“To the Editor”
In clinical practice it is sometimes observed that allergic patients
exhibit allergic symptoms or positive skin prick test results in the
absence of detectable allergen-specific IgE in serum. In this study,
twenty five subjects, age from 13 to 59 years [Mean 32.6±13.6], 16
males and 9 females, with birch pollen-related allergic
rhinoconjunctivitis (ARC) with or without allergic asthma (AA), with
(group 1: n=13) or without cross-reactive food allergy to apple (group
2: n=12) (oral allergy syndrome, OAS) were enrolled (Table E1) and
investigated at three time points (i.e., before the birch pollen
season-time point 1, shortly after the birch pollen season-time point 2
and thereafter in autumn-time point 3) (Figure 1). The diagnosis of
birch pollen allergy and OAS, demographic, serological and clinical
features of the patients are described in the Supplemental materials
(Table E1). Two patients from group 1, (patients 1 and 2) with
apple-related OAS stood out. They contained low but clearly positive Bet
v 1-specific IgE levels before the birch pollen season (time point 1,
patient 1: 0.16 kUA/L; patient 2: 0.11 kUA/L), showed increases of Bet v
1-specific IgE shortly after the birch pollen season (time point 2,
patient 1: 0.75 kUA/L, patient 2: 0.53kUA/L) which then declined again
until September at time point 3 (patient 1: 0.53 kUA/L; patient 2: 0.43
kUA/L) (Table E2, Table E3, Figures 2 and 3). Interestingly, Mal d
1-specific IgE could not be detected in the sera obtained from patients
1 and 2 at any of the three time points even by sensitive and
quantitative ImmunoCAP technology (Table E2; Figure 2 and 3).
The two patients were young females who suffered from birch
pollen-related allergic rhinoconjunctivitis and one (patient 1) suffered
also from birch pollen-induced asthma (Table E4). Importantly, both
patients suffered from OAS upon consumption of apples, especially after
the birch pollen season and were positive in open food challenge with
apple (Table E4). The two patients were sensitized only to few other
allergens besides Bet v 1 and Mal d 1 and sensitization to other
cross-reactive food allergens (e.g., profilin, lipid transfer proteins
could be excluded (Table E5).
An increase of Bet v 1-specific IgE levels was observed in both patients
1 and 2 at time point 2 shortly after the birch pollen season (Figure 2
and 3, Table E2) which was associated with a strong increase in Bet v
1-specific basophil sensitivity in patient 2 (Figure 3, Table E6). The
Bet v 1-specific basophil sensitivity in patient 1 at time points 1 and
2 was comparable whereas at time point 3 patient 1 showed basophil
activation with a tenfold lower Bet v 1 concentration indicating
increased Bet v 1-specific basophil sensitivity at time point 3 (Figure
2, Table E6). Exposure of basophils to anti-IgE antibodies indicated
strongly reduced IgE-mediated basophil sensitivity in patient 1 at time
point 3 as compared to time points 1 and 2 (Table E6).
We could not detect at any of the three time points Mal d 1-specific IgE
in the sera of patients 1 and 2 (Table E2, Figures 2 and 3) but we found
a strongly increased Mal d 1-specific basophil sensitivity in both
patients at time points 2 and 3 (Figures 2 and 3, Table E6) after the
birch pollen season indicating increases of Mal d 1-specific IgE on the
patients basophils after birch pollen exposure. The increase of Mal d
1-specific basophil sensitivity was associated with marked increased
apple-induced skin responses at time points 2 and 3 after the birch
pollen season as compared to time point 1 before the birch pollen season
(Figures 2 and 3, Table E7). By contrast, skin responses to histamine
were comparable at all three time points in patients 1 and 2 (Table E7).
Using purified allergen molecules, our study has demonstrated that
basophils and mast cells of allergic patients contain already
allergen-specific IgE before it can be detected in serum in the form of
free-allergen-specific antibodies. This finding is important because it
demonstrates that allergen-specific IgE sensitization may already occur
before allergen-specific IgE can be detected as free IgE in serum
(Figure E1). This indicates that the early detection of
allergen-specific IgE sensitization may require basophil activation,
skin testing and/or provocation testing with defined allergen molecules
to detect IgE bound to cellular IgE receptors before it can be traced in
serum (Figure E1). Our conclusions are supported by an earlier study
which has demonstrated that after administration of omalizumab, a
monoclonal anti-IgE antibody which prevents allergen-specific IgE from
binding to the high and low affinity IgE receptor, IgE sensitizations to
allergen sources became detectable in serum which were undetectable
before administration of omalizumab 1. This effect
would be explained by the fact that omalizumab blocked the binding of
low levels of IgE specific for the allergen sources to cellular IgE
receptors so that they became measurable in serum which is in agreement
with the notion that application of omalizumab generally increases
levels of allergen-specific free IgE in serum.
Our results are important for diagnosis of IgE-mediated allergic
sensitization in general because they reveal that a certain threshold of
allergen-specific IgE levels must be reached which exceeds the capacity
of cellular IgE receptors in the body to bind IgE before it can become
detectable as free IgE in serum. This must be taken into consideration
when patients suffer from allergic symptoms but IgE specific for the
corresponding allergens cannot be detected. In such cases basophil
activation or skin testing with defined allergen molecules should be
considered.
Our findings are also important when it comes to the investigation of
the inception (i.e., time window of sensitization) of allergic
sensitization in children because they indicate that early
allergen-specific IgE sensitization may not be accurately detected by
serology. This may apply for subjects with low allergen-specific IgE
levels who are sensitized only to few allergen molecules. In fact,
monitoring of allergen-specific IgE to multiple allergen molecules in
birth cohorts indeed showed that children are often sensitized only to
few dominant allergen molecules 2, 3, 4. This may have
important consequences for strategies for allergen-specific prevention
because it may affect the definition of primary versus secondary
prevention depending on the accurate definition at what time point in
life allergic sensitization has indeed occurred. Finally, our data may
explain why certain patients show allergic symptoms and/or
allergen-specific positive challenge test (e.g., skin test) and basophil
activation results although no specific IgE can be detected in serum. It
is a limitation of our study that we have investigated only 25 birch
pollen allergic patients but among those approximately 10% of patients
showed a discrepancy between detection of specific IgE by tests based on
effector cell activation versus IgE serology. Larger studies will be
needed to determine the percentages of patients who present only
effector cell bound specific IgE without detectable specific IgE in
serum. Since we are currently running out regarding high quality
allergen preparations for skin testing 5 such studies
will need to be performed with purified allergen molecules performed
under Good Manufacture Practice (GMP) conditions suitable for clinical
trials but they are not available at the moment.
The strength of our study is that it was conducted with highly purified
allergen molecules and hence delivered unambiguous results because they
demonstrate the presence of allergen-specific IgE on basophils and in
the tissues of allergic patients without detectable allergen-specific
IgE in serum.
KEYWORDS: Basophil activation test, allergen-specific IgE,
basophils, birch pollen allergy, cross-reactive food allergy, Bet v 1,
Mal d 1, oral allergy syndrome.
WORD COUNT : 1222 words, 3 figures.
Alla O. Litovkina, MD 1,2 §
Maria G. Byazrova, PhD 1,2,3 §
Evgenii V. Smolnikov, MD 1,2
Alexandra A. Nikonova, PhD 1,2
Olga G. Elisyutina, MD 1,2
Elena S. Fedenko, MD 1
Nataliya I. Ilina, MD 1
Oluwatoyin Akinfenwa, PhD 4
Raffaela Campana, PhD 4
Dmitry A. Kudlay, MD 1,5
Rudolf Valenta, MD 1,4, 5, 6, ¶
Musa R. Khaitov, PhD 1,7 ¶ .
1 National Research Center – Institute of Immunology
FMBA of Russia, Moscow, Russian Federation
2 Peoples’ Friendship University of Russia (RUDN
University), Moscow, Russian Federation
3 Lomonosov Moscow State University, Moscow, Russian
Federation
4 Division of Immunopathology, Department of
Pathophysiology and Allergy Research, Center for Pathophysiology,
Infectiology and Immunology, Medical University of Vienna, Vienna,
Austria
5 Department of Clinical Immunology and Allergy,
Sechenov First Moscow State Medical University, Moscow, Russian
Federation
6 Karl Landsteiner University of Health Sciences,
Krems, Austria
7 Pirogov Russian National Research Medical
University, Moscow, Russian Federation
§Contributed equally as first authors,¶Contributed equally as senior authors
*Address correspondence to: Rudolf Valenta, MD,
Division of Immunopathology, Department of Pathophysiology and Allergy
Research, Center for Pathophysiology, Infectiology and Immunology,
Medical University of Vienna, Vienna, Austria. Email:rudolf.valenta@meduniwien.ac.at
Author Contributions: RV, AOL, AAN, MRK, DAK, OGE designed the
research studies. AOL, SEV, OGE, ESF, NII performed clinical work. AOL,
MGB, SEV, AAN, OA, RC performed experiments. RV, AOL, MGB, SEV analyzed
and interpreted the data. AOL wrote the manuscript with contributions
from RV and SEV.
Acknowledgements: This study was supported by a Megagrant of
the Government of the Russian Federation, grant number 14.W03.31.0024,
by the Danube ARC grant of the country of Lower Austria, by the
FWF-funded project P34472-B from the Austrian Science Foundation (FWF),
and by the RUDN University Strategic Academic Leadership Program.
Disclosures and potential conflicts of interest: Rudolf Valenta
has received research grants from Viravaxx AG, Vienna, Austria, HVD
Biotech, Vienna, Austria and Worg Pharmaceuticals, Hangzhou, China. He
serves as a consultant for Viravaxx and Worg. The other authors have not
conflicts of interest to declare.