ORIGINAL ARTICLE
Bacterial Mucosal Immunotherapy with MV130 Prevents Recurrent
Wheezing in Children
A Randomized, Double-Blind, Placebo-controlled Clinical Trial
Antonio Nieto1, Angel Maz
on1, Mar
ıa Nieto1, Rafael Calder
on2, Susana Calaforra1, Blanca Selva1, Sonia Uixera1,
Maria Jos
e Palao2, Paola Brandi3, Laura Conejero4, Paula Saz-Leal4, Cristina Fern
andez-P
erez5, David Sancho3,
Jos
e Luis Subiza4, and Miguel Casanovas4
1Unidad de Neumolog
ıa y Alergia Pedi
atrica, Instituto de Investigaciones Sanitarias, Hospital Universitario La Fe, Valencia, Spain;
2Departmento de Pediatr
ıa, Manises Hospital, Valencia, Spain; 3Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain;
4Inmunotek S.L., Alcal
a de Henares, Spain; and 5Instituto de Investigaci
on Sanitaria del Hospital Cl
ınico San Carlos, Universidad
Complutense, Madrid, Spain
ORCID IDs: 0000-0002-6302-6115 (A.N.); 0000-0001-5639-1037 (A.M.); 0000-0001-6515-9221 (L.C.); 0000-0002-6263-4709 (P.S.-L.);
0000-0003-2890-3984 (D.S.); 0000-0002-0134-5321 (J.L.S.); 0000-0003-2330-3963 (M.C.).
Abstract
Rationale: Recurrent wheezing in children represents a severe
public health concern. Wheezing attacks (WA), mainly associated
with viral infections, lack effective preventive therapies.
Objectives: To evaluate the efficacy and safety of mucosal
sublingual immunotherapy based on whole inactivated bacteria
(MV130) in preventing WA in children.
Methods: A Phase 3 randomized, double-blind, placebo-controlled,
parallel-group trial including a cohort of 120 children ,3 years old
with>3WA during the previous year was conducted. Children with
a positive skin test to common aeroallergens in the area where the
clinical trial was performed were excluded from the trial. Subjects
received MV130 or placebo daily for 6 months. The primary
endpoint was the number of WA within 1 year after the first dose
comparing MV130 and placebo.
Measurements and Main Results: There was a significant lower
number of WA in MV130 versus the placebo group, 3.0
(interquartile range [IQR], 2.0–4.0) versus 5.0 (IQR, 3.0–7.0)
(P, 0.001). As secondary outcomes, a decrease in the duration of
WA and a reduction in symptoms and medication scores in the
MV130 versus placebo group were found. No adverse events were
reported related to the active treatment.
Conclusions: Mucosal bacterial immunotherapy with MV130
shows safety and clinical efficacy against recurrent WA in children.
Clinical trial registered with www.clinicaltrials.gov (NCT 01734811).
Keywords: clinical trial; wheezing attacks; mucosal vaccination; MV130
(Received in original form March 5, 2020; accepted in final form March 11, 2021)
This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0
(https://creativecommons.org/licenses/by-nc-nd/4.0/). For commercial usage and reprints, please contact Diane Gern (dgern@thoracic.org).
Supported by the INNPACTO grant IPT-2012-0639-090000; the Spanish Ministry of Economy, Industry, and Competitiveness (MINECO), Spain, to
Inmunotek S.L.; MINECO grant BES-2014-069933 (“Ayudas para Contratos Predoctorales para la Formaci
on de Doctores 2014”) (P.B.); and the
European Respiratory Society Fellowship grant RESPIRE2-2013-3708 (L.C.). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is
supported by the Ministerio de Ciencia e Innovaci
on and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).
Author Contributions: A.N., J.L.S., and M.C. conceived and/or designed the clinical trial. A.N., A.M., M.N., R.C., S.C., B.S., S.U., and M.J.P.
performed the clinical trial. C.F.-P. performed the statistical analysis of the clinical trial. A.N., A.M., P.B., L.C., P.S.-L., D.S., J.L.S., and M.C. wrote the
manuscript. All authors reviewed and revised the manuscript before submission.
Data collected for the study will be made available for researchers whose proposed use of the data has been approved for specified purposes and
on a signed data access agreement. Inmunotek will take all necessary measures to ensure that patient privacy is safeguarded. Request for this
information must be sent to the corresponding author.
Correspondence and requests for reprints should be addressed to Antonio Nieto, M.D., Ph.D., Unit of Pediatric Allergy and Pneumology, Health Research
Institute La Fe, La Fe University Hospital, Avinguda de Fernando Abril Martorell, n
 106, 46026 Valencia, Spain. E-mail: antonio.nieto@me.com.
This article has a related editorial.
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
Am J Respir Crit Care Med Vol 204, Iss 4, pp 462–472, Aug 15, 2021
Copyright © 2021 by the American Thoracic Society
Originally Published in Press as DOI: 10.1164/rccm.202003-0520OC on March 11, 2021
Internet address: www:atsjournals:org
462 American Journal of Respiratory and Critical Care Medicine Volume 204 Number 4 | August 15 2021
Wheezing attacks (WA) or
bronchospasms are defined as episodes of
cough with difficult breathing and/or
wheezing, lasting at least 6 hours, with or
without fever and without chest
radiographic abnormalities (1). WA
constitute an important health threat in
children worldwide (2), affecting a high
percentage (30–50%) of them within their
first 6 years of life. Recurrent WA show a
high (17%) prevalence (3, 4). Although
most of the affected children with
bronchiolitis eventually outgrow this
disorder, recurrent WA constitute a
public health problem, with a
considerable social, quality of life, and
economic impact (2, 5).
Most wheezing episodes in young
children have a viral etiology, mainly
rhinovirus and respiratory syncytial virus (2,
6). The prevention of recurrentWA is amajor
concern, as vaccines targeting these viruses are
not yet available (7) and effective antiviral
therapies are still lacking (6). Current
prevention strategies with antiinflammatory
drugs are far from being optimal (2). Some
studies propose the use of daily inhaled
corticosteroid therapy among preschool
children with recurrent wheezing and high-
dose intermittent inhaled corticosteroids
amongpreschool childrenwithviral-triggered
wheezing (8). The use of oral corticosteroids
for acute wheeze exacerbation in preschool
children has also been suggested, but the level
of clinical benefit demonstrated remains to be
low(9).Azithromycinhasbeenalsopostulated
as a therapeutic option in children with
recurrent wheezing in the context of lower
respiratory tract infections for acute
exacerbations or to prevent severe
exacerbations (9, 10); however, potential
benefits must be weighed against further
selection of antimicrobial-resistant
microorganisms and possible detrimental
effects on the commensal or beneficial airway
or gut microbiota. Montelukast has been also
studied as a potential alternative in the
prevention of preschool recurrent wheezing
withcontroversial results (11,12).All inall, the
lack of clearly effective therapeutic
interventions and preventive strategies are a
reality.
An increasingnumber of studies indicate
that certainmicrobial stimuli acting on innate
immunecells canpromote aquite long-lasting
nonspecific protection against different
pathogens, a phenomenon termed trained
immunity (13–15).
MV130 is a sublingual polybacterial
preparation able to stimulate innate
immune cells and to enhance certain T cell
responses to related and unrelated
(bystander) antigens (16, 17). In previous
studies in adults, MV130 was effective in
preventing recurrent infections (18, 19),
including someof viral origin (17).Here,we
have addressed the efficacy and safety of
MV130 in children with recurrent WA in a
double-blind, placebo-controlled (DBPC)
study. Some of the results of this study have
been previously reported in the form of
abstracts (20, 21).
Methods
Study Design, Randomization,
and Masking
A Phase 3 randomized, DBPC, parallel-
group clinical trial was performed in two
hospitals in Spain (La Fe University
Hospital and Manises Hospital, both in
Valencia) to assess the safety and efficacy of
MV130. From October 2012 through May
2015, subjects were enrolled in the clinical
trial. The study included 120 children,3
years old with recurrent WA, defined as
three or more episodes of WA during the
previous year (2). WA were defined as
episodes of cough with difficult breathing
and/or wheezing, lasting at least 6 hours,
with or without fever and without chest
radiographic abnormalities in the case that
a radiograph was made (1). All children
were referred by their primary care
pediatrician because of recurrent wheezing
during the previous year. The exclusion
criteria were other chronic respiratory
diseases, malnutrition, positive skin tests to
common aeroallergens in the area where
the clinical trial was performed, or
treatment with g-globulin,
immunostimulants, or
immunosuppressants in the previous 12
months. The WA were confirmed by the
review of themedical records. Aeroallergen
sensitization at baseline was used as an
exclusion criterion to avoid eventual WA
due to allergic asthma (2, 22, 23). Details on
the characteristics of the study and
inclusion and exclusion criteria are
described in Table 1.
The clinical trial was conducted within
the ethical and legal framework established by
the Spanish Agency forMedicines andHealth
Products (RD 223/2004) following good
clinical practice (ICH E6: Good Clinical
Practice: Consolidated Guideline, CPMP/
ICH/135/95) in accordance with European
Union Directive 2005/28/CE. Parents or legal
guardiansprovidedwritten informedconsent.
Regulatory approvals and consent procedures
are described in the online supplement.
Participantswere allocated bymeans of a
list generated by Random software (Random
Software Ltd.) in a 1:1 ratio to receive either
MV130 or placebo. Both participants and
physicians (who enrolled the patients) were
masked to treatment assignments. The active
product (MV130) (Bactek; Inmunotek; S.L.)
consists of a suspension of heat-inactivated,
whole-cell gram-positive (90%) and gram-
At a Glance Commentary
Scientific Knowledge on the
Subject:Wheezing attacks constitute
a worldwide health threat for
children. Almost 50% of children
during their first 6 years of life
present episodes of wheezing attacks.
The etiology of wheezing attacks is
diverse, although rhinovirus and
respiratory syncytial virus are the
main causes of wheezing episodes in
young children. Although wheezing
attacks can be treated with
antiinflammatory drugs, the lack of
vaccines remains the major concern
in the prevention of the disease.
Based on the evidence to date,
bacterial immunostimulants provide
clinical benefit in patients with
recurrent respiratory tract infections,
including pediatric wheezing.
What This Study Adds to the Field:
For the first time, in this Phase 3
randomized, double-blind, placebo-
controlled clinical trial, the safety and
efficacy of the sublingual heat-
inactivated whole-cell bacterial
formulation MV130 were
demonstrated. An important
reduction in the number and
duration of wheezing attacks in the
MV130 group compared with the
placebo group is shown. The
heterologous immunity conferred by
MV130 provides persistent
protection without the limitations of
antigen specificity being useful in
case of coinfection or against
pathogens with high mutation rates.
ORIGINAL ARTICLE
Nieto, Maz
on, Nieto, et al.: MV130 Prevents Children’s Wheezing Attacks 463
negative (10%) bacteria (300 formazin
turbidity units/ml109 bacteria/ml) in
glycerol, sodium chloride, and artificial
pineapple flavoring with the following
formula: Streptococcus pneumoniae (60%),
Staphylococcus aureus (15%), S. epidermidis
(15%), Klebsiella pneumoniae (4%),
Moraxellacatarrhalis (3%),andHaemophilus
influenzae (3%). The placebo preparation
containedall excipientswithoutbacteria.The
treatment was administered sublingually by
spraying under the tongue two puffs of 100
ml each daily for 6 months. To check the
correct administration of themedication, the
firstdosewasadministered inthehospital; the
following doses were administered at home.
To assess the degree of compliance to the trial
medication, the volume of medication
Table 1. Design Characteristics of the Trial
Type of study Prospective, two-center, randomized, double-blind, placebo-controlled, parallel-group
Patients Children ,3 yr old with recurrent wheezing
Exclusion criteria Other chronic respiratory diseases
Malnutrition
Positive skin tests to any of Dermatophagoides pteronyssinus, Dermatophagoides farinae,
Alternaria alternata, dog dander, cat dander, olive tree pollen, and/or grass pollen
Treatment with g-globulin, immunostimulants, or immunosuppressants in the previous
12 mo
Sample size N=120; difference in expected number of WA: 1.7; SD: 2.55
a-error: 0.05, b-error (two tailed): 0.1 estimated drop-out rate: 20%
Intervention Active treatment (MV130): Placebo:
Glycerol
Sodium chloride
Glycerol
Sodium chloride
Artificial pineapple flavoringArtificial pineapple flavoring
300 FTU/ml (109 whole-cell
inactivated bacteria/ml):
Streptococcus pneumoniae (60%)
Staphylococcus aureus (15%)
Staphylococcus epidermidis (15%)
Klebsiella pneumoniae (4%)
Moraxella catarrhalis (3%)
Haemophilus influenzae (3%)
Dose and duration of treatment Two puffs sublingually daily for 6 mo
(108 bacteria/100 ml per puff)
Two puffs sublingually daily for 6 mo
Allocation randomization Allocation (1:1) by simple randomization
Randomization list generated using Random software
Blinding Parents, physicians involved in recruiting and follow-up, and any other care providers were
blinded
Treatment concealment Identical bottles with active treatment or placebo were supplied. Each bottle was marked with
the individual patient numeric exclusive code generated by the randomization list
Treatment adherence The volume of medication remaining was measured from the bottles returned at each
scheduled visit
Code breaking For analysis, after the last visit of the last patient
Main outcome Number of WA during a whole year
(6 mo of treatment1 6 additional mo of follow-up)
Other outcomes Duration and severity of WA, symptom and medication scores, use of health and social
resources, time to first WA
Statistical analysis Shapiro-Wilk test. Chi-square or Fisher’s exact tests. Student’s t test and Mann-Whitney U
test. Hodges-Lehmann estimator. The number needed to treat. Kaplan-Meier estimator
and Cox model. P, 0.05 was considered significant
Type of analysis Per intention-to-treat. Sensitivity analysis using the “worst-case scenario”
Ethics Approved by Ethics Committee and Spanish health authorities
Run according to the Helsinki declaration, the European laws, and the rules of good clinical
practice
Written information. Informed consent signed by parents
Trial registration EudraCT number 2012-002450-24
Clinicaltrials.gov identifier NCT01734811
Definition of abbreviations: FTU= formazin turbidity units; WA=wheezing attacks.
ORIGINAL ARTICLE
464 American Journal of Respiratory and Critical Care Medicine Volume 204 Number 4 | August 15 2021
remaining was measured from the bottles
returned at each scheduled visit. The whole
study lasted 12 months, with 6 months of
treatment plus 6 additional months of
follow up.
Patients could receive additional
medication as needed according to the Global
Initiative for Asthma guidelines. Visits to
primary care pediatricians or an emergency
department were allowed as needed for the
child’s clinical condition. A symptom score
andmedication score were rated, calculated,
and recorded. WA that occurred during the
trial were diagnosed by the primary care
pediatrician or in emergency room visits.
Parentsor legalguardianswereduly instructed
to record symptom andmedication scores in
daily diary cards. These were carefully
reviewed in a blinded manner by the
investigators who defined for each patient the
initiation and the endof eachwheezing attack.
In these cards, health and social resources
during the study (including complementary
tests and unscheduled visits to a pediatrician)
were also recorded.
Outcomes
The primary endpoint was to compare the
meannumberofWAbetweenactive (MV130)
and placebo groups during a follow up of 12
months after receiving the first dose. A
reductionintheactivegroupascomparedwith
the placebo group will indicate that MV130
maypreventWAinchildrenat risk.Secondary
endpoints included the number of days with
wheezing during the study, duration (days) of
WA, time (days) until the appearance of the
first WA, number of patients with recurrent
WA during the study, symptom score and
medication score and their combination
related toWA, and, during the whole study
period, the use of health and social resources
anddayswith fever.Patientswerevisitedat the
clinics every 3 months, and diaries were
reviewed together with the parents. Chest
symptoms (bronchialmucus secretion, cough,
difficult breathing, and wheezing), nose
symptoms (nasal mucus secretion), and
discomfort were rated as follows: 0= absent
(no sign/symptom evident), 1 =mild (sign/
symptom clearly present but easily tolerated),
2 =moderate (definite awareness of sign/
symptom that is bothersome but tolerable),
and 3= severe (sign/symptom that is hard to
tolerate and causes interference with activities
of daily life and/or sleeping). Fever was scored
as thenumberofdayswithaxillar temperature
over 37
C. The total symptom score was
calculated as the sum of all individual scores.
We did not address specifically whether some
symptoms, like cough, were mainly due to
upper or lower tract infections, as they can be
shared inmany cases and therefore difficult to
distinguish. The medication was rated
according toDreborg and colleagues (24)with
slightmodifications: twopointswere given for
one tablet of 4mgofMontelukast, for onepuff
of 50 mg budesonide equivalent, or for one
dose of oral prednisolone, and three points
weregiven for the inhalationofonepuff of 200
mg budesonide equivalent. In the case of
antibiotics and antipyretic and/or
antiinflammatory drugs, the number of daily
doses was recorded.
The evaluation of the worst-case
scenario, which assumes that all missing
participants have a poor outcome in the
MV130 groupand a favorable outcome in the
placebo group, was further performed as
described in the online supplement.
Safetywasassessedvia therecordingofall
adverse events that occurred during the trial.
Adverse events were individually examined to
evaluate their severity and to classify them as
probably related or unrelated to the study
medication. Adverse events were coded with
the use of theMedical Dictionary for
Regulatory Activities. Respiratory tract
infections, the objective of the study, were not
considered an adverse event.
Statistical Analysis
The sample size for the clinical trial was
calculated using data fromGuti
errez-Tarango
and colleagues related to the number of acute
respiratory tract infections in children (25). In
this study, we found a difference of means for
episodes of bronchospasm of 2.96 (95%
confidence interval [CI],24.22 to21.70). To
detect a difference of 1.7 (the lowest 95% CI
limit) or higher in episodes of bronchospasm
at 12 months between both groups, assuming
ana-errorof0.05andapowerof90% ina two-
sided comparison, a minimum of 59 subjects
per group was required. A loss to follow up of
20% was assumed.
All randomizedpatientswere included in
our analysis (i.e., an intention-to-treat
analysis). We present summary statistics as
frequency (percent) for categorical data and
median (interquartile range [IQR]) or 95%CI
for continuous data, according to the normal
distributionanalyzedbytheShapiro-Wilktest.
Chi-square or Fisher’s exact tests and paired
Student’s t tests were used to analyze the
significant differences of the variables. When
data did not fit a normal distribution, a
nonparametric Mann-WhitneyU test and
Hodges-Lehmann estimator were used. The
Kaplan-Meier estimator was used to compare
time until the appearance of the firstWAafter
the initiation of treatment and after the
discontinuationof treatment6months later.A
Coxmodel was adjusted to evaluate the real
effect expressed as a hazard ratio and the 95%
CI. TheHodges-Lehmann estimatorwas used
to measure the effect size of the differences
betweenMV130 and placebo groups. The
number needed to treat was calculated based
on the number of patients to be treated to
prevent one case of recurrent wheezing (three
or moreWA) during the study. In any case, a
levelofsignificanceofP, 0.05wasestablished
for all tests performed.
For those patients who stopped study
medication prematurely, the values recorded
at the last visit postbaseline (last observation
carried forward) were used to maintain the
intention to treat.
Statistical analyses were performed
using the STATA 12.0 (StataCorp) software
or Prism Software 6.01 (GraphPad). Outliers
were identified and removed by means of
Tukey’s range test.
Results
Trial Participants
Of the 373 children (age range 6–35mo) who
underwent screening, 120 were enrolled. A
total of 62 patients were assigned to MV130
(active) and 58 to the placebo. Seven patients
abandoned the study (three active, four
placebo): five cases (three active, two placebo)
could not be followed up and two placebo
requested withdrawal (one placebo because of
an epileptic seizure at the beginning of the
study, and the other one due to vomiting and
diarrhea on the third day of treatment). The
flowchart of patients is shown inFigure 1.The
groups appear balanced at baseline.
Efficacy
The median of WA before the study was 8.0
(IQR, 7.0–10.0) and 9.0 (IQR, 7.0–11.3) for
theMV130 and placebo groups, respectively
(P= 0.102) (Table 2).Notably, themedian of
WA per child during the 12-month clinical
trial period (primary outcome) was 3.0
(IQR, 2.0–4.0) versus 5.0 (IQR, 3.0–7.0) for
MV130 and placebo, respectively
(P, 0.001). Thus, a significant reduction in
the number of WA was observed in the
subjects who received MV130 when
compared with the placebo. The total
number ofWAwas 176 in theMV130 group
ORIGINAL ARTICLE
Nieto, Maz
on, Nieto, et al.: MV130 Prevents Children’s Wheezing Attacks 465
and 299 in the placebo group (Figure 2A and
see Table E1 in the online supplement). The
total median number of days withWA (19.0
[IQR, 8.5–35.0] vs. 42.0 [IQR, 17.0–57.5])
and the median duration in days ofWA (6.0
[IQR, 4.0–9.5] vs. 7.9 [IQR, 6.2–11.0]), both
secondary efficacy endpoints, were also
significantly lower in the MV130 group
(P, 0.001 and P= 0.005, respectively)
(Figures 2B and 2C). These data are shown
for thewhole year study and for the different
time points (Table E1). A post hoc statistical
analysis showed that the age (as a covariate)
of the population included in the study has
no effect (P= 0.749) in the highly significant
clinical improvement (P, 0.001) obtained
with MV130 when compared with placebo.
Because the number of children younger
than 18 months was low (13 in the placebo
and 16 in the active group), we analyzed the
model adjusted with age as a categorical
variable (younger or older than 18 mo) and
its interaction with the group; the effect of
the intervention on the number of WA
remains highly significant (P, 0.001), the
effect of the age (as a categorical variable) on
the number of WA between groups is not
significant (P= 0.114), and the P value of the
interactionof age–group is 0.048, suggesting
that the effect of MV130 compared with the
placebo is different between those younger
than 18 months and those older.
Patients free of newWAwere 6 in the
MV130 and 0 in the placebo group (P=0.029)
(Figure 2D), whereas children continuing to
have recurrentWA (>3WA/yr) were 36
(58%) in theMV130 and 45 (80%) in the
placebo group (P=0.009). The number
neededto treat topreventonecaseofrecurrent
wheezing was five (95%CI, 2.6–16.1). Among
the participants, the median time until the
appearance of the firstWA after the initiation
of treatment was significantly delayed in the
MV130 group compared with the placebo
(41.0 [15.0–94.5] vs. 5.0 [2.0–28.8] d,
respectively). Likewise, during the
observational posttreatment period, there
were differences in the same outcome (180
[48.5–180.0] vs. 44 [20.0–121.5] d,
respectively) (Figures 2D and 2E).
Other secondary efficacy endpoints
were the symptom score, medication score,
and their combination considered during
the WA and throughout all the study
periods. These were also significantly lower
in the MV130 group when considering
global combinations (Figure 3 andTable 3).
In addition, in the evaluation of the worst-
case scenario, the MV130 group still
showedahighly significant difference in the
analyzed outcomes (Table E2). The results
of additional secondary endpoints
including health and social resources are
detailed in Table E3.
Safety
Neither local nor systemic reactions occurred
during the clinical trial. One patient in the
placebo group had a serious adverse event
(epileptic seizure), and this subject withdrew
on the recommendation of his/her
neuropediatrician. A total of 166 adverse
eventswere registered, 81 in theMV130group
and85 in theplacebogroup, andnoneof them
were considered to be related to the active
product. These adverse events were common
pathologies in infants, including
gastroenteritis (21 [12.7%] of 166),
conjunctivitis (20 [12.0%]of166),diarrhea (17
[10.2%] of 166), and dermatitis (17 [10.2%] of
166). The adverse reactions were classified as
mild (155 [93.3%]),moderate (10 [6.0%]), and
severe (1 [0.6%]). The most frequent adverse
events are shown in Table 4.
Discussion
Based on the available clinical data, there is
evidence supporting the benefit of bacterial
mucosal immunotherapy in the prevention of
recurrent respiratory tract infections in
children, includingWA.However, the diverse
trial results, together with poor
methodological quality, give rise to a need to
conduct randomized controlled trials to
generate high-quality data (26, 27).Herein,we
have shown in a randomized DBPC parallel-
group clinical trial that MV130 is safe and
reducesWA in infants and toddlers by 40%
over a placebo.No adverse reactions related to
the active product were reported. In addition,
theperiodwithout respiratorysymptomsuntil
the next WAwere statistically different
between groups, even whenMV130 was
discontinued (second half-year), pointing to a
long-lasting effect inferring clinical
significance. The robustness of our study was
further supported by a “worst-case scenario”
analysis (28). Finally, in the active group, there
was a significant reduction in social resources,
including daycare, absenteeism, and
caregivers, that might have a direct impact on
the economy of families.
The current clinical trial is, to the best of
our knowledge, the first one in which a heat-
inactivated, whole-cell bacterial, sublingual
preparation is evaluated in a DBPC study for
efficacy againstWA in children. Compared
with gastrointestinal administration,
sublingual and intranasal delivery induce a
superiormucosal immune response in a range
of tissues, including the airways, and present
similar magnitude and anatomic
373 patients assessed for eligibility
120 randomized
253 excluded
•    65 declined
•  114 presented exclusion criteria
•    74 other reasons
54 completed59 completed
2 lost to follow-up
(unable to contact)
2 discontinued
•  1 epilepsy seizure
•  1 vomiting/diarrhea
3 lost to follow-up
(unable to contact)
62 assigned to MV130 58 assigned to placebo
Figure 1. Trial profile.
ORIGINAL ARTICLE
466 American Journal of Respiratory and Critical Care Medicine Volume 204 Number 4 | August 15 2021
dissemination of the induced immune
responses (29).Moreover, thesublingual route
is effective for vaccine delivery, inducing both
systemic and mucosal immunity (29, 30) for
MV130 (16–18) andotherwhole-cell bacterial
formulations (31–33). Improving mucosal
immunity is an important aspect when
considering pathogens that infect through
mucosal tissues (34, 35).Our results are in line
with a previous study of Razi and colleagues
in preschool children who received oral
capsules of bacterial lysates, showing a
reduction of 38% of WA over placebo (36).
Despite similar efficacy in preventing WA,
no reduction in systemic steroids was
achieved in that study (36), incontrast toour
results showing reduced medication score
(including inhaled budesonide and oral
prednisolone). In a more recent study, the
same product used by Razi and colleagues
appeared to be effective in preventing severe
lower respiratory illness in at-risk infants
during the first winter (37) yet without any
carryover protection, in contrast to the
previous results (36) and current study with
MV130, which might be related to the
younger age of the children studied (37). In
A
0
5
10
15
P< 0.001
Placebo MV130
N
um
be
r o
f W
A
C
0
4
8
12
16
20
P= 0.005
Placebo MV130
D
ur
at
io
n 
of
 W
A 
(da
ys
)
B
0
30
60
90
120
P< 0.001
Placebo MV130
D
ay
s 
wi
th
 W
A
Time until first WA
Wheezing attacks (WA)
0 50 100 150 200 250 300 350 400
0
20
40
60
80
100
Placebo
MV130
Placebo
MV130
Time (Days)
Pa
tie
nt
s 
fre
e 
of
 W
A 
(%
)
D Whole period of study (1 year)
Number at Risk
MV130
Placebo
62
58
15
3
8
2
7
1
6
0
28
12
11
3
7
1
8
1
Hazard ratio 0.45
(95% CI 0.30 – 0.67); P<0.001 
0 50 100 150 200
0
20
40
60
80
100
Time (Days)
Pa
tie
nt
s 
fre
e 
of
 W
A 
(%
)
E Post-treatment period (6 months)
Number at Risk
MV130
Placebo
62
58
48
28
37
22
35
13
34
12
Hazard ratio 0.57
(95% CI 0.39 – 0.82); P<0.001
Figure 2. MV130 reduces the number and duration of wheezing attacks (WA). (A–C) WA episodes during the whole study: (A) number of WA, (B)
days with WA, and (C) duration of WA. The results in the scatter plots represent values from single patients; the horizontal lines indicate
medians, and error bars show the interquartile range. The P values were calculated using a Mann-Whitney U test. (D and E) A survival analysis
(Kaplan-Meier estimator) representing the number of days until the appearance of the first WA (D) during the whole study period (1 yr) and (E)
during the posttreatment period of the study (6 mo). The P values were calculated using log-rank (Mantel-Cox) test. CI =confidence interval.
ORIGINAL ARTICLE
Nieto, Maz
on, Nieto, et al.: MV130 Prevents Children’s Wheezing Attacks 467
our study, a post hoc analysis suggested that
the effect of MV130 is different between
those younger than 18 months and those
older. However, this finding would need
further confirmation because only 24% of
the participants in our study were younger
than 18 months, as the main inclusion
criterion (at least three WA during the
previous year) was more difficult to achieve
in the youngest children.
Allergen-sensitized children have an
increased riskof recurrent and severewheezing
in slightly older ages (4, 6). In our study,
children with positive skin tests were excluded
to avoid enrolling subjects whose recurrent
wheezingmight be triggered by aeroallergens
instead of viral infection.Moreover, although
the inability of patientswith asthma to generate
an adequate type I IFN response during viral
infection leading to greater or prolonged
infection is well established (38), this could
interfere with the correct interpretation of the
results. Although some of these children
eventually develop allergic sensitization, we
assume that they were equally represented in
our two groups; however, this could be
considered as a limitation of the study.
Confirmation of this would require longer
follow up, and the opportunity to initiate
treatment would be lost.
Symptom and medication scores during wheezing attacks
P< 0.001
Placebo MV130
0
500
1,000
1,500
2,000
Co
m
bi
na
tio
n 
of
 S
M
S
A
P< 0.001
Placebo MV130
0
150
300
450
600
O
ve
ra
ll 
sy
m
pt
om
s
B
Symptom and medication scores during the whole study
P< 0.001
Placebo MV130
0
900
1,800
2,700
3,600
4,500
Co
m
bi
na
tio
n 
of
 S
M
S
D
P< 0.001
Placebo MV130
0
300
600
900
1,200
O
ve
ra
ll 
sy
m
pt
om
s
E
P< 0.001
Placebo MV130
0
600
1,200
1,800
2,400
3,000
 
M
ed
ica
tio
n 
sc
or
es
F
P< 0.001
Placebo MV130
0
200
400
600
800
1,000
1,200
1,400
 
M
ed
ica
tio
n 
sc
or
es
C
Figure 3. MV130 decreases symptom and medication scores (SMS). (A–C) The combination of SMS (A), overall symptom scores (B), and
medication scores (C) during wheezing attacks. (D–F) The combination of SMS (D), overall symptom scores (E), and medication scores (F)
throughout the study. Data are displayed in scatter dot plots in which values from single patients are represented. The line indicates the median, and
error bars show the interquartile range. The P values were calculated using Mann-Whitney U test.
ORIGINAL ARTICLE
468 American Journal of Respiratory and Critical Care Medicine Volume 204 Number 4 | August 15 2021
As observed in previous trials (36), the
number ofWA in the placebo group declined
during thewhole study period comparedwith
the baseline. Viral-induced wheezing has a
good prognosis and spontaneous
improvement is expected, but our study was
designed to assess the saving effect of MV130
while the condition is present, both in clinical
symptoms andmedication consumption, and
it was significantly better than the placebo in
children with similar risk characteristics at
baseline.
Table 2. Demographic and Clinical Characteristics of the Study Populations at Baseline
Characteristics MV130 (n=62) Placebo (n=58) P Value
Sex, M/F 37/25 33/25 0.757
Median age, mo (IQR) 24.0 (17.3–28.0) 24.0 (16.5–29.0) 0.475
,1 yr 2 (3.2%) 7 (12.1%) 0.060
Between 1 and 2 yr 30 (48.4%) 23 (39.7%) 0.335
.2 yr 30 (48.4%) 28 (48.3%) 0.990
Mean weight, kg (95% CI) 12.6 (12.0–13.2) 12.2 (11.6–12.7) 0.398
Mean height, cm (95% CI) 85.1 (83.3–87.0) 84.9 (82.8–87.0) 0.878
Median previous WA (IQR) 8.0 (7.0–10.0) 9.0 (7.0–11.3) 0.102
Median previous monthly WA (IQR) 0.67 (0.58–0.83) 0.79 (0.67–1.00) 0.053
Atopic dermatitis 22 (35.5%) 25 (43.1%) 0.393
Smoking during pregnancy 11 (17.8%) 15 (25.9%) 0.280
Gestational age
,37 wk 9 (14.5%) 11 (19.0%) 0.513
37–41 wk 52 (83.9%) 45 (77.6%) 0.382
.42 wk 1 (0.02%) 2 (0.03%) 0.517
Definition of abbreviations: CI = confidence interval; IQR= interquartile range; WA=wheezing attacks.
Table 3. Symptom and Medication Scores Alone and in Combination during the Whole Period of the Study (1 yr)
MV130
(n=62)
Placebo
(n=58)
P Value
(Mann-
Whitney
U Test) Hodges-Lehmann
Symptoms
Overall symptom score 276.5 (151.0 to 426.0) 421.0 (303.0 to 673.0) 0.001 2147 (2248 to 263)
Respiratory symptoms 115.0 (53.8 to 165.5) 157.0 (110.0 to 295.3) 0.002 253 (297 to 221)
Other symptoms 152.0 (73.3 to 262.0) 253.5 (175.3 to 353.8) 0.001 284 (2151 to 238)
Score of individual symptoms and
days with symptoms
Days with nasal mucus secretion 71.0 (32.0 to 101.3) 100.5 (61.8 to 146.8) 0.005 233 (255 to 210)
Score of nasal mucus secretion 82.0 (42.0 to 147.8) 123.0 (83.8 to 199.5) 0.004 244 (275 to 212)
Days with bronchial mucus secretion 21.0 (8.0 to 42.5) 41.0 (14.8 to 73.0) 0.008 214 (228 to 24)
Score of bronchial mucus secretion 29.0 (11.0 to 59.0) 58.5 (24.0 to 102.5) 0.005 221 (241 to 26)
Days with cough 54.5 (26.0 to 87.0) 78.5 (53.0 to 113.0) 0.013 222 (237 to 24)
Score of cough 83.0 (35.5 to 119.5) 104.5 (72.0 to 153.5) 0.020 230 (254 to 25)
Days with difficult breathing 8.5 (1.3 to 18.8) 19.5 (6.8 to 43.3) 0.003 28 (216 to 22)
Score of difficult breathing 11.0 (2.0 to 26.5) 25.5 (10.0 to 62.8) 0.001 211 (221 to 23)
Days with wheezing 5.0 (0.0 to 14.0) 19.0 (7.0 to 44.0) ,0.001 211 (218 to 26)
Score of wheezing 11.0 (2.0 to 33.5) 25.5 (8.5 to 61.5) ,0.001 215 (224 to 26)
Days with discomfort 11.0 (3.3 to 32.3) 20.0 (13.8 to 42.3) 0.010 210 (216 to 22)
Score of discomfort 14.5 (4.0 to 47.5) 31.0 (18.8 to 59.5) 0.010 214 (223 to 23)
Fever 9.5 (5.0 to 14.0) 11.5 (7.0 to 20.5) 0.068 23 (26 to 0)
Medication
Overall medication 618.5 (318.3 to 1,035.5) 1,043.5 (669.5 to 1,696.0) ,0.001 2399 (2642 to 2188)
Antibiotics 22.5 (6.0 to 44.5) 41.5 (18.8 to 77.3) 0.013 216 (228 to 22)
Antipyretic and antiinflammatory drugs 31.0 (18.0 to 50.5) 38.5 (19.5 to 98.3) 0.064 212 (229 to 0)
Salbutamol 92.0 (40.3 to 224.0) 252.5 (120.0 to 387.0) ,0.001 2130 (2193 to 265)
Inhaled budesonide 38.0 (0.0 to 272.0) 323.0 (80.0 to 539.0) ,0.001 2185 (2282 to 262)
Oral prednisolone 0.0 (0.0 to 8.0) 7.5 (0.0 to 18.0) 0.002 23 (27 to 0)
Montelukast, 4 mg 212.5 (129.8 to 254.0) 206.0 (141.5 to 257.0) 0.957 0 (230 to 34)
Other medications* 58.0 (129.8 to 254.0) 103.0 (19.0 to 194.5) 0.334 211 (258 to 14)
Combination of symptom and medication
scores
1,092.2 (925.8 to 1,258.5) 1,760.9 (1,505.9 to 2,016.0) 0.001 2508 (2828 to 238)
*Includes antihistamines and mucolytics.
ORIGINAL ARTICLE
Nieto, Maz
on, Nieto, et al.: MV130 Prevents Children’s Wheezing Attacks 469
Despite the effect onWA, some
secondary variables on health resource
consumption did not differ between the active
treatment and the placebo. This might mean
that parents learn to cope with their child’s
symptoms, use rescue medication, and limit
the use of those resources.However, therewas
asignificantreductioninsomesocialresources
that might have a direct impact on the
economy of families.
The safety data showed a good general
profile. Adverse events were not different
between the active and placebo preparations.
Theyweremild(except forepilepticseizures in
a child with the placebo) and subsided during
the treatment; the only two dropouts because
of possible side effects appeared in the placebo
group. In this line, the most common adverse
event in the active group was conjunctivitis,
withnoobviousexplanation.Thepatientsfully
recovered, and although it was not statistically
different from placebo and causality
assessment did not estimate any relation with
the medication, there was a numerical
difference that would need further assessment
with larger groups.
Wedidnotdirectlyaddressamechanistic
evaluation of MV130 on samples from the
children included in the DBPC by assessing
immunological parameters that might
correlate with the clinical outcome (39). This
would have shed further light on the
mechanism of protection of MV130 (16, 40,
41). On the other hand, whether
immunotherapywithMV130may impact the
airway microbiome would have been very
interesting to address, as it may influence
susceptibility versus resilience to viral
infection (42). These are limitations of our
study because both approaches would have
raised new insights about howmucosal
bacterial immunotherapy may modulate the
airway response against viral infections.
Further studies will need to be performed to
clarify these issues.
Although the beneficial role of bacterial
preparations in the protection against
recurrentrespiratorytract infections, including
thoseof viral origin, hasbeenalready evaluated
in different studies (27, 43, 44), their
mechanism of action remains unclear.
AlthoughMV130doesnot affectoral epithelial
cell activation (41), itmodulates the functionof
human dendritic cells (16). In addition, it
enhances both specific and nonspecific T-cell
responses in vitro and in vivo, including Th1
and Th17 responses, known to be involved in
host resistance against intracellular and
extracellular pathogens, respectively.
Furthermore,MV130promotesthegeneration
of IL-10–producing T cells (16), which are
essential for pathogen clearance and to keep
tissuehomeostasis (45). Inthissense, induction
ofIL-10byregulatoryTcellshasbeenproposed
asanantiinflammatorymechanismofbacterial
lysates having a role in preventing wheezing-
asthma in childhood (46).
Inexperimentalrespiratoryvirusinfection
models, MV130 has recently been shown to
confer protection by inducing trained
immunity (21). Trained immunity endows
innate immunitywith immunologicalmemory
based on stable epigenetic modifications (47,
48). As a result, nonspecific innate immune
responses are enhancedduring a relatively long
time, so the host responds rapidly and robustly
in a nonspecific way to subsequent challenges,
providing cross-protection against different
infections (14, 40). In this regard, it has been
postulated that the underlyingmechanism
behind the heterologous immunity induced by
mucosal bacterial vaccines (35), bacillus
Calmette-Gu
erin, and other vaccines can be
mediated by trained immunity (49, 50). Thus,
the term “trained immunity based vaccines”
has been proposed for those formulations
containing trained immunity inducers that
confer broad and durable protection against
infections far beyond the nominal
microorganismantigens theymaycontain(40).
Of note, the protection achieved byMV130 in
thecurrentclinical trialwasalsoobserved in the
following 6months after treatment
discontinuation, inlinewithamemoryascribed
to a trained immunity-mediatedmechanism
(50–54).WhetherMV130may be acting this
way inhumans iscurrentlyunder investigation.
In summary, our study reveals, for the
first time, thatmucosal immunotherapywith
a sublingual polybacterial preparation
formulated with a defined composition of
heat-inactivated, whole-cell bacteria
(MV130) prevents wheezing episodes in
young children and reduces symptom and
medication scores, which together with a
good safety profile makes it an alternative to
other treatments currently used. A further
understanding of its underlying mechanism
of action may support the development of
prophylactic and therapeutic options,
particularly in conditions associated with
recurrent infections or new emerging
pathogens, for which conventional vaccines
are not available yet.

Author disclosures are available with the text
of this article at www.atsjournals.org.
Acknowledgment: The authors thank the
patients and their families for participating in this
study. They are grateful to the members of the
David Sancho laboratory, especially to Carlos
del Fresno, for helpful critical discussion.
Table 4. Adverse Events
MV130 (n= 62) Placebo (n=58)
Adverse Event(s) Events (n=81) Events (n=85)
Most frequent adverse events*
Gastroenteritis 5 (8%) 16 (28%)
Conjunctivitis 14 (23%) 6 (11%)
Diarrhea 6 (10%) 11 (19%)
Dermatitis 6 (10%) 11 (19%)
Head injury 0 (0%) 4 (7%)
Chickenpox 4 (6%) 1 (2%)
General malaise 3 (5%) 2 (3%)
HFMD 5 (8%) 1 (2%)
Iron-deficiency anemia 2 (3%) 3 (5%)
Oral candidiasis 3 (5%) 0 (0%)
Scarlet fever 3 (5%) 3 (5%)
Synovitis 3 (5%) 2 (3%)
Urticaria 2 (3%) 3 (5%)
Vomiting 2 (3%) 3 (5%)
Severe adverse event(s)† 0 (0%) 1 (2%)†
Definition of abbreviation: HFMD=hand-foot-and-mouth disease.
Data are n (%) of patients with one or more such events in each group. No variable showed
significant differences (P .0.05).
*Adverse events reported in more than 5% of patients in either treatment group are shown.
†
Epileptic seizure; the patient withdrew from the clinical trial.
ORIGINAL ARTICLE
470 American Journal of Respiratory and Critical Care Medicine Volume 204 Number 4 | August 15 2021
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ORIGINAL ARTICLE
472 American Journal of Respiratory and Critical Care Medicine Volume 204 Number 4 | August 15 2021