Pediatrics 134(2): e362-e372

PMC: PMC4187229

PMID: 25002669

Stool Microbiota and Vaccine Responses of Infants

OBJECTIVE:

Oral vaccine efficacy is low in less-developed countries, perhaps due to intestinal dysbiosis. This study determined if stool microbiota composition predicted infant oral and parenteral vaccine responses.

METHODS:

The stool microbiota of 48 Bangladeshi infants was characterized at 6, 11, and 15 weeks of age by amplification and sequencing of the 16S ribosomal RNA gene V4 region and by Bifidobacterium-specific, quantitative polymerase chain reaction. Responses to oral polio virus (OPV), bacille Calmette-Guérin (BCG), tetanus toxoid (TT), and hepatitis B virus vaccines were measured at 15 weeks by using vaccine-specific T-cell proliferation for all vaccines, the delayed-type hypersensitivity skin-test response for BCG, and immunoglobulin G responses using the antibody in lymphocyte supernatant method for OPV, TT, and hepatitis B virus. Thymic index (TI) was measured by ultrasound.

RESULTS:

Actinobacteria (predominantly Bifidobacterium longum subspecies infantis) dominated the stool microbiota, with Proteobacteria and Bacteroidetes increasing by 15 weeks. Actinobacteria abundance was positively associated with T-cell responses to BCG, OPV, and TT; with the delayed-type hypersensitivity response; with immunoglobulin G responses; and with TI. B longum subspecies infantis correlated positively with TI and several vaccine responses. Bacterial diversity and abundance of Enterobacteriales, Pseudomonadales, and Clostridiales were associated with neutrophilia and lower vaccine responses.

CONCLUSIONS:

Bifidobacterium predominance may enhance thymic development and responses to both oral and parenteral vaccines early in infancy, whereas deviation from this pattern, resulting in greater bacterial diversity, may cause systemic inflammation (neutrophilia) and lower vaccine responses. Vaccine responsiveness may be improved by promoting intestinal bifidobacteria and minimizing dysbiosis early in infancy.

Supplementary Material

Supplemental Information:

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^{US Department of Agriculture Western Human Nutrition Research Center, Davis California;}

^{International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh; and}

Departments of ^{Viticulture and Enology, and}

^{Pediatrics, University of California, Davis, Davis, California}

^{Corresponding author.}

Address correspondence to Charles B. Stephensen, USDA Western Human Nutrition Research Center, 430 West Health Sciences Dr, University of California, Davis, Davis, CA 95616. E-mail: vog.adsu.sra@nesnehpets.selrahc

Accepted 2014 May 1.

Abstract

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords: vaccine, intestinal, microbiota, Bifidobacterium, Actinobacteria, Proteobacteria, Bangladesh, T lymphocyte, antibody, polio, tetanus, tuberculosis, hepatitis

Abstract

What’s Known on This Subject:

Oral vaccine responses are low in children from less-developed countries perhaps as a result of intestinal dysbiosis. New high-throughput DNA-based methods allow characterization of intestinal microbiota as a predictor of vaccine responses.

What This Study Adds:

High abundance of stool Actinobacteria, including Bifidobacterium, was associated with higher responses to oral and parenteral vaccines and a larger thymus in Bangladeshi infants. Conversely, high abundance of Clostridiales, Enterobacteriales, and Pseudomonadales was associated with neutrophilia and lower vaccine responses.

The composition of the community of microbes that inhabits the gastrointestinal tract (the gut microbiota) has a profound influence on the developing infant.¹ Dysbiosis, defined as deviation from an optimal, health-promoting microbial community,² may cause necrotizing enterocolitis and sepsis in premature infants³ and allergic disease in term infants.⁴ A gut microbiota dominated by appropriate commensal bacteria promotes infant health by a variety of mechanisms,⁵6 including appropriate development of the immune system.⁷8

Infant immunization is an important measure for decreasing morbidity and mortality from infectious diseases.⁹ However, oral vaccines are often less effective than expected when used in developing countries, as described in a recent review,¹⁰ perhaps as a result of malnutrition, intestinal dysbiosis, or the presence of other inhibitory factors related to the local environment. Evidence that dysbiosis may influence vaccine responses include direct effects of intestinal bacteria on the ability of polio virus to infect target cells in the intestine¹¹ and direct impairment by dysbiosis of the host immune response.¹² Studies showing that probiotic interventions increase immune responses to oral vaccines in adults¹³ and animals¹⁴ support this hypothesis, although results in infants and children are equivocal.¹⁵16

New DNA-based methods that use direct sequencing of short regions of the bacterial 16S ribosomal RNA (rRNA) gene produce a detailed picture of the gut microbiota, providing the relative abundance of taxa at multiple phylogenetic levels (ie, phylum, class, order, family, and genus).¹⁷ The intestinal microbiota of term infants is dominated by 4 major phyla: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The relative abundance of taxa within these phyla is influenced early in infancy by mode of delivery,¹⁸ gestational age,¹⁹ type of feeding,²⁰ and locality.²¹22 Early colonizers typically include commensal facultative anaerobes such as Escherichia coli and other Enterobacteriaceae (phylum Proteobacteria), followed by an increased relative abundance of strict anaerobes including Bifidobacterium (phylum Actinobacteria), Bacteroides (phylum Bacteroidetes), and Clostridium (phylum Firmicutes).¹Firmicutes includes several genera of importance to infant health including Streptococcus, Staphylococcus, and Lactobacillus, whereas the phylum Proteobacteria includes pathogens from the genera Escherichia, Shigella, and Campylobacter. The relative abundance of these taxa is used to define the gut microbiota.

In the current study we hypothesized that the composition of the gut microbiota would affect responses to oral and perhaps parenteral vaccines. To test this hypothesis, we characterized the microbiota from the stool of 48 Bangladeshi infants and measured responses to 4 vaccines: oral polio virus (OPV), bacille Calmette-Guérin (BCG; given to protect against tuberculosis), tetanus toxoid (TT), and hepatitis B virus (HBV) by using 2 methods for each vaccine. Vaccine-specific T-cell proliferation was measured for all vaccines; the delayed-type hypersensitivity (DTH) skin test to purified protein derivative (PPD) of Mycobacterium tuberculosis was measured as a second, functional indicator of response to BCG vaccination; and vaccine-specific immunoglobulin (Ig) G levels were measured for OPV, TT, and HBV using the antibody in lymphocyte supernatant assay²³ as an index of the memory B-cell response.

N = 48.

Data are presented as n (%).

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Glossary

BCG	bacille Calmette-Guérin
DTH	delayed-type hypersensitivity
HBV	hepatitis B virus
Ig	immunoglobulin
OPV	oral polio virus
PCR	polymerase chain reaction
PPD	purified protein derivative of Mycobacterium bovis
qPCR	quantitative polymerase chain reaction
rRNA	ribosomal RNA
SEB	Staphylococcus enterotoxin B
SI	stimulation index
T-RFLP	terminal restriction fragment length polymorphism
TI	thymus index
TT	tetanus toxoid

Glossary

Footnotes

Dr Stephensen, working with coauthors, conceptualized and implemented the major hypothesis of the study (evaluating the association of microbiota with immune function), conceptualized and implemented the work to evaluate immune function, conducted statistical analysis, and wrote the first draft of the manuscript; Dr Mills worked to conceptualize, design, and implement the major hypothesis of the study (evaluating the association of microbiota with immune function); supervised acquisition of microbiota data; and participated in analysis and interpretation of the overall data set; Dr Underwood worked to conceptualize, design, and implement the major hypothesis of the study (evaluating the association of microbiota with immune function) and participated in analysis and interpretation of the overall data set; Drs Ahmad, Raqib, and Qadri and Mr Rashid contributed substantially to acquisition of immunologic data and participated in the analysis and interpretation of the overall data set; Mr Huda carried out laboratory studies to analyze microbiota and to characterize immune function, conducted bioinformatic analysis of microbiota data, conducted statistical analysis, and collaborated in writing the first draft of the manuscript; Mr Lewis and Dr Kalanetra carried out laboratory and bioinformatic analysis of stool microbiota data and contributed to the first draft of the manuscript; all authors participated in drafting the manuscript and/or revising it critically for important intellectual content, had final approval authority for the version to be published, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

This trial has been registered at www.clinicaltrials.gov (identifier {"type":"clinical-trial","attrs":{"text":"NCT01583972","term_id":"NCT01583972"}}NCT01583972).

FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.

FUNDING: This work was funded by grant UL1 RR024146 from the National Center for Research Resources, US Department of Agriculture–Agricultural Research Service project 5306-51530-018-00, and World Health Organization project 2010168947, which was funded by the Bill and Melinda Gates Foundation. Funded by the National Institutes of Health (NIH).

POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

Footnotes

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