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\begin{document}

\title{Safety and immunogenicity of the second and third doses of COVID-19
vaccine in adolescents with rheumatic diseases}



\author[1]{Merav Heshin-Bekenstein}%
\author[2]{Amit Ziv}%
\author[3]{Natasa Toplak}%
\author[1]{Siman Lazauskas}%
\author[4]{Danielle Kadishevich}%
\author[4]{Efrat Ben-Nun Yaari}%
\author[5]{Adi Miller-Barmak}%
\author[5]{Yonatan Butbul Aviel}%
\author[1]{Esther Saiag}%
\author[1]{Sara Pel}%
\author[1]{Ori Elkayam}%
\author[2]{Yosef Uziel}%
\author[1]{Victoria Furer}%
\affil[1]{Tel Aviv Sourasky Medical Center}%
\affil[2]{Tel Aviv University Sackler Faculty of Medicine}%
\affil[3]{Univerzitetni klinicni center Ljubljana Pediatricna klinika}%
\affil[4]{Meir Medical Center}%
\affil[5]{Rambam Health Care Campus}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background}: To explore the long-term safety and dynamics of the
immune response induced by the second and third doses of the BNT162b2
mRNA COVID-19 vaccine in adolescents with juvenile-onset autoimmune
inflammatory rheumatic diseases (AIIRD) compared with healthy controls.
\textbf{Methods}: This international prospective study included
adolescents with AIIRD and controls vaccinated with two (AIIRD n=124;
controls n=80) or three (AIIRD n=64; controls n=30) doses of the
BNT162b2 vaccine, evaluated for vaccine side-effects, disease activity,
COVID-19 breakthrough infection rates and severity, and anti-spike S1/S2
IgG antibody titres in a sample from both groups. \textbf{Results}: The
vaccination safety profile was favourable, with most patients reporting
mild or no side-effects. The rheumatic disease remained stable among
98\% and 100\% after the second and third doses, respectively. The
two-dose vaccine induced comparable seropositivity rates among patients
(91\%) and controls (100\%), (p=0.55), which declined within 6 months to
87\% and 100\%, respectively (p=0.3), and increased to 100\% in both
groups, after the third vaccine dose. The overall post-vaccination
COVID-19 infection rate was comparable between patients and controls,
47.6\% (n=59) and 35\% (n=28), respectively; p=0.5278, with most
infections occurring during the Omicron surge. In relation to the last
vaccination, time-to-COVID-19 infection was similar between patients and
controls, at a median of 5.5 vs. 5.2 months, respectively (log-rank
p=0.1555). \textbf{Conclusion}: The safety profile of three doses of the
BNT162b2 mRNA vaccine was excellent, with an adequate humoral response
and similar efficacy among patients and controls. These results support
the recommendation for vaccinating adolescents with juvenile-onset AIIRD
against COVID-19.%
\end{abstract}%



\sloppy


\textbf{Safety and immunogenicity of the second and third doses of
COVID-19 vaccine in adolescents with rheumatic diseases}

Merav Heshin-Bekenstein\textsuperscript{1,2}, Amit
Ziv\textsuperscript{2,3}, Natasa Toplak\textsuperscript{4,5}, Siman
Lazauskas\textsuperscript{6}, Danielle Kadishevich\textsuperscript{7},
Efrat Ben-Nun Yaari\textsuperscript{7}, Adi
Miller-Barmak\textsuperscript{8}, Yonatan Butbul
Aviel\textsuperscript{8}, Esther Saiag\textsuperscript{9}, Sara
Pel\textsuperscript{10}, Ori Elkayam\textsuperscript{2,10} Yosef
Uziel\textsuperscript{2,3}\textsubscript{,} Victoria
Furer\textsuperscript{2,10}

\textsuperscript{1}Pediatric Rheumatology Service, Dana-Dwek Children's
Hospital, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

\textsuperscript{2}Sackler Faculty of Medicine, Tel Aviv University, Tel
Aviv, Israel

\textsuperscript{3}Pediatric Rheumatology Unit, Meir Medical Center,
Kfar Saba, Israel

\textsuperscript{4}Department of Allergology, Rheumatology and Clinical
Immunology, University Children's Hospital, University Medical Center
Ljubljana, Ljubljana, Slovenia

\textsuperscript{5}Faculty of Medicine, University of Ljubljana,
Ljubljana, Slovenia

\textsuperscript{6}Pediatric Department, Dana-Dwek Children's Hospital,
Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

\textsuperscript{7}Pediatric Department, Meir Medical Center, Kfar Saba,
Israel

\textsuperscript{8}Pediatric Rheumatology Unit, Rambam Medical Center,
Haifa, Israel

\textsuperscript{9}Hospital Management, Information and Operation
Branch, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

\textsuperscript{10}Rheumatology Department, Tel Aviv Sourasky Medical
Center, Tel Aviv, Israel

\textbf{Running title:} COVID vaccine in rheumatic adolescents.

\textbf{Corresponding author}

Merav Heshin Bekenstein, MD

Pediatric Rheumatology Service

Dana Dwek Children's Hospital

Tel Aviv Sourasky Medical Center

6 Weizmann Street

Tel Aviv 6423906 Israel

Email: \emph{meravheshin@gmail.com}

ORCID ID:\emph{https://orcid.org/0000-0002-9488-6476}

\textbf{Word count:} 3429 words.

\textbf{Conflict of interest statement}

The authors declare no conflicts of interest.

\textbf{Funding support}

This study was partially funded by a research grant from the Israel
Society for Clinical Pediatrics. No other funding was received from any
commercial entity to carry out the work described in this article.

\textbf{ABSTRACT}

\textbf{Background} : To explore the long-term safety and dynamics of
the immune response induced by the second and third doses of the
BNT162b2 mRNA COVID-19 vaccine in adolescents with juvenile-onset
autoimmune inflammatory rheumatic diseases (AIIRD) compared with healthy
controls.

\textbf{Methods} : This international prospective study included
adolescents with AIIRD and controls vaccinated with two (AIIRD n=124;
controls n=80) or three (AIIRD n=64; controls n=30) doses of the
BNT162b2 vaccine, evaluated for vaccine side-effects, disease activity,
COVID-19 breakthrough infection rates and severity, and anti-spike S1/S2
IgG antibody titres in a sample from both groups.

\textbf{Results} : The vaccination safety profile was favourable, with
most patients reporting mild or no side-effects. The rheumatic disease
remained stable among 98\% and 100\% after the second and third doses,
respectively. The two-dose vaccine induced comparable seropositivity
rates among patients (91\%) and controls (100\%), (p=0.55), which
declined within 6 months to 87\% and 100\%, respectively (p=0.3), and
increased to 100\% in both groups, after the third vaccine dose. The
overall post-vaccination COVID-19 infection rate was comparable between
patients and controls, 47.6\% (n=59) and 35\% (n=28), respectively;
p=0.5278, with most infections occurring during the Omicron surge. In
relation to the last vaccination, time-to-COVID-19 infection was similar
between patients and controls, at a median of 5.5 vs. 5.2 months,
respectively (log-rank p=0.1555).

\textbf{Conclusion} : The safety profile of three doses of the BNT162b2
mRNA vaccine was excellent, with an adequate humoral response and
similar efficacy among patients and controls. These results support the
recommendation for vaccinating adolescents with juvenile-onset AIIRD
against COVID-19.

\textbf{Keywords:} vaccines, COVID-19, rheumatology, safety,
immunomodulation, adolescent.

\textbf{Key message:}

3-dose COVID-19-vaccine has excellent safety profile in adolescents with
rheumatic diseases; minimal risk of exacerbation. Adequate long-term
immunogenicity in healthy controls and most rheumatic adolescents; the
third dose restored titres. Breakthrough COVID-19 infection rates and
time from last vaccination to infection were similar between groups.
BNT162b2 mRNA COVID-19 booster vaccine is recommended for adolescents
with rheumatic diseases

\textbf{INTRODUCTION}

Patients with juvenile-onset autoimmune inflammatory rheumatic diseases
(AIIRD) on immunomodulatory treatment are prone to infections, including
SARS CoV-2 infection. Thus far, vaccination, along with personal
protective measures have been the mainstay approach to preventing
COVID-19 infection.

Studies on adults with AIIRD demonstrated that the two-dose BNTb262
regimen was associated with comparable immunogenicity in patients and
healthy controls. Most patients developed an adequate immunogenic
response to the vaccines, except for patients on B-cell-depleting
therapy.

Data regarding the safety and immunogenicity of COVID-19 vaccination in
adolescents with AIIRD on immunomodulation was initially lacking, as
these individuals were excluded from the vaccine trials. Since 2022, a
few studies demonstrated that the two-dose COVID-19 vaccine regimen was
safe and immunogenic in adolescents with AIIRD as compared to healthy
adolescents.

The waning of vaccine-induced immunity after the two-dose BNT162b2 mRNA
vaccine regimen was well-documented. In October 2021, the American
College of Rheumatology recommended a booster vaccine dose for AIIRD
patients receiving any immunosuppressive treatment. This was also
supported by the EULAR and PReS recommendations.

Since then, several studies have shown that the third vaccine dose
induce an augmented immunogenic response among patients with AIIRD. The
booster vaccination was also shown to reduce the rates of both confirmed
COVID-19 and severe COVID-19 in a large Israeli population of
participants ages60 years or older.

However, longitudinal data on the safety and the dynamics of anti-S
antibody titre following the second and third doses of COVID-19 vaccine
in adolescents with AIIRD receiving different immunomodulatory regimens,
is lacking. Here, we report the results of a multicentre, prospective
longitudinal study, conducted to investigate the safety and dynamics of
the immunogenic response following the second and third doses of the
BNT162b2 COVID-19 vaccine in adolescents with AIIRD and healthy
controls.

\textbf{METHODS}

This international, prospective, multicentre study was conducted from
April 2021 to July 2022 at the Pediatric Rheumatology Clinics of
Dana-Dwek Children's Hospital (Tel Aviv), Meir Medical Center (Kfar
Saba), and Rambam Medical Center (Haifa) in Israel, and at the
University of Ljubljana Children's Hospital in Slovenia.

\textbf{Ethical approval information}

The study was approved by the Institutional Review Board (IRB) of Tel
Aviv Sourasky Medical Center, TLV-0175--21 (coordinating centre), and by
the IRBs of the participating centres. The study was performed in
accordance with the principles of the Declaration of Helsinki. Written
informed consent was obtained from all study participants.

\textbf{Study endpoints}

The primary endpoint of the study included the long-term safety of the
BNT162b2 mRNA vaccine, as evaluated by side-effects following the second
and third doses in adolescents with AIIRDs compared with healthy
controls, and the impact of vaccination on clinical disease activity in
the patient group. The secondary endpoints were long-term immunogenicity
of the BNT162b2 mRNA vaccine in adolescents with AIIRDs measured at
2--8 weeks and 6 months after the second vaccine and at 2--8 weeks after
the third vaccine dose, compared with controls. Breakthrough COVID-19
infections were recorded based on positive SARS-CoV-2 polymerase chain
reaction (PCR) tests, documented [?]14 days following vaccination.

\textbf{Study population}

The study population included adolescents (ages 12--18 years) with
juvenile-onset of the following AIIRDs: JIA according to the EULAR 2001
classification criteria; systemic lupus erythematosus (SLE) according to
the 1997 ACR or 2012 SLICC criteria; systemic vasculitis, i.e.
ANCA-associated vasculitis (AAV), including granulomatosis with
polyangiitis (GPA), according to the Chapel Hill Consensus Conference
definitions; Beh\selectlanguage{ngerman}çet's disease, idiopathic uveitis, IBD-related
arthritis, systemic or localized scleroderma or juvenile dermatomyositis
according to the EULAR/ACR classification criteria; or autoinflammatory
syndromes. Patients were instructed to continue all medications during
the vaccination period. Patients were recruited from four medical
centres, including 97 (78\%) participants from Israel and 27 (22\%) from
Slovenia. The control group consisted of healthy adolescent volunteers
recruited by Dana Dwek Children's Hospital, who provided blood samples
during a well-being visit. Exclusion criteria were previous COVID-19
infection and for controls, history of an AIIRD or immunosuppressive
treatment.

All study participants were vaccinated with two doses of BNT162b2 mRNA
vaccine (30\selectlanguage{english} \selectlanguage{greek}μ\selectlanguage{english}g per dose, administered intramuscularly, 3 weeks apart).
The third vaccine dose was administered at least 6 months after the
first vaccine dose, as indicated by the national guidelines of Israel.

\textbf{Safety and clinical assessment of the AIIRD}

The participants were contacted by telephone within 2--8 weeks following
the second and third vaccine doses and completed a questionnaire
regarding local and systemic side-effects.

Medical history and the use of medications were recorded.
Immunomodulating medications were continued throughout the study period.
Treatment was not withheld before or after the vaccine dose. Data
regarding disease activity up to 3 months before vaccination were
retrieved from the patients' medical records. Post-vaccination disease
activity was assessed by in-person clinical examination within 3 months
after each vaccine dose and at 1-year intervals after the first vaccine.

The following disease activity indices were included: Juvenile Arthritis
Disease Activity Score 10 for JIA, SLEDAI for SLE, and patients' and
physicians' global assessments (PGA, PhGA respectively), using a visual
analogue scale of 0--10 mm, for JIA, SLE, vasculitis, inflammatory
myositis, scleroderma, uveitis, and autoinflammatory syndromes.

\textbf{Vaccine immunogenicity}

Vaccine immunogenicity was evaluated for a subgroup of patients and
controls by measuring serum IgG antibody levels against SARS-CoV-2
trimeric spike S1/S2 glycoproteins, 2--8 weeks after the second and the
third vaccine doses. We used the FDA-authorized LIAISON (DiaSorin,
Sallugia, Italy) quantitative assay. The assay provides an indication
for the presence of neutralizing IgG antibodies against SARS-CoV-2 and
its clinical sensitivity and specificity is \textgreater{}98\%. A value
of \textgreater{}15 binding antibody units (BAU) was considered positive
according to the manufacturer's instructions.

\textbf{Breakthrough COVID-19 infection rate}

Breakthrough infection among fully vaccinated individuals was defined
according to the US Centers for Disease Control and Prevention, as
infection occurring [?]14 days after the second dose in a two-dose series
or [?]14 days after a single-dose vaccine. Participants were queried for
evidence of breakthrough COVID-19 infection following each vaccine dose
and electronic medical records were reviewed for the evidence of
infection 6 months after each vaccination, based on the results of
positive SARS-CoV-2 PCR tests. Follow-up was 1 year after the first
vaccine dose.

\textbf{Patient and public involvement}

The study research question and outcome measures were developed in
collaboration with the parent representatives of the adolescents with
AIIRD, based on a shared priority to investigate the long-term immune
response to the mRNA BNT162b2 vaccine. Parents and patients with AIIRD
under the care of the medical centres conducting the trial were actively
informed regarding the study and offered enrolment. The main study
results will be disseminated to the parent representatives, who will
help in the disseminating them through parent networks locally and
abroad.

\textbf{Statistical analysis}

Differences between continuous variables were tested for significance
using the independent-samples~\emph{t} -test. Differences between
categorical variables were tested for significance using the chi-squared
test or Fisher's exact test, each as appropriate. All tests applied were
two-tailed and a~\emph{P} -value [?]0.05 was considered statistically
significant. Survival was analysed with Kaplan-Meier plots. Median
survival durations were reported with 95\% confidence intervals (CI).
Differences between groups were tested using the Log-Rank test. The data
were analysed using R version 4.0.5 (R Foundation for Statistical
Computing, Vienna, Austria).

\textbf{RESULTS}

\textbf{Study sample}

A total of 124 adolescents with AIIRDs and 80 healthy adolescents,
vaccinated with two doses of BNT162b2 mRNA vaccine, participated in the
study (Table 1). Among the participants, 64 (54.6\%) patients and 30
(37.5\%) controls were also vaccinated with the third dose. Both patient
and control groups had comparable gender distributions of 60\% and 51\%
females, respectively (p=0.27). Adolescents with AIIRD were
significantly older than controls, mean age 15.5 years (SD 1.99)
compared with 13.7 years (SD 1.36), p\textless{}0.0001, respectively.
JIA was the most common disease (n=54, 44\%), followed by SLE (n=20,
16\%). The mean disease duration was 4.12 years (SD. 3.79).

Most patients, n=92 (74\%), were receiving immunomodulatory therapies
(Table S1). Conventional synthetic DMARDs (csDMARDs) were used by 57
(45.9\%) patients, with methotrexate and plaquenil the most frequent
(n=27 (21.8\%) and n=26 (21.1\%), respectively), followed by
mycophenolate mofetil (MMF) (n=13, 10.5\%). Glucocorticoids were used by
21 (16.9\%) patients, mean dose 11.6 mg/day (SD 11.88). Biologic DMARDs
(bDMARDs) were used by 48 (38.7\%) patients. TNF inhibitors (TNFi) were
the most common bDMARD, used by 39 (31.5\%) patients, followed by the
IL-6 inhibitor (n=7, 5.7\%) and CD-20 depleting therapy (rituximab
(RTX); n=4, 3.2\%). Janus kinase inhibitors were used by 5 (4\%)
patients.

\textbf{Safety of the BNT2b2 vaccine}

The safety profile of the 3 vaccine doses was favourable, with most
participants experiencing either no or mild side-effects (Table 2).
Local reactions to vaccination were more common among AIIRD patients
compared with controls at 2--8 weeks after the second vaccination, 71\%
(n=88) and 55\% (n=44), p=0.0293, respectively; whereas after the third
vaccine dose, AIIRD patients had significantly fewer local reactions
than controls did, 39\% (n=25) vs. 73\% (n=22), p=0.004, respectively.
The prevalence of systemic side-effects was comparable between patients
with AIIRD and controls. The most common systemic side-effects were
fatigue, myalgia, headache and arthralgia, with a comparable prevalence
among AIIRD patients and controls. Importantly, there were no serious
side-effects in either group. No cases of pericarditis or myocarditis
were observed. Five patients (4\%) experienced an exacerbation of
rheumatic disease shortly after the first vaccine dose and 3 patients
(2\%) after the second dose. Two patients\textbf{,} both with AAV, were
hospitalized shortly after the first vaccine dose, as previously
reported by our group. None of the patients who received the third
vaccine reported subsequent rheumatic disease exacerbation or
hospitalization. Disease activity remained stable at 1-year clinical
follow-up in all patients.

\textbf{Humoral response to the BNT162b2 second and third vaccine doses}

After the second vaccine dose, 37 patients and 23 controls provided
serum samples for immunogenicity; 36 patients and 14 controls provided
serum samples 6 months later. A total of 9 patients and 9 controls
provided samples after the third vaccine. The dynamics of the S1/S2
antibody level among both study groups are presented in Figures 1 and 2.
Among the control group, the seropositivity rate was 100\% at all three
time points. Anti-S1/S2 antibody titres declined at 6 months after the
second vaccine from a mean of 388.3 (SD 56.09) BAU/ml to 212.93 (SD
93.69) BAU/ml, and was restored shortly after the third vaccine dose, to
400 (SD 0) BAU/ml. As expected, the seropositivity rate was lower among
AIIRD patients compared to controls, 91\% at 2-8 weeks and 87\% at 6
months after the second vaccination, and returned to 100\% following the
third dose. Anti-S1/S2 antibody titres declined at 6 months after the
second vaccine from a mean of 243.1 (SD 140.8) BAU/ml to 216.9 (SD
157.6) BAU/ml, and increased shortly after the third vaccine dose to
355.8 (SD 126.4) BAU/ml. In comparison to the control group, anti-S1/S2
antibody titres among the patients were significantly lower only at 2--8
weeks after the second vaccine dose (p\textless{}0.0001) and comparable
at the subsequent 6-month follow-up after the second, and 2-8 weeks
after the third dose. Notably, the decline of the anti-S1/S2 antibody
titres within 6 months after the second vaccine dose was significantly
steeper among the controls compared to patients, with a mean of -197 (SD
87.4) vs. -61.2 (SD 151.4), p=0.0197, respectively. In the limited
sample of controls (n=5) and patients (n=2) with available serologic
data, the increase in the antibody titre after the third vaccine dose
was comparable in both groups.

Patients with JIA maintained the highest level of anti-S1/S2 antibody at
all time-points, whereas the lowest anti-S1/S2 antibody levels were
found among patients with vasculitis and scleroderma (Supplement Table
2).

Analysis of humoral response according to the use of anti-rheumatic
medications showed that patients treated with csDMARDs and bDMARDs
developed a comparable humoral response at all points tested. Among 17
patients treated with TNFi, those treated with TNFi monotherapy achieved
numerically higher anti-S1/S2 antibody levels than those treated with
combination therapy. MMF was most strongly associated with blunted
humoral response to vaccination after the second vaccine dose (115\selectlanguage{ngerman}± 89).
The humoral response following the third vaccine dose evaluated in 2 of
6 patients treated with MMF was completely restored. Notably, 4 patients
treated with anti-CD20 therapy (RTX), 2 patients with AAV, 1 patient
with SLE, and 1 with systemic sclerosis developed an adequate
seropositive response after the second vaccine dose, with the steepest
decline in anti-S1/S2 antibody levels within 6 months, compared to
antibody kinetics of patients treated with other medications. The
humoral response following the third vaccine dose evaluated in one of
these patients was completely restored. Patients treated with RTX were
vaccinated 6 months after the last RTX dose and before the next dose.

\textbf{Efficacy of the second and third BNT162b2 vaccine doses}

The prevalence of COVID-19 infection observed from the second vaccine
dose and forward was 47.6\% (59/124) among adolescents with AIIRD and
35\% (28/80) among controls, p=0.5278 (Table 3). Most of the
breakthrough infections (88\%) occurred during the Omicron surge. During
the Delta surge, the prevalence of infection was numerically higher in
the patient group than in the healthy controls (n=7, 11\% vs. n=1, 3\%,
p=0.1555).

The prevalence of a breakthrough infection was similar among patients
with JIA and SLE. Time-to-infection following the last vaccination
was~similar~among AIIRD patients compared to controls, at a median of
5.5 vs. 5.2 months,~respectively; log-rank p=0.6499 (Figure 3). In both
patient and control groups, all cases of COVID-19 infection were mild,
with no severe infections or hospitalizations and no COVID-19 related
complications.

\textbf{DISCUSSION}

To the best of our knowledge, this prospective, multicentre,
international study is the first to report the long-term safety of the
BNT162b2 mRNA COVID-19 vaccine and the immunogenicity over time, induced
by two and three doses of the vaccine,~in a cohort of adolescents with
juvenile-onset AIIRD, treated with a variety of immunomodulating agents.

The safety profile of the second and third vaccine doses was highly
favourable, with most participants experiencing either no or mild
side-effects and no serious side-effects in either group. Two percent of
the AIIRD cohort experienced an exacerbation of the rheumatic disease
shortly after the second vaccine dose and none of the patients who
received the third vaccine reported subsequent rheumatic disease
exacerbation or hospitalization.

The initial 2-dose vaccine was shown to be immunogenic in 91\% of the
AIIRD adolescents vs. 100\% of healthy controls. However, anti-S1/S2
titres were significantly lower in the AIIRD group; similar to reports
in adults with AIIRD.

Previous studies have shown waning antibody response to COVID-19
vaccination in the general population and AIIRD. In our patient and
control groups, we observed waning of the anti-S1/S2 antibody titres
over 6 months, with a faster decline in the control group compared to
patients. This finding needs further clarification in larger trials.

Following the third vaccine, the seropositivity rate increased to 100\%
in adolescents with AIIRD. Furthermore, anti-S1/S2 titres reached a
higher level as compared to those measured following the 2-dose vaccine,
supporting the recommended booster vaccine dose in adolescents with
AIIRD and also consistent with the response observed in adults with
AIIRD. Notably, in the largest cohort of AIIRD adults, seropositivity
after the third vaccine (82.3\%) increased to a rate similar to that
documented after the second dose (84.7\%). The more vigorous response to
the third dose in our adolescent patient cohort compared to adult
patients may be explained by a stronger immunogenic response at younger
ages and/or the more prevalent use of combination anti-rheumatic therapy
in the adult cohort.

A meta-analysis published in 2022 showed that pooled seroconversion
rates after a two-dose SARS-CoV-2 vaccination regimen were lower in
patients with immune-mediated inflammatory disease compared to healthy
controls. Importantly, certain therapies did not impact the
seroconversion rates, including anti-TNF, anti-integrin, anti-IL-17,
anti-IL6, and anti-12/23, while other therapies resulted in poorer
responses including anti-CD20 and anti-CTLA-4. The current study was not
large enough to determine the impact of the anti-rheumatic medication on
humoral response to vaccination. We reported an adequate humoral
response in patients treated with methotrexate and anti-TNF, and a
blunted humoral response after the second vaccine dose in a small group
of patients treated with MMF, which is consistent with a previous
report. According to the literature regarding adults with AIIRD, RTX is
a dominant factor for the lack of antibody response to the COVID-19
vaccine at all time points, especially among patients treated with RTX
within 6 months prior to vaccination. B-cell depletion also consistently
correlates with negative immunological response. Our small cohort of
adolescents treated with RTX had a good humoral response. This might be
because the vaccines were given at the appropriate times or because of
the favourable immunogenic response at young ages.

It is important to note that there is still no consensus regarding the
correlation between anti-spike antibody levels and protection from
COVID-19 infection. Vaccination against COVID-19 induces both humoral
and cellular responses, but it is widely thought that vaccine-induced
neutralizing antibodies to the receptor binding domain of the SARS-CoV-2
S protein are a plausible mechanism of protection. A few studies in
healthy adults demonstrated a significant correlation between
neutralizing antibody titres and vaccine efficacy, with most of the
breakthrough infections reported as mild or asymptomatic. Sakir et al.
studied 630 patients with AIIRD and reported an association between
breakthrough infections and sero-negativity following a COVID-19
vaccination. This provides a basis for exploring postvaccination
antibody titres as a potential predictor of breakthrough infection in
patients with AIIRD.

The breakthrough infection rate in this study was increased overall, but
still comparable between patients with AIIRD and controls. Most of the
breakthrough COVID-19 infections occurred either before or after the
third vaccine. The median time from last vaccine dose to the COVID-19
infection, was 5 months in both groups. We suggest this as a means to
estimate the actual protection from the vaccines. Most cases (88\%) were
documented during the Omicron surge, which is not surprising, as it is
now evident that the original COVID-19 vaccine did not provide enough
protection from the Omicron strain. It is notable that during the Delta
surge, the breakthrough infection rates were low, similar to a previous
report in adolescents with AIIRD. Even though the COVID-19 infection
rates during the Delta and Omicron waves were higher among the AIIRD
patients as compared to the healthy controls, the differences were not
statistically significant.

These data are encouraging to the community of adolescents with
juvenile-onset AIIRD and can help reduce hesitancy to vaccinations. The
finding that no severe COVID-19 cases were documented can be explained
by the effectiveness of the vaccine, that most of the young population
experienced a mild form of the disease. In addition, the Omicron variant
mostly caused mild illness.

There were several limitations to this study. The number of participants
who provided serum samples for humoral response evaluation at 6 months
and following the third vaccine decreased significantly, despite the
ongoing COVID-19 pandemic. This is probably because the population was
less compliant with the third vaccine dose.

In addition, due to the relatively small sample size, and specifically
the diversity of rheumatic diseases and medications included in this
juvenile-onset cohort, it was not possible to investigate the impact of
immunomodulatory medications and type of disease on the anti-S1/S2
titres. In addition, the matching by age was not optimal, as the control
group was younger than the patient group, with mean ages of 13.7 and
15.5 years, respectively. However, this might actually strengthen our
findings of high seropositivity rates, as younger control children could
have more robust immune activity. This study did not include
neutralization assays or cellular immunity.

To summarize, this is the first longitudinal study of adolescents with
AIIRD to report long-term safety, immunogenicity dynamics and
breakthrough COVID-19 infections after the second and third doses of the
BNT162b2 mRNA COVID-19 vaccine. We found an excellent safety profile
with minimal to no side effects after the vaccinations overall and a
minimal risk of rheumatic disease exacerbation. In addition, the rates
of breakthrough COVID-19 infections and time from the last vaccination
to infection were similar between groups. All the healthy controls and
most of the adolescents with AIIRD were seropositive following the
second vaccine, followed by a decline in anti-spike S1/S2 antibody
titres in both groups over the 6 month period after the second vaccine.
The titres were restored in all patients and controls after the third
vaccine dose.

The findings of this study can help decrease vaccine hesitancy in
adolescents with juvenile-onset AIIRD. Based on our results, it is
recommended to booster adolescents with rheumatic disease with the
BNT162b2 mRNA COVID-19 vaccine. Additional studies with larger numbers
of adolescents with AIIRD are needed to evaluate the impact of
anti-rheumatic/immunomodulatory therapies on the long-term immune
response to the BNT162B2 vaccine.

\textbf{Acknowledgments}

We thank the families and adolescents who participated in the study. We
thank Yishai Friedlander, MPH, for performing the statistical analysis.
We thank Faye Schreiber, MS, for editing the manuscript.

\textbf{Contributors}

MHB and YU are responsible for the study conception. MHB has directed
and coordinated the study. MHB, YU and OE designed the study. MHB, AZ,
NT, SL, DK, EBY, AMB, YAB, ES recruited participants into the study. SP
helped with sample handling and coordination. MHB and VF wrote the
manuscript, that was critically reviewed by YU, OE, AZ, NT, YAB, ES.

\textbf{Data availability statement}

All the data relevant to this study are included in the manuscript,
tables, and figures. Additional data will be provided upon request.

\textbf{References}

\textbf{Table 1: Baseline characteristics of adolescents with AIIRD and
controls}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Variable} & \textbf{AIIRD (124)} & \textbf{Controls (80)} &
\textbf{P-Value}\tabularnewline
\midrule
\endhead
Demographics & & &\tabularnewline
Age, mean (SD) & 15.48 (1.99) & 13.74 (1.36) &
\textless{}.0001\tabularnewline
Female, N (\%) & 74 (60\%) & 41 (51\%) & 0.2682\tabularnewline
Country & & &\tabularnewline
Israel, N (\%) & 97 (78\%) & 80 (100\%) &
\textless{}.0001\tabularnewline
Slovenia, N (\%) & 27 (22\%) & &\tabularnewline
Disease duration, mean (SD) & 4.12 (3.79) & &\tabularnewline
AIIRD, N (\%) & 124 (100\%) & &\tabularnewline
JIA, N (\%) & 54 (44\%) & &\tabularnewline
SLE, N (\%) & 20 (16\%) & &\tabularnewline
Autoinflammatory diseases, N (\%) & 11 (9\%) & &\tabularnewline
Scleroderma, N (\%) & 7 (6\%) & &\tabularnewline
Vasculitis, N (\%) & 6 (5\%) & &\tabularnewline
Uveitis, N (\%) & 8 (6\%) & &\tabularnewline
Myositis, N (\%) & 5 (4\%) & &\tabularnewline
IBD related arthritis, N (\%) & 7 (6\%) & &\tabularnewline
Other, N (\%) & 6 (5\%) & &\tabularnewline
Immunosuppressive treatment, N (\%) & 92 (74\%) & &\tabularnewline
Methotrexate treatment, N (\%) & 27 (22\%) & &\tabularnewline
Biologic treatment, N (\%) & 48 (39\%) & &\tabularnewline
\bottomrule
\end{longtable}

AIIRD, autoimmune inflammatory rheumatic diseases; JIA juvenile
idiopathic arthritis; SLE, systemic lupus erythematosus; IBD,
inflammatory bowel diseases.

\textbf{Table 2: Table 2. Adverse events following BNT162b2 mRNA
COVID-19 vaccine among adolescents with AIIRD and controls}\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\textbf{Variable} & \textbf{2-weeks after 1st vaccination} &
\textbf{2-weeks after 1st vaccination} & \textbf{2-weeks after 1st
vaccination} & \textbf{2-8 weeks after 2nd vaccination} & \textbf{2-8
weeks after 2nd vaccination} & \textbf{2-8 weeks after 2nd vaccination}
& \textbf{2-8 weeks after 3rd vaccination} & \textbf{2-8 weeks after 3rd
vaccination} & \textbf{2-8 weeks after 3rd vaccination}\tabularnewline
\midrule
\endhead
& \textbf{AIIRD (n=124)} & \textbf{Controls (n=80)} & \textbf{P value} &
\textbf{AIIRD (n=124)} & \textbf{Controls (n=80)} & \textbf{P value} &
\textbf{AIIRD (n=64)} & \textbf{Controls (n=30)} & \textbf{P
value}\tabularnewline
Local reactions & 88 (71\%) & 44 (55\%) & 0.03 & 89 (72\%) & 44 (55\%) &
0.02 & 25 (39\%) & 22 (73\%) & 0.004\tabularnewline
Systemic reactions, n (\%) & Systemic reactions, n (\%) & Systemic
reactions, n (\%) & Systemic reactions, n (\%) & Systemic reactions, n
(\%) & Systemic reactions, n (\%) & Systemic reactions, n (\%) &
Systemic reactions, n (\%) & Systemic reactions, n (\%) & Systemic
reactions, n (\%)\tabularnewline
Fever & 6 (5\%) & 2 (2\%) & 0.64 & 18 (15\%) & 8 (10\%) & 0.47 & 7
(11\%) & 1 (3\%) & 0.40\tabularnewline
Chills & 5 (4\%) & 2 (2\%) & 0.84 & 17 (14\%) & 3 (4\%) & 0.04 & 4 (6\%)
& 1 (3\%) & 0.92\tabularnewline
Runny nose & 4 (3\%) & & & 6 (5\%) & 1 (1\%) & 0.32 & 1 (2\%) &
&\tabularnewline
Cough & 3 (2\%) & & & 5 (4\%) & 1 (1\%) & 0.46 & 1 (2\%) &
&\tabularnewline
Nausea & 5 (4\%) & 2 (2\%) & 0.84 & 6 (5\%) & 2 (2\%) & 0.63 & 3 (5\%) &
&\tabularnewline
Vomiting & 1 (1\%) & 2 (2\%) & 0.69 & 3 (2\%) & 2 (2\%) & 1 & 0 (0\%) &
&\tabularnewline
Fatigue/ weakness & 14 (11\%) & 6 (8\%) & 0.51 & 33 (27\%) & 12 (15\%) &
0.07 & 9 (14\%) & 2 (7\%) & 0.48\tabularnewline
Myalgia & 12 (10\%) & 6 (8\%) & 0.77 & 23 (19\%) & 7 (9\%) & 0.08 & 6
(9\%) & 2 (7\%) & 0.96\tabularnewline
Arthralgia & 7 (6\%) & 1 (1\%) & 0.22 & 14 (11\%) & 2 (2\%) & 0.04 & 2
(3\%) & &\tabularnewline
Headache & 10 (8\%) & 8 (10\%) & 0.82 & 29 (23\%) & 9 (11\%) & 0.04 & 8
(12\%) & 2 (7\%) & 0.61\tabularnewline
Chest pain & 0 (0\%) & & & 0 (0\%) & & & 3 (2\%) & &\tabularnewline
Pericarditis or myocarditis & 0 (0\%) & & & 0 (0\%) & & & 0 (0\%) &
&\tabularnewline
Herpes zoster & 0 (0\%) & & & 0 (0\%) & & & 0 (0\%) & &\tabularnewline
Allergic reaction & 0 (0\%) & 1 (1\%) & 0.82 & 0 (0\%) & & & 0 (0\%) &
&\tabularnewline
Exacerbation of rheumatic disease symptoms & 5 (4\%) & & & 3 (2\%) & & &
0 (0\%) & &\tabularnewline
Hospitalization within 2 weeks post-vaccine & 2 (2\%) & & & 0 (0\%) & &
& 0 (0\%) & &\tabularnewline
\bottomrule
\end{longtable}

AIIRD, autoimmune inflammatory rheumatic diseases

\textbf{Table 3:} COVID-19 infection events following vaccination among
patients with AIIRD compared with controls\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Group} & \textbf{Total} & \textbf{P-value} & \textbf{Between 2nd
and 3rd doses} & \textbf{P-value} & \textbf{After 3rd dose} &
\textbf{P-value}\tabularnewline
\midrule
\endhead
Controls & 80 & & 80 & & 30 &\tabularnewline
& 28 (35\%) & & 13 (16.3\%) & & 15 (50\%) &\tabularnewline
AIIRD patients & 124 & & 124 & & 64 &\tabularnewline
& 59 (47.6) & 0.1033 & 33 (26.6\%) & 0.1193 & 26 (40.6\%) &
0.5278\tabularnewline
JIA & 54 & & 54 & & 23 &\tabularnewline
& 26 (48.2) & 0.1794 & 17 (31.5\%) & 0.0624 & 9 (39.1\%) &
0.6104\tabularnewline
SLE & 20 & & 20 & & 10 &\tabularnewline
& 7 (35\%) & 1 & 3 (15\%) & 1 & 4 (40\%) & 0.855\tabularnewline
\bottomrule
\end{longtable}

AIIRD, autoimmune inflammatory rheumatic diseases; JIA, Juvenile
Idiopathic Arthritis; SLE, Systemic Lupus Erythematosus.

\textbf{Figure legends:}

\textbf{Figure 1.} Kinetics of anti-S1/S2 IgG titres after the second
BNT162b2 vaccine dose, 6 months later, and after the third dose in AIIRD
patients and controls.

AIIRD, autoimmune inflammatory rheumatic diseases

\textbf{Figure 2.} Kinetics of anti-S1/S2 IgG titres after the second
BNT162b2 vaccine dose, 6 months later, and after the third dose in AIIRD
patients grouped by diagnosis and in controls.

AIIRD, autoimmune inflammatory rheumatic diseases; JIA, Juvenile
Idiopathic Arthritis; SLE, Systemic Lupus Erythematosus.

\textbf{Figure 3.} Time from last BNT162b2 vaccine dose to COVID-19
breakthrough infection in healthy adolescents and those with AIIRD.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Group} & \textbf{records} & \textbf{events} & \textbf{median} &
\textbf{0.95LCL} & \textbf{0.95UCL}\tabularnewline
\midrule
\endhead
Group=Healthy & 80 & 32 & 5.49 & 4.4 & 6.24\tabularnewline
Group=AIIRD & 124 & 61 & 5.16 & 4.7 & 6.18\tabularnewline
\bottomrule
\end{longtable}

Log-Rank P-Value is 0.4836\selectlanguage{english}
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