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\begin{document}

\title{The association of different enteroviruses with atopy and allergic
diseases in early childhood}



\author[1]{Tiina Palmu}%
\author[1]{Jussi Lehtonen}%
\author[2]{Laura Korhonen}%
\author[3]{Suvi Virtanen}%
\author[4]{Onni Niemelä}%
\author[5]{Jorma Toppari}%
\author[6]{Jorma Ilonen}%
\author[7]{Riitta Veijola}%
\author[8]{Mikael Knip}%
\author[1]{Olli Laitinen}%
\author[1]{Maria Lönnrot}%
\author[1]{Heikki Hyöty}%
\affil[1]{University of Tampere}%
\affil[2]{Tampere University Hospital}%
\affil[3]{Finnish Institute for Health and Welfare}%
\affil[4]{Seinäjoki Central Hospital and University of Tampere}%
\affil[5]{Turun yliopisto Kliininen laitos}%
\affil[6]{University of Turku}%
\affil[7]{University of Oulu}%
\affil[8]{University of Helsinki}%


\vspace{-1em}



  
  \date{June 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Enterovirus (EV) infections, being among the most prevalent
viruses worldwide, have been associated with reduced risk of allergic
diseases. We sought to determine the association of EVs with allergic
sensitization and disease in early childhood. Methods: The study was
carried out in a nested case-control setting within a prospective birth
cohort in Finland. We included 138 case children who had specific IgE
(s-IgE) sensitization at the age of 5 years and 138 control children
without s-IgE sensitization. Allergic disease was recorded at study
visits and asked with ISAAC questionary. We screened for the presence of
serotype specific antibodies against 41 EVs at 1 to 5 years of age and
assessed their association with allergic sensitization and disease.
Results: The overall number of EV infections did not differ between
s-IgE-sensitized children and non-sensitized control children. However,
there was a tendency of case children with an allergic disease having
less EV infections than their controls. This observation was
statistically significant for species A EVs in case children with atopic
dermatitis vs. control children: OR 0.6 (95 \% CI 0.36-0.99), P = 0.048.
Conclusion: This study supports the evidence that EV exposure and
development of allergic disease are inversely associated. Interestingly,
the inverse association was not observed for bare atopic IgE
sensitization, but for IgE sensitization coupled with clinical atopic
disease. This suggests that environmental factors influencing IgE
sensitization may differ from those influencing progression to clinical
allergic disease.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Atopy is defined as a tendency to produce IgE antibodies in response to
ordinary harmless exposures to allergens. The prevalence of atopic IgE
sensitization in developed countries is extremely high, rising from
around 12\% at the age of 1 year to around 30\% at the age of 6
years\textsuperscript{1} and staying at around 20-50\% throughout
adulthood \textsuperscript{2}. The rapid increase in the prevalence
during the last half a century \textsuperscript{3} suggests involvement
of environmental factors.

According to the hygiene/biodiversity hypothesis
\textsuperscript{4,5}the increase in atopy and allergic diseases is
caused by reduction in microbial exposures in childhood and thus,
skewing of the immune responses towards allergy prone Th2-based
reactions. Viruses guide the immune system towards Th1-direction
\textsuperscript{6}. One could thus hypothesize that virus infections
reduce the risk of atopy and allergy. Accordingly, low-grade
inflammation, induced for example by microbial exposure during infancy,
has been shown to decrease allergen specific IgE (s-IgE)
sensitization\textsuperscript{7}. Multiple viruses have been linked to
reduction of allergic diseases and/or IgE
sensitization\textsuperscript{8--13} but there are also reports
contradicting the role of viruses \textsuperscript{3,9,14,15}.

Enterovirus infections (EVs) are among the most common human virus
infections worldwide. The classic human EVs can be classified into four
species (EV-A, -B, -C and -D) comprising more than 100 serotypes. The
pathophysiology of different serotypes differs depending on their
tropisms to different tissues and cells. Enterovirus infections are
mostly asymptomatic or mild such as common cold or hand, foot and mouth
disease, but also serious diseases such as meningitis, myocarditis, and
paralysis may occur \textsuperscript{16}. In \emph{in vitro} models EVs
have been shown to bias the immune reactions towards the mature Th1-like
pathway \textsuperscript{17,18}. They have also been linked with reduced
risk of allergic diseases/atopy \textsuperscript{11,12,19}, but the
results are contradictory \textsuperscript{20,21}.

We contribute new insight into the association between EVs and atopy
with respect to the individual EV serotypes. This study is the most
comprehensive of its kind, containing 41 different human EV serotypes
analyzed in a large case-control setting.

\textbf{Methods}

\emph{Study population}

The study population was derived from the prospective population-based
Type 1 Diabetes Prediction and Prevention (DIPP) birth-cohort study
described in detail previously \textsuperscript{22}. Briefly, children
born in the Tampere, Turku, and Oulu University Hospitals in Finland are
screened at birth for HLA genotypes conferring to increased risk of type
1 diabetes. Written informed consent was obtained from caretakers both
for HLA screening and follow-up. The study protocol was approved by the
ethics committees of the participating hospitals. The present study
included children born between years 1995 and 2004 in Tampere and Oulu
where children carrying the HLA-DQB1 genotypes *02/*0302 and *0302/x (x
[?]*02), *0301 or *0602 were invited to follow-up. Blood samples were
drawn at 3, 6, 12, 18, and 24 months of age and once a year thereafter.
Atopic case and non-atopic control subjects were identified among
children with an available serum sample for both EV antibody assay
(serum sample between 1 and 5 years of age) and s-IgE antibody assay
(serum sample at 5 years of age). Altogether 138 case children with a
positive s-IgE test and 138 matched control children with negative s-IgE
test were identified. Of the 138 case children 65\% were sensitized to
the food allergen mixture and 71\% to the inhalant allergen mixture. Of
those positive for foods 57\% tested positive for cow's milk, 32\% for
egg, 22\% for wheat, and 6\% for fish. Of those positive for inhalants
57\% were positive for birch, 37\% for timothy, 3 \% for cat, and 10\%
for house dust mite. The pairs were matched by gender and age at the EV
antibody measurement (+/- 3 months). The proportion of boys was 65\%.

Allergic diseases were identified at 5 years of age by ISAAC
questionnaire questions: ``Has your child ever had asthma/hay fever or
some other allergic rhinitis/atopic eczema diagnosed by a doctor?''.
ISAAC-data was missing in 39 children. Their allergic disease diagnoses
were retrieved from the DIPP database: atopic dermatitis (ICD-code
L20.x), asthma (J45.x) and allergic rhinitis/hay fever (J30.x) reported
and confirmed by a physician by 5 years of age. Sixty-four (46\%) out of
the 138 IgE-sensitized case children reported an allergic disease (47
atopic dermatitis, 27 allergic rhinitis and 15 asthma) and formed a case
subgroup for analysis of enteroviruses' association with clinical
disease. These 64 case children were compared to their IgE-negative
matched control children. Some of the 64 IgE-negative control children
also reported an allergic disease (n = 17), but were eligible as
controls, as IgE sensitization was the feature defining case vs. control
status. Thirty-one (22.5\%) out of the total of 138 IgE-negative control
children reported an atopic disease (25 atopic dermatitis, 2 allergic
rhinitis and 8 asthma reports).

Background data of the participants retrieved from the DIPP database
included information of the age at the entry of daycare, duration of
breastfeeding, level of maternal and paternal education,
HLA-DQB1-genotype, the presence of type 1 diabetes associated islet
autoantibodies, pets at home, older siblings and maternal smoking during
pregnancy. Parental atopy background information was available in the
ISAAC-data for all but 39 children. Maternal smoking was excluded from
the analyses as it was reported by only five mothers.

\emph{Testing for allergen specific IgE}

The s-IgE antibodies were tested at the age of 5 years against a mixture
of airborne and food allergens (a multi-allergen test Phadiatop\selectlanguage{ngerman}®, Thermo
Fisher Scientific, Uppsala, Sweden) using the ImmunoCAP® enzyme
immunoassay (Thermo Fisher Scientific, Uppsala, Sweden) according to the
manufacturer's protocol. The test was regarded positive if the food or
inhalant allergen mixture showed a result of 0.35 kU/L or more.
Individual s-IgE results were available for cow's milk, hen's egg, fish,
wheat, house dust mite, cat, birch, and timothy grass.

\emph{Enterovirus seroneutralization assays}

Neutralizing antibodies were measured in serum or plasma against 41 EV
serotypes listed in Table 2 as previously described\textsuperscript{23}.
Viruses were isolated and cultivated, and seroneutralization assays were
performed using the A549, Vero, RD (all bought from American Type
Culture Collection) and GMK (from the National Institute for Health and
Welfare, Finland) cell lines. Most of the viruses were analyzed using a
standard plaque neutralization assay\textsuperscript{24}, whereas
viruses that did not form clear plaques were analyzed using a
microneutralization assay (CAV5, CAV6, CAV10, CAV16, EV71, EV78 and
EV94). The neutralization assay was considered positive when the serum
dilution 1:4 reduced the number of plaques by more than 75\% (plaque
assay) or inhibited the ability of the virus to kill cells
(microneutralization assay). The serum/plasma samples analyzed were
taken between 1 and 5 years of age. The sample volume was adequate for
the analysis of neutralizing antibodies against all 41 serotypes in 183
children. In 83 children the volume was adequate for the analysis of
39-40 serotypes and in nine children for 4-21 serotypes.

\emph{Statistical Methods}

The statistical analyses were performed using R version 4.0.2
(2020-06-22, The R Foundation for Statistical Computing,
https://www.R-project.org). Data are presented as medians, interquartile
ranges (IQRs), and ranges, as the continuous variables were not normally
distributed. Wilcoxon test was used to assess differences in continuous
variables. For determining the risk effect of different exposures
conditional logistic regression models were applied. The results are
presented as odds ratios (OR) and 95\% confidence intervals
(CI).\emph{P} value was considered significant when \textless{} 0.05.

\textbf{Results}

\textbf{Prevalence} \textbf{of neutralizing antibodies}

The median age for the EV antibody analysis was 2.0 years and the median
age difference between case and control children was 7 days. All
children were seropositive for at least one EV serotype. The median
number of positive serotypes was eight (range 1-21). Most common
serotypes were Echo33 (antibody prevalence 77\%), Echo30 (74\%), EV74
(58\%), CAV10 (56\%), and CVB1 (52\%).

\textbf{Background factors}

None of the background factors differed statistically significantly
between the case and control children (\textbf{Table 1} ).

\textbf{Exposure to individual EV serotypes and risk of atopy}

Seropositivity for individual EV serotypes is shown in \textbf{Table 2}
. Antibodies against Echo3 and Echo14 were less prevalent among case
children as compared to control children (OR 0.11, 95\% CI
0.01-0.88,\emph{P} = 0.037 and OR 0.17, 95\% CI 0.04-0.75, \emph{P} =
0.019, respectively). EV74 antibodies were more prevalent among case
children (OR 1.68, 95\% CI 1.02-2.76\emph{, P} = 0.04). In a subgroup
analysis of cases with higher likelihood of clinical symptoms
(s-IgE-levels over 0.7 kU/L) CAV6 was a strong protective element for
atopic sensitization (OR 0.22, 95\% CI 0.08-0.66, \emph{P} = 0.007)
while EV74 continued to show a risk association (OR 1.82, 95\% CI
1.01-3.29, \emph{P} = 0.047).

When case children with an allergic disease were compared to their
matched controls CAV6, CAV16, and Echo14 seropositivity were
significantly less frequent in cases (OR 0.23, 95\% CI
0.07-0.81,\emph{P} = 0.022; OR 0.33, 95\% CI 0.12-0.92, \emph{P} = 0.033
and OR 0.11, 95\% CI 0.01,0.88, \emph{P} = 0.037, respectively).

The prevalence of EV seropositivity was analysed separately in case boys
vs. control boys and case girls vs. control girls. Two EV serotypes
showed significant inverse assocation with atopic sensitization among
boys: CAV6 (OR 0.27, 95\% CI 0.09-0.80, \emph{P} = 0.019) and Echo14 (OR
0.10, 95\% CI 0.01-0.78, \emph{P} = 0.028). In addition, CAV6, CAV16,
and Echo5 showed significant inverse assocation with allergic disease
among case boys compared to their controls (OR 0.18, 95\% CI
0.04-0.82,\emph{P} = 0.027; OR 0.21, 95\% CI 0.06-0.75, \emph{P} = 0.015
and OR 0.31, 95\% CI 0.1-0.94, \emph{P} = 0.039, respectively). None of
the serotypes showed assocation with atopic sensitization or allergic
disease among girls.

\textbf{Cumulative enterovirus exposure}

The cumulative exposure to different EV serotypes illustrates the total
number of different EVs that the child has encountered. We hypothesized
that cumulative EV exposure would protect from allergic sensitization
but found no such association (\textbf{Figure 1} ). However, there was a
trend of an inverse association between cumulative number of EV
infections and allergic disease (\textbf{Figure 1} ). This association
was statistically significant between the cumulative exposure to group A
EVs and cases with atopic dermatitis (OR 0.6, 95\% CI 0.36-0.99,\emph{P}
= 0.048).

\textbf{Discussion}

For the first time, antibodies against a vast panel of different EVs
were analyzed to shed light on the possible role of EVs as
risk-modifying factors in the development of allergic sensitization
and/or disease. The cumulative number of infections by different EV
serotypes was not associated with IgE sensitization. However, the
cumulative number of species A EV infections showed a trend for
protective association with the development of allergic disease. This
inverse association was significant for IgE-sensitized cases with atopic
dermatitis, whereas for hay fever and asthma only a statistically
insignificant inverse tendency was observed. Especially asthma is a
heterogeneous disease and many patients lack atopic
background\textsuperscript{25}, which could explain the lack of
association. Another explanation could be limited statistical power,
since the number of cases with asthma or hay fever were lower than those
with atopic dermatitis.

Certain individual EV serotypes showed an association with IgE
sensitization and/or allergic disease, including Echo3 and 14 which both
showed an inverse association and EV74 which showed a risk association.
Interestingly, Echo3 has been reported to associate with a reduced risk
of allergic diseases also in a previous study \textsuperscript{11}.
Additionally, we wish to highlight CAV6, CAV16, Echo5, and Echo14 as
intriguing protective players in the development of allergic diseases.
Especially CAV6 exposure, a common virus causing hand, foot and mouth
disease, was significantly less frequent in cases with allergic disease
than in their matched controls and CAV6 was also less frequent in cases
with stronger (\textgreater{} 0.7kU/L) IgE sensitization. The inverse
CAV6 association was observed for boys with IgE sensitization and boys
with atopic disease, but not for girls. This sex difference is parallel
with our previous report of rhinoviruses' association with reduced risk
of atopy within boys but not in girls\textsuperscript{21}, and indicates
that viruses' influence on atopy risk may be more pronounced in males.
The fact that only few of the 41 EV serotypes showed association with
atopy may relate to immunofenotype differences between virus serotypes
and strains, as suggested by our previous report showing very different
immune system activation by individual CBV1 strains\textsuperscript{27}.
It is also known that EVs differ in their ability to infect various
white blood cell populations\textsuperscript{26}. Thus, it is possible
that certain EV types, such as CAV6, have a unique ``anti-allergic''
immunophenotype due to their specific interaction with the immune
system. This hypothesis requires further studies.

Previous studies have not identified any specific microbes driving the
inverse association between microbial exposures and the risk of allergic
disease. Instead, it seems that a vast variety of microbes, perhaps even
the total microbiome, is involved\textsuperscript{4}. Our finding of an
inverse association between the cumulative exposure to different EV
types and allergic disease fits with the hypothesis of more than a
single culprit microbe involved. Accordingly, even though we found some
significant associations when analyzing the individual serotypes, we
want to encourage opting for as large virus panel as possible in future
studies on EVs and atopy.

Strengths of this study are the comprehensive panel of the most common
circulating EV serotypes and the utilization of a highly serotype
specific neutralization assay, which can be considered the gold standard
for determining serotype specific EV antibodies. The endpoints of our
study were also solid, as the IgE sensitization was determined with a
wide allergen panel including both inhalant and food allergens, and
information on allergic diseases was based on either a validated atopy
questionnaire (ISAAC) or a diagnosis made by a physician.

The limitations of the study include the lack of correction of
\emph{P}values for the great number of comparisons performed when
analyzing the individual EV serotypes. In our study using a Bonferroni
coefficient of 41 (number of EV serotypes studied) leads to a
statistical significance cutoff value P \textless{} 0.0012 and wipes out
the significant associations for individual serotypes. Bonferroni
correction has been criticized for being overly conservative, and
therefore we held to the generally accepted \emph{P} value
\textless{}0.05. One must keep in mind that without correction for
multiple comparisons the results for individual EV serotypes must be
kept preliminary and they need confirmation in further studies.

A further limitation is that the study subjects did not represent the
general population but carried specific type 1 diabetes-associated HLA
haplotypes. However, no consistent relationship has been shown between
allergic diseases and type 1 diabetes \textsuperscript{27,28}, and
neither islet autoimmunity nor HLA was associated with atopic
sensitization in the current cohort. Lastly, we were unable to ascertain
the chronological order of the EV infections and IgE sensitization, as
the latter was measured only at the age of 5 years.

In conclusion, we show that there is an inverse association between
cumulative EV infections and allergic disease but not with allergic
sensitization alone. Whether this relationship is causal and how EVs
could obstruct the disease progression from sensitization to allergic
disease needs to be further studied. We also want to highlight serotypes
Echo3, Echo5, Echo14, CAV6 and CAV16 as possible protective serotypes
and EV74 as a possible risk factor in allergic sensitization and
disease.

\textbf{Acknowledgements:} The authors would like to thank the families
who have participated in the DIPP study as well as the study nurses and
other personnel running the study.

\textbf{Impact statement:} Hygiene hypothesis has long been proposed to
explain the increasing incidence of allergic sensitization and disease.
Enteroviruses, the most common viruses infecting human beings, have been
associated with the risk of allergy and asthma, but it is not known
whether this association links to certain enterovirus types. The present
study utilized the most comprehensive serotype panel thus far applied
and was able to strengthen previous indications of a possible role of
enteroviruses in the regulation of the risk of allergic diseases.

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\textbf{Table1.} Background data of case children with allergic
sensitization and controls\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Table 2.} Prevalence of neutralizing antibodies against
enterovirus serotypes in cases and controls and the risk for allergic
sensitization associated with enterovirus serotypes.

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/399897/articles/512303-the-association-of-different-enteroviruses-with-atopy-and-allergic-diseases-in-early-childhood}

\textbf{FIGURE LEGENDS}

\textbf{Figure 1.} Association between cumulative enterovirus exposure
for any enterovirus (EV) serotype / species A-serotype (A group) /
species B-serotype (B group) and allergic sensitization or allergic
disease in cases vs. controls (left) and for cases with a specific
allergic disease vs. their matched controls (right).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}

\selectlanguage{english}
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