The Vaccine Coverage and Vaccine Immunity Status and Risk Factors of Non-Protective Levels of Antibodies Against Vaccines in Children with Juvenile Idiopathic Arthritis: Russian Tertiary Centre Study.

Background: Immunosuppressive drugs, decreased vaccine coverage, aberrant immunity might be factors of low anti-vaccine antibodies in JIA patients. The study aimed to evaluate vaccine coverage, post-vaccine immunity and risk factors of non-protective levels of antibodies against measles, mumps, rubella, hepatitis B and diphtheria in JIA patients. Methods: A prospective study included 170 children diagnosed with JIA aged 2 to 17 years, who received routine vaccinations against measles, rubella, mumps (MMR) diphtheria and hepatitis B. In all patients, the levels of post-vaccination antibodies (IgG) for measles, rubella, mumps, hepatitis B and diphtheria measured with ELISA. Results: Protective level of antibodies were 50% against hepatitis B, 52% - diphtheria, 58% - measles, 80% - mumps, 98% rubella. The best coverage for MMR had patients with enthesytis-related arthritis-85%, compare to oligoarthritis-70%, polyarthritis-69%, systemic arthritis-63%. Diphtheria coverage was 50%, 51%, 46%, 63%, respectively. Incomplete MMR vaccination had 39% patients, treated with biologics, 22% with methotrexate and 14% with NSAID (p=0.025), and 61%, 46%, 36% for diphtheria (p=0.021). Incomplete vaccination was a risk factor of non-protective level of antibodies against measles (HR=2.03 [95%CI: 1.02; 4.0], p=0.042), parotitis (HR=6.25 [95%CI: 2.13; 17.9], p=0.0008) and diphtheria (HR=2.39 [95%CI: 1.18; 4.85], p=0.016) vaccines, as well as JIA category, biologics, corticosteroids and long-term methotrexate treatment for distinct vaccines. Conclusion: check, incomplete vaccination, biologics, systemic arthritis and long-term methotrexate treatment. corticosteroids, methotrexate, biologics. Russian national vaccine schedule supposes diphtheria-tetanus-pertussis vaccination in 3, 4½, 6 and 18 months and further diphtheria-tetanus vaccination in 14 MMR and six and hepatitis B in 6 Depending цуre into two


Introduction
Juvenile idiopathic arthritis (JIA) is the most common pediatric rheumatic disease (PRD) with a prevalence of between 16 and 150 per 100,000 children [1]. Both innate and adaptive immune systems are associated with several other factors, contributing to the pathological development of chronic in ammation in patients with JIA [2]. The main goal of the JIA treatment is control of immune-mediated in ammation with different medication, e.g. corticosteroids, methotrexate and biologics [1]. Patients with JIA are at greater risk of infections than healthy children due to their aberrant immunity and using immunosuppressive drugs [3]. They might have more infections required hospital admissions (e.g. pneumonia or sepsis) [4]. Biologic agents, especially TNF-a inhibitors, change the structure of infection diseases in JIA patients [5][6][7]. The infection episodes are the main reason for missing the biologic and methotrexate with subsequent JIA ares, impaired JIA remission and outcomes [8,9]. Vaccinations can decrease the number of infection episodes and maintain treatment of the disease and hold remission [10]. Escalation of treatment regimens might alter "protective" immune response (e.g., suppression of T and B cells and impaired antibody production) and increases the risk of infections [11,12]. Many children with PRD stop vaccinating when a diagnosis of rheumatic disease is established [13]. Many practising pediatricians and pediatric rheumatologists continue to believe that the immune response of JIA patients disrupted by immunosuppressive drugs and does not lead to the proper level of seroprotection [14,15]. Some physicians fear that vaccines may cause a persistent autoimmune response, lead to severe disease, or relapse to existing diseases [14,15]. The fear of parents before the risk of a are of rheumatic disease due to vaccination leads to the omitting vaccinations, usually recommended by the health authorities for healthy children [13].
Children who have younger-onset age of JIA are in the risk of incomplete vaccination and often miss revaccination against measles, rubella, mumps and diphtheria [16]. As a result, we have a rather large cohort of immune-compromised children with incomplete vaccination.

Methods
To evaluate vaccine coverage, post-vaccine immunity and risk factors of non-protective levels of antibodies against measles, mumps, rubella, hepatitis B and diphtheria in JIA patients, we have performed our study.
A prospective pilot study included data from 170 children diagnosed with juvenile idiopathic arthritis -JIA (55 boys and 115 girls) aged 2 to 17 years, who received routine vaccinations against measles, rubella, mumps (MMR) and diphtheria. The diagnosis of JIA based on the ILAR criteria (1997) [17].
In all patients, the levels of post-vaccination antibodies (IgG) for measles, rubella, mumps, hepatitis B and diphtheria measured with ELISA. IgG concentrations determined from calibration curves constructed using Dynex Technologies Inc. software (USA). The protective level of antibodies was established in accordance with the criteria speci ed in the manufacturer's instructions: for measles IgG − 0.18 IU/ml (coe cient of variation, CV, 8%; analytical sensitivity 0.07 IU/ml), for antibodies to rubella − 10 IU/ml (8%; 2 IU / ml), for hepatitis B (anti-HBs antibodies) − 10 mME / ml (8%; 2 mIU/ml), for diphtheria − 0.09 IU / ml (7, 5%; 0.004 IU/ml).The minimal protective level of IgG against mumps established with a positivity coe cient > 1.0. To detect measles, rubella, mumps and hepatitis B antibodies, we used the commercial kit, created by Vector-Best JSC, Russia and IBL International GMBH (Germany) for diphtheria antibodies. Information about the scheduled vaccination against MMR, hepatitis B and diphtheria, JIA course and treatment obtained from the patient's charts. The following classes of immunosuppressive medications, which were used by the patients during study recruitment, were taken into account: corticosteroids, methotrexate, biologics. Russian national vaccine schedule supposes diphtheria-tetanuspertussis vaccination in 3, 4½, 6 and 18 months and further diphtheria-tetanus vaccination in 6-7 and 14 years and MMR vaccine at the age of 1 year and six years, and hepatitis B vaccination in 0, 1, 6 months. Depending on the number of scheduled vaccines for subsequent analysis, patients цуre divided into two groups with complete and incomplete vaccination.
According to the national vaccine schedule, incomplete vaccination means fewer vaccines or vaccine doses to age.

Statistical analysis
Statistical analysis was performed with the software STATISTICA, version 10.0 (StatSoft Inc., USA) and MedCalc (MedCalc Software, Belgium). Description of quantitative variables was done with median and percentiles (25; 75) for continuous variables and in terms of absolute frequencies and percentages for categorical variables. For comparison, the categorical variables Pearson's χ2 test or the Fisher's exact test in case of expected frequencies < 5 was used, and comparison of two quantitative variables was carried out using the Mann-Whitney test.
Survival analysis in each group, with a non-protective level of antibodies against vaccine as the event of interest, was conducted using the Kaplan-Meier method. The log-rank test compared survival curves. Factors signi cantly associated with a time when the non-protective level was detected or not then tested in a Cox proportional hazards regression model, calculating the Hazard-ratio (HR). P-value < 0.05 was considered as statistically signi cant.

Demographics and vaccine coverage
The characteristics of patients with JIA included in the study presented in Table 1. We included patients with different ages and JIA categories to evaluate the vaccine coverage. The distribution in the JIA categories was similar to a common population. High proportion of children received corticosteroids (25%) and near the half received biologics: etanercept − 44%, adalimumab − 29%, tocilizumab − 20% and abatacept − 7%. Sixteen children (9.4%) received more than one biologic, consequently.

Incomplete vaccination
The majority of patients had a restricted number of vaccines which can explain a relatively h proportion of the JIA patients without non-protective levels of antibodies against vaccines. Data are in Table 2. Protective level of antibodies in the whole studied population of JIA patients ranged from 50% (against hepatitis B) to 98.2% (rubella). An incomplete complex of vaccines against MMR had 50 (42%) patients with JIA, against diphtheria 85 (50%) patients with JIA. All patients received a whole complex of vaccines against hepatitis B. The time between last vaccination and study recruitment is relatively big and ranged from 6.0 years for diphtheria to 10.9 years for hepatitis B. MMR vaccine coverage was similar between oligoarthritis (70%), polyarthritis (69%) and systemic arthritis (63%) except enthesitis-related arthritis (85%), who were elder and received more vaccine doses before the onset of the disease. The vaccination coverage pattern against diphtheria showed similar results between JIA categories and were 51%, 46%, 63% and 50%, relatively. Comparing children with complete and incomplete vaccination showed the lower levels of antibodies against mumps and diphtheria and the number of patients with protective levels of antibodies against diphtheria were found in patients with incomplete vaccination. Incomplete vaccination was associated with lower levels of antibodies against parotitis and diphtheria (Table 3). , p = 0.016) vaccines, which was con rmed with Cox proportional regress models ( Fig. 1).
Incomplete vaccination was strongly associated with the severity of arthritis and the degree of immunosuppression. Incomplete MMR vaccination had 39% of patients, treated with biologics, 22% with MTX and 14% with NSAID. A similar pattern was observed in diphtheria vaccination: 61%, 46% and 36% respectively in distinct treatment groups.

Risk factors of non-protective levels of antibodies against vaccines
The possible factors might in uence the level of antibodies against vaccine were JIA category, treatment modalities, vaccine coverage, and time since last vaccination, so best options were time-dependent statistical methods. In survival analysis, we have found differences in patients with protective and non-protective levels of antibodies only against hepatitis B (LogRank test, p = 0,018). The lowest probability of having a protective level of antibodies was observed in systemic arthritis compared to oligoarthritis (p = 0.008) and polyarthritis (p = 0.005).
JIA patients, with non-protective levels of antibodies against measles, had more extended methotrexate treatment ( Table 4.

Discussion
Our study aimed to describe vaccine coverage status and vaccine immunity status in JIA patients in a Russian tertiary centre.
The launch of vaccination drastically decreased the incidence of infections around the world and prevented many deaths [18]. The European League Against Rheumatism recommended the national guidelines for the vaccination of PRD [19]. High vaccine coverage in the population may prevent blocking infection in the circulation. It is necessary to have a very high proportion of the people with protective antibodies level. The population threshold level required for blocking the virus's circulation is 95% for measles, 90% for mumps and 85% for rubella [20]. [22]. In our study, incomplete vaccination and treatment with biologic affect the antibodies' level against measles (Fig. 1, 2, Table 4). The seroprotection level against mumps was relatively high, at 80%, but lower than local epidemiological data [22].
Incomplete vaccination and biologics were the main predictors of anti-mumps antibodies' non-protective level, similar to measles ( Fig. 1, 2, Table 4).
The situation with rubella is much better as well in our cohort, as in the population. In 400 JIA patients, the level of antibodies against diphtheria (OR 0.1; 0.06 to 0.2) and tetanus (OR 0.1; 0.05 to 0.3) were lower in JIA patients compare to 2176 healthy controls [21]. Methotrexate and corticosteroids did not signi cantly in uence the geometric median antibody concentration, except for the weak negative correlation between methotrexate usage and the antibody levels against diphtheria (r = − 0.28). In our study, incomplete vaccination, duration of methotrexate and biologics, affected the level of antibodies against diphtheria. No differences in the seroprotection rates for diphtheria were observed between JIA categories in our study and previous [21].
Vaccine Immunity Against Hepatitis B The vaccine against hepatitis B (HB) is recombinant and may be recommended for vaccination to all immune-compromised children because of safety and e cacy [30][31][32]. Japanese College of Rheumatology and the Japanese College of Hepatology considered anti-HB vaccination for unimmunized patients with JIA as soon as JIA has been under control for three months [31,32]. The protective level of anti-HBs antibodies had 50% of our studied JIA group, and all patients had complete vaccination due to the earliest course (before six months). In the Polish study, 60.7% of JIA patients with similar ages had protective levels of antibodies against hepatitis B. Girls, and patients with polyarticular JIA had the lowest levels of antibodies [33]. Only half of the patients with different PRD receiving immunosuppressive treatment had a protective anti-HBs level compare to controls -4% [33]. In Maritsi D. et al. in 89 patients with a different JIA form, only 55% had a protective level of anti-HBs antibodies and 92% in healthy controls [34]. According to the literature data, the vaccination of JIA patients against HB is safe and effective [30,31]. Kasapcopur et al. showed that the level of seroprotection was achieved by 38/39 (98%) in patients with different forms of JIA treated with methotrexate or methotrexate with prednisolone. No vaccine-associated reaction or disease ares occurred [30]. In the study of 18 JIA children without a history of anti-HB vaccination treated with different biologic drugs, in 13, the protective level con rmed. Patients received 0, 1, 6 months scheduled vaccination, and the adverse reaction was in 5 patients who were similar to healthy children. No JIA ares observed in the studied group [31]. In our group, the main predictors affecting antibodies against hepatitis B were a systemic-onset category of JIA and biologics treatment.
Several issues in the vaccination process and maintenance of anti-vaccine antibodies have identi ed. Many patients (near 40%) who developed arthritis early (before 6-7 years) often miss the following scheduled vaccinations, e.g. measles, mumps, diphtheria, tetanus [35]. Usually, most patients with oligoarthritis and half of the RF-negative polyarthritis patients are in the risk of incomplete vaccination [36]. Incomplete vaccination is a usual problem for JIA and other PRD patients. Incomplete vaccination against MMR had 42% and against diphtheria 50% of our patients. In Slovenian study, 35% of 187 PRD children had incomplete vaccination. The mostly omitted vaccines were hepatitis B and the second dose of MMR similar to our group [37]. In Canadian study with 200 JIA children, the incomplete vaccination had 48%, 32%, and 39% of patients at 2.5 years, 10.5 years and their last clinic visit, respectively [38]. In 715 JIA children from Germany, incomplete vaccination status had one-third of patients. The main reason for incomplete vaccination was physicians' suggestion. The vaccination coverage in preschool children was similar to healthy matches and lower in adolescents (24-79% for diphtheria and tetanus and 60-75% for MMR) with JIA. More incomplete vaccination cases were among polyarticular and systemic JIA patients than oligoarticular and who received immunosuppressive therapy compared to children without immunosuppression [39]. Our results are similar. Minimal vaccine coverage has been observed in patients with immunosuppression therapy (biologics, methotrexate) compared to patients with NSAID alone (Table 4).
Interestingly, incomplete vaccination affects predominantly anti-measles, anti-mumps, and anti-diphtheria antibodies maintenance without signi cant in uence on other vaccine-speci c antibodies. In our cohort, the main reasons for interrupted or omitted vaccination were parental fear of JIA are. Similar professional behaviour of Russian healthcare providers was observed too, as in Canada and in Brazil, where 38% of 82 and 43.5% of 207 PRD consequently have omitted the vaccination due to fear of parents or the recommendation of the physicians [40,41]. There are many concerns from patients, their parents, and healthcare providers about vaccination safety in immunocompromised children, e.g. JIA [13, 15; 42]. Many physicians had concerns and uncertainty about the role of vaccines in the JIA ares, which realized in the interruption or discordance in the vaccine practice. Physicians make some delays in the scheduled vaccination before a certain period (e.g., stabilizing the disease or more prolonged remission) or neglecting some vaccine [13,42]. Our primary care physicians and some pediatric rheumatologists, unfortunately, acted in the same way.
The main risks of incomplete vaccination related to JIA's onset age and parents or healthcare providers have started to omit vaccination. In our study, patients with onset age less than four years had a risk of incomplete vaccination of MMR (OR = 12.2 The second issue is related to anti-rheumatic drugs' effect on the immune system functioning, especially B-cells and memory B-cells. Children with PRD may have reduced levels of anti-vaccine antibodies against measles, mumps, rubella, diphtheria, tetanus and hepatitis B compared to age-matched healthy controls [21,34]. Children with PRD may have lower levels of antivaccine antibodies if they have not even received any medications [43]. Several studies in PRD patients showed a similar response to healthy controls, but sometimes the antibody levels might be lower. Disease activity usually does not affect the antibody generation and maintenance if vaccinated patients were not in the remission [44][45].
The main unresolved question is related to the different ability of anti-rheumatic medications affects the levels of antibodies against the vaccine. In previous studies, methotrexate and corticosteroids' negative effect on the antibodies against measles, mumps, rubella, diphtheria, and tetanus was not shown [21,27,28,46,47]. However, other studies have shown the opposite data [31,33,48].
Revaccination of JIA patients is effective and safe and should be encouraged, especially in patients with incomplete vaccination or low anti-vaccine antibody levels. In the randomized study, the antibody against measles was higher in the JIA patients, who received revaccination compare to control group (JIA patients have not received revaccination). No cases of measles, mumps and rubella were detected after the vaccination [26]. According to the EULAR recommendations-2011 inactivated vaccines are safe, independently to a type of treatment and live attenuated vaccines should be used individually on the case-to-case basis, regarding disease activity, treatment modalities and the risk-bene t ratio. However, more precise recommendations and guidelines are required [19].
In real practice, the situation with the vaccination of immunocompromised children looks safe and effective. No increased are rate was shown in JIA patients during the vaccination in several studies. In Australia, the are rate during 90-days after vaccination was lower than patients' baseline risk (RR = 0.59 (95% CI 0.39-0.89, p = 0.012). Authors explained the reduced risk that "children may be healthier than usual' by the time of vaccination, a vaccine was delayed before the time "free of viral infection" and so on [50]. In the literature, there are at least 29 studies about vaccination of JIA patients. Among them were different types of articles, ranged from case reports to randomized trials. In all studied vaccines against in uenza, MMR, varicella, pneumococcus, human papillomavirus and hepatitis B in JIA patients no increased ared risk, related to vaccination was identi ed [25,[49][50].
In our study, the main predictors affected the anti-vaccine antibody levels were incomplete vaccination, JIA category, duration of methotrexate treatment and biologics. Corticosteroids did not affect the antibodies levels, except the anti-diphtheria antibodies, possibly due to using them in a short-term manner, as the bridge therapy. According to EULAR recommendation and published data about vaccine status we can strongly recommend routinely check patient's vaccine schedule and in patients with incomplete vaccination and patients having a risk of non-protective levels of antibodies should routinely check the anti-vaccine antibody levels and encourage patients and their families to continue vaccination with individual vaccination program [19]. It is necessary to provide simple algorithms for primary care physicians about the management of vaccinations, routine antibodies checking. The vaccine coverage in our population is non-optimal compare to other countries and our healthy population. Interdisciplinary communication between rheumatologist, immunologist, primary care physicians and healthcare providers is still required to improve vaccine coverage in JIA patients.

Limitations Of The Study
The present study's main limitations are related to JIA patients' differences in age, JIA categories, treatment approaches, and the time gap between last vaccination and study recruitment. The different onset age and duration of the disease before the obtaining samples are the additional factors that in uence anti-vaccine antibodies' survival to diseases pathogenesis and treatment.

Conclusion
Children with JIA have lower antibody levels, and many JIA patients have non-protective levels of antibodies against vaccines and require a routine check. Incomplete vaccination, JIA category, biologics, corticosteroids, and long-term methotrexate treatment might be supposed to be the risk factors of aberrant vaccine immunity. Individual vaccination schedule required for JIA patients without protective antibody levels and should be tailored by individual basis with antibody levels sampling. It is necessary to decrease the level of parental fair and healthcare providers against vaccination in Russia. Further studies about the safety and e cacy of vaccination in JIA patients required.

Declarations
Ethics: Written consent has obtained according to the declaration of Helsinki. The Saint Petersburg State Pediatric Medical University's local Ethics Committee approved the trial protocol (protocol number 9/2 from 02.09.2019).
Consent for publication: All authors are agree to publish the manuscript Availability of data and material: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. M. M. Kostik: Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; nal approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. ORCID: (0000-0002-1180-8086), e-mail: kost-mikhail@yandex.ru; mikhail.kostik@gmail.com N. A. Lubimova: Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; nal approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. ORCID: (0000-0002-3187-8997), e-mail: natali18111989@gmail.com I.V. Fridman: Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; nal approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. ORCID: (0000-0002-2633-491X) e-mail: fridiv@mail.ru Olga V. Goleva: Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; nal approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. ORCID: (0000-0003-3285-9699) e-mail: golev.ao@mail.ru S. M. Kharit: Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; nal approval of the version to be published; Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. ORCID: (0000-0002-2371-2460), e-mail: kharit-s@mail.ruAll authors read and approved the nal manuscript. JIA patients, with non-protective levels of antibodies against measles, had more extended methotrexate treatment (2.8 [1.3; 6.4] vs 2.2 [0.9; 3.9] years, p<0.05) and increased applying of the biologics (76% vs 52%, p<0.05) compare to the patients with protective levels of antibodies. Patients treated with biologics had the lowest probability of having protective levels of antibodies against measles, mumps, hepatitis B, and diphtheria than MTX and NSAID