438 (48%) of patients after the third dose as compared to 471 (54%) after the second dose and 193 (78 %) of controls. Disease flares were reported by 42 (5%) and 69 (8%) patients after the second and third dose, respectively.ConclusionThis study suggests that a third vaccine dose for immunosuppressed patients closes the gap in serological response between patients and the healthy population. Antibody levels following the three-dose regimen in IMID patients were comparable to healthy controls vaccinated twice, and no new safety issues emerged. This finding was consistent across all diagnoses and treatment groups, supporting the implementation of a three-dose vaccine regimen as standard in the IMID population.Disclosure of InterestsIngrid Jyssum: None declared, Anne Therese Tveter: None declared, Joe Sexton: None declared, Ingrid E. Christensen: None declared, Trung T. Tran: None declared, Siri Mjaaland: None declared, David J Warren: None declared, Tore K. Kvien Speakers bureau: Amgen,Celltrion, Egis, Evapharma, Ewopharma, Hikma, Oktal, Sandoz, Sanofi, Consultant of: Abbvie, Amgen, Biogen, Celltrion, Eli Lilly, Gilead, Mylan, Novartis, Pfizer, Sandoz, Sanofi, Grant/research support from: Grants to institution (Diakonhjemmet Hospital): Abbvie, Amgen, BMS, MSD, Novartis, Pfizer, UCB, Kristin Hammersbøen Bjørlykke: None declared, Grete B. Kro: None declared, Jørgen Jahnsen Speakers bureau: AbbVie, Astro Pharma, Boerhinger Ingelheim, BMS, Celltrion, Ferring, Gilead, Hikma, Janssen Cilag, Meda, MSD, NappPharma, Novartis, Orion Pharma Pfizer, Pharmacosmos, Roche, Takeda, Sandoz, Consultant of: AbbVie, Boerhinger Ingelheim, BMS, Celltrion, Ferring, Gilead, Janssen Cilag MSD, Napp Pharma, Novartis, Orion Pharma, Pfizer, Pharmacosmos, Takeda, Sandoz, Unimedic Pharma, Grant/research support from: Abbvie, Pharmacosmos, Ferring, Ludvig A. Munthe Speakers bureau: Novartis, Cellgene, Espen A Haavardsholm: None declared, John Torgils Vaage: None declared, Gunnveig Grodeland Speakers bureau: Bayer, Sanofi Pasteur, Thermo Fisher, Consultant of: Consulting fees from the Norwegian System of Compensation to Patients and AstraZeneca, Fridtjof Lund-Johansen: None declared, Sella Aarrestad Provan: None declared, Kristin Kaasen Jørgensen Speakers bureau: Roche, BMS, Consultant of: Celltrion, Norgine, Guro Løvik Goll Speakers bureau: AbbVie, Pfizer, UCB, Sandoz, Orion Pharma, Novartis, Consultant of: Pfizer, AbbVie, Silje Watterdal Syversen: None declared

The leukocyte carbohydrate (CHO) Ag CD15, sialyl-CD15, and CDw65 have recently been found to function as ligands for CD62 and ELAM-1 cell adhesion molecules on platelets and endothelium, respectively. Cell adhesion ligands also may act as receptors capable of signal transduction. We therefore investigated the possibility that these CHO Ag and CDw17, a glycolipid Ag whose expression is regulated by leukocyte activation, may have receptor-like characteristics. The effects of antibody cross-linking of CHO Ag on phagocyte activation were measured by using flow cytometry and fluorescent indicators for cytoplasmic calcium ions, oxidative burst, and the granule-associated proteins CD11b and CD67. Cross-linking of CD15, sialyl-CD15, CDw65, or CDw17 induced a moderate release of calcium ions into the cytoplasm of granulocytes, a strong activation of oxidative burst, and a low up-regulation of CD11b and CD67 compared to the effects of treatment with 4 microM FMLP. The results suggest a role for CHO Ag in leukocyte signal transduction and support the view that these molecules are involved in phagocyte activation.

Activation-induced protein phosphorylation can be studied by Western blotting, but this method is time consuming and depends on the use of radioactive probes for quantitation. We present a novel assay for the assessment of protein phosphorylation based on latex particles and flow cytometry.This method employs monoclonal antibodies coupled to latex particles to immobilize protein kinase substrates. Their phosphorylation status is assessed by reactivity with phosphoepitope-specific antibodies. The amount of immobilized protein on the particles was analyzed by direct or indirect immunofluorescence with antibodies to nonphosphorylated epitopes.The assay allowed measurement of phosphorylation of multiple protein kinase substrates in stimulated T cells, including the zeta chain of the T-cell receptor, ZAP-70, CD3, CD5, SHP-1, and ERK-2, using 1-3 microg of total cell protein per sample. The assay provided high resolution of kinetics of phosphorylation and dephosphorylation. Interactions of protein kinase substrates with associated signaling molecules were demonstrated.The novel assay allows high-throughput quantitative measurement of protein modifications during signal transduction.

In the present study, we investigated whether apoptosis in hematopoietic cells is associated with changes in cellular phosphotyrosine content. Murine thymocytes and B cells, human leukemia cells, and normal peripheral blood leukocytes were induced to undergo apoptosis by treatment with specific stimuli or by incubation in growth factor-deprived medium. Multiparameter flow cytometry was used to measure changes in phosphotyrosine content that correlated with the appearance of features of programmed cell death, such as cell shrinkage, DNA fragmentation, and loss of membrane integrity. The results show that conditions that induced apoptosis also induced a dramatic decrease in cellular phosphotyrosine levels. Tyrosine dephosphorylation preceded the loss of plasma membrane integrity and, in most cases, was temporally correlated with the onset of DNA fragmentation. The protein tyrosine phosphatase antagonist pervanadate had a dose-dependent inhibitory effect on both dephosphorylation and apoptosis in murine thymocytes, which were treated with dexamethasone or with the topoisomerase II inhibitor etoposide. The results suggest that extensive tyrosine dephosphorylation is an intrinsic part of the apoptotic process of hematopoietic cells and may be involved mechanistically in the apoptosis induced by certain stimuli.