Helicobacter pylori upregulates PAD4 expression via stabilising HIF-1α to exacerbate rheumatoid arthritis.

OBJECTIVE: Helicobacter pylori infection has been reported to aggravate rheumatoid arthritis (RA), but the relevant mechanism remains unclear. This study aimed to investigate the underlying pathogenic mechanism of H. pylori infection in the progression of RA. METHODS: The Disease Activity Score (DAS-28) and serum anticitrullinated protein antibody (ACPA) levels were compared between H. pylori-negative and H. pylori-positive patients with RA. MH7A cells were stimulated with polyclonal ACPA purified from the peripheral blood of patients with RA. The citrullination levels were assessed by western blot in GES-1 cells and sera. ChIP, luciferase reporter assays, mass spectrometry and ELISA were applied to explore the molecular mechanism of H. pylori infection in RA progression. RESULTS: The DAS-28 and ACPA levels of patients with RA in the H. pylori-positive group were significantly higher than those in the H. pylori-negative group. Polyclonal ACPA derived from H. pylori-positive patients promoted cell proliferation and induced secretion of IL-6 and IL-8. For the first time, we found that H. pylori infection induces cellular protein citrullination by upregulating protein arginine deiminase type 4 (PAD4). Furthermore, we confirmed a direct functional binding of hypoxia-inducible factor 1α on the PADI4 gene promoter. We demonstrated that PAD4 interacts with and citrullinates keratin 1 (K1), and serum and synovial fluid levels of anti-Cit-K1 antibody were markedly increased in H. pylori-infected patients with RA. CONCLUSION: Our findings reveal a novel mechanism by which H. pylori infection contributes to RA progression. Therapeutic interventions targeting H. pylori may be a viable strategy for the management of RA.

Integrating pretest probability for rheumatoid arthritis with likelihood ratios of RF and ACPA to improve clinical utility of rheumatoid arthritis autoantibody testing.

BACKGROUND AND AIMS: Rheumatoid arthritis (RA) manifests through various symptoms and systemic manifestations. Diagnosis involves serological markers like rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA). Past studies have shown the added value of likelihood ratios (LRs) in result interpretation. LRs can be combined with pretest probability to estimate posttest probability for RA. There is a lack of information on pretest probability. This study aimed to estimate pretest probabilities for RA. MATERIALS AND METHODS: This retrospective study included 133 consecutive RA patients and 651 consecutive disease controls presenting at a rheumatology outpatient clinic. Disease characteristics, risk factors associated with RA and laboratory parameters were documented for calculating pretest probabilities and LRs. RESULTS: Joint involvement, erosions, morning stiffness, and positive CRP, ESR tests significantly correlated with RA. Based on these factors, probabilities for RA were estimated. Besides, LRs for RA were established for RF and ACPA and combinations thereof. LRs increased with antibody levels and were highest for double high positivity. Posttest probabilities were estimated based on pretest probability and LR. CONCLUSION: By utilizing pretest probabilities for RA and LRs for RF and ACPA, posttest probabilities were estimated. Such approach enhances diagnostic accuracy, offering laboratory professionals and clinicians insights in the value of serological testing during the diagnostic process.

Immunophenotypic Landscape of synovial tissue in rheumatoid arthritis: Insights from ACPA status.

Objective: To examine the clinical features and synovial pathologies in rheumatoid arthritis (RA) patients across varying titers of circulating anti-citrullinated protein antibodies (ACPA). Methodology: We devised a negative pressure suction and rebound synovial biopsy tool to enhance the yield of synovial biopsies, noted for its ease and safety of use. This research involved a retrospective examination of 60 active RA patients who underwent synovial biopsies with this tool from June to November 2013 at our institution. A range of disease activity markers were collected, including DAS28-CRP, ESR, CRP, count of swollen and tender joints, VAS pain scale, and so forth. Synovial tissue underwent HE staining and immunohistochemistry, including synovitis grading (GSS) and counting of B cells (CD20), T cells (CD3), macrophages (CD68), and plasma cells (CD138). Participants: were categorized into three groups as per ACPA titers: ACPA-negative (0-5U/mL), low-titer (5-20U/mL), and high-titer (above 20U/mL). The study compared the clinical features and synovial pathologies across these groups. Results: Of the 60 RA patients, they were segregated into three groups based on ACPA titers: 20 in ACPA-negative, 9 in the low-titer group, and 31 in the high-titer group. No significant differences were observed in GSS scores, synovial cell proliferation and loss, matrix activation, inflammatory infiltration, and neovascularization among these groups (P > 0.05). The high-titer ACPA group demonstrated significantly increased counts of CD3+ T cells, CD20+ B cells, and CD68+ macrophages in synovial tissues compared to the ACPA-negative and low-titer groups (p < 0.05), along with a higher incidence of ectopic lymphoid neogenesis (p < 0.05). Ordinal logistic regression revealed that rheumatoid factor (RF), and counts of synovial T cells, B cells, macrophages, and ectopic lymphoid neogenesis correlated with ACPA titers (P < 0.05), particularly lymphoid neogenesis (OR = 3.63, P = 0.023). Conclusion: RA patients with high-titer ACPA demonstrate elevated levels of inflammatory cell infiltration in synovial tissues, with ectopic lymphoid neogenesis showing a strong correlation with high ACPA positivity.

Acetylated bacterial proteins as potent antigens inducing an anti-modified protein antibody response.

OBJECTIVE: Gut-residing bacteria, such as Escherichia coli, can acetylate their proteome under conditions of amine starvation. It is postulated that the (gut) microbiome is involved in the breach of immune tolerance to modified self-proteins leading to the anti-modified protein antibodies (AMPAs), hallmarking seropositive rheumatoid arthritis (RA). Our aim was to determine whether acetylated bacterial proteins can induce AMPA responses cross-reactive to modified self-proteins and be recognised by human AMPA (hAMPA). METHODS: E. coli bacteria were grown under amine starvation to generate endogenously acetylated bacterial proteins. Furthermore, E. coli proteins were acetylated chemically. Recognition of these proteins by hAMPA was analysed by western blotting and ELISA; recognition by B cells carrying a modified protein-reactive B cell receptor (BCR) was analysed by pSyk (Syk phosphorylation) activation assay. C57BL/6 mice were immunised with (modified) bacterial protein fractions, and sera were analysed by ELISA. RESULTS: Chemically modified bacterial protein fractions contained high levels of acetylated proteins and were readily recognised by hAMPA and able to activate B cells carrying modified protein-reactive BCRs. Likely due to substantially lower levels of acetylation, endogenously acetylated protein fractions were not recognised by hAMPA or hAMPA-expressing B cells. Immunising mice with chemically modified protein fractions induced a strong cross-reactive AMPA response, targeting various modified antigens including citrullinated proteins. CONCLUSIONS: Acetylated bacterial proteins are recognisable by hAMPA and are capable of inducing cross-reactive AMPA in mice. These observations provide the first conceptual evidence for a novel mechanism involving the (endogenous) acetylation of the bacterial proteome, allowing a breach of tolerance to modified proteins and the formation of cross-reactive AMPA.

Association of rheumatoid factor, anti-citrullinated protein antibodies and shared epitope with clinical response to initial treatment in patients with early rheumatoid arthritis: data from a randomised controlled trial.

OBJECTIVES: To investigate whether rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs) and shared epitope (SE) allele-related genetic markers associate with treatment response to abatacept, certolizumab pegol or tocilizumab versus active conventional treatment (ACT). METHODS: Patients with treatment-naïve early rheumatoid arthritis were randomised in the NORD-STAR trial to ACT, certolizumab pegol, abatacept or tocilizumab, all with methotrexate. Centralised laboratory analyses for ACPA, RF and SE were performed. Clinical Disease Activity Index remission was analysed longitudinally with logistic generalised estimating equations. Differences in treatment effect across RF, ACPA and SE subgroups were assessed with interaction terms at 24 and 48 weeks, adjusted for sex, country, age, body mass index, Disease Activity Score of 28 joints based on C-reactive protein and smoking. RESULTS: In total, 778 patients were included. At 24 weeks, abatacept treatment showed a better response than ACT in the RF and/or ACPA-positive subgroups, but this effect was not significantly different from the negative subgroups. By 48 weeks, abatacept treatment showed better response regardless of RF/ACPA status. No differences were found across RF, ACPA, SE allele, valine at amino acid position 11 or valine-arginine-alanine haplotype subgroups for any biological treatment at 48 weeks. CONCLUSIONS: Based on this randomised controlled trial, abatacept treatment was associated with a better response than ACT in the RF and/or ACPA-positive subgroup at 24 weeks, but this was no longer seen at 48 weeks; adding SE allele-related genetic markers did not strengthen the association. Moreover, ACPA, RF and SE allele-related genotypes were not, alone or in combination, associated with clinical responses of importance sufficiently strongly to warrant implementation in clinical practice. TRIAL REGISTRATION NUMBER: EudraCT 2011-004720-35; ClinicalTrials.gov NCT01491815.

Higher levels of markers for early atherosclerosis in anti-citrullinated protein antibodies positive individuals at risk for RA, a cross sectional study.

OBJECTIVE: To identify differences in levels of serum biomarkers associated with atherosclerosis between anti-citrullinated protein antibodies (ACPA) positive groups. METHODS: Cross-sectional data were used from the Dutch Lifelines Cohort Study combined with data derived from RA risk and early RA studies conducted at the University Medical Center Groningen (UMCG). Serum biomarkers of inflammation, endothelial cell activation, tissue remodeling and adipokine, which were previously associated with atherosclerosis, were measured with Luminex in four ACPA positive groups with different characteristics: without joint complaints, with joint complaints, RA risk and early RA groups. RESULTS: Levels of C-reactive protein (CRP), Interleukin-6 (IL-6), Tumor Necrosis Factor Receptor 1 (TNFR1) and vascular endothelial growth factor (VEGF) were significantly higher in the RA risk and early RA groups compared to the joint complaints and the no joint complaints groups. The difference remained statistically significant after correcting for renal function, smoking and hypertension in multivariate logistic regression analysis, with focus on ACPA positive with joint complaints group versus RA risk group: CRP OR = 2.67, p = 0.033; IL-6 OR = 3.73, p = 0.019; TNFR1 OR = 1.003, p < 0.001; VGEF OR = 8.59, p = 0.019. CONCLUSION: Individuals at risk for RA have higher levels of inflammatory markers and VEGF, which suggests that they might also have a risk of higher cardiovascular disease (CVD); however, this does not apply to individuals with ACPA positivity with self-reported joint complaints or without joint complaints only. Therefore, it is important that individuals with RA risk are referred to a rheumatologist to rule in or out arthritis/development of RA and discuss CVD risk.

Early identification of rheumatoid arthritis: does it induce treatment-related cost savings?

OBJECTIVE: Early diagnosis and treatment-start is key for rheumatoid arthritis (RA), but the economic effect of an early versus a later diagnosis has never been investigated. We aimed to investigate whether early diagnosis of RA is associated with lower treatment-related costs compared with later diagnosis. METHODS: Patients with RA consecutively included in the Leiden Early Arthritis Clinic between 2011 and 2017 were studied (n=431). Symptom duration was defined as the time between symptom onset and first presentation at the outpatient clinic; early treatment start was defined as symptom duration <12 weeks. Information on disease-modifying anti-rheumatic drug use per patient over 5 years was obtained from prescription data from patient records. Prices were used from 2022 and 2012 (proxy of time of prescription) to study the impact of changes in drug costs. Autoantibody-positive and autoantibody-negative RA were studied separately because differences in disease severity may influence costs. RESULTS: Within autoantibody-negative RA, costs were 316% higher in the late compared with the early group (ß=4.16 (95% CI 1.57 to 11.1); €4856 vs €1159). When using 2012 prices, results were similar. For autoantibody-positive RA, costs were 19% higher in the late group (€9418 vs €7934, ß=1.19, 0.57 to 2.47). This effect was present but smaller when using 2012 prices. Within patients with autoantibody-positive RA using biologicals, late treatment start was associated with 46% higher costs (ß=1.46 (0.91 to 2.33)); higher costs were also seen when using 2012 prices. CONCLUSION: When RA is detected within 12 weeks after symptom onset, treatment-related costs were lower in both autoantibody-negative and autoantibody-positive RA. This study is the first to report how early diagnosis and treatment start impact treatment-related costs.

Clinical Features of ACPA-Negative and ACPA-Positive Variants of Rheumatoid Arthritis.

The aim of the study was to investigate the features of the clinical picture of the disease in patients with ACPA-negative and ACPA-positive variants of rheumatoid arthritis. MATERIALS AND METHODS: : The study included patients with a reliable diagnosis of rheumatoid arthritis (RA) according to the criteria of ACR/EULAR 2010. Depending on the ACPA values, two groups of patients were recruited: ACPA-positive and ACPA-negative, comparable in gender, age, duration of the disease, and therapy. The nature of the onset and course of the disease and the activity of RA were evaluated (according to the DAS28, SDAI, CDAI indices). RESULTS AND DISCUSSION: : The study involved 79 patients with ACPA-negative variant of RA and 79 ACPA-positive patients. The age of patients (Me [IR] (in years)) with the ACPA(-) variant was 52 [39; 62]; with the ACPA(+) variant, 54 [42; 62]; the duration of the disease (in months) was 59 [23; 122] and 48 [17; 84], respectively. In ACPA(+) patients, a higher disease activity was determined (by the indices DAS 28crp, DAS28esr, SDAI, CDAI), higher values of C-reactive protein and erythrocyte sedimentation rate, and a greater number of painful and swollen joints (p < 0.05). According to the localization of the involved joints, arthritis of the proximal interphalangeal, metacarpal, wrist and shoulder joints was more often determined in ACPA(+) patients. Systemic manifestations of RA at the time of examination and in the anamnesis were statistically significantly more common in ACPA(+) (32.9%) than in ACPA(-) (17.7%) patients. Of the systemic manifestations, rheumatoid nodules were more common in ACPA(+) patients, whereas a tendency to a higher frequency of neuropathy, sclerites, and episcleritis was revealed in ACPA(-) patients. CONCLUSIONS: . In patients with ACPA(-) subtype, clinical signs of joint damage and the inflammatory component are less pronounced compared to ACPA(+). However, the mixed picture of manifestation, the less "bright" course of the disease, the absence of characteristic immunological biomarkers necessitate long-term and careful monitoring of this group of patients. At the same time, the subjective severity of the disease and dysfunction due to ankylosing joints do not differ from the ACPA(+) variant of RA.

Nipocalimab, an anti-FcRn monoclonal antibody, in participants with moderate to severe active rheumatoid arthritis and inadequate response or intolerance to anti-TNF therapy: results from the phase 2a IRIS-RA study.

OBJECTIVES: To investigate the efficacy, safety, pharmacokinetics and pharmacodynamics of nipocalimab in participants with moderate to severe active rheumatoid arthritis (RA) and inadequate response or intolerance to ≥1 antitumour necrosis factor agent. METHODS: In this phase 2a study, participants with RA seropositive for anticitrullinated protein antibodies (ACPA) or rheumatoid factors were randomised 3:2 to nipocalimab (15 mg/kg intravenously every 2 weeks) or placebo from Weeks 0 to 10. Efficacy endpoints (primary endpoint: change from baseline in Disease Activity Score 28 using C reactive protein (DAS28-CRP) at Week 12) and patient-reported outcomes (PROs) were assessed through Week 12. Safety, pharmacokinetics and pharmacodynamics were assessed through Week 18. RESULTS: 53 participants were enrolled (nipocalimab/placebo, n=33/20). Although the primary endpoint did not reach statistical significance for nipocalimab versus placebo, a numerically higher change from baseline in DAS28-CRP at Week 12 was observed (least squares mean (95% CI): -1.03 (-1.66 to -0.40) vs -0.58 (-1.24 to 0.07)), with numerically higher improvements in all secondary efficacy outcomes and PROs. Serious adverse events were reported in three participants (burn infection, infusion-related reaction and deep vein thrombosis). Nipocalimab significantly and reversibly reduced serum immunoglobulin G, ACPA and circulating immune complex levels but not serum inflammatory markers, including CRP. ACPA reduction was associated with DAS28-CRP remission and 50% response rate in American College of Rheumatology (ACR) criteria; participants with a higher baseline ACPA had greater clinical improvement. CONCLUSIONS: Despite not achieving statistical significance in the primary endpoint, nipocalimab showed consistent, numerical efficacy benefits in participants with moderate to severe active RA, with greater benefit observed for participants with a higher baseline ACPA. TRIAL REGISTRATION NUMBER: NCT04991753.

Classification of rheumatoid arthritis: is it time to revise the criteria?

Classification criteria have been developed for rheumatoid arthritis (RA) and other rheumatic diseases in order to gather a homogeneous patient population for clinical studies and facilitate the timely implementation of therapeutic measures. Although classification criteria are not intended to be used for diagnosis, they are frequently used to support the diagnostic process in clinical practice, including clinical decision-making. The 2010 American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) classification criteria for RA are capable of identifying the majority of symptomatic patients with RA already in the earliest stages of the disease who are not yet showing radiographic changes. These patients will also profit from the early implementation of therapy with disease-modifying antirheumatic drugs (DMARDs). However, the risk of misclassification is higher as compared with the former 1987 ACR criteria, which were considerably less sensitive to the recognition of patients with early RA. Of note, the presence of rheumatoid factors (RFs) and anticitrullinated protein antibodies (ACPAs) has been attributed equal weight in the 2010 ACR/EULAR criteria and may contribute up to 50% of the score needed for being classified as RA. However, while ACPAs have been proven to be the most specific serological markers of RA, the specificity of RF is moderate, especially at lower titres. This may lead to the misclassification of RF-positive patients and, consequently, the unjustified implementation of DMARD therapy. Therefore, issues arise on how comprehensive the criteria should be and whether they should be updated and adapted to findings from the past two decades that might increase both their specificity and sensitivity.

Utility of testing for third-generation anticyclic citrullinated peptide (anti-CCP3) antibodies in individuals who present with new musculoskeletal symptoms but have a negative second-generation anticyclic citrullinated peptide (anti-CCP2) antibody test.

OBJECTIVES: To investigate the role of third-generation anticyclic citrullinated peptide (anti-CCP3) antibodies in predicting progression to inflammatory arthritis (IA) in individuals with new musculoskeletal (MSK) symptoms and a negative second-generation anti-CCP antibody test (anti-CCP2-). METHODS: 469 anti-CCP2- individuals underwent baseline anti-CCP3 testing (QUANTA Lite CCP3; Inova Diagnostics) and received a post enrolment 12-month questionnaire. A rheumatologist confirmed or excluded diagnosis of IA. Univariable/multivariable analyses were performed to assess the value of anti-CCP3 in predicting IA development in these anti-CCP2- individuals. RESULTS: Only 16/469 (3.4%) anti-CCP2- individuals had a positive anti-CCP3 test. Of these 16 individuals, 4 developed IA. In addition, 61/469 (13.0%) anti-CCP2- individuals self-reported, to have developed, IA. Progression was confirmed in 43/61 of them (70.5%); of whom 30/43 (69.8%) and 13/43 (30.2%) were given a diagnosis of IA and rheumatoid arthritis (RA), respectively. In qualitative univariable analysis, anti-CCP3 positivity was associated with self-reported progression (p<0.01) and IA (p=0.03), but not with RA. Anti-CCP3 levels differed significantly between progressors and non-progressors (p<0.01) for all three categories. At the manufacturer's cut-off, OR for progression ranged from 2.4 (95% CI 0.5 to 18.6; RA) to 7.5 (95% CI 2.3 to 24.0; self-reported progression). Interestingly, when cut-offs for anti-CCP3 were optimised, lower values (≥5 units) significantly increased the OR for progression in all three categories. In multivariable analysis, anti-CCP3 positivity at the manufacturer's cut-off did not remain associated with IA progression, while this lower cut-off value (≥5 units) was associated with diagnosis of RA (p=0.02). CONCLUSIONS: Anti-CCP3 testing could improve the prediction of IA development in anti-CCP2- individuals with new MSK symptoms.

Clinical significance of anti-mutated citrullinated vimentin antibodies in rheumatoid arthritis patients.

BACKGROUND: Anti-mutated citrullinated vimentin (MCV) antibodies have recently been recommended as a better arthritis diagnostic marker. OBJECTIVES: To investigate the association between anti-MCV antibodies and the clinical, functional, and radiographic characteristics of rheumatoid arthritis (RA) patients. METHODS: This case-control study was conducted on 40 RA patients and 40 healthy subjects. All patients were subjected to an assessment of disease using the 28-joint DAS (DAS28) and Clinical Disease Activity Index (CDAI), function by HAQ-DI, physical activity by International Physical Activity Questionnaire (IPAQ), fatigue by Functional Assessment of Chronic Illness Therapy (FACIT), serological tests as well as anti-MCV Abs measurement. A plain X-ray of both hands and wrists was done. RESULTS: The anti-MCV Abs level was significantly higher in RA patients than in healthy controls (P< 0.001). The anti-MCV Abs had a significant positive correlation with DAS, CDAI, HAQ, RF, Anti-CCP, and CRP (P= 0.006, 0.013, 0.005, < 0.001, < 0.001and 0.041 respectively) and a significant negative correlation with FACIT (p= 0.007). Positive anti-MCV RA patients had significantly higher erosions, JSN, and a total sharp score. CONCLUSIONS: Anti-MCV Abs may contribute to poor physical activity and more fatigue in RA patients beyond their established role in disease activity and erosion.

Macrophage extracellular traps require peptidylarginine deiminase 2 and 4 and are a source of citrullinated antigens bound by rheumatoid arthritis autoantibodies.

Introduction: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis, but the sources of citrullinated antigens as well as which peptidylarginine deiminases (PADs) are required for their production remain incompletely defined. Here, we investigated if macrophage extracellular traps (METs) could be a source of citrullinated proteins bound by APCAs, and if their formation requires PAD2 or PAD4. Methods: Thioglycolate-induced peritoneal macrophages from wild-type, PAD2-/-, and PAD4-/- mice or human peripheral blood-derived M1 macrophages were activated with a variety of stimulants, then fixed and stained with DAPI and either anti-citrullinated histone H4 (citH4) antibody or sera from ACPA+ or ACPA- rheumatoid arthritis subjects. METs were visualized by immunofluorescence, confirmed to be extracellular using DNase, and quantified. Results: We found that ionomycin and monosodium urate crystals reliably induced murine citH4+ METs, which were reduced in the absence of PAD2 and lost in the absence of PAD4. Also, IgG from ACPA+, but not ACPA-, rheumatoid arthritis sera bound to murine METs, and in the absence of PAD2 or PAD4, ACPA-bound METs were lost. Finally, ionomycin induced human METs that are citH4+ and ACPA-bound. Discussion: Thus, METs may contribute to the pool of citrullinated antigens bound by ACPAs in a PAD2- and PAD4-dependent manner, providing new insights into the targets of immune tolerance loss in rheumatoid arthritis.

Immune response to post-translationally modified proteins in rheumatoid arthritis: what makes it special?

Rheumatoid arthritis (RA) exhibits common characteristics with numerous other autoimmune diseases, including the presence of susceptibility genes and the presence of disease-specific autoantibodies. Anti-citrullinated protein antibodies (ACPA) are the hallmarking autoantibodies in RA and the anti-citrullinated protein immune response has been implicated in disease pathogenesis. Insight into the immunological pathways leading to anti-citrullinated protein immunity will not only aid understanding of RA pathogenesis, but may also contribute to elucidation of similar mechanisms in other autoantibody-positive autoimmune diseases. Similarly, lessons learnt in other human autoimmune diseases might be relevant to understand potential drivers of RA. In this review, we will summarise several novel insights into the biology of the anti-citrullinated protein response and their clinical associations that have been obtained in recent years. These insights include the identification of glycans in the variable domain of ACPA, the realisation that ACPA are polyreactive towards other post-translational modifications on proteins, as well as new awareness of the contributing role of mucosal sites to the development of the ACPA response. These findings will be mirrored to emerging concepts obtained in other human (autoimmune) disease characterised by disease-specific autoantibodies. Together with an updated understanding of genetic and environmental risk factors and fresh perspectives on how the microbiome could contribute to antibody formation, these advancements coalesce to a progressively clearer picture of the B cell reaction to modified antigens in the progression of RA.

New-onset Seropositive Rheumatoid Arthritis Post-mRNA COVID-19 Vaccine: A Case Report.

Growing evidence in the medical literature has linked the COVID-19 vaccine as a potential trigger for the development or exacerbation of various autoimmune rheumatic diseases. To the best of our knowledge, we report one of the first cases of seropositive rheumatoid arthritis diagnosed after the messenger RNA COVID-19 vaccine.