Abstract
Background: Autoimmune Rheumatic Diseases (ARDs) patients are at high risk of COVID-19 re-infection even after vaccination. However, since the majority of studies in the related literature are focused on the efficacy of messenger RNA in rheumatic patients, the present study aimed to assess the efficiency of the inactivated whole virus vaccines and viral vector COVID-19 vaccines in rheumatic diseases in Mashhad, Iran.
Methods: This prospective cross-sectional study was conducted on ARDs patients who were referred to private clinics and rheumatologic clinics of Ghaem and Imam Reza Hospitals, Mashhad, Iran, during 2021-22. Anti-neutralizing antibodies have been considered to check antibodies of Sinopharm and Barkat vaccines, and an anti-spike antibody was considered to check Sputnik V and AstraZeneca vaccine antibodies. Humoral immunity was investigated using the anti-spike Enzyme-linked immunosorbent assay and anti-neutralizing antibodies.
Results: The obtained results showed that humoral immunity after COVID-19 vaccination was observed in 73.9% of the patients with ARDs. However, humoral immunity was lower in ARDs patients who took tacrolimus and higher in patients with a history of COVID-19 infection (χ2=5.84, p=0.01). The infection after the second COVID-19 vaccination was lower in patients with humoral immunity (χ2=5.69, p=0.01).
Conclusion: This study demonstrated that a homologous second dose of an inactivated whole viral vector SARS-CoV-2 vaccine was safe and provided a remarkable antibody response in ARDs patients. 
Keywords: Autoimmune rheumatic diseases, COVID-19, Humoral immunity, Vaccination  

Introduction
The outbreak of the SARS-CoV-2 virus, the causative agent of a type of acute respiratory syndrome with a viral agent from the beta coronavirus family, was a crisis phenomenon in the world and affected all aspects of the lives of billions of people in recent years (1). Vaccines are one of the most important global strategies for controlling the SARS-CoV-2 pandemic, especially in high-risk groups (2,3).
Humoral immunity is a type of acquired immune response caused by the activity of antibodies in the extracellular fluids (4-6). In most forms of vaccination, the antigen of a pathogen or weakened pathogens is injected into the body and the immune system is stimulated to produce antibodies. There are five main categories of SARS-CoV-2 vaccines. These include inactivated whole virus vaccines, viral vector vaccines, live attenuated whole virus vaccines, messenger RNA (mRNA) and DNA-based vaccines, and recombinant protein vaccines (7). Many vaccines interact with the surface proteins of viruses directly or indirectly (8).
Many patients with Autoimmune Rheumatic Diseases (ARDs) are treated with immunosuppressive drugs. The mortality risk of these patients is higher compared to the general population if they are affected by the SARS-CoV-2 virus. Therefore, the efficacy and safety of emerging COVID-19 vaccines are very important in the rheumatology population, as a high-risk group of patients (9,10). Few trials have assessed the best-selected COVID-19 vaccine in patients with immune system deficiency treated with immunosuppressive drugs (11). 
It appears that vaccines are currently safe to be used in the rheumatology population, except for live attenuated vaccines that seem to induce an adequate immune response (12). However, the effect of the COVID-19 vaccine on patients with ARDs should be monitored, especially if they are under treatment with immunosuppressive drugs (13,14). Soy et al showed a weaker immune response after COVID-19 vaccination in rheumatic patients who used immunosuppressive drugs and concluded that these patients were at risk of infection with COVID-19 even after vaccinations (15). Patients with ARDs may experience a slow reaction or a rapid decrease in antibody titer after the COVID-19 vaccine. However, there is currently no reason to exclude ARDs patients under treatment from getting vaccinated (16). 
Some studies have shown an increase in the antibody level after vaccination of ARDs patients, at the threshold level of creating immunity against the COVID-19 vaccine, which is less than that in healthy people. To increase the rate of the immune response, it seems that the right time of vaccination should be considered in the case of patients treated with immunosuppressive drugs (16). ARDs patients are at risk of COVID-19 re-infection, even after vaccination (17). In some studies, inactivated whole-virus COVID-19 vaccines have been considered effective vaccines with mild, predictable, and non-life-threatening side effects in transplant patients (18,19). However, no studies have been conducted on the effect of the vaccine type in patients with ARDs. 
Regarding the lack of studies on the effectiveness of COVID-19 vaccines for ARDs patients in Iran, and considering the fact that studies conducted in other countries are mainly focused on some specific types of vaccines, including mRNA, the present study aimed to evaluate the effectiveness of inactivated whole virus vaccines and viral vector COVID-19 vaccines for ARDs patients in Mashhad, Iran.

Materials and Methods 
This prospective cross-sectional study was conducted on patients with ARDs who were referred to private clinics and the rheumatology center of Ghaem and Imam Reza Hospitals, Mashhad, Iran, during 2021-2022.

Inclusion and exclusion criteria
The rheumatic patients who received two doses of the COVID-19 vaccine were entered into this study. However, the patients who only received one dose of the COVID-19 vaccine, non-cooperative patients (during the follow-up), and those with insufficient information in the follow-up were excluded from the study.  

Study design 
Eligible COVID-19-vaccinated ARDs patients with were entered into the study, without restrictions for age and gender. The sample size was estimated based on previous studies (20). To increase the study power, the number of samples was increased to 88.
Initially, the demographic characteristic data and clinical information were gathered and recorded in a checklist. These included the type of rheumatic disease, type of immunosuppressive drugs used, discontinuation of immunosuppressive drugs, co-morbidity, and the history of COVID-19 infection. The patients were referred for COVID-19 vaccination by a recommendation letter from a rheumatologist, taking into account the consumption of immunosuppressive drugs. If necessary, the drugs were discontinued under the supervision of a rheumatologist and considering the 2021 American College of Rheumatology (ACR) guidelines for COVID-19 vaccination (21). Patients who received two doses of vaccine were referred to the laboratory (at the beginning of the 3rd week after administration of the second dose) to take a venous blood sample (5 ml) and check the level of humoral immunity. 
The majority of patients were vaccinated with inactivated whole virus vaccines. Anti-neutralizing antibodies were adopted to check Sinopharm and Barkat vaccine antibodies and anti-spike antibodies were used to check Astrazeneca vaccine antibodies. Humoral immunity was investigated using the anti-spike enzyme-linked immunosorbent assay (ELISA) and anti-neutralizing antibodies.

Statistical analysis 
The data were entered into the SPSS 24 (IBM Corp., Armonk, NY, USA). Mean and frequency indices were used to describe the descriptive data. The Kolmogorov-Smirnov and Chi-square tests were used to evaluate the normality of the data and assess the categorical variables, respectively. The interval variables were evaluated using the t-test and its non-parametric equivalent. p-values lower than 0.05 (p<0.05) were considered significant. 

Ethical considerations 
The study protocol was approved by the Ethical Committee of Mashhad University of Medical Sciences, Mashhad, Iran (Ethics code: IR.MUMS.MEDICAL.REC.1401.319). The present study was extracted from a thesis to receive the specialist degree in rheumatology (Code:4000620). All the data were coded and recorded in a checklist anonymously to maintain confidentiality. Informed written consent was obtained from all the patients.  

Results
A total of 88 ARDs patients with a mean±SD age of 44.07±11.06 years (age range of 20-76 years) were entered into this study. The majority of patients (n=76, 86.4%) were female. The mean±SD duration of rheumatic disease was 7.03±6.66 years (ranging from 6 mounts to 32 years). Table 1 presents the demographic and clinical information of patients with ARDs. In total, 39.8% of the patients had underlying diseases including diabetes mellitus, hypertension, and hyperlipidemia. Moreover, 3.4% (n=2) were suffering from anxiety, deep vein thrombosis, and irritable bowel syndrome. Asthma, benign prostatic hyperplasia, fatty liver, brain aneurysm, and minor thalassemia were reported in only 2.3% of the patients. Table 2 presents the frequency of medications used in patients with ARDs. 

Table 1. Demographic and clinical information of patients with autoimmune rheumatic diseases

Table 2. Frequency of medications used in patients with autoimmune rheumatic diseases

Out of 88 participations, 47.7% were previously infected with COVID-19, and COVID-19 recurrence was reported in 23.9% of these patients after vaccination. The mean±SD time interval between COVID-19 vaccination and COVID-19 re-infection was 64±44.82 days (ranging from 5 to 180 days). Adverse effects after vaccination were reported in 47.7% of the patients. Local pain, swelling, and redness were the most commonly reported adverse effects in the patients (n=17, 19.3%). Headache, fever, malaise, myalgia, and dizziness were observed in 17.1% (n=15), 11.3% (n=10), 10.2% (n=9), 4.5% (n=4), and 2.3% (n=2) of the patients, respectively. Hair loss and nausea were observed in only 1.15% (n=1) of the patients. Table 3 indicates the patients’ clinical information related to COVID-19. 
In general, the mean±SD level of humoral immunity after COVID-19 vaccination in patients with ARDs was 82.9±68.64 (ranging from 0.4 to 180). Humoral immunity after COVID-19 vaccination was observed in 73.9% (n=65) of ARDs patients.
The rate of humoral immunity was not different in patients of various ages (t=-1.65, p=0.101) and was not associated with the duration of rheumatic disease (Z<0.01, p>0.99). The association between the use of rheumatic drugs and humoral immunity is presented in table 4. Based on the obtained results, the use of rheumatic drugs (e.g., corticosteroids, cytotoxics, biologic disease-modifying antirheumatic drugs [DMARDs], traditional DMARDs), except for tacrolimus (χ2=5.78, p=0.01), was not associated with the humoral immunity in the rheumatic patients.

Table 3. The patients’ clinical information related to COVID-19

Table 4. The association between the use of rheumatic drugs and humoral immunity

Table 5 shows the association between humoral immunity and other variables. Based on the results, gender (χ2=64, p=0.42), type of rheumatic disease (χ2=17.82, p=0.27), the presence of underlying diseases (χ2=0.32, p=0.56), patients’ social history (χ2=0.8, p=0.77), type of vaccination (χ2=5.01, p=0.17), and rheumatoid disease severity (χ2=0.1.9, p=0.59) were not related to humoral immunity. Humoral immunity was higher in patients with a history of COVID-19 infection (χ2=5.84, p=0.01). The rate of relapse after COVID-19 vaccination was not significantly different in patients with and without humoral immunity, meaning that vaccination did not prevent COVID-19 re-infection after the first dose (p=0.98). However, COVID-19 infection after the second vaccination was higher in patients without humoral immunity, indicating that humoral immunity could not prevent COVID-19 re-infection after the second dose (χ2=5.69, p=0.01). Table 6 presents humoral responses in patients, based on the type of rheumatic diseases.

Table 5. The association between humoral immunity and other variables

Table 6. Humoral response based on the type of rheumatic diseases

Discussion 
This is the first study to provide evidence of humoral immunity of two doses of inactivated whole virus and viral vector SARS-CoV-2 vaccines in patients with ARD in Iran. According to the results, humoral immunity after COVID-19 vaccination was found in 73.9% of ARDs patients. This immunity was lower in rheumatic patients who took tacrolimus, which could be attributed to their age, duration of rheumatic disease, gender, type of rheumatic disease, the presence of underlying diseases, patients’ social history, type of vaccine, and rheumatoid disease severity. Humoral immunity was higher in patients with a history of COVID-19 infection. Humoral immunity could not prevent COVID-19 re-infection after the first dose; however, it could decrease COVID-19 re-infection after the second COVID-19 vaccination. 
It seems that patients with rheumatic diseases may benefit from an additional heterologous dose to achieve their full potential of immunogenicity by inactivated whole virus vaccines such as the Sinovac-CoronaVac vaccine (22,23). Based on the study conducted by Hagen et al, each DNA-based vaccine (BNT162b2) can remarkably increase neutralizing capacity for patients with slow-responding ARD. They showed that these patients could benefit from up to three partly dose-intensified booster vaccinations, even with low or no peripheral B cells. The different outcomes seem to support the hypothesis that even individual differences play an important role in humoral response (24). Most patients with ARD included in this study were positive for humoral immunity. It should be noted that the second SARS-CoV-2 vaccination resulted in a significant increase in overall antibody response. 
Humoral response rates in immunocompromised patients with rheumatic diseases have been reported to be between 49 and 100% in various studies (25-28), which may be explained by differences in the study design, type of vaccines, and consumed medications. In some studies, the seropositivity rate was reported to be 94% in patients who did not receive RTX, abatacept, or Mycophenolate Mofetil (MMF) (26), while the humoral response has been lower in studies focusing on RTX therapy (49-59.3%) (27,28). 
In one study performed by Krasselt et al, the seropositivity rate after COVID-19 vaccination was estimated to be 78.5% in patients with inflammatory rheumatic diseases who consumed different anti-rheumatic drugs (29).  Although they found a significant vaccination response in subjects with and without immunosuppressive therapy, no such relationship was found between the use of immunosuppressive drugs and humoral immunity except for tacrolimus in the current study. Moreover, no difference was found in humoral immunity in terms of the vaccine type. It should also be noted that there were limitations in choosing vaccines due to their unavailability in Iran. Therefore, only inactivated whole virus vaccines (Sinopharm and Barecatco) and viral vector vaccines (AstraZeneca and Sputnik V) were used in the present study due to their availability.
 In the present study, no significant difference was reported between patients with and without humoral immunity among rheumatic patients who received Methotrexate (MTX) and MMF. However, the results may have been affected by the small sample size, which causes to cast doubts on the apparent immunogenicity increase. Moreover, the detrimental effect of immunosuppressive therapy on antibody production has been previously demonstrated in patients with ARD (2, 30-32).
Some studies showed that prednisone (in doses ≥7.5 mg/day) and rituximab had a negative impact on immune response (31-33). 
It has been suggested that rituximab treatment and low B cell counts are the two main suppressors of humoral response to vaccination (34-37). Krasselt et al reported that humoral immunity was prevented by the use of rituximab, abatacept, glucocorticoids, mycophenolate-mofetil, and even TNFi (29), while other studies showed no negative impact of TNFi on the humoral response (25). The decreased humoral response has been reported after one vaccination due to the consumption of TNFi in combination with MTX (38). In general, it seems that combination therapy (e.g., MTX+TNFi) is more immunosuppressive compared to monotherapy. However, the difference in humoral response after vaccination was not reported in the present study. Similar to our study, the results of another study demonstrated that humoral immunity was not weakened in psoriasis patients under MTX monotherapy who were vaccinated for COVID-19 (39). It is noteworthy that the majority of aforementioned studies have investigated vaccination with the mRNA vaccines (BNT162b2), while the present study was focused on the inactivated whole virus and viral vector vaccines.
A high humoral response has been reported in patients treated with AZA, MTX monotherapy, and JAK inhibitors. These results may indicate that there is no need to withhold immunosuppressive medication therapy prior to vaccination. However, suspending immunosuppressive medication therapy should be considered when MTX is used in combination with other DMARDs (29). Due to the relationship between humoral response rate and the time since the last application of immunosuppressive agents, it is recommended that the interval be lengthened in rheumatic patients if clinically feasible.
The results of the present study showed no significant difference between the types of rheumatic diseases and humoral response. However, since some patients had two or more rheumatic diseases simultaneously and it was not possible to separate them, this result cannot be considered definite. Based on one study, Systemic Lupus Erythematosus (SLE) patients tended to have higher rates of positive antibody responses compared to patients with Sjogren’s syndrome and Rheumatoid Arthritis (RA) (29). The separate assessment of the patients with rheumatic diseases indicated that 82.2% of the RA patients had humoral immunity, while the humoral response was observed in 76.4% of SLE patients and in only 40% of the patients with Sjogren’s syndrome. Considering the small number of studies that have either rejected or confirmed these findings, further studies in this regard can yield more definite results. 

Advantages and limitations
This was the first study to provide evidence of humoral immunity of two doses of inactivated whole virus and viral vector SARS-CoV-2 vaccines in patients with ARDs. The results of this study can provide useful information on the effect of vaccine type on rheumatic patients. The small size and cross-sectional observational nature of this study are its main limitations, which prevent us from generalizing the findings to other populations. Failure to follow up and evaluate the patients after receiving the third dose of vaccine can be considered another limitation of this study. Moreover, the cellular immunity was not assessed in the current study. The results of other studies showed that rituximab taking could not remarkably compromise T cell response, which provided a further argument for vaccinations despite B cell depletion (40,42). Therefore, it is suggested that future studies assess the longitudinal determination of anti-S IgG antibody levels for measuring individual courses of decreasing antibody responses.

Conclusion
The obtained results in this study demonstrated that a homologous second dose of an inactivated whole viral vector SARS-CoV-2 vaccine was safe and provided a remarkable antibody response in patients with ARDs. 

Funding 
Mashhad University of Medical Sciences (Code: 4000620).

Conflict of Interest
The authors have stated that they had no interests which might be perceived as posing a conflict or bias