Probiotic Supplementation in Breastfed Newborn Infants

The proposed phase I clinical trial is a parallel-group, placebo-controlled, randomized,
double-blind ascending dose study of dietary supplementation with Bifidobacterium longum
subsp. infantis (B. infantis) in healthy breastfed infants to evaluate its safety as well as
determine the pharmacologically effective dose (ED) of B. infantis producing at least 50% gut
colonization at six weeks of age. Infants will be enrolled sequentially in groups of five
(three randomized to receive B. infantis and two to receive placebo). For each group, infants
will be dosed with B. infantis or placebo on day 7 and day 14 of life. A calculated Maximally
Recommended Starting Dose (MRSD) will be used to initiate the dose escalation and is defined
below. Every two weeks, an additional group of five infants (randomized 3:2 to B. infantis
and placebo) will be enrolled to receive progressively higher doses of B. infantis.
Calculation of the appropriate dose escalation will be performed using a modified Fibonacci
series as described below in an effort to identify the ED of B. infantis.

After the ED of B. infantis has been identified (defined as the dose capable of producing 50%
gut colonization by six weeks of age) two additional sequential dose escalations will be
performed. The purpose of the final two dose escalations is to determine if successively
higher doses of B. infantis result in increased gut colonization or barrier protection; or,
alternatively, if a Maximum Effective Dose (MaxED) for B. infantis exists above which there
is no further increase in gut colonization or barrier protection. Following the final dose
escalation, Hanley's Rule of Three will be applied in order to determine if lower-frequency
adverse events are caused by B. infantis. Hanley's Rule of Three states that in order to
identify any adverse events occurring at a frequency of 1:10 or greater with a 95% confidence
interval, at least 30 subjects must be enrolled.

Study visits will be scheduled for weeks 1, 2, 6, 24, 36, 52 and 78. Parents will complete
surveys at each study visit to monitor the infants for potential adverse events associated
with probiotic administration including feeding intolerance, fevers, or bowel irregularities
including constipation and diarrhea.

Stool samples will be collected twice weekly for the first six weeks of life then once weekly
at weeks 24, 36, 52 and 78. Stool samples will be analyzed to determine the relative
abundance of B. infantis over time, and the overall diversity of the gut microbiota with and
without B. infantis supplementation. Stool will also be analyzed for milk oligosaccharides to
verify consumption of breast milk and to correlate proportion of human milk oligosaccharides
and free sugar monomers seen in the infant stool at various levels of B. infantis
colonization.

At each study visit, infants will receive a full skin examination to evaluate for signs of
atopic dermatitis (AD). The Infant Dermatitis Quality of Life Index (IDQOLI) will be
administered to parents at each study visit to screen for possible signs of AD such as infant
irritability, skin rashes and hypersensitivity. If AD is present, the Scoring Atopic
Dermatitis (SCORAD) grading system will be used to assess severity. Urine samples will be
collected at each study visit to measure levels of fatty acid binding proteins (FABPs) and
glutathione-S-transferase (alpha-GST), which are non-invasive markers of gastrointestinal
permeability that may indicate the presence of food allergies. Blood will be collected via
finger or heel stick at weeks 6 and 52 for immune phenotyping (including measuring
inflammatory cytokines and food-specific IgEs). In addition, levels of serum fatty acid
binding proteins (FABPs) and glutathione-S-transferase (alpha-GST) will be measured as
markers of gastrointestinal permeability and potential food allergy. Parents will also
complete surveys at each study visit to monitor the infants for potential adverse events
associated with probiotic administration including feeding intolerance, fevers, or bowel
irregularities including constipation and diarrhea.

Entry into the study requires the intent to breastfeed exclusively for a minimum of six
months. If mothers decide to discontinue breastfeeding during the study, the investigators
will note that in the infant's chart and obtain an additional series of weekly stool samples
for six weeks after discontinuation of breastfeeding. The purpose of this additional stool
sample collection is to determine if discontinuation of breastfeeding has an impact on the
level of existing B. infantis colonization in the infant gut.

Inclusion Criteria:

- Healthy newborn infants between 1 and 7 days old with intent to be exclusively
breastfed for a minimum of six (6) months

Exclusion Criteria:

- Infants given dietary supplementation, including other probiotics.

- Infants born prior to 34 weeks gestation.

- Infants below 10th percentile for body weight.

- Postnatal use of antibiotics (oral, intramuscular or intravenous) by either the mother
or the infant. Of note, prenatal maternal Group B streptococcus prophylaxis is not a
criterion for study exclusion.

- Family history of immunodeficiency syndrome(s).

- Infants with signs of a clinically apparent underlying immunodeficiency.

- Intent to use non-breast milk infant formula for feeding during the first six months.

- History of GI tract abnormality or infection.