key: cord-0841711-m0qb1t5k
authors: Araujo, C. S. R.; Medeiros-Ribeiro, A. C.; Saad, C. G. S.; Bonfiglioli, K. R.; Domiciano, D. S.; Shimabuco, A. Y.; Silva, M. R.; Yuki, E. F. N.; Pasoto, S. G.; Pedrosa, T.; Kupa, L. d. V. K.; Zou, G.; Pereira, R. M. R.; Silva, C. A.; Aikawa, N. E.; Bonfa, E.
title: A RANDOMIZED CLINICAL TRIAL OF 2-WEEK METHOTREXATE DISCONTINUATION IN RHEUMATOID ARTHRITIS PATIENTS VACCINATED WITH INACTIVATED SARS-COV-2 VACCINE
date: 2021-11-24
journal: nan
DOI: 10.1101/2021.11.23.21266785
sha: d7cb09c46730db3218713d5d0a17d1b632f3f52b
doc_id: 841711
cord_uid: m0qb1t5k

Objectives: To evaluate the effect on immunogenicity and safety of 2-week methotrexate(MTX) discontinuation after each dose of the Sinovac-CoronaVac vaccine versus MTX maintenance in rheumatoid arthritis(RA) patients. Methods: This was a single-center, prospective, randomized, investigator-blinded, intervention study (#NCT04754698, CoronavRheum), including adult RA patients(stable CDAI<10, prednisone<7.5mg/day), randomized(1:1) to withdraw MTX(MTX-hold) for 2 weeks after each vaccine dose or maintain MTX(MTX-maintain), evaluated at D0, D28 and D69. Co-primary outcomes were anti-SARS-CoV-2 S1/S2 IgG seroconversion(SC) and neutralizing antibody(NAb) positivity at D69. Secondary outcomes were GMT and changes in disease activity scores. For immunogenicity analyses, we excluded patients with baseline positive IgG/NAb, and, for safety reasons, those unable to hold MTX twice(CDAI>10 at D28). Results: Randomization included 138 patients with 9 exclusions(5 COVID-19, 4 protocol violations). Safety evaluation included 60(MTX-hold) and 69(MTX-maintain) patients. Further exclusions consisted of 27 patients[13(21.7%) vs. 14(20.3%),p=0.848] with positive baseline IgG/NAb and 10 patients(21.3%) in MTX-hold with CDAI>10 at D28. At D69, a higher increase in SC[29(78.4%) vs 30(54.5%),p=0.019] was observed in MTX-hold(n=37) in comparison to MTX-maintain(n=55), with parallel augmentation in GMT[34.2(25.2-46.4) vs 16.8(11.9-23.6),p=0.006]. No differences were observed for NAb positivity[23(62.2%) vs 27(49.1%),p=0.217]. Longitudinal variations in disease activity scores were alike in both groups(CDAI,p=0.144; DAS28-CRP,p=0.718). Conclusion: We provided novel data that 2-week MTX withdrawal after each vaccine dose improves anti-SARS-CoV-2 immunogenicity. The comparable longitudinal variations of disease activity in both groups suggest that discontinuation is a feasible and efficient strategy in well-controlled RA patients, and may be even safer for vaccines with longer interval between doses or single dose schedules.

The SARS-CoV-2 virus has caused a worldwide health, social and economic crisis with a death toll reaching millions, [1] . Brazil has been one of the most impacted countries, with mortality surpassing 600.000 subjects in October 2021, [ UCI admission and COVID-related death was over 85%, [5] .

Rheumatoid arthritis (RA) patients are at higher risk of hospitalization and death by COVID-19 due to RA itself, [6] , comorbidities, [7] [8] , or immunosuppressive treatments, [6] [7] [8] [9] . Moreover, patients with RA have reduced immunogenicity to COVID-19 vaccines, [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] , particularly those on glucocorticoids (GC), [10] [11] [12] [13] , methotrexate (MTX), [11] [12] [13] [14] [15] [16] [17] , rituximab (RTX), [11] [12] [17] [18] [19] [20] [21] and abatacept (ABA), [11] [12] 21] therapies.

Temporary immunosuppressant withdrawal is suggested as a possible strategy to enhance vaccine immunogenicity in autoimmune rheumatic diseases (ARD) patients, [14] [15] 22] . In this context, Park et al. demonstrated that the discontinuation of MTX improved immunogenicity of the annual influenza vaccines in RA patients, [14] [15] , concluding that the interruption of two MTX doses after vaccination was safe and equally effective as holding four MTX doses, [23] [24] . Due to these results, recommendations have emerged . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.23.21266785 doi: medRxiv preprint favoring the withdrawal of MTX for 1-2 weeks after COVID vaccines, [25] [26] . However, to this date, no comparative study has assessed the impact of this intervention on immunogenicity and disease activity after any SARS-CoV-2 vaccination schedule.

Therefore, this trial aimed to evaluate the immunogenicity and safety of 2-week MTX discontinuation after each dose of the Sinovac-CoronaVac vaccine in remission/low disease activity RA patients compared to age and sex balanced RA group who maintained the drug.

This was a single-center, randomized, investigator-blind, intervention study performed at the rheumatology outpatient clinic of a tertiary center. The study was conducted in accordance with the Declaration of Helsinki and local regulations, and was approved by the institutional and national ethics committee (CAAE: 42566621.0.0000.0068). All RA patients fulfilled the American College of Rheumatology/European League Against Rheumatism criteria for the classification of RA, [27] , and agreed to participate in the study and signed informed consents. The protocol is part of a larger study of immunosuppressed patients with ARD (Clinicaltrials.gov#NCT04754698), [12] .

The co-primary outcomes were seroconversion (SC) rates for anti-SARS-CoV-2 S1/S2 IgG and neutralizing antibodies (NAb) positivity at D69. Secondary immunogenicity . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.23.21266785 doi: medRxiv preprint outcomes were: seroconversion rates for anti-S1/S2 IgG and NAb positivity at D28; geometric mean titer (GMT) and factor increase of GMT (FI-GMT) for anti-SARS-CoV-2 S1/S2 IgG and NAb activity at D28 and D69.

Secondary safety outcomes were longitudinal variations in disease activity scores:

Clinical Disease Activity Index (CDAI), [28] , Simplified Disease Activity Index (SDAI), [28] , Disease Activity Score in 28 joints with C-reactive protein (DAS28-CRP), [29] , and frequency of adverse events related to vaccine. Exploratory outcomes were the frequency of patients with flare at D28 and D69 defined by CDAI>10, [30, 31] or by an increase in DAS28-CRP >1.2 (or >0.6 if the baseline DAS28 was >3.2), [32] [33] . Moreover, patient perception of disease activity worsening was also evaluated.

We recruited adult (≥ 18 years old) patients with RA diagnosis [27] with low disease activity or remission (CDAI ≤10) [28] at first vaccination day and stable MTX dose for at least 4 weeks in monotherapy or in association with synthetic or biologic diseasemodifying antirheumatic drugs (DMARD). Maximum allowed prevaccination oral prednisone dose was 7.5 mg/day. Patients were invited to participate after the review of their electronic records in the last 3 months (recruitment up to 3 weeks before enrollment).

Exclusion criteria were acute febrile illness/symptoms of COVID-19 at vaccination, history of anaphylaxis to vaccine components, demyelinating disease, decompensated heart failure (class III/IV), blood transfusion ≤6 months, inactivated virus vaccine ≤14 days, live virus vaccine <4 weeks, denial to participate, hospitalization, previous vaccination with any SARS-CoV-2 vaccine, reverse-transcriptase-polymerase-chain-reaction (RT-PCR) confirmed COVID-19 during the study, and rituximab use in the previous 12 months.

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The copyright holder for this this version posted November 24, 2021. ;  Patients with prevaccination positive COVID-19 serology (anti-S1/S2 IgG and/or NAb) were excluded from immunogenicity analysis but kept for safety evaluation.

Patients were evaluated in three visits: D0 (first dose of the vaccine), D28 (second dose) and D69 (6 weeks after the 2 nd dose). First dose was given on February, 9 th -10 th , 2021 (D0), while the second dose was on March 9-10 th , 2021 (D28).

The vaccination protocol included two doses of ready-to-use syringes with Sinovac-CoronaVac vaccine (Sinovac Life Sciences, Beijing, China, batch #20200412), consisting of 3 µg in 0.5 mL of β-propiolactone inactivated SARS-CoV-2 with aluminum hydroxide as an adjuvant. Vaccine was administered intramuscularly in the deltoid area.

Investigators responsible for disease activity measures, statisticians and laboratory personnel were blinded to the allocation groups. Only 2 researchers (C.S.R.A. and M.R.S.) were not blinded and were responsible for safety surveillance and patient followup by telephone for adherence purposes. These 2 investigators were not involved in disease activity measures, laboratory analysis or patient vaccination.

At D0, before the first vaccine dose, patients were evaluated by blinded experienced rheumatologists who assessed disease activity by CDAI and rechecked inclusion and exclusion criteria. Patients with CDAI<10 proceeded to the enrollment station, where the unblinded researchers revised the protocol, explained the procedures, collected the informed consent and conducted the randomization, which was performed on the web-. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 24, 2021. ;  based software "The REDcap Project version 10.5.0". Allocation was generated instantaneously in a 1:1 ratio to one of the following groups: withdraw MTX for 2 weeks after each dose of the CoronaVac (MTX-hold group) or to maintain MTX continuously (MTX-maintain group).

At D28 and D69, patients were initially assessed by the unblinded researchers, checked for protocol violation, and instructed not to inform their allocation groups to anyone else.

Then, they proceeded for the blinded disease activity evaluation. Subsequently, they returned to the unblinded researchers and were instructed accordingly.

At D0, the two unblinded researchers instructed patients in the MTX-hold group not to take 2 doses of MTX after vaccination, according to the last MTX dose. They provided a date diagram (Supplementary figure 1) informing the dates in which they would skip MTX, and the date to resume its usage. At D28, patients in the MTX-hold group with CDAI<10 were instructed to withdraw MTX again, and a new date diagram was produced. Patients with CDAI>10 in the MTX-hold group were instructed not to withdraw MTX again after vaccine 2 nd dose. Patients in the MTX-maintain group were instructed to continue MTX on the same day and dose throughout the study. The 2 unblinded researchers checked adherence to protocol by telephone contact with all patients in the weeks following both vaccine doses.

Adding or changing DMARD therapy was not allowed until D69, though patients were permitted to use analgesics, non-steroidal anti-inflammatory drugs or prednisone up to 10 mg/day in case of disease activity worsening.

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Blood samples (30 mL) were collected immediately before each vaccine dose (D0 and D28) and 6 weeks after the 2 nd dose (D69). Serum samples were stored at -70 ºC. IgG antibodies against the SARS-CoV-2 S1/S2 proteins were measured using a chemiluminescent immunoassay (Indirect ELISA, LIAISON®, DiaSorin, Italy). SC was defined as positive serology (≥ 15.0 UA/mL). GMT and 95% confidence intervals were calculated attributing the value of 1.9 UA/mL to undetectable levels (<3.8 UA/mL). FI-GMT was calculated as the ratio of the IgG titer after vaccination to the IgG titer before vaccination. Detection of NAb was performed using the SARS-CoV-2 sVNT Kit (GenScript, Piscataway, NJ, USA). Positivity was defined as ≥30% inhibition of this linkage, [34] . Medians (interquartile range) of the percentage of neutralizing activity were only calculated for positive samples. CRP (by the nephelometric method), was also measured.

Disease activity was checked by experienced rheumatologists, blinded to allocation groups, who assessed the following parameters: number of tender joints, number of swollen joints (both in 28 joint count), patient global assessment of disease activity (PGA, by visual analog scale -VAS) and evaluator global assessment of disease activity (EGA -by VAS). With this data, CDAI was calculated. With CRP from sera collected on the same day, SDAI and DAS28-CRP were also calculated.

Patients were instructed to fill a structured diary of local (pain, erythema, swelling, bruise, pruritus, and induration at the vaccine site) and systemic symptoms (fever, malaise, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 24, 2021. ;  somnolence, lack of appetite, nausea, vomit, diarrhea, abdominal pain, vertigo, tremor, headache, fatigue, myalgia, muscle weakness, arthralgia, back pain, cough, sneezing, coryza, stuffy nose, sore throat, shortness of breath, conjunctivitis, pruritus, and skin rash) after each vaccination to explore potential vaccine side effects. Adverse effect severity was classified according to WHO definition, [35] . Patients who had symptoms suggestive of COVID-19 infection had nasopharyngeal RT-PCR tests done.

The sample size calculation was based on the 2009 non-adjuvanted influenza A/H1N1 primo vaccination in a large cohort of RA patients under MTX, which induced SC rate of 46%, [36] . Expecting an increment of 20% in the MTX-hold group, [14] [15] , that should achieve 66% SC rate, with a 5% alpha error and 80% power (1:1 ratio), the minimum sample would be of 96 patients per group.

Categorical variables were presented as number (percentage) and compared using chisquare or Fisher's exact tests, as appropriated. Continuous general data were presented as medians (interquartile ranges) and compared using t-test or Mann-Whitney test, as appropriate. Data regarding IgG titers and disease activity scores at different time points were analyzed using generalized estimating equations (GEE) with normal marginal distribution and gamma distribution respectively and identity binding function assuming first order autoregressive correlation matrix between moments (D0, D28 and D69) in the comparison of the 2 groups (MTX-hold and MTX-maintain), followed by Bonferroni's multiple comparisons. IgG titers were analyzed as neperian logarithm (ln)-transformed data. Multiple regression analyses were performed including SC or presence of NAb at . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. MTX-hold and MTX-maintain groups had similar age and female sex frequencies (p>0.05). Other demographic characteristics, comorbidities, disease duration, baseline disease activity, rheumatoid factor and anti-CCP positivity, current therapy did not differ between the two groups (p>0.05), except for the association with prednisone which was more frequent in the MTX-hold group regarding safety analyses (p=0.021) ( Table 1) .

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The copyright holder for this this version posted November 24, 2021. ; Results are expressed in median (interquartile ranges) and n (%). Continuous data are compared using the Mann-Whitney U-test, and categorical variables with the chi-square or Fisher's exact test, as appropriate, as two-sided analyses. For safety analyses, all patients who adhered to protocol were included. For immunogenicity analyses, patients with baseline positive S1/S2 IgG were excluded from both groups, and patients with CDAI>10 at D28 that withdrew MTX only once were excluded only from the MTX-hold group; * -indicates that percentages calculated according to available data. MTX-methotrexate; RF -rheumatoid factor; anti-CCPanti-cyclic citrullinated peptides; CDAI -clinical disease activity index; SDAI -simplified disease activity index; DAS28-CRP -disease activity score with 28 joints and C-reactive protein; TNF -tumor necrosis factor.

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The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.23.21266785 doi: medRxiv preprint

Baseline anti-SARS-CoV-2 S1-S2 IgG GMT (Table 3) .

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The copyright holder for this this version posted November 24, 2021. ; MTX-hold -baseline seronegative patients randomized to interrupt MTX after the 1 st and 2 nd doses, excluding those who had CDAI>10 at D28 and withdrew MTX only once. MTX-maintain -baseline seronegative patients randomized to maintain methotrexate throughout the study and who adhered to the protocol. SC-seroconversion, GMT-geometric mean titers, FI-GMT-factor increase of geometric mean titers, RA -rheumatoid arthritis, MTX -methotrexate. Seroconversion (SC) is defined as post-vaccination titer ≥15 AU/ml by indirect ELISA, LIAISON SARS-CoV-2 S1/S2 IgG. Frequencies of SC are presented as number (%) and were compared using a two-sided chi-square or Fisher's exact test, as appropriate, at prespecified time points (D28 and D69). IgG antibody titers and FI-GMT are expressed as geometric means (GMT) with 95% CI. Data regarding IgG titers were analyzed in neperian logarithm (ln-) transformed data using generalized estimating equations (GEE) with normal marginal distribution and gamma distribution respectively, and identity binding function assuming first order autoregressive correlation matrix between moments (D0, D28 and D69) in the comparison of the 2 groups (MTX-hold and MTX-maintain), followed by Bonferroni's multiple comparisons. The mean behavior of the ln-transformed IgG titers was different in MTX-hold and MTX-maintain (p interaction=0.001). Mean titers increased at each time point for MTX-hold group (*p<0.001 both from D0 to D28 and from D28 to D69). For MTX-maintain group, the titers did not increase from D0 to D28 (p=0.423) but increased from D28 to D69 (*p<0.001). FI-GMT values were compared using the Mann-Whitney U-test for intergroup comparisons in ln-transformed data at prespecified time points (D28 and D69). All analyses were twosided.

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The copyright holder for this this version posted November 24, 2021. ; In a further analysis combining both groups, the comparison of patients who had SC and those who did not seroconvert showed that older age, age > 60 years and combination with LEF were negatively associated with SC, while MTX withdrawal twice was positively related to it. For NAb, only older age and age > 60 years were negatively associated with absence of NAb (Supplemental Table 1 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 24, 2021. ; For safety analyses, all patients who adhered to protocol were included. Results are expressed in n (%) and compared with the chi-square or Fisher's exact test, as appropriate, as two-sided analyses. MTX -methotrexate; ΔDAS28-CRP -variation of DAS28-CRP score; DAS28-CRP -disease activity score with 28 joints and C-reactive protein; CDAI -clinical disease activity index.

Approximately half of the patients reported mild side effects, without differences between the groups. After the 2 nd vaccine dose, myalgia [10(16.7%) vs. 3(4.3%), p=0.037] and vertigo [7(11.7%) vs. 1(1.5%), p=0.024] were more frequent in the MTX-hold group (Supplemental Table 2 ).

To the best of our knowledge, this is the first randomized study to compare the impact of MTX withdrawal in the immunogenicity and disease activity of any COVID-19 vaccine in RA patients. We demonstrated that temporary suspension is effective in increasing IgG SC and GMT levels. The observed comparable disease activity variation in MTX-hold and MTX-maintain groups suggests that this strategy is effective and safe to boost vaccine immunogenicity in well-controlled RA patients under MTX therapy.

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The copyright holder for this this version posted November 24, 2021. ; https://doi.org /10.1101 /10. /2021 The study has some strengths such as the inclusion of patients in remission/low disease activity and low prednisone doses, providing a safer condition for MTX withdrawal, [24] . In addition, the randomized clinical design with allocation concealment, the blind evaluation of disease activity status and use of validated RA scores, [28] [29] allowed a precise analysis of flares. Moreover, a balanced distributions of demographic profile, disease features and treatment were relevant since these are known factors to influence vaccine immunogenicity and flares, [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . The final small sample size of the study is related to the high rate of refusals to participate and the rigorous exclusion criteria, and is an important limitation of this trial. However, the larger than expected benefit of MTX withdrawal allowed the identification of a significant difference between groups for SC and GMT.

We provide herein novel evidence of an increment of approximately 25% in anti-SARS-CoV-2 antibodies induced by Sinovac-CoronaVac vaccine with temporary MTX withdrawal. Such improvement is very similar to the 20% increase first described regarding MTX discontinuation for 2 weeks after influenza vaccine, [15] , and could therefore partially reduce the deleterious effects in seroconversion induced by MTX reported for Sinovac-CoronaVac vaccine, [12] and BNT162b2 mRNA COVID-19, [13, 16] . This immunogenicity enhancement was observed even with a high frequency of combined DMARD therapy and corticosteroids, factors that could further impair immune response to COVID vaccine, [12] [13] . Importantly, MTX dose was comparable between the groups and all patients had doses above 10mg/week, in line with the observation that only patients with doses greater than 7.5mg/week benefited from MTX withdrawal after influenza vaccine, [15] .

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The copyright holder for this this version posted November 24, 2021. ; https://doi.org /10.1101 /10. /2021 Concerning combination therapy, the distribution of drugs was alike between the groups, equalizing possible additional harmful effects of different DMARD. We also deliberately excluded patients under rituximab, due to well-known effect on humoral immunogenicity and the heterogeneity of phases of treatment cycles, [11] [12] [17] [18] [19] [20] [21] . In this context, multiple regression analyses revealed that neither combined DMARD nor prednisone impacted the benefit of MTX temporary discontinuation.

Safety related to vaccine and MTX withdrawal intervention was carefully assessed and included several composite measures. Longitudinally, CDAI, SDAI, DAS28-CRP and CRP had similar behaviors between the groups, increasing after the first dose and remaining stable after the second dose. In fact, the increase in disease activity measures, even in MTX-maintain group, is in accordance with the 20% flare rate of SDAI after BNT162b2 mRNA vaccination, [11] .

Considering the rate of flares, MTX-hold and MTX-maintain groups were also comparable regarding DAS28-CRP criteria. The similar criteria with DAS28-ESR was used in previous influenza vaccine MTX withdrawal studies, [14] [15] and performed better than other DAS28 flare definitions according to OMERACT, [32] . In our trial, however, CDAI>10 showed to be more sensitive than DAS28-CRP, detecting significantly more flares in MTX-hold in comparison to MTX-maintain group. Of note, the magnitude of CDAI variation was similar between the groups.

The Sinovac-CoronaVac vaccine was well tolerated, with no severe side effects.

However, MTX-hold group reported a higher frequency of myalgia and vertigo. The former manifestation may be associated with the vaccine or related to underlying disease activity.

In conclusion, this is the first randomized study to show a benefit of MTX withdrawal in . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 24, 2021. ;  the immunogenicity of COVID-19 vaccine. In light of these results, we recommend MTX withdrawal for 2 weeks after each vaccine shot in patients with CDAI<10, with close disease activity surveillance. This strategy may be even safer for other vaccines with longer interval between doses or single dose schedules.

This study was sponsored by grants from Fundação de Amparo à Pesquisa do Estado 

MTX-hold -baseline seronegative patients randomized to interrupt MTX after the 1 st and 2 nd doses, excluding those who had CDAI>10 at D28 and withdrew MTX only once. MTX-maintain -baseline seronegative patients randomized to maintain methotrexate throughout the study and who adhered to the protocol. Analyses were performed with neperian logarithm (ln)-transformed data using generalized estimating equations (GEE) with normal marginal distribution and gamma distribution respectively, and identity binding function assuming first order autoregressive correlation matrix between moments (D0, D28 and D69) in the comparison of the 2 groups (MTX-hold and MTX-maintain), followed by Bonferroni's multiple comparisons. The mean behavior of the lntransformed IgG titers was different in MTX-hold and MTX-maintain (p interaction=0.001). Groups were comparable at baseline (p>0.999), but MTX-hold group had higher mean titers at D28 (p=0.002) and at D69 (p=0.006). Mean titers increased at each time point for MTX-hold group ( †p<0.001 from D0 to D28 and from D28 to D69). For MTX-maintain group, the titers did not increase from D0 to D28 (p=0.423) but increased from D28 to D69 ( ‡p<0.001). All analyses were two-sided. Dotted line denotes the cut-off level for positivity (ln 15 AU ml-1 = 2.71 by Indirect ELISA, LIAISON SARS-CoV-2 S1/S2 IgG).

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MTX-hold -baseline seronegative patients randomized to interrupt MTX after the 1 st and 2 nd doses, excluding those who had CDAI>10 at D28 and withdrew MTX only once. MTX-maintain -baseline seronegative patients randomized to maintain MTX throughout the study and who adhered to the protocol. Data regarding disease activity parameters are shown as means and were analyzed using generalized estimating equations (GEE) with normal marginal distribution and gamma distribution respectively, and identity binding function assuming first order autoregressive correlation matrix between moments (D0, D28 and D69) in the comparison of the 2 groups (MTX-maintain and MTX-hold), followed by Bonferroni's multiple comparisons. CDAI -clinical disease activity index; SDAI -simplified disease activity index; DAS28-CRP -disease activity score with 28 joints and C-reactive protein; CRP -C-reactive protein. The mean behavior of CDAI ( Figure 3A ), SDAI ( Figure 3B ), DAS28-CRP ( Figure 3C ), and CRP ( Figure 3D ) was similar in MTX-hold and MTX-maintain throughout the study (p=0.144, p=0.117, p=0.718, and p=0.410, respectively), increasing after the first dose (p<0.001, p<0.001, p<0.001, and p=0.021, respectively) and remaining stable after the second dose (p>0.999, p>0.999, p=0.602, and p>0.999, respectively).

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Antibody response to inactivated COVID-19 vaccine (CoronaVac) in immune-mediated diseases: a controlled study among hospital workers and elderly

Disease activity and humoral response in patients with inflammatory rheumatic diseases after two doses of the Pfizer mRNA vaccine against SARS-CoV-2. Ann Rheum Dis

The effect of tofacitinib on pneumococcal and influenza vaccine responses in rheumatoid arthritis

Optimal time between the last methotrexate administration and seasonal influenza vaccination in rheumatoid arthritis: post hoc analysis of a randomised clinical trial

Effect of short-term methotrexate discontinuation on rheumatoid arthritis disease activity: post-hoc analysis of two randomized trials

American College of Rheumatology Guidance for COVID-19 Vaccination in Patients With Rheumatic and Musculoskeletal Diseases: Version 3. Arthritis Rheumatol

COVID-19 vaccination and antirheumatic therapy

rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative

The Simplified Disease Activity Index (SDAI) and the Clinical Disease Activity Index (CDAI): a review of their usefulness and validity in rheumatoid arthritis

Validation of the 28-joint Disease Activity Score (DAS28) and European League Against Rheumatism response criteria based on C-reactive protein against disease progression in patients with rheumatoid arthritis, and comparison with the DAS28 based on erythrocyte sedimentation rate

Predictors of disease flare after discontinuation of concomitant methotrexate in Japanese patients with rheumatoid arthritis treated with tocilizumab

Low rates of biologic-free clinical disease activity index remission maintenance after biologic disease-modifying anti-rheumatic drug discontinuation while in remission in a Japanese multicentre rheumatoid arthritis registry

Construct and criterion validity of several proposed DAS28-based rheumatoid arthritis flare criteria: an OMERACT cohort validation study

Disease activity guided dose reduction and withdrawal of adalimumab or etanercept compared with usual care in rheumatoid arthritis: open label, randomized controlled, non-inferiority trial

A New SARS-CoV-2 Dual-Purpose Serology Test: Highly Accurate Infection Tracing and Neutralizing Antibody Response Detection

Draft Guidelines for Adverse Event Reporting and Learning Systems

Reduced seroprotection after pandemic H1N1 influenza adjuvant-free vaccination in patients with rheumatoid arthritis: implications for clinical practice