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\begin{document}

\title{Transcriptome changes during peanut oral immunotherapy and omalizumab
treatment}



\author[1]{Sofia Bjorkander}%
\author[1]{Simon Kebede Merid}%
\author[1]{David Brodin}%
\author[1]{Josef Brandstrom}%
\author[1]{Fredrik Fagerstrom-billai}%
\author[2]{Marieke van der Heiden}%
\author[3]{Jon Konradsen}%
\author[4]{Michael Kabesch}%
\author[5]{Cornelis  Drunen}%
\author[5]{Korneliusz  Golebski}%
\author[6]{Anke-Hilse Maitland - van der Zee}%
\author[7]{Uros Potocnik}%
\author[6]{Susanne Vijverberg}%
\author[1]{Anna Nopp}%
\author[1]{Caroline Nilsson}%
\author[1]{Erik Melén}%
\affil[1]{Karolinska Institute}%
\affil[2]{University Medical Centre Groningen}%
\affil[3]{Astrid Lindgren Children's Hospital}%
\affil[4]{University of Regensburg}%
\affil[5]{University of Amsterdam}%
\affil[6]{Amsterdam University Medical Centres}%
\affil[7]{University of Maribor}%


\vspace{-1em}



  
  \date{May 20, 2024}


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\textbf{Transcriptome changes during peanut oral immunotherapy and
omalizumab treatment}

To the Editor,

Peanut allergy is a common food allergy and the main cause of
anaphylaxis among children\textsuperscript{1}. In recent years, oral
immunotherapy has emerged as a promising treatment for children with
different IgE-mediated food allergies, although safety issues must be
considered\textsuperscript{2}. The main aim of immunotherapy is to
induce tolerance or desensitization to an allergen which otherwise
causes an allergic reaction. For oral immunotherapy this means ingesting
the allergen in a controlled manner with gradually increasing dosages.
Specifically, peanut oral immunotherapy (pOIT) is able to induce
tolerance/desensitization\textsuperscript{3}. While the pathogenesis of
food allergy in general is relatively well-studied\textsuperscript{4},
mechanisms of OIT-induced tolerance are not well understood. Omalizumab
(anti-IgE) used as treatment for severe allergic asthma and other
IgE-driven allergies, can facilitate OIT initiation\textsuperscript{5},
however, little is known about the involved mechanisms, including
possible changes at the transcriptional level. We therefore investigated
transcriptional changes in whole blood using RNA-sequencing profiles
during omalizumab treatment and pOIT in participants from the FASTX
(Food Allergen Suppression Therapy with Xolair \selectlanguage{ngerman}®) study previously
described in detail elsewhere\textsuperscript{5}.

In brief, peanut-allergic adolescents (n=23 of whom 17 completed the
study, age 12-18 years) were started on omalizumab (baseline) and
treated for at least 8 weeks before starting pOIT (pOIT start) while on
omalizumab. The peanut-dose was gradually increased during the 8 weeks
until reaching a maintenance dose. Guided by a basophil activation test
(BAT/CD-sens)\textsuperscript{6} after 8 weeks on the maintenance dose,
participants decreased the omalizumab dose by 50\% (maintenance) and
continued to decrease the omalizumab dose if pOIT was tolerated. Eleven
patients were able to tolerate pOIT without omalizumab protection for
\textgreater{}8 weeks and then passed an open peanut food challenge
(final); 6 patients could not discontinue omalizumab, but blood samples
were obtained for analysis after 2-3 years of omalizumab treatment
(final); 6 patients dropped out of the study. RNA-sequencing was
performed on whole blood at baseline, pOIT start, maintenance and final
time-points using the NovaSeq 6000 platform. DESeq2 was used for
differential expression analysis of the omalizumab effect and a linear
mixed-effect model for analyses during pOIT in combination with
omalizumab (pOIT+O) after adjustment for treatment outcome and cell
type. A complete description of the treatment protocol and method is
given in Appendix S1.

General characteristics of the study participants at baseline can be
found in Table S1 . To elucidate if omalizumab treatment alone induced
alterations in peripheral blood gene expression, we investigated the two
first timepoints, baseline and pOIT start, however no significant
differences were observed (Figure S1 ). In the longitudinal analysis
(pOIT start to final), 680 genes associated with pOIT+O at nominal p
\textless{}0.005 (Table S2 ). The Gene Ontology (GO) biological process
of the up- and down-regulation of these 680 genes are presented in
Figure 1A,B . Upregulation of 337 genes were linked to GO terms
``protein regulation and modification'', while ``neutrophil
degranulation, immune response, phagocytosis, and metabolic process''
were among the top terms for the downregulated 343 genes. Out of the 680
genes, 16 were differentially expressed at false discovery rate (FDR)
adjusted p\textless{}0.05 (Table 1, Figure S2 ). The three genes with
the largest negative and positive coefficients, respectively, are
displayed in Figure 1C,D ; downregulation of \emph{ASGR2} ,\emph{GPBAR1}
and \emph{HM13,} and upregulation of \emph{USP44} ,\emph{ICOS} and
\emph{CDKN2AIP} . Finally, we evaluated the enrichment of 680
pOIT+O-associated genes, relative to peripheral blood gene expression
associated with acute peanut allergic reactions in a recently published
clinical study by Watson et al using the same p-value cut-off
(p\textless{}0.005)\textsuperscript{7}. Out of our 680 significant
genes, 108 genes overlapped with the differentially expressed genes in
Watson et al\textsuperscript{7}, mostly with opposite direction,
P\textsubscript{enrichment}\selectlanguage{english} = 0.0095 (Figure 2 ).

Our results demonstrate that omalizumab treatment alone does not induce
alterations in whole blood gene expression in patients with severe food
allergy. This is not surprising given that these patients were unexposed
to peanut allergen at the time of blood sampling, and any concomitant
asthma, rhinitis or eczema were well controlled. However, the
longitudinal analysis during pOIT+O identified up- and downregulation of
several immune-related genes. CD278/ICOS (Inducible T-cell
costimulatory) is expressed on activated T-cells and appears to play a
role in directing effector T-cell differentiation and responses during
inflammatory conditions\textsuperscript{8}. ICOS-expression on T
regulatory cells and T follicular helper cells may be involved in the
allergic disease mechanism\textsuperscript{9}. In the pathway analyses,
we observed significant enrichment for several GO biological process
terms related to T-cell function and immune responses. Notably, we have
previously described alterations in T-cell polyclonal \emph{in
vitro}activation during pOIT +O in the FASTX study\textsuperscript{10}.
Comparing our findings with data described by Watson \emph{et
al\textsuperscript{7}} , suggests that pOIT+O may alter the expression
level of many genes that were found activated during an acute peanut
allergy reaction.

The main limitations of this study are lack of any control subjects and
small sample size. Moreover, further studies are needed to evaluate the
long-term biological effect of pOIT+O.

In conclusion, omalizumab treatment alone does not alter the
transcriptional signature in peripheral blood of peanut allergic
patients, but during pOIT+O, several immune-related signatures were
observed. These results may provide insights into mechanisms of allergen
tolerance.

\textbf{ACKNOWLEDGEMENTS}

We thank all FASTX patients for their participation and the research
staff at Forskningscentrum, Sodersjukhuset.

\textbf{CONLFICT OF INTEREST}

None.

\textbf{ETHICAL APPROVAL}

Approved by the ethics committee in Stockholm: 2013/827-31/3,
2014/1980-32, 2016/1390-32, 2020-00807 and the Swedish Drug Agency:
5.1-2013-46183; The trial is registered at EudraCT: 2012-005625-78,
ClinicalTrails.gov; NCT02402231. Patients and caregivers provided
written informed consent.

\textbf{FUNDING}

The PERMEABLE consortium, supported by ZonMW (456008004), the Swedish
Research Council (2018-05619), the Ministry of Education, Science and
Sport of the Republic of Slovenia (C3330-19-252012), the German Ministry
of Education and Research (BMBF) (FKZ01KU1909A), under the frame of the
ERA PerMed JTC 2018 Call. Additional funding in the Appendix.

Sophia Bjorkander\textsuperscript{1\#}

Simon Kebede Merid\textsuperscript{1\#}

David Brodin\textsuperscript{2}

Josef Brandstrom\textsuperscript{3,4}

Fredrik Fagerstrom-Billai\textsuperscript{2}

Marieke van der Heiden\textsuperscript{5}

Jon R Konradsen\textsuperscript{4}

Michael Kabesch\textsuperscript{6}

Cornelis M van Drunen\textsuperscript{7}

Korneliusz Golebski\textsuperscript{8,9}

Anke H Maitland-van der Zee\textsuperscript{9}

Uro\selectlanguage{polish}š \selectlanguage{english}Poto\selectlanguage{polish}č\selectlanguage{english}nik\textsuperscript{10,11}

Susanne J H Vijverberg\textsuperscript{9}

Anna Nopp\textsuperscript{1,12}

Caroline Nilsson\textsuperscript{1,12}

Erik Mel\selectlanguage{ngerman}én\textsuperscript{1,12}

\begin{enumerate}
\tightlist
\item
  Department of Clinical Science and Education, Karolinska Institutet,
  Södersjukhuset, Stockholm, Sweden
\item
  Bioinformatics and Expression Core Facility, Karolinska Institutet,
  Huddinge, Sweden
\item
  Clinical Epidemiology Division, Department of Medicine, Solna,
  Karolinska Institutet, Stockholm, Sweden
\item
  Astrid Lindgren Children´s Hospital, Karolinska University Hospital,
  Stockholm, Sweden
\item
  Department of Medical Microbiology and Infection Prevention,
  University Medical Centre Groningen, University of Groningen, The
  Netherlands
\item
  Department of Pediatric Pneumology and Allergy, University Children's
  Hospital Regensburg (KUNO), Regensburg, Germany
\item
  Department of Otorhinolaryngology, Amsterdam UMC, University of
  Amsterdam, Amsterdam, The Netherlands
\item
  Department of Experimental Immunology, Amsterdam UMC, University of
  Amsterdam, Amsterdam, The Netherlands
\item
  Department of Respiratory Medicine, Amsterdam UMC, University of
  Amsterdam, Amsterdam, The Netherlands
\item
  Faculty of Medicine, Center for Human Molecular Genetics and
  Pharmacogenomics, University of Maribor, Maribor, Slovenia
\item
  Laboratory for Biochemistry, Molecular Biology and Genomics, Faculty
  for Chemistry and Chemical Engineering, University of Maribor,
  Maribor, Slovenia
\item
  Sachs Children and Youth Hospital, Stockholm, Sweden
\end{enumerate}

\textsuperscript{\#}Equal contribution

\textbf{Correspondence}

Professor Erik Melén Telephone: +46-(0)8-524-875-08.

E-mail: erik.melen@ki.se

\textbf{Key words:} peanut allergy, oral immunotherapy, Omalizumab,
Xolair, RNA-sequencing, gene expression, adolescences

\textbf{REFERENCES}

1 Vetander, M. \emph{et al.} Anaphylaxis to foods in a population of
adolescents: incidence, characteristics and associated
risks.\emph{Clinical and experimental allergy : journal of the British
Society for Allergy and Clinical Immunology} \textbf{46} , 1575-1587,
doi:10.1111/cea.12842 (2016).

2 Chu, D. K. \emph{et al.} Oral immunotherapy for peanut allergy (PACE):
a systematic review and meta-analysis of efficacy and safety. \emph{The
Lancet} \textbf{393} , 2222-2232,
doi:https://doi.org/10.1016/S0140-6736(19)30420-9 (2019).

3 Fiocchi, A. \emph{et al.} Oral immunotherapy for peanut allergy: The
con argument. \emph{World Allergy Organization Journal} \textbf{13} ,
100445, doi:https://doi.org/10.1016/j.waojou.2020.100445 (2020).

4 Ramsey, N. \& Berin, M. C. Pathogenesis of IgE-mediated food allergy
and implications for future immunotherapeutics. \emph{Pediatric allergy
and immunology : official publication of the European Society of
Pediatric Allergy and Immunology} , doi:10.1111/pai.13501 (2021).

5 Brandström, J. \emph{et al.} Individually dosed omalizumab facilitates
peanut oral immunotherapy in peanut allergic adolescents. \emph{Clinical
and experimental allergy : journal of the British Society for Allergy
and Clinical Immunology} \textbf{49} , 1328-1341, doi:10.1111/cea.13469
(2019).

6 Nopp, A., Cardell, L. O., Johansson, S. G. \& Oman, H. CD-sens: a
biological measure of immunological changes stimulated by
ASIT.\emph{Allergy} \textbf{64} , 811-814,
doi:10.1111/j.1398-9995.2008.01900.x (2009).

7 Watson, C. T. \emph{et al.} Integrative transcriptomic analysis
reveals key drivers of acute peanut allergic reactions. \emph{Nature
communications} \textbf{8} , 1943, doi:10.1038/s41467-017-02188-7
(2017).

8 Wikenheiser, D. J. \& Stumhofer, J. S. ICOS Co-Stimulation: Friend or
Foe? \textbf{7} , doi:10.3389/fimmu.2016.00304 (2016).

9 Li, D. Y. \& Xiong, X. Z. ICOS(+) Tregs: A Functional Subset of Tregs
in Immune Diseases. \emph{Frontiers in immunology} \textbf{11} , 2104,
doi:10.3389/fimmu.2020.02104 (2020).

10 van der Heiden, M. \emph{et al.} A pilot study towards the
immunological effects of omalizumab treatment used to facilitate oral
immunotherapy in peanut-allergic adolescents. \emph{Scandinavian journal
of immunology} \textbf{93} , e13005, doi:10.1111/sji.13005 (2021).

\textbf{FIGURE LEGENDS}

\textbf{Figure 1.} \emph{(A, B)} Gene ontology (GO) biological process
analysis of upregulated \emph{(A)} and downregulated \emph{(B)}genes.
The x-axis shows the gene ratio of the overlapping genes of our gene
list with the pathway gene set. The colour bar represents the adjusted
p-value and the circle size is the count of overlapping genes.\emph{(C,
D)} Boxplots display log2 expression values for six pOIT genes (\emph{C}
: upregulated, \emph{D} : downregulated) throughout the treatment
protocol at FDR p\textless{}0.05. Red box: pOIT start, green box:
Maintenance, blue box: Final. ORA = Over-representation analysis.

\textbf{Figure 2.} Overlap of 108 genes from the FASTX pOIT study with
the peanut-related genes found in \emph{Watson et al} . at nominal
p\textless{}0.005. The y-axis represents changes in gene expression
between the means at baseline and the four-hour time point of the peanut
challenge in \emph{Watson et al} , and the x-axis is effect size
estimates from the mixed-effect model in the FASTX pOIT study. The green
colour shows opposite direction, and the pink same direction.

\textbf{Hosted file}

\verb`PAI Table 1.docx`  available at \url{https://authorea.com/users/424423/articles/529517-transcriptome-changes-during-peanut-oral-immunotherapy-and-omalizumab-treatment}

\selectlanguage{english}
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