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
background25, 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 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 (> 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 girls21, 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 strains27.
It is also known that EVs differ in their ability to infect various
white blood cell populations26. 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 involved4. 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 Pvalues 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 < 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 P value <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 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.
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.
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.