Protocol design for the ACTIVATE clinical trial: Exposure to vaginal microbiome in cesarean-delivered infants at high risk for allergies

Abstract

Background

Food allergy is increasingly common in the United States. Studies suggest that rising cesarean delivery rates are associated with many immune disorders, including allergic diseases. A preceding proof-of-concept study showed that the microbiota of infants born by cesarean delivery could be partially restored via vaginal microbiome exposure at birth via “vaginal seeding”.

Objectives

Described here is the design of a clinical trial to evaluate the effects of vaginal seeding in infants born by cesarean delivery on food allergen sensitization (egg, milk, and peanut) at 12 months of age.

Methods

This study is supported by the Immune Tolerance Network in collaboration with the National Institute of Allergy and Infectious Diseases. ACTIVATE is a single-center, randomized, double-blind, placebo-controlled trial enrolling pregnant women and their newborns who have a first-degree relative with atopic disease (NCT03567707). Forty infants born vaginally and 80 infants born by cesarean delivery, randomized 1:1 to receive vaginal or placebo seeding, will be enrolled. Families are followed for 1 year, with an option to extend the follow-up for a total of 3 years.

Results

The study is currently underway with an enrollment goal of 120 mother–infant pairs. Surveys and samples are collected from mothers and infants during the follow-up period including blood, stool, skin swabs, oral swabs, nasal swabs, maternal vaginal swabs, and breast milk.

Conclusions

This pilot study will provide important data on the effects of vaginal seeding on allergen sensitization, the microbiome, and the development of immune responses in the first 3 years of life.

Food allergy is estimated to affect 5% to 10% of children in the United States,1, 2, 3, 4 and studies have suggested that this percentage is rising.⁵ Food allergies cause significant morbidity and are the leading cause of anaphylaxis in children.⁶ Infants with a family history of allergies have a higher risk for development of food and environmental allergies.⁷ In recent years, early introduction of food allergens in the infant’s diet, in particular peanut and egg, has been shown to decrease the risk8, 9, 10, 11, 12, 13, 14 but does not prevent all food allergy.¹⁵

Differences in stool microbiota in early life have been linked with sensitization to food allergies. Neonatal stool microbiome composition at 1 month of age has been shown to be significantly associated with a higher risk of sensitization to food (including milk, egg, and peanut) and/or inhalant allergens.¹⁶ Infants with lower abundance of bacteria generally considered beneficial and enrichment of proinflammatory metabolites at 1 month had a 2- to 3-fold risk increase of sensitization to allergens at 2 years of age compared to other infants.

Starting at birth, the microbial environment varies significantly depending on mode of delivery, which can affect the development of the immune system.¹⁷ Infants born vaginally are initially colonized with bacteria from the maternal vaginal canal, while those born by cesarean delivery acquire bacteria similar to skin microbiota.18, 19, 20 With cesarean delivery rates rising,²¹ it has been hypothesized that aberrant neonatal microbiome could impair immune development and thereby contribute to the increase in allergic disorders.¹⁷ In epidemiologic studies, cesarean delivery has been associated with atopic diseases including atopic dermatitis,²² asthma,23, 24, 25 allergic rhinitis,²⁶ allergic sensitization,^27,28 and food allergy.29, 30, 31, 32

In a proof-of-principle study, the microbiome of infants born by cesarean delivery was shown to be partially restored through exposure to maternal vaginal fluid (known as vaginal seeding) at the time of birth.³³ This study included 18 mother–infant pairs, with 7 infants delivered vaginally and 11 by cesarean delivery, including 4 who received vaginal seeding. The microbiome of the cesarean-delivered infants that received vaginal seeding resemble that of vaginally delivered infants. These findings have been further confirmed in a more recent study that included 35 infants exposed to vaginal seeding.³⁴ The safety profile or effects of vaginal seeding in immune development and allergic outcomes has not yet been assessed, and its use in clinical practice is not currently recommended until sufficient data are available.³⁵ Here, we present the design of a randomized, double-blind, placebo-controlled trial to evaluate whether vaginal seeding has a protective effect against sensitization to common food and environmental allergens in a high-risk population.

Methods

Study design

This is a pilot study aiming to enroll 120 pregnant women and their infants born at two hospitals in the Mount Sinai Health System in New York. Eighty women delivering by elective, unlabored cesarean and 40 delivering vaginally will be enrolled (Fig 1). The infants born via cesarean delivery will be randomized 1:1 to receive vaginal seeding with their mother’s vaginal microbiota versus placebo seeding with a saline-soaked gauze, within 10 minutes after birth. Infants who are randomized to receive placebo seeding serve as the control population. Infants born via vaginal delivery will also be included as a second control group.

Fig 1.

Study design.

The infants enrolled into the study are followed for the first year of life. Maternal participants, study investigators, and research staff are blinded to the study intervention (active seeding gauze vs placebo) for the duration of the study. Only the obstetric study clinicians performing the seeding procedure on the infant at the time of birth are aware of the study intervention. These individuals are not a part of any subsequent study assessments.

Biospecimens are collected from maternal and child participants, including maternal samples to assess vaginal, skin, stool, placenta, breast milk, and oral microbiome, and infant samples to assess allergen sensitization and immune markers from blood, and gut, skin, nasal, and oral samples to characterize their microbiome (Fig 2). Maternal participants are asked to complete questionnaires and send in breast milk and stool samples (maternal and infant) during the follow-up period. In-person study visits take place at 3 and 12 months of age of the infant. The primary end point is assessed at 12 months of age. While deliveries take place at two hospitals within the same New York City health system, all infant assessments starting at 3 months of age occur at a single research location with one research team. Therefore, this clinical trial is considered to be a single-site study.

Fig 2.

Assessment and specimen collection overview.

After the 12-month study visit, participants can choose to consent to participate in the extended follow-up, until the child turns 3 years of age, because other atopic conditions such as environmental allergies and wheezing can develop during this time. Additional questionnaires and biospecimens are collected every 6 months, with in-person study visits occurring at 24 and 36 months of age. At these in-person visits, additional assessments for food and environmental aeroallergen sensitization are collected through blood and skin prick testing.

Throughout the study, participants receive monetary compensation for their time and effort including at-home sample collections and survey completion, in-person study visits, and travel costs.

Study end points and objectives

The primary end point is sensitization to at least one of the following food allergens in infants at 12 months of age in the following order of priority: egg, milk, and peanut (Table I). These are the 3 most common food allergens observed in young children.² Sensitization is defined as serum IgE ≥ 0.1 kU_A/mL for each food allergen.

Table I.

Study objectives and end points

Characteristic	Objectives	End points
Primary	•Assess how vaginal seeding in neonates delivered by cesarean alters allergic sensitization to food allergens and development of immune responses at 52 weeks of age.	•Sensitization to at least one of the following food allergens in infants at 52 weeks of age (listed in order of priority): egg, milk, and peanut. Sensitization is defined by serum IgE ≥ 0.1 kUA/mL for each allergen.
Secondary	•Assess safety of vaginal seeding in neonates delivered by cesarean. •Assess how vaginal seeding in neonates delivered by cesarean alters allergic sensitization to food allergens at 104 and 156 weeks of age. •Assess how vaginal seeding in neonates delivered by cesarean alters allergic sensitization to aeroallergens at 52, 104, and 156 weeks of age. •Assess degree of atopy at 13, 52, 104, and 156 weeks of age	•AEs. •Sensitization to at least one of the following food allergens in infants at 104 and 156 weeks of age (listed in order of priority): egg white, cow’s milk, and peanut, as determined by serum IgE assessment. •Sensitization to at least one of the following aeroallergens at 52, 104, and 156 weeks of age (listed in order of priority): house dust mite (Dermatophagoides pteronyssinus), cat dander, dog dander, and German cockroach (Blattella germanica), as determined by serum IgE assessment. •Degree of allergen-specific atopy at 52, 104, and 156 weeks of age, defined as magnitude of individual serum IgE levels to: egg white, cow’s milk, peanut, house dust mite (D pteronyssinus), cat dander, dog dander, and German cockroach (B germanica), as determined by serum IgE assessment. •Degree of combined atopy at 52, 104, and 156 weeks of age, defined as sum of serum IgE levels to egg white, cow’s milk, peanut, house dust mite (D pteronyssinus), cat dander, dog dander, and German cockroach (B germanica). •Number of food and aeroallergens each infant is sensitized to at 52, 104, and 156 weeks of age, determined by serum IgE assessment. •Sensitization (yes/no characterization) to food allergens and aeroallergens at 104 and 156 weeks of age, as determined by skin prick testing. Sensitization is defined as having mean wheal diameter ≥3 mm greater than negative control. •Number of clinical IgE-mediated food allergies diagnosed by physician at 52, 104, and 156 weeks of age. •Severity of atopic dermatitis as measured by EASI at 13, 52, 104, and 156 weeks of age.
Exploratory	•To investigate differences in proportion of infants sensitized to food and aeroallergens at 52, 104, and 156 weeks of age between vaginal-seeded cesarean delivery infants and placebo-seeded cesarean delivery infants, as well as between vaginal-seeded, placebo-seeded, and vaginal-delivery infants. •Assess impact of TEWL on allergic sensitization at 52, 104, and 156 weeks of age and atopic dermatitis at 13, 52, 104, and 156 weeks of age. •To determine impact of cesarean delivery as well as vaginal seeding on innate and adaptive immune responses during first 156 weeks of life. •To determine how cesarean delivery and vaginal seeding influences subsequent development of infant microbiome for first 156 weeks of life. •Assess correlation of microbiome characteristics with allergen sensitization during first 156 weeks of life. •Assess impact of TEWL on microbiome composition during first 156 weeks of life.	•Bacterial/fungal composition of infant microbiome (gut, oral, skin, nasal). •Profiling and functional assessment of T cells and innate immune system. •Composition and concentration of metabolites of fecal metabolome. •Immunomodulatory influences of fecal metabolome. •Quantitative and qualitative assays to assess maternal vaginal microbiome transfer. •TEWL.

Key secondary end points are summarized in Table I and include: adverse events (AEs), sensitization by serum IgE assessment to at least one food (egg, milk, peanut) at 24 and 36 months of age, sensitization by serum IgE assessment to environmental aeroallergens (house dust mite [Dermatophagoides pteronyssinus], cat dander, dog dander, and German cockroach [Blattella germanica]) at 12, 24, and 36 months of age, and eczema severity as assessed by the Eczema Area and Severity Index (EASI) score at 3, 12, 24, and 36 months of age.

Main mechanistic end points include: the microbiome composition of the infant from stool, oral, skin and nasal samples, T-cell and innate immune system profiling and functional assays, and transepidermal water loss (TEWL) measurements (Table I).

Study population

Pregnant women are enrolled during their second or third trimester of pregnancy. The infant participants must have a first-degree relative (biological mother, biological father, or full sibling) with a history of food allergy or atopic disease, including atopic dermatitis, environmental allergies, and/or asthma.

A detailed list of all inclusion and exclusion criteria can be found in Table II. To minimize potential disruptions to the microbiome, women planning to have a cesarean delivery cannot be in labor, defined as no evidence of labor with cervical changes and no rupture of membranes. Women who receive cervical prostaglandins or have a Foley catheter placed with topical antiseptics for the purpose of labor induction are excluded. Pregnant women receiving systemic antibiotics or vaginal antibiotics/antifungals during the third trimester are also excluded, with the exception of standard-of-care antibiotics provided for the cesarean surgery.

Table II.

Inclusion and exclusion criteria

Type	Subject	Criteria
Inclusion criteria	Maternal	1.Must be able to understand and provide informed consent
		2.Pregnant women aged 18-45 years with singleton pregnancy with nonanomalous, appropriately grown fetus.
		3.Atopic disease (asthma, allergic rhinoconjunctivitis, atopic dermatitis) or food allergy in first-degree relative of infant to be delivered (for exceptions, see exclusion criterion 6).
	Infant	4.Infants who are medically stable (requiring no more than standard neonatal resuscitation including tactile stimulation, bulb suctioning, and drying) at time of delivery are eligible.
Exclusion criteria	Cesarean-delivery mothers	1.In labor with evidence of cervical change before scheduled cesarean delivery.
		2.Rupture of amniotic sac.
		3.Vaginal pH > 4.5 on day of delivery.
	Vaginal-delivery mothers	4.Use of cervical prostaglandins or Foley catheter placed with topical antiseptics as induction agents for cervical ripening.
	All mothers and their infants	5.Inability or unwillingness of participant to give written informed consent or comply with study protocol.
		6.History of moderate to severe atopic dermatitis within past 1 year in mother.
		7.Express no intention to breast-feed.
		8.Diabetes mellitus or gestational diabetes mellitus during current pregnancy.
		9.History of inflammatory bowel disease (eg, Crohn disease, ulcerative colitis).
		10.Evidence of active STI (eg, primary herpes, genital warts) or vaginal lesions consistent with herpes infection on day of delivery.
		11.Evidence of prior or current hepatitis B or C infection as demonstrated by presence of hepatitis B surface antigen, antibody positivity against hepatitis B core antigen, or antibody positivity against hepatitis C virus. Assessment for hepatitis B and C infection will be repeated for this study during third trimester of pregnancy even if prior testing during current pregnancy was negative.
		12.Evidence of HIV infection (eg, positive HIV serology, detectable virus load). Testing will be required during third trimester of pregnancy, even if prior testing during current pregnancy was negative.
		13.Positive GBS test results by rectovaginal swab performed at or after 36 weeks’ gestation or within 5 weeks of delivery, prior infant with invasive GBS disease, or GBS bacteriuria at any point during current pregnancy.
		14.Evidence of Neisseria gonorrhoeae or Chlamydia trachomatis infection by testing performed at or after 36 weeks’ gestation or within 5 weeks of delivery.
		15.Positive test for syphilis (screening and reflex confirmatory testing according to institutional standards). Testing will be required during third trimester of pregnancy, even if prior testing during current pregnancy was negative.
		16.History of systemic antibiotic administration during third trimester of current pregnancy, except for routine antibiotics administered for cesarean delivery procedure.
		17.History of vaginal antibiotic or vaginal antifungal receipt during third trimester of current pregnancy.
		18.Mothers with serious chronic conditions during pregnancy (eg, systemic lupus erythematosus, history of organ transplant).
		19.Mothers with complicated pregnancies including preeclampsia, chorioamnionitis, placenta previa, vasa previa, placental abruption, and active vaginal bleeding.
		20.Maternal fever on day of delivery.
		21.Infants with complications during delivery, such that infant requires more than standard neonatal resuscitation after delivery.
		22.Infants delivered before 37 weeks’ gestation.
		23.Thick particulate meconium noted on delivery of infant.
		24.Presence of congenital abnormality in infant for which study participation is not recommended.
		25.Current diagnosed mental illness, or current diagnosed or self-reported drug or alcohol abuse in mother that, in investigator’s opinion, would interfere with participant’s ability to comply with study requirements.
		26.Use of investigational drugs during third trimester of pregnancy, except for SARS-CoV-2 vaccination.
		27.Past or current medical problems or findings from physical examination or laboratory testing that are not listed above, which, in investigator’s opinion, may pose additional risks from participation in study, may interfere with participant’s ability to comply with study requirements, or may affect quality or interpretation of data obtained from study.

SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2.

This study had strict exclusion criteria to ensure the safety of infants and to prevent potential transmission of infectious diseases from the vaginal seeding procedure (Table II). Infants born before 37 weeks’ gestation are excluded. Serious complications during pregnancy (eg, preeclampsia, placental abruption, active vaginal bleeding) or at time of delivery, such as thick meconium, are reasons for exclusion. Infants must be medically stable at the time of delivery. Maternal participants are required to test negative for human immunodeficiency virus (HIV), syphilis (Treponema pallidum), and hepatitis B and C infection during the third trimester of pregnancy, even if earlier testing was negative during the current pregnancy. Testing for Neisseria gonorrhoeae and Chlamydia trachomatis infection (via vaginal swab) and group B Streptococcus (GBS) (via rectovaginal swab) must also be negative within 5 weeks of delivery or after 36 weeks’ gestation. A prior infant with invasive GBS disease or maternal GBS bacteriuria at any point during the pregnancy were reasons to exclude a pregnant woman from the study. Pregnant women were also assessed on the day of delivery and were excluded if there was evidence of an active sexually transmitted infection (STI) such as herpes lesions or genital warts, if vaginal pH was greater than 4.5, or if there was a maternal fever on the day of delivery suggestive of active infection.

Additionally, pregnant women with a history of moderate to severe atopic dermatitis in the past year are excluded. These women are at higher risk of Staphylococcus aureus carriage and are excluded to avoid possible transfer of S aureus during the seeding procedure because S aureus colonization is linked to development of atopic disease.^36,37

Intervention

The study intervention is the vaginal seeding of cesarean-delivered infants using a gauze soaked in maternal vaginal microbiota. On the day of delivery, a sterile piece of gauze is folded and wet with sterile saline. For all maternal participants planning a cesarean delivery, the gauze is inserted in the pregnant woman’s vagina for about an hour (45-75 minutes). Before the cesarean surgery, the gauze is removed and kept at room temperature for no longer than 6 hours. The cesarean-delivered infants are then randomized 1:1 to receive either the vaginal seeded gauze or a sterile piece of gauze folded and soaked in sterile saline as the placebo.

Once the infant is delivered and it is determined that all study inclusion criteria are met and there are no exclusion criteria, the infant is swabbed with either the vaginal microbiota-soaked gauze or the placebo gauze. Swabbing occurs as soon as possible and no longer than 10 minutes after delivery. The swabbing technique is based on a prior proof-of-principle study,³¹ starting from the infant’s lips, then face, and followed by the rest of the body including the thorax, arms, back, legs, genitals, and anal region (Fig 3). A prior study by Dominguez-Bello et al³³ demonstrated that the same technique led to the partial transfer of vaginal microbiota to infants born by cesarean delivery. If any exclusion criteria are met by the time of delivery, the infant is not swabbed, and the mother–infant pair do not continue in the study.

Fig 3.

Illustration of vaginal seeding procedure. Reprinted with permission from Dominguez-Bello et al (Nat Med 2016;22:250-3).³³

Infants delivered vaginally receive no intervention. For both vaginally and cesarean-delivered infants, the first bath is delayed for at least 12 hours after delivery. In all other matters, care of the infants is routine and per hospital standard policy.

Stratification, randomization, and blinding

Pregnant women who consent to the study, meet eligibility criteria, and plan to have an elective cesarean delivery are randomly assigned for their infant to have vaginal seeding or placebo seeding. Randomization is performed through a Web-based, password-protected randomization system maintained by the Division of Allergy, Immunology, and Transplantation Statistical and Clinical Coordinating Center using a validated system that automates the 1:1 random assignment of treatment groups. Randomization is accomplished using a stratified, permuted block randomization design with varying block size.

Only the unblinded obstetric study clinician can administer the study intervention per the randomization assignment. All other staff members, research investigators, and parents of infants are blinded to the study intervention. Unblinding will occur when the 12-month study visit and related study activities are completed by all participants, and the data are cleaned and locked.

Potential risks and safety

The risks of vaginal seeding have not been previously studied in a large clinical trial. The main risk of vaginal seeding is infection of the infant from transfer of microorganisms from the mother’s vaginal fluid, including GBS, hepatitis B, hepatitis C, or other STIs.³⁸ Therefore, all pregnant women in the study will be screened for GBS, N gonorrhoeae, C trachomatis, HIV, hepatitis B, hepatitis C, and syphilis. All pregnant women will be examined for active genital herpes lesions at time of delivery. Furthermore, vaginal pH will be tested for pregnant women undergoing cesarean delivery to screen for trichomonas and vaginosis. Women with any evidence of the above infections are excluded from the study as a precaution.

Because of potential risks, the vaginal fluid for each pregnant woman that is used for vaginal seeding is considered to be an Investigational New Drug by the US Food and Drug Administration. An investigator’s brochure was created for the study product being evaluated (maternal vaginal fluid) despite variability among vaginal fluid between different maternal participants.

There are no prespecified study stopping rules. However, an ad hoc Data and Safety Monitoring Board (DSMB) review will take place if any of the following occurs: any death that is possibly related to the study intervention; the occurrence of any infection due to vaginal pathogens in an infant who received study intervention; a moderate or severe vaginal infection in ≥2 mothers who receive vaginal gauze placement; or a severe AE in any participant determined to be possibly related to the study intervention. Additionally, an infant born by cesarean delivery who is undergoing an evaluation for sepsis in the first 60 days of life who is found to have an infection with an organism that might be related to the study intervention or that required antibiotics for ≥72 hours (and the need for antibiotics might be related to the study intervention) would also trigger a DSMB safety review. Any ad hoc DSMB review would result in a temporary halt in vaginal seeding in the study.

Maternal study procedures and assessments

Study assessments are summarized in Fig 2. Consented pregnant women are seen for a screening visit, during which blood is collected to assess for hepatitis B (surface antigen and core antibody), hepatitis C (antibody), HIV (serology or detectable virus load), and syphilis (screening and reflex confirmatory testing) during the third trimester. The following are also collected from 36 weeks’ gestation onward or within 5 weeks of delivery: vaginal swab to test for N gonorrhoeae and C trachomatis infection and rectovaginal swab to test for GBS. A maternal stool sample is collected between 36 weeks until day of delivery.

On the day of delivery, the following maternal samples are collected: vaginal swab, vaginal gauze (cesarean delivery only), and swabs (oral and skin—chest, abdomen, dominant hand); in addition, vaginal pH is measured (Fig 2). Placental biopsy samples are obtained shortly after delivery for maternal participants who opted into the collection of placental tissues at the time of consent.

For maternal participants, breast milk samples are collected 1 week and 4 weeks postpartum, while maternal stool samples are collected approximately every 3 months until 12 months postpartum (Fig 2). Maternal stool and breast milk samples are placed in the freezer at home for at least 8 hours (but not more than 96 hours) and then packed with frozen ice packs and shipped overnight to the research site for processing. Maternal AEs are tracked until 4 weeks postpartum, while questionnaires and concomitant medications are assessed through an electronic app on the maternal participant’s phone (MyOwnMed) throughout the follow-up period. Maternal surveys also included questions on maternal diet and tobacco exposure.

Infant study procedures and assessments

For infants, cord blood (optional per maternal participant), swabs (oral, nasal, skin—forehead, forearm, palm of right hand), and meconium are collected on the day of delivery (Fig 2). Data are collected on the infant’s gestational age, duration of hospitalization, and antibiotic exposure. During the postdelivery hospital stay, stool samples are collected daily if available, and the same oral, nasal, and skin swabs are collected before discharge from the hospital. Infant stool is collected at home at weeks 1, 2, 3, 4, 8, 13, 26, 29, and 52, and, if in the extension, at weeks 78, 104, 130, and 156. The time points of infant stool collection are more frequent in the first few months of life when the microbiome changes more quickly,³³ after which infant stool collections become more spaced out over time. Similar to maternal samples, infant stool samples are placed in the freezer at home by the caregiver for at least 8 hours (but not more than 96 hours) and then packed with frozen ice packs and shipped overnight to the research site. AEs, concomitant medications, and survey questions are assessed via an app (MyOwnMed) on the parent’s cellphone during the first year of life, and up to 3 years of age if consented in the extended follow-up of the study. Infant surveys include questions on infant diet (breast-feeding, formula,. solids), bathing habits, environmental exposures (pets, other children, pests, tobacco exposure), and home environment (type of dwelling, neighborhood).

In-person visits take place at 13 and 52 weeks of age (Fig 2). During these visits, swabs are collected (oral, nasal, skin— forehead, forearm, palm of right hand) as well as blood. Skin assessments via TEWL and EASI score are also completed. TEWL measurements are taken over the forearm in close proximity to the location of the forearm skin swab. At the 13-week visit, guidance on prevention of peanut allergies according to National Institute of Allergy and Infectious Diseases guidelines is provided by an allergist.³⁹ During the extended follow-up period at 2 and 3 years of age, the same in-person assessments are done with the addition of skin prick testing for the following: egg white, cow’s milk, peanut, dust mite (D pteronyssinus), cat dander, dog dander, and German cockroach (B germanica).

Mechanistic assays

We will determine the effect of exposure to the maternal vaginal microbiome at birth on the development of the infant’s microbiome, metabolome, and systemic immune system. The microbiome will be assessed by 16S rRNA sequencing and shotgun metagenomics to evaluate the microbial composition, diversity, and function. Vaginal swabs and stool samples collected from all mothers before delivery will be used to evaluate the transfer of maternal microbes to the infants. Stool samples from the mother and infant are assessed over the first year after delivery to evaluate changes in the microbiome. Skin and oral samples are collected from mothers and infants to determine the extent of microbial transfer from mother to infant. Breast milk will be profiled to determine its influence of the infant’s stool microbiome.

Metabolome profiles of infant stool and plasma and maternal breast milk will be assessed using ultrahigh-performance liquid chromatography–tandem mass spectrometry and gas chromatography–mass spectrometry. Raw metabolomics data will be processed to obtain calibration equations and to identify the concentration of each metabolite in the individual samples. Fecal metabolome of infant stool will also be evaluated to determine how it may differ on the basis of mode of delivery, exposure to vaginal seeding or placebo, and development of allergies.

Cytometry by time of flight will be utilized on blood samples to determine T-cell subpopulations including pathogenic T_H2 (T_H2A) cells and innate immune cells that include basophils and eosinophils. We will evaluate allergen-specific T-cell frequency and phenotype using allergen-stimulated peripheral blood mononuclear cells.

Response to vaccines and vaccine components will also be assessed to determine whether cellular changes in response to vaccines or vaccine components vary depending on the microbiome and/or mode of delivery.

Additional immunoglobulin assays may be performed to measure IgE, IgG, IgA, and IgG₄ to allergens using ImmunoCAP, ImmunoCAP ISAC, ELISA, or similar methods to determine the full allergic profile of the infants.

Statistical analysis plan

Analyses of all end points measured on or before week 52 will be performed when all participants have completed the 12-month visit and all data have been collected. Until this time point, all study personnel will remain unaware of the randomization status of the cesarean delivery infants (placebo vs vaginal seeding), with the exception of the unblinded obstetric study clinician who performed the seeding procedure. Any secondary or exploratory end points after the 12-month visit will be conducted when the participant has completed the final extended follow-up visit at 3 years of age.

The primary end point is sensitization to at least one of the following food allergens at 52 weeks of age: egg, milk, or peanut. Participants with at least one valid specific IgE measurement to at least one of these food allergens at 52 weeks of age will be considered evaluable. Participants missing all 3 IgE measurements are considered nonevaluable for the primary end point.

This is a pilot study that is not powered for comparison of proportion of sensitized infants between the intervention groups. The primary analyses will be descriptive, estimating the proportion of infants who meet the primary end point. Formal comparisons between study intervention groups will still be estimated as exploratory analyses to help inform future studies. CIs will be calculated by the Clopper-Pearson interval method. Risk ratios and 95% CIs will be determined between the vaginally seeded versus placebo-seeded cesarean-delivered infants and between the vaginally seeded cesarean-delivered infants and the vaginally delivered infants.

Statistical comparisons will be made between 3 distinct study groups for secondary and exploratory analyses as summarized in Table I: infants born vaginally, infants born by cesarean delivery with vaginal seeding, and infants born by cesarean delivery with placebo seeding. Frequency and proportions will be calculated to assess the following: AEs, sensitization to at least one aeroallergen at 52 weeks of age (serum IgE >= 0.1 kU_A/mL), and number of clinical food allergies diagnosed by a physician. Geometric means and associated 95% log-normal CI of IgE levels will be determined to compare allergen-specific IgE levels at 52 weeks of age (to each of the 7 food and aeroallergens in the evaluable sample) and combined IgE levels at 52 weeks of age (sum of the IgE levels to the 7 food and aeroallergens in the evaluable sample). Descriptive statistics (median, mean, and associated 95% CI) will also be calculated of EASI scores, a measurement of atopic dermatitis severity. Regression modeling will be used to estimate the rate of food and aeroallergen sensitizations at 52 weeks of age as well as the rate of physician-diagnosed clinical food allergies at 52 weeks of age.

The same analysis as described above will also be evaluated for participants completing the 24-and 36-month study visits. In addition, skin testing is performed to food and aeroallergens at the 24- and 36-month study visits. Analyses of skin testing will include evaluation of sensitization to at least one food allergen at 24 and 36 months of age and sensitization to at least one aeroallergen at 24 and 36 months of age. Sensitization on skin prick testing is defined as a mean wheal diameter that is ≥3 mm larger than the negative control. The total number of food and aeroallergen sensitizations per participant as determined by skin prick testing at 24 and 36 months of age will also be analyzed.

Sample size considerations

A total of 120 mothers and their infants will be accrued into the study. There will be 40 mother–infant pairs in each group. As a participating site in the Inner-City Asthma Consortium’s Urban Environment and Childhood Asthma trial (URECA), this study population is estimated to be similar to the URECA cohort. On the basis of estimates of prevalence of sensitization from the URECA trial, about 60% of infants from atopic mothers born by cesarean delivery are sensitized to at least one food allergen (egg, milk, or peanut) at 52 weeks of age.⁴⁰ On the basis of a sample size of 40 participants in each investigational group, the 95% Clopper-Pearson CI for the proportion of infants who are sensitized to food allergens at 12 months of age, assuming 60% sensitization, would be (43%, 75%).

Pregnant women with history of moderate to severe atopic dermatitis in the past year are excluded to avoid possible transmission of S aureus to the infant. Therefore, it is worth noting that this exclusion criterion, while important for safety, may result in a decreased likelihood of infants developing atopic dermatitis and/or food allergies among this study population compared to URECA.

The exploratory objective to detect differences in the proportion of sensitization between the two intervention groups will be evaluated by the Fisher exact test for two proportions at a level of significance of 0.05. A sample size of 80 infants born by cesarean delivery will allow for detection in differences in the prevalence of food sensitization between the vaginally seeded versus placebo-seeded infants born by cesarean delivery of 0.33 or larger with at least 80% power (Tables III and IV).

Table III.

Sample size and power estimates for comparison of two proportions

Prevalence∗	Reduction between groups†	Risk ratio	No. of participants per group with 80% power	Power with 40 participants per group
60%	50%	0.17	17	99%
60%	40%	0.33	28	94%
60%	33%	0.45	41	80%
60%	30%	0.50	49	71%
60%	20%	0.67	107	35%
60%	10%	0.83	407	10%

^∗Prevalence of food sensitization in nonseeded cesarean-delivery infants at 52 weeks of age, from mothers who underwent delivery in Inner City Asthma Consortium’s URECA trial.⁴⁰

^†Reduction in prevalence of food sensitization between nonseeded cesarean-delivery infants and seeded cesarean-delivery infants.

Table IV.

Hypothetical risk ratio of food sensitization between cesarean delivery infants with vaginal seeding and non-seeded cesarean delivery infants and associated 95% CIs

Hypothetical prevalence of food sensitization in cesarean delivery infants with vaginal seeding	Risk ratio	95% CI
Assuming no dropout∗
10%	0.17	0.06, 0.44
20%	0.33	0.17, 0.65
30%	0.50	0.29, 0.86
40%	0.67	0.42, 1.05
50%	0.83	0.56, 1.24
Assuming 15% dropout†
9%	0.15	0.05, 0.46
21%	0.35	0.17, 0.72
29%	0.50	0.28, 0.90
41%	0.70	0.43, 1.14
50%	0.85	0.55, 1.32

^∗Assumes 60% (24/40) prevalence of food sensitization in infants at 52 weeks of age from mothers who underwent cesarean deliveries without vaginal seeding (based on unpublished data obtained from Inner City Asthma Consortium’s URECA trial).⁴⁰

^†Assumes 59% (20/34) prevalence of food sensitization in infants at 52 weeks of age from mothers who underwent cesarean deliveries without vaginal seeding.

Discussion

The ACTIVATE study is the first randomized, double-blind placebo-controlled clinical trial evaluating whether the microbiome of cesarean-delivered infants can be modified by vaginal seeding and lead to changes in immunologic development that may protect the infant from developing allergic sensitization in a high-risk population. The study has an extensive list of exclusion criteria in order to ensure safety of the maternal and infant participants as well to minimize other potential influences on the microbiome. For example, any pregnant women exposed to antibiotics during the third trimester of pregnancy are excluded from the study because of the effect of antibiotics on the microbiome. While the strict exclusion criteria are necessary for the clinical trial, this may affect the generalizability of the results.

This pilot study will closely examine the early immune effects of vaginal seeding on infants born by cesarean delivery compared to infants receiving placebo seeding and infants born vaginally. The results of this study may serve to power a future larger-scale prospective clinical trial.

Clinical implication.

This pilot study will evaluate the effects of vaginal seeding in infants born by cesarean delivery on allergic sensitization and immune development by 1 year of age.

Disclosure statement

Disclosure of potential conflict of interest: H. A. Sampson reports grants from Immune Tolerance Network, NIAID/NIH, and the Food Allergy Research & Education foundation (FARE); personal fees and stock options from N-Fold and DBV Technologies; and personal fees from Alpina Biotech AG, RAPT Therapeutics, and Siolta Therapeutics unrelated to this study and outside the current study. M. L. Sever reports receipt of funding from Rho (UM2AI117870); and a current award at PPD (U01AI178756). The rest of the authors declare that they have no relevant conflicts of interest.