Long-lasting effects of sublingual immunotherapy
according to its duration: A 15-year prospective study
Maurizio Marogna, MD,aIgino Spadolini, MD,bAlessandro Massolo, BS,cGiorgio Walter Canonica, MD,d
and Giovanni Passalacqua, MDd
Varese, Florence, and Genoa, Italy, and Calgary, Alberta, Canada
Background: Data on the long-term effects of sublingual
immunotherapy (SLIT) are sparse, and the optimal duration of
treatment is a matter of debate.
Objective: We sought to prospectively evaluate the long-term
effect of SLIT given for 3, 4, or 5 years and to compare the effect
of those different durations.
Methods: In this prospective open controlled study we
followed up patients with respiratory allergy who were
monosensitized to mites for 15 years. The subjects were
divided in 4 groups receiving drug therapy alone or SLIT for
3, 4, or 5 years. Clinical scores, skin sensitizations,
methacholine reactivity, and nasal eosinophil counts were
evaluated every year during the winter months. The clinical
effect was considered to persist until clinical scores remained
at less than 50% of the baseline value, and then patients
underwent another course of SLIT.
Results: Seventy-eight patients were enrolled, and 59 completed
the study. In the 12 control subjects no relevant change in
clinical scores was seen throughout the study. In the patients
receiving SLIT for 3 years, the clinical benefit persisted for 7
years. In those receiving immunotherapy for 4 or 5 years, the
clinical benefit persisted for 8 years. New sensitizations occurred
in all the control subjects over 15 years and in less than a
quarter of the patients receiving SLIT (21%, 12%, and 11%,
respectively). The second course of vaccination induced a benefit
more rapidly than the first course. The behavior of bronchial
hyperreactivity and nasal eosinophils paralleled the clinical
Conclusion: Under the present conditions, it can be suggested
that a 4-year duration of SLIT is the optimal choice because it
induces a long-lasting clinical improvement similar to that seen
with a 5-year course and greater than that of a 3-year
vaccination. (J Allergy Clin Immunol 2010;nnn:nnn-nnn.)
Key words: Sublingual immunotherapy, allergic rhinitis, long-last-
ing effect, house dust mite
Specific immunotherapy is one of the cornerstones of the
treatment of respiratory allergy, together with pharmacologic
treatment and allergen avoidance.1Since its introduction a cen-
neously (subcutaneous immunotherapy [SCIT]), but during the 2
troduced in clinical practice, with the primary aim of improving
safety and convenience. The efficacy of SLIT has been demon-
strated in numerous clinical trials and confirmed by several
meta-analyses.2Despite some aspects still needing clarification,3
SLIT is regarded as a suitable therapeutic option,2,4and it is
widely used in Europe and other countries, whereas no product
is still approved in the United States. SCITand SLIT induce pro-
found modifications in the immune response to allergens involv-
ing regulatory T cells, cytokines, and effector cells.5This
complex mechanism of action results in the reduction of inflam-
matory phenomena in the target organs. In addition, the changes
induced in the immune response lead to special properties not
shared by drugs, such as the preventative effect and long-lasting
persistence of the clinical benefit after discontinuation.6-8
Since the introduction of SLIT in everyday practice, some
clinical questions have been raised. One of the most important
questions is the optimal duration of a SLIT course to maintain a
long-lasting effect, if any, and the opportunity to repeat the
vaccination once the effect has vanished. In recent years, the long-
lasting effect has been demonstrated for SLIT,9,10and it is now
also known that the duration of the effect is partially dependent
on the duration of immunotherapy itself. Nonetheless, in the ear-
liest years of the use of SLIT, those aspects were totally unknown,
and experimental data were urgently needed. For this reason, we
undertook this long-term prospective study to assess the extent
of the long-lasting effects of SLIT, if any, according to its duration
(3,4,or5 years).Acomplementary and additionalassessment was
carried out on the effect of revaccination after the clinical benefit
had vanished. Despite new data being published in the meantime
and the classification and treatment strategy of respiratory allergy
having changed, the prospective study initiated in 1992 was active
for more than 15 years. We describe herein the results of this long-
term trial comparing different durations of SLIT.
For more data on study design, see Fig 1. We performed a prospec-
tive, open, controlled, 4-parallel-group, partially randomized study in-
volving patients with allergic rhinitis and bronchial hyperreactivity
Medical Department, Anallergo, Florence;cthe Department of Ecosystem and Public
Health, Faculty of Veterinary Medicine, University of Calgary; anddAllergy and Res-
piratory Diseases, DIMI, University of Genoa.
Supported in part by ARMIA (Associazione Ricerca Malattie Immunologiche
Disclosure of potential conflict of interest: I. Spadolini is employed by Anallergo. The
rest of the authors have declared that they have no conflict of interest.
Received for publication May 31, 2010; revised July 22, 2010; accepted for publication
August 19, 2010.
Reprint requests: Giovanni Passalacqua, MD, Allergy and Respiratory Diseases, Depart-
ment of Internal Medicine, Padiglione Maragliano, L.go R. Benzi 10, 16132 Genoa,
Italy. E-mail: email@example.com.
? 2010 American Academy of Allergy, Asthma & Immunology
PD20: Provocative dose causing a 20% decrease in FEV1
SCIT: Subcutaneous immunotherapy
SLIT: Sublingual immunotherapy
SLIT3: SLIT for 3 years
SLIT4: SLIT for 4 years
SLIT5: SLIT for 5 years
SMS: Symptoms plus medications score
with or without overt asthma solely because of mites. After a baseline
evaluation, patients were assigned to the control group (drugs only) or to the
SLIT group (SLIT plus drugs). This assignment was made according to
patients’ preferences. Patients receiving SLITwere then randomized according
to the birth date (decade of month) to receive the treatment for 3, 4, or 5 years.
Assessments were made yearly. The clinical diary card for the symptoms plus
medications score (SMS) was recorded from September to February. The clin-
ical efficacy of SLITwas arbitrarily assumed to persist until the SMS remained
at less than50%ofthe baselinevalue. Otherwise,a new courseofSLIT(revac-
cination) was prescribed. The duration of the second course of vaccination was
set a posteriori at 4 years based on the outcome of the first course. Additional
evaluations were as follows: (1) the number of patients who had new skin sen-
sitizations, (2) the methacholine provocative dose causing a 20% decrease in
FEV1(PD20), and (3) the percentage of nasal eosinophils. The study lasted
from1992to2007. Theethic committee approvedthe studybut denied permis-
sion to blind the treatment because of ethical reasons mainly related to the du-
ration of follow-up.
Patients and diagnosis
Adult outpatients (18-65 years) of both sexes who signed an informed
consent form were enrolled. Inclusion criteria were as follows: (1) allergic
(2) single sensitization to house dust mites, as assessed by skin prick tests or
RASTs; (3) normal spirometric results with an FEV1of greater than 79% of
the predicted value; (4) positive methacholine challenge result with a PD20
of less than 400 mg; and (4) percentage of eosinophils in the nasal smear of
at least 19%. Exclusion criteria were as follows: (1) multiple skin sensitiza-
tion; (2) previous immunotherapy courses; (3) FEV1less than 80% of pre-
dicted value or asthma requiring systemic steroids; (3) major anatomic
throphy); and (4) major systemic or autoimmune diseases, malignancies, psy-
chiatric disorders, and pregnancy.
Rhinitis was diagnosed on a clinical basis according to the presence of the 4
typical symptoms of rhinorrhea, sneezing, obstruction, and itching. Asthma
dyspnea, or cough responding to inhaled salbutamol. Because Global Initiative
for Asthma guidelines did not exist in 1991, patients were enrolled if their
tests11were performed atbaseline and at eachsubsequent year at the end ofthe
winter period. The standard panel of allergens (ALK-Abello ´, Milan, Italy) in-
cluded mites, grass, olive, Parietaria species, birch, cat and dog dander, mug-
wort, ragweed, and Alternaria and Cladosporium species.
Immunotherapy and concomitant treatments
the morning before breakfast, keeping the drops under the tongue for 2
minutes and then swallowing. The extract was biologically standardized,
titrated in RAST units per milliliter, and prepared in 5 vials at increasing
concentrations (100, 300, 1,000, 3,000, and 10,000 RAST units/mL). The
build-up phase (about 50 days) was followed by a continuous maintenance
regimen (SLIT given during the whole year), with 5 drops from the vial
containing 10,000 RAST units/mL 3 times per week. The mean cumulative
dose per year was 390 mg of Der p 1/Der p 2, which is about 10 times greater
side effects, if any, and a physician was always available by telephone for
questions or problems related to SLIT. Adherencewas assessed by measuring
the remaining volume of the extract in returned vials and, by means of
subtraction, the actually consumed volume. Adherence was expressed as a
percentage of actual versus expected consumption. All patients were pre-
scribed pharmacotherapy to be taken regularly in the period from September
through February, including cetirizine (10 mg in 1 tablet per day), nasal
cromolyn, and salbutamol (100-200 mg) on demand. Nasal beclomethasone
(400 mg/d), inhaled salmeterol (50 mg twice daily), and inhaled beclometha-
sone (500 mg twice daily) were given on medical judgment. These treatments
were chosen in 1992, when the Global Initiative for Asthma12and Allergic
Rhinitis and Its Impact on Asthma2guidelines for the treatment of asthma
and rhinitis did not exist. In the following years, after the introduction of
guidelines, the treatment was adequately modified for all patients. In particu-
lar, new inhaled or nasal steroids (budesonide and fluticasone) were intro-
duced once commercially available.
FIG 1. Patients’ disposition and dropouts.
J ALLERGY CLIN IMMUNOL
2 MAROGNA ET AL
Clinical diary for symptoms and drug intake
All patients were required to fill a diary card recording symptoms and
medication intake every year from September to February. This specific period
was chosen because a maximum exposure to mites was expected during this
time of the year and because a year-long recording was not feasible. Ten
symptoms were considered (nasal itching, sneezing, obstruction, rhinorrhea,
of them could be scored from 0 (absent) to 3 (very troublesome); the possible
maximum monthly scoreofsymptoms was therefore 900. Moreover, eachdose
of drug was scored as 1 point (eg, 2 puffs of inhaled beclomethasone 5 500
mg 5 1 point). A cumulative SMS was calculated, and the meanmonthly score
(September-February) was then used for statistical analysis.
Pulmonary function and methacholine challenge
Pulmonary function testing was performed with a plethysmograpic cabin
and a computerized spirometer (Yaeger, Wurtzburg, Germany). The methacho-
line challenge was carried out by using a dosimeter (Yaeger) with output
activated by the inhalational effort. Increasing doses of methacholine (30, 60,
FEV1was reached.12,13Spirometry with methacholine challenge was carried
out yearly in all subjects at the end of the winter season (approximately Febru-
ary), with the patients free of inhaled bronchodilators for at least 12 hours.
Nasal eosinophil counts were measured at baseline and at each subsequent
year offollow-up. The assay was performed between September and February.
Patients were instructed to withdraw nasal steroids, if taken, at least 10 days
tip. The smear was put on a glass slide, air-dried, and stained with May-
Grunwald-Giemsa stain. Samples were read with optical microscopy, and the
eosinophil count was expressed as a percentage of 100 cells in 10 fields. The
No formal sample size calculation was made, but at the time the study was
planned in 1991, based on a previouswork,14the numberof approximately20
patients per group was considered adequate. Equality of sex ratios in different
differences in the baseline level of clinical parameters were tested by using t
tests and Kruskal-Wallis tests. The intragroup and intergroup comparisons
were carried out by using the Student t test (paired or unpaired samples).
The patientswith newskin sensitizations in the 4 groupswere analyzed by us-
yses on the effect of treatments and the duration of the effect were carried out
by using a modified ANOVA for repeated measures (repeated-measures gen-
tested with a generalized linear model as well.
criteria were identified. Of them, 78 patients (44 male and 34
female patients with a mean age of 22.2 6 5.2 years) agreed to
participate in the study and entered the baseline evaluation from
September 1992 to February 1993. Twenty-one patients refused
the control group. The remaining 57 patients were prescribed
SLIT in addition to drugs and were subsequently randomized
(according to birth date decade) to receive immunotherapy for 3
(the SLIT5 group, n 5 17). The 4 groups were homogeneous at
baseline for demographics and symptom scores. The PD20was
significantly higher in the SLIT5 group versus the others, and
nasal eosinophil counts were higher in the SLIT3 and SLIT5
groups than in the SLIT4 and control groups (Table I).
During the 15 years of observation, 19 patients dropped out (5
from the SLIT3 group, 5 from the SLIT4 group, and 9 from the
control group; Fig 1). All the dropouts were due to protocol devi-
ations (ie, patients lost because they refused to attend the clinical
of evaluations after 15 years (14 from the SLIT3 group, 16 from
the SLIT4 group, 17 from the SLIT5 group, and 12 from the con-
trol group). The adherence to SLIT, evaluated by measuring the
volume of extract in the returned vials, was greater than 80% in
all the groups.
Clinical evaluation and duration of effect
the control group no significant change versus baseline was seen
for 6 years. Starting from the seventh year, the reduction become
significant (P 5 .03) and remained so afterward. In the 3 SLIT
nificant at every year until the end of the study. The difference
versus the control group remained significant since the first year
for the SLIT3 and SLIT4 groups (P 5.004 and P 5.02, respec-
tively) and since the second year for the SLIT5 group (P 5.04).
in the remaining groups (Fig 2). The SMS increased to more than
8 years in the SLIT4 and SLIT5 groups. At these points, revacci-
nation was started, achieving a significant reduction in the SMS
versus that seen in the control group after 1 year (P <.01 for all
groups). The medication intake score represented about 20% of
the SMS. If analyzed separately, their behavior strictly paralleled
that of the SMS, as shown in Fig 3.
(F5 25.350,df5 2.919,P <.001)wassignificantlyinfluencedby
treatment (F 5 9.968, df 5 8.757, P <.001) but not by the symp-
toms’ levels at baseline (F 5 1.324, df 5 2.919, P > .05). Con-
versely, the differences in SMSs among groups in the years after
SLIT were significantly influenced by the treatment duration
(F 5 33.327, df 5 3, P <.001), as well as by baseline symptoms
(F 5 9.589, df 5 1, P 5 0.003) and their interaction (F 5 6.141,
df 5 1, P 5.001). Among treated groups, the only significant dif-
ference was between the SLIT3 and SLIT5 groups (P 5.024).
During the 15 years of follow-up, all the patients in the control
group had at least 1 new skin sensitization. In the other groups at
theend ofthestudy,newsensitizationshadappeared in3(21.4%)
of 14 patients of the SLIT3 group, 2 (12.5%) of 16 patient of the
SLIT4 group, and 2 (11.7%) of 17 patients of the SLIT5 group. In
particular, thedifference becomesignificant at year 6 (P 5.03, x2
test). The yearly percentage of patients with at least 1 new sensi-
tization over the 15-year period is summarized in Fig 4.
The behavior of nasal eosinophils mirrored, in part, that of
symptoms, with a decrease that became significant versus base-
line values at the first year in the SLIT3 group (P 5.03), at the
J ALLERGY CLIN IMMUNOL
VOLUME nnn, NUMBER nn
MAROGNA ET AL 3
second year in the SLIT5 group (P 5.02), and at the third year in
the SLIT4 group (P 5.04, Fig 5). Starting from the third year, a
significant difference versus baseline was maintained in all the
SLIT groups during the entire study. In the control group a tran-
sient but significant decrease in eosinophil counts was detectable
at the sixth and seventh years of the study. Notably, in the SLIT4
and SLIT5 groups, the eosinophil counts remained significantly
lower than in the control group for the entire study. On the
contrary, in the SLIT3 group the significant difference versus
the control group was lost 4 years after discontinuation and
achieved again immediately after revaccination (Fig 5).
All patients had a baseline PD20of approximately 200 mg. This
value did not change significantly over time in the control group,
TABLE I. Clinical characteristics at baseline
Control group (n 5 21) SLIT3 group (n 5 19) SLIT4 group (n 5 21) SLIT5 group (n 5 17)P value
Age (y), mean 6 SEM
Age range (y)
SMS, mean 6 SEM
PD20(mg), mean 6 SEM
Nasal eosinophils (%)
23.8 6 1.5
385 6 17.0
186.2 6 23.2
33.8 6 0.6
21.1 6 1.4
417 6 14.8
163.6 6 21
40.9 6 1.5
21.3 6 1.6
383 6 15.3
124.0 6 15.4
35.0 6 0.8
22.8 6 1.6
412 6 16.3
250.5 6 24.1
39.1 6 1.7
Values in boldface are significantly different from the other groups.
*Defined retrospectively according to the Global Initiative for Asthma.
FIG 2. Mean monthly SMSs (means and SDs) throughout the 15 years of the study in patients in the SLIT3
(A), SLIT4 (B), and SLIT5 (C) groups. The duration of SLIT treatment is indicated by arrows at the bottom.
The asterisks indicate a significant difference versus the control group.
J ALLERGY CLIN IMMUNOL
4 MAROGNA ET AL
except for the years 1997, 2002, and 2006, when PD20was signif-
gressively increased, and a statistical difference versus baseline
was seen at the first year in the SLIT4 group (P 5.05) and at the
second year in the SLIT3 and SLIT5 groups (P 5.008 and P 5
(Fig 6). Starting from the third year of the study, the PD20in the 3
SLIT groups remained significantly higher than in the control
group, with the exception of the SLIT3 group, in which the differ-
ence versus the control group disappeared 6 years after discontin-
uation and was then reachieved after the second SLIT course. The
same happened in the SLIT4 group 8 years after discontinuation
(Fig 6). Of note, in the SLIT5 group the PD20slowly decreased
over the years, but it remained always significantly different
from baseline and from that seen in the control group.
During the first courses of treatment, 5 patients (2 in the SLIT3
group, 1 in the SLIT4 group, and 1 in the SLIT5 group) had
the SLIT3 group and 1 in the SLIT5 group) reported 1 episode of
generalized itching on maintenance. All events occurred 30
minutes after dosing and spontaneously disappeared without
FIG 3. Monthly drug intake scores throughout the study in the SLIT3 (blue line), SLIT4 (red line), SLIT5
(green line), and control (black line) groups. The SLIT treatments are indicated by colored horizontal arrows.
The asterisks indicate a significant difference between the control group and the 3 SLIT groups.
FIG 4. Percentage of patients with at least 1 new skin sensitization in the SLIT3 (blue line), SLIT4 (red line),
SLIT5 (green line), and control(black line) groups. The asterisk indicates the significant difference versus the
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VOLUME nnn, NUMBER nn
MAROGNA ET AL 5
The most important distinguishing feature between drug ther-
apy and specific immunotherapy is that the latter can profoundly
it has been shown that the mechanism of action involves
numerous components of the immune response, such as regula-
tory T cells, IgG4 ‘‘blocking’’ antibodies, and THcell subsets.5
Some of those effects have been recently confirmed also for
tion is that immunotherapy possesses special properties not
shared by drugs, namely the long-lasting efficacy after discontin-
uation and the modification of the natural course of the disease.
The long-lasting effect is of primary relevance, not only from a
specific immunotherapy course to achieve a long-lasting benefit.
About 15 years ago, there were only sparse data on the long-
lasting effect of SLIT, and this aspect was considered one of the
whether a long-lasting effect exists and, if so, to determine the
nature of its relationship with the treatment’s duration. Our long-
lasting survey showed that (1) a long-term effect (>4 years) of
SLITexistsand correlates to the duration of treatment; (2) theop-
timal duration of SLIT to achieve a long-lasting effect is 4 years
because a duration of 5 years adds only marginal additional ben-
FIG 5. Percentages of nasal eosinophils throughout the study in the SLIT3 (blue line), SLIT4 (red line), SLIT5
(green line), and control (black line) groups. SLIT treatments are indicated by colored horizontal arrows. The
asterisks indicate a significant difference between the control group and the 3 SLIT groups. NS, No signif-
icant difference between the control group and the SLIT3 group.
FIG 6. Methacholine PD20(in micrograms) throughout the study in the SLIT3 (blue line), SLIT4 (red line),
SLIT5 (green line), and control (black line) groups. The duration of SLIT treatments is indicated by colored
horizontal arrows. The asterisks indicate a significant difference between the control group and the 3 SLIT
groups. NS, No significant difference between the control and SLIT3 groups.
J ALLERGY CLIN IMMUNOL
6 MAROGNA ET AL
involves local inflammation (nasal eosinophil counts) and bron- Download full-text
chial responsiveness; and (4) a second course of SLIT, once the
clinical benefit has vanished, has a prompt effect.
Overall, the clinical results obtained in this study are in
agreement with other reports concerning SLIT and SCIT.6,9,10
For instance, the carry-over effect 1 year after discontinuation of
SLIT has been confirmed in a large randomized trial.19Another
trial with SLIT has shown that a coseasonal treatment given for 3
benefit.20Also, the effect of SLIT on bronchial hyperresponsive-
ness has been demonstrated both in children21and adults.22In
this regard it can be speculated that the effect on PD20reflects
the anti-inflammatory effect of the treatment, although inflamma-
tion is only one of the components of bronchial hyperreactivity.
Similarly,theeffect onnasal inflammation (ie,number ofinfiltrat-
in the SLIT3 group, the increase ineosinophilcounts afterdiscon-
tinuation of SLIT seems to anticipate the clinical worsening.
The main weakness of this study is the lack of an initial
randomization (eg, patients receiving SLIT vs control subjects),
criteria of evidence-based medicine24were not yet well estab-
lished in clinical research. Also, there is not a placebo group,
which is considered mandatory for immunotherapy trials25be-
cause the placebo effect could account for up to 30% of the ob-
served changes.26On the other hand, it was neither ethical nor
feasible to have a placebo group in such a long-term study. This
drawback is partly counterbalanced by the fact that the control
and active groups were homogeneous at baseline. Another weak
point is the high rate of dropouts, but this could not be avoided
in a study that lasted many years. In fact, all the dropouts were
due to lack of compliance with the protocol. Another possible
weakness is that the criteria for diagnosis and treatment (at least
for asthma) changed over the years. The choice of the cumulative
SMS was based on the fact that the 2 parameters are strictly inter-
dependent,and infact, themostrecent recommendationsindicate
of our work is its long duration, which allowed us to well appre-
ciate the changes in the parameters over time. Another strength is
the careful selection of patients, who were monosensitized and
homogeneous for disease severity. In addition, the objective pa-
rameters, such as bronchial reactivity, nasal eosinophil counts,
and skin sensitizations, reasonably support the clinical results.
In conclusion, under the described conditions, a 4-year dura-
of the clinical benefit. In addition, a second course of vaccination
achieves an even more rapid benefit.
a 4-year course seems to be a reasonable choice. When the long-
lasting effect attenuates, a second course promptly achieves the
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