The protocol was conducted according to the Declaration of Helsinki and local regulations and approved by the National and Institutional Ethical Committee of Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil (CAAE: 42566621.0.0000.0068). Written informed consent was obtained from each participant before enrollment.
This phase 4 prospective controlled clinical trial (clinicaltrials.gov #NCT04754698) was conducted at a single tertiary center in Brazil.
Patients and controls
ARD patients ≥ 18 years old from the Outpatient Rheumatology Clinics at our center were included, with the following diagnosis: RA38; SLE39; AxSpA40; PsA41; primary vasculitis42–43; pSSj44; SSc45; IIM46 and PAPS47.
After confirming ARD patient’s participation, CG were invited, matching by gender and sex (up to ± 5 years differences). None of them were previously vaccinated in the hospital's regular campaign. Well-controlled medical conditions were allowed in the CG, except ARD, use of immunosuppressive drugs or HIV infection.
Overall exclusion criteria were: history of anaphylactic response to vaccine components, acute febrile illness or symptoms compatible to COVID-19 at vaccination, Guillain-Barré syndrome, decompensated heart failure (class III or IV), demyelinating disease, previous vaccination with any SARS-Cov-2 vaccine, history of live virus vaccine up to four weeks before, virus vaccine inactivated up to two weeks before, history of having received blood products up to six months before the study, individuals who did not accept to participate in the study, hospitalized patients, and pre-vaccination positive COVID-19 serology and/or Nab.
After receiving the first vaccine dose, participants with RT-PCR confirmed COVID-19 were excluded from the immunogenicity analysis, but included in the evaluation of incident cases.
The vaccination protocol for ARD patients and GC consisted of a two-dose schedule of the COVID-19 vaccine. The first dose with blood collection was given on February 9-10th 2021 (D0), the second dose with blood collection on March 9th and 10th 2021 (D28) and the last blood collection on April 19th 2021 (D69) at the Hospital Convention Center. This protocol was delayed 4 weeks for participants with incident COVID-19 during the study. Ready-to-use syringes loaded with CoronaVac (Sinovac Life Sciences, Beijing, China, batch #20200412), that consists of 3 µg in 0.5 mL of β-propiolactone inactivated SARS-CoV-2 (derived from the CN02 strain of SARS-CoV-2 grown in African green monkey kidney cells - Vero 25 cells) with aluminum hydroxide as an adjuvant were administered intramuscularly in the deltoid area.
Immunogenicity's primary outcome was assessed by two criteria at D69: seroconversion rates of total anti-SARS-Cov-2 S1/S2 IgG and presence of NAb. Secondary immunogenicity criteria were: anti-S1/S2 IgG seroconversion and presence of Nab at D28 (after vaccine first dose); geometric mean titers of anti-S1/S2 IgG and their factor-increase in GMT (FI-GMT) at D28 and D69; and median (interquartile range) neutralizing activity of NAb at D28 and D69. In order to assess these outcomes, blood samples (20mL) from all participants were obtained at days D0 (baseline - immediately before first vaccine dose), D28 (immediately before the second dose), and D69 (six weeks after the second dose). Sera were stored in a -70 ºC freezer.
Anti-SARS-CoV-2 S1/S2 IgG antibodies
A chemiluminescent immunoassay was used to measure human IgG antibodies against the S1 and S2 proteins in the RBD (Indirect ELISA, LIAISON® SARS-CoV-2 S1/S2 IgG, DiaSorin, Italy). Seroconversion rate (SC) was defined as positive serology (≥ 15.0 UA/mL) post vaccination taking into consideration that only patients with pre-vaccination negative serology were included. Geometric mean titers (GMT) and 95% confidence intervals of these antibodies were also calculated at all time points, attributing the value of 1.9 UA/mL (half of the lower limit of quantification 3.8 UA/mL) to undetectable levels (< 3.8 UA/mL). The factor increase in GMT (FI-GMT) is the ratio of the GMT after vaccination to the GMT before vaccination, showing the growth in titers. They are also presented and compared as geometric means and 95% confidence intervals (CI).
SARS-CoV-2 cPass virus-neutralization antibodies (Nab)
The SARS-CoV-2 sVNT Kit (GenScript, Piscataway, NJ, USA) was performed according to manufacturer instructions. This analysis detects circulating neutralizing antibodies against SARS-CoV-2 that block the interaction between the receptor binding domain (RBD) of the viral spike glycoprotein with the angiotensin-converting enzyme 2 (ACE2) cell surface receptor. The tests were performed on the ETI-MAX-3000 equipment (DiaSorin, Italy). The samples were classified as either "positive" (inhibition ≥ 30%) or "negative" (inhibition < 30 %), as suggested by the manufacturer.48 The frequency of positive samples was calculated at all time points. Medians (interquartile range) of the percentage of neutralizing activity only for positive samples were calculated at all time points.
Vaccine adverse events and incident cases of COVID-19
Patients and control groups were advised to report any side effects of the vaccine and they received on D0 (first dose) and on D28 (second dose) a standardized diary for local and systemic manifestations. Local manifestations included in the diary were local pain, erythema, swelling, bruise, pruritus and induration at the vaccine site. Systemic reactions included were: fever, malaise, 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. Vaccine AE severity was defined according to WHO definition49.
Environmental factors associated with high risk of exposure to SARS-CoV-2 were recorded from all participants, including adherence to social isolation, number of people living in the same house, household contact with infected people and use of public transportation.
Additionally, all ARD patients and controls were instructed to communicate any manifestation associated or not with COVID-19 through telephone, smartphone instant messaging or email. Our medical team was divided to provide a proper follow-up for the assigned group of patients/controls including the need for medical care, hospitalizations, severity of infections, sick days, and treatment. Suspicious cases of COVID-19 were instructed to seek medical care near the residence and if recommended to come to our tertiary hospital to have the PCR exam or in-person visit. If tertiary care was required, the participant was transferred to a referenced Hospital. The standardized diary of adverse events was carefully reviewed with each participant on the day of the second dose (D28) and at the last visit (D69). COVID19 incident cases were followed for 40 days [from D0 to 10 days after the second dose (D39)] and thereafter for the following 40 days [from D40 to D79].
Study data were collected and managed using REDCap electronic data capture tools hosted at our Institution50,51.
RT-PCR for SARS-CoV-2 and analysis of variants of concern
Clinical samples for SARS-CoV-2 RT-PCR consisted in naso- and oropharyngeal swabs, using a laboratory developed test52. Participants with positive samples collected at our hospital were further analyzed for variants of concern using the previously described protocol53, including the deletion in the NSP6 gene that is observed in the P1, B.1.1.7, and B.1.351 lineages. Distinction of B1.1.7 from P1 and B 1.351 lineages was performed as described54.
The sample size calculation was based on the previous 15% reduction of seroconversion rate after primo vaccination with the 2009 non-adjuvanted influenza A/H1N1 vaccine in a large cohort of ARD patients35. Expecting seroconversion rates of 63% in the ARD patient’s cohort and 78% in the control group, considering an alpha error of 5% and power of 80%, in 5:1 ratio in order to include more ARD patients, the minimum sample required would be 445 ARD patients and 89 healthy subjects, sex-matched and with similar ages. Expecting a higher SC rate of 98% for this vaccine,27 such sample size had a power greater than 99% to detect a 15% reduction in SC of ARD patients. Due to the peak of pandemics ongoing in Brazil during the vaccination period, we invited more patients and controls, expecting a high incidence of previously infected people and a high rate of infections.
Continuous general data are presented as medians (interquartile ranges) and compared using Mann-Whitney test for intergroup comparisons. Longitudinal analyses within the same group were performed using ANOVA on ranks. Continuous data regarding anti-S1/S2 serology titers are presented as geometric means (95% CI) and compared with the same tests, but in neperian logarithm (ln) transformed data. Categorical variables are presented as number (percentage) and compared using the chi-square or Fisher's exact tests, as appropriate. Multivariate logistic regression analyses were performed using as dependent variables seroconversion or presence of Nab, and as independent variables those with p < 0.2 in univariate analysis. Statistical significance was defined as p < 0.05. All statistical analyses were performed using GraphPad Prism (version 8.0.2–159, serial number GPS-1360936-TLPU-E3F30).