Anti-SARS-CoV-2 immunogenicity decay and incident cases six months after Sinovac-CoronaVac inactivated vaccine in autoimmune rheumatic diseases patients: phase 4 prospective trial

We provide novel data on anti-SARS-CoV-2 immunogenicity decay and incident cases six months after the 2nd dose of Sinovac-CoronaVac inactivated vaccine(D210) in 828 autoimmune rheumatic diseases(ARD) patients compared with 223 age/sex-balanced control group(CG). From D69 to D210, anti-S1/S2IgG positivity and GMT reduced 23.8% and 38% in ARD(p<0.001/p<0.001) and 20% and 51% in CG(p<0.001/p<0.001). From D69 to D210 NAb positivity and activity declined 41% and 54% in ARD(p<0.001/p<0.001) and 39.7% and 47% in CG(p<0.001/p<0.001). Multivariate logistic regression analysis showed that male(OR=0.56;95%CI0.40-0.79;p<0.001), prednisone(OR=0.56; 95%CI0.41-0.76;p<0.001), anti-TNF(OR=0.66;95%CI0.45-0.96;p=0.031), abatacept(OR=0.29; 95%CI0.15-0.56;p<0.001) and rituximab(OR=0.32;95%CI0.11-0.90;p=0.031) use were associated with a substantial reduction on IgG response at D210 in ARD patients. A decrease of COVID-19 cases(from 27.5 to 8.1/100 person-years;p<0.001) occurred during the study despite the Delta variant spread. In conclusion, after 6-months of Sinovac-CoronaVac 2nd dose, immunogenicity of ARD patients was markedly reduced, particularly in males and those under prednisone/biological therapies, without a concomitant rise in COVID-19 cases(NCT04754698).


Introduction
Mass vaccination is the main measure to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread and emergence of new viral variants of concern 1 .While the pandemic drags on, de nition of immunogenicity durability is an essential step to establish booster doses strategies.
Data on persistence of post-vaccination immunogenicity are, however, scarce with few cases series on messenger RNA (mRNA) and viral vector vaccines demonstrating in the general population a variable decline of antibody levels in a period of two to six months after two doses of SARS-CoV-2 vaccination [2][3][4][5][6][7][8] .In addition, a large prospective study in health care workers reported a substantial decrease of mRNA vaccine induced antibodies in 6-months 8 .Some of these studies identi ed age and sex as associated with reduced durability of vaccine humoral immune response 3,8 .
The inactivated Sinovac-CoronaVac vaccine is currently used in the most populated countries of the world, and its protective effect against hospitalization and death related to coronavirus infectious disease 2019 (COVID-19) was demonstrated in more than 10 million subjects 9 .The short-term waning of antibody response to this vaccine was evaluated in 159 health care workers with a persistent seropositivity up to 98 days after vaccination, although with signi cant reduction in levels after 42 days 10 .
The vulnerable population of immunocompromised individuals was also evaluated for durability of vaccine immunogenicity in few cancer patients under active therapy.The follow-up after the second COVID-19 vaccination lasted solely 3-4 months, with a reported decay of IgG titers or inability to sustain IgG levels above the threshold 11,12 .
With regard to autoimmune diseases one study assessed 242 patients with a wide range of different conditions using a general computer-based questionnaire.They identi ed that participants with immunosuppression had a 65% reduction in IgG levels and 70% in neutralizing antibody concentrations compared to those without these therapies up to 6 months after mRNA vaccination 8 .
The deleterious impact of immunosuppressive therapy in a large autoimmune rheumatic diseases (ARD) population was reported for primary Sinovac-CoronaVac vaccination in a prospective study 13,14 .However, there is no report evaluating the long-term durability of anti-SARS-CoV-2 immunogenicity in COVID-19 vaccinated ARD patients.
Here, we described the analysis of a large ARD population, that was conducted to assess prospectively the 6-month durability of SARS-CoV-2 immunogenicity in fully vaccinated adults with Sinovac-CoronaVac compared with age-and sex-balanced control group.We further evaluated incident symptomatic COVID-19 cases con rmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).We also assessed risk factors for reduced 6-month durability of anti-SARS-CoV-2 immunogenicity.
For NAb analysis at D210, SLE diagnosis was less frequent in seronegative ARD patients (p=0.019)whereas biologic therapy (p=0.031),particularly abatacept use (p=0.018) was higher in patients without NAb (Table 2).After multivariate logistic regression analysis using NAb positivity as the dependent variable and as independent variables those with p<0.2 in univariate analysis, only abatacept use (OR=0.24;95%CI 0.13-0.46,p=0.041) remained signi cantly associated with absence of NAb at D210 in ARD patients.
Deaths due to COVID-19 occurred in n=4 ARD patients: n=1 in T1 and n=3 in T2 (p=0.694), after the 2 nd dose of Sinovac-CoronaVac.The rst death occurred in a 58 years-old female rheumatoid arthritis patient with bronchiolitis, diabetes mellitus, arterial hypertension, obesity, and hypothyroidism, under rituximab (last dose 5 months earlier) and prednisone 2.5 mg/day.The second death was on a 72 years-old male rheumatoid arthritis patient with chronic renal failure, pleural effusion, asthma, and dyslipidemia, under hydroxychloroquine and prednisone 2.5mg/day.Third death occurred in a 73 years-old female, with idiopathic in ammatory myopathy and interstitial lung disease, arterial hypertension, diabetes mellitus, and dyslipidemia, under mycophenolate mofetil, and another in a 65 years-old female patient, with granulomatosis with polyangiitis and arterial hypertension, under azathioprine and prednisone (2.5 mg/day).
Further comparative survival analysis of ARD and CG (p=0.152), with random selection of age and sex comparable subjects pointed that n=33 (ARD n=31 and CG n=2) RT-PCR con rmed COVID-19 incident symptomatic cases were reported during T1 (40 days) and n=52 cases (ARD n=44 in CG n=8) during T2 (180 days) evenly distributed along this period in ARD patients (Supplementary Figure 2).

Discussion
To our knowledge this is the rst study to speci cally demonstrate a substantial decline of anti-SARS-CoV-2 immunogenicity in ARD patients 6-months after the full inactivated vaccine schedule without a simultaneous increase in breakthrough cases.We further identi ed male sex and immunosuppression as deleterious for long-term antibody persistence in these patients.
The large number of ARD patients with a balanced age and sex CG included in this study was a main strength since it provided a unique opportunity to de ne more accurately risk factors for vaccine induced anti-SARS-CoV-2 durability.In fact, age and sex were identi ed previously as determinants of inability to sustain SARS-CoV-2 antibody levels in health population 3 .The use of established classi cation criteria for each disease allowed a more accurate de nition of the impact of different conditions and their therapy.The inclusion of a broad spectrum of non-rheumatologic immunosuppressed participants and the use of a generic computer-based questionnaire about existing conditions and treatment precluded a de nitive conclusion about the subgroup of ARD and their treatment in a previous study 8 .
The period of 6 months with a parallel observation of incident cases is endorsed by the report of breakthrough infection 4-month post-BTN162b2 vaccination associated with reduced levels of antibodies peri-infection and a signi cantly diminished humoral response in 6-months 1,8 .The uniform post-vaccination follow-up was an essential parameter for a more accurate de nition of vaccine induced antibodies persistence at 6 months.In fact, previous studies have demonstrated that vaccine humoral response waning dynamics varies overtime and is also distinct for IgG and NAb level 8 .An important limitation of the present study is the non-assessment of cellular immunity in this population, but neutralizing antibodies evaluated herein were reported to be associated with protective immunity 15 .
We provide novel data of a reduction of anti-SARS-CoV-2 antibodies positivity over 6-months post Sinovac-CoronaVac second dose in ARD patients with a magnitude of approximately 20% for patients and controls.We also con rmed the observation of a robust (≥ 4 times) decrease in SARS-CoV-2 mRNA vaccine IgG levels after 6-months in health care workers 8 .We further demonstrated that the same phenomena, although less intense, occurred for the Sinovac-CoronoVac with a more expressive decrease in controls (51%) than in ARD patients (38%) at 6 months post second dose.
The analysis of NAb activity, reported to be a strong correlate of protection 15 , revealed a substantial waning of 54% after 6-months of Sinovac-CoronaVac vaccination in ARD patients.A similar nding of 70% reduction in NAb titer 6months post-mRNA vaccination was reported for participants with immunosuppression 8 .In contrast, a less prominent decline in NAb positivity of approximately 40% for ARD patients and controls was found herein.
Regarding deleterious factors for vaccine-induced immunogenicity durability, we have identi ed in the multivariate logistic regression analysis that male sex, prednisone and anti-TNF have a moderate impact in immunogenicity persistence whereas a major harmful effect was evidenced for abatacept and rituximab after 6-months.For NAb positivity, only abatacept was associated with absence of these antibodies at D210 in ARD patients.Likewise, a signi cant lower NAb activity level was reported in men and immunosuppressed health worker participants 6-months after receipt of the second dose 8 .
The predominance of incident cases after the vaccine rst shot up to 10 days after second dose (T1) contrast with the signi cant drop in infection and hospitalization in the study participants in the subsequent 40 days which coincided with the second peak of COVID-19 cases in São Paulo city (45% increase in the same time period) 16 .
Interestingly, the emerging of Delta (B.1.617.2) variant in Sao Paulo in July 17 with a rapid spread in the following months did not lead to a parallel upsurge of COVID-19 breakthrough cases among our cohort which remained with homogeneous distribution of cases.Our data supports the notion that Sinovac-CoronaVac may sustain a 6-month effectiveness against Delta strain.
In conclusion, we provide novel data on the long-term Sinovac-CoronaVac immunogenicity in ARD patients demonstrating a signi cant decrease in IgG and NAb levels 6 months after the second dose without a corresponding rise in symptomatic COVID-19 cases in the same period.Male, prednisone and biological therapy were identi ed as the main contributing factors to reduced durability of vaccine induced humoral response.GMT -Geometric mean titers (AU/mL); Frequencies of seropositivity are presented as number (%) and they were compared between groups (ARD and CG) and between time points (D69 vs. D210) using generalized estimating equations (GEE) with binomial distribution and logit link function, assuming autoregressive correlation matrix between moments.IgG antibody titers are expressed as geometric means with 95% confidence interval (95%CI).Data regarding IgG titers were analyzed at neperian logarithm (ln)-transformed basis using Serology parameters were compared between groups (ARD and CG) and timepoints (D69 and D210) using GEE with normal marginal distribution and gamma distribution respectively and identity binding function assuming first order autoregressive correlation matrix between moments.

Tables
Results were followed by Bonferroni multiple comparisons to identify differences between groups and timepoints.All analyses were two-sided.

Supplementary Files
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