To the Editor:
In vivo provocation test methods (eg, skin testing and conjunctival provocation testing)
1
are useful surrogates for clinical improvement, but the identification of in vitro
markers for monitoring the effects of specific immunotherapy (SIT) has been a long-sought
goal. It has been shown that allergen-specific blocking IgG antibodies inhibit allergen-induced
mast cell and basophil degranulation as well as IgE-facilitated allergen presentation
to T cells and is associated with a reduction of in vivo sensitivity.
1-4
Cellular assays (eg, basophil activation assays and FAB assay)
5,6
may allow uncovering and measuring the effects of allergen-specific blocking antibodies
on the allergen-IgE interaction and to correlate in vitro results with clinical outcomes
but are quite cumbersome.
We recently found that measurements of allergen-specific IgE levels performed in the
presence of low allergen concentrations in the solid phase, for example, allergen
microarrays, allow visualizing the inhibition of IgE binding in the presence of blocking
IgG antibodies when allergen-specific blocking IgG antibodies are present.
7
Therefore, it may be hypothesized that IgE measurements performed using low allergen
concentrations such as in allergen microarrays may better reflect the in vivo patients'
situation (ie, the patients' sensitivity).
We aimed to study the influence of SIT-induced allergen-specific IgG antibodies on
IgE binding in microarray and CAP assays and to determine whether IgE levels measured
by microarray are associated with clinical parameters. For this purpose, residual
serum samples from a double-blind placebo-controlled immunotherapy trial performed
in birch pollen–allergic patients with recombinant hypoallergenic Bet v 1 derivatives
were analyzed.
2
Sera were obtained before and immediately after treatment, shortly after the following
birch season, and 1 year after starting the treatment (see timeline in Fig E1 in this
article's Online Repository at www.jacionline.org) (placebo group, n = 27; recombinant
Bet v 1 fragments, n = 17; recombinant Bet v 1 trimer, n = 21). A demographic characterization
of the patients and their treatment (ie, cumulative doses administered and numbers
of injections) can be found in Table E1 in this article's Online Repository at www.jacionline.org.
Recombinant Bet v 1 fragments and trimer administered in this study are described
in this article's Online Repository at www.jacionline.org.
Sera (Fig E1) were analyzed for Bet v 1–specific IgE and IgG levels by ImmunoCAP and
ISAC, a multiallergen chip that contained 103 allergen-specific components to record
kinetics of IgE and IgG responses (Thermo Fisher/Phadia AB, Uppsala, Sweden). Linear
contrasts after ANOVA and correlations were calculated using Statistica 10.0 (StatSoft,
Tulsa, Okla) and SPSS 22.0 (IBM, Armonk, NY).
In those patients who received active treatment and thus developed high levels of
allergen-specific IgG, the detected Bet v 1–specific IgE antibodies differed strongly
between ImmunoCAP and ISAC measurements in the sera obtained after but not before
treatment. ImmunoCAP measurements showed significant increases in Bet v 1–specific
IgE antibodies after treatment/before pollen season in both actively treated groups
(Fig 1, A; see Table E2 in this article's Online Repository at www.jacionline.org),
whereas detected Bet v 1–specific IgE levels decreased significantly when measured
by ISAC compared to placebo-treated patients. Boosts of allergen-specific IgE production
caused by seasonal allergen exposure were found in the “after-season” samples from
all patients by ISAC and ImmunoCAP measurements, but, as earlier reported, increases
were lower for actively treated patients than for placebo-treated patients
2
(Fig 1, B). The decrease in Bet v 1–specific IgE measured by ISAC in the actively
treated groups was associated with a strong increase in Bet v 1–specific IgG found
by both CAP and ISAC measurements (Fig 1, C and D) and thus may be explained by blocking
of Bet v 1–specific IgE binding by therapy-induced IgG in the ISAC. Immunization experiments
performed with rBet v 1 fragments and trimer in animals following an immunization
scheme close to the one used for this study showed that the trimer is more immunogenic
than the fragments.
8
This fits the observation that the trimer induced higher Bet v 1–specific IgG levels
after vaccination as determined by quantitative CAP measurements (Fig 1, C, CAP: IgG
increase comparing before treatment with after treatment; P < .05) than the fragments
in the patients. Fragment-treated and trimer-treated patients had received comparable
cumulative doses of the vaccines (Table E1). Therefore, the higher increase in Bet
v 1–specific IgG in the trimer group was not due to different cumulative doses injected.
In contrast to the ISAC measurements, an increase in Bet v 1–specific IgE was found
by CAP measurements because allergen is present in excess in the solid phase and therefore
SIT-induced Bet v 1–specific IgE becomes visible. In fact, it is known that SIT also
induces a rise in allergen-specific IgE.
9
No relevant alterations in Bet v 1–specific IgG antibodies were observed for placebo-treated
patients (Fig 1, C and D).
Rises in Bet v 1–specific IgE were observed for all groups as a result of seasonal
allergen exposure after the pollen season and allergen-specific IgE then declined
again 1 year after treatment before the next pollen season (Fig 1, A and B). Similar
results of IgE and IgG antibody reactivities to Bet v 1–related pollen and plant food
allergens (rAln g 1: alder; rCor a 1: hazel; rMal d 1: apple; rPru p 3: peach) were
noted, but responses were lower than for Bet v 1, mirroring the degree of sequence
similarity with Bet v 1 (Aln g 1 > Cor a 1 > Mal d 1 > Pru p 3) (see Figs E2 and E3
in this article's Online Repository at www.jacionline.org).
Next, we compared alterations in nasal allergen sensitivity as determined by active
anterior rhinomanometry with changes in allergen-specific IgE levels measured by ISAC
for those patients for whom nasal provocation data were available before treatment
and after the pollen season (placebo, n = 22; rBet v 1 fragment, n = 12; rBet v 1
trimer, n = 16) (Fig 2). Results obtained 1 year after starting the treatment (Fig
E1) were not analyzed because IgG levels had declined almost to baseline at this time
point (Fig 1) and no differences between groups were found by nasal provocation.
10
Nasal provocation was performed using increasing doses of natural birch pollen extract
containing defined Bet v 1 concentrations. Changes in nasal allergen tolerance in
the patients are represented by positive (increased nasal allergen tolerance) or negative
(decreased nasal allergen tolerance) points, where 1 point indicates a 10-fold change
to the results measured before treatment (Fig 2, y-axes).
10
When changes in nasal sensitivity and allergen-specific IgE were plotted against each
other, it became visible that patients with increases in Bet v 1–specific IgE without
improvement or deterioration in nasal sensitivity were mainly found in the placebo
group (Fig 2, placebo: right lower quarter) whereas patients with reduced Bet v 1–specific
IgE were frequently observed in the actively treated group and often tolerated higher
allergen doses during nasal provocation (Fig 2, left upper quarter; fragments: 25%,
3 of 12 patients, and trimer: 31.3%, 5 of 16 patients). Fig 2 shows that there is
a significant correlation of the reduction in Bet v 1–specific IgE binding measured
by ISAC with increased nasal allergen tolerance in the trimer-treated group (r = −0.620;
P = .012). No significant correlation of the reduction in IgE binding to Bet v 1 determined
by ISAC was found with increased nasal allergen tolerance in fragment-treated patients,
which may be explained by the lower induction of allergen-specific IgG by fragments
as compared to trimer (Fig 1, C and D).
Considering all treatment groups, decreases in IgE measured by ISAC seemed to be useful
for the prediction of clinical improvement because we found a clinical improvement
prediction of 90% (ie, 100% for placebo and trimer groups and 71% for the fragment
group). This was not the case for increases in ΔIgE as measured by the ISAC, which
was associated with a clinical worsening prediction of only 25% for the placebo group
and 20% for trimer and fragment groups, respectively. A limitation of our study is
that data were available only for a relatively small number of patients but our results
indicate that decreases in allergen-specific IgE as measured on the chip are associated
with reduced nasal allergen sensitivities. This effect was not at all observed when changes
in allergen-specific IgE were measured under conditions of allergen excess by CAP because
IgE levels increased in the placebo- and actively treated patients (see Fig E4 in
this article's Online Repository at www.jacionline.org).
The results of our study thus indicate that allergen microarrays are useful to monitor
the development of allergen-specific IgG responses during SIT, both against the allergen
present in the SIT vaccine as well as against cross-reactive allergens. Moreover,
the reduction in allergen-specific IgE binding measured by microarray analysis may
be a useful surrogate marker for clinical effects of SIT, warranting more extensive
prospective studies designed to analyze the association of IgE levels measured by
microarray with results from in vivo allergen provocation and clinical end points.