Global Vaccine and Immunization Research Forum
Johannesburg, South Africa 2016
Intestinal Microbiota and Enteric Vaccine Design
James M. Fleckenstein M.D.
Department of Medicine, Division of Infectious Diseases
& The Molecular Microbiology Microbial Pathogenesis Program
Washington University School of Medicine
modulation of intestinal immunity by commensal microorganisms
Perez-Lopez, et al Nature Rev Immunology 2016
microbiota prepare us for attack
• stimulate TLRs
• sustain gut “innate immune tone”
• stimulate regulatory T cells
• induce IgA production• modulate pro-inflammatory cytokines
TLR4 sensing changes the sugar landscape in the intestine
Pickard, et al Nature, 2014
use byproducts of microbiota
• for anaerobic respiration
• as carbon sources
• alternative energy source (H2)
• to modulate gene expression
pathogens exploit microbiota to colonize
Perez-Lopez, et al Nature Rev Immunology 2016
nexus of nutrient metabolism and immunity
Andy Kau, Nature 2011
• nutrients shape microbial community• nutrients shape microbial community
• microbes alter nutritional value• microbes alter nutritional value
• MAMPs>modify local immunity• MAMPs>modify local immunity
• immune system responds to microbial products• immune system responds to microbial products
host
genotypes
host
genotypes
food
insecurity
food
insecurityMalnutrition
(undernutrition)
impairedimmunity
infection with enteropathogens
environmentalenteropathy gut microbiota
Andy Kau, Nature 2011
microbiota considerations for vaccines
• nutritional interventions to optimize vaccine efficacy?• severe acute malnutrition impacts on microbiota
• can we optimize nutrient supplementation?
Subramanian, et al. Nature 2014
immaturity in gut microbiotapersists after nutritional replacement
severe acute malnutrition� microbiota immaturity
� only partially ameliorated by nutritional interventions
human milk oligosaccharideseffects on microbiota
Charbonneau et al. Cell 2016
HMOs as prebiotics
• promote beneficial bacteria
• compete for pathogen binding
• shape microbiota development
De Leoz J Proteome Research 2014
Ty21a live-attenuated oral typhoid vaccine
Eloe-Fadrosh, et al PlOS One 2013
vaccine effects on microbiota microbiota effects on vaccine
from Kotloff et al. Lancet 2013; 382: 209-222
ETEC and Shigella are predominant bacterial diarrheal pathogens
0 1 2 3 4 5 6 7 8
0-11
12-23
24-59
rotavirus
cryptosporidium
Shigella
ETEC
attributable incidence per 100 child-years
ag
e in
mo
nth
sGlobal Enteric Multicenter Study (GEMS)
Pathogens associated with deaths due to diarrhea
Mirpur Dhaka, Bangladesh
0 0.65 1.3 1.95 2.6 3.25
ETEC
EPEC
cryptosporidia
Hazard ratio
GEMS data
Kotloff et al. Lancet 2013; 382: 209-222
ETEC pathogenesisclassical paradigm
Cl-
LT(A1)
PKA
ST
p*^cGMP
H+
Na+
cftr
^cAMP
GC
-C
pkg
2
Gut microbial succession after ETEC infection
Lawrence A. David et al. mBio 2015; doi:10.1128/mBio.00381-15
Clonality of ETEC in stool cultures from
patients with severe diarrheal illness
Parkland, 1975
Amazon, village 1_1998
Amazon, village 2_1998
Dhaka, Bangladesh 2011
Matlab, Bangladesh 2011
mBio 2015
Gut microbiota are “glycophiles”
Sonnenburg, et al. Science 307 (25) March, 2005
seminal studies on B. “theta”� gut glycans induce OMP lectins,
hydrolases� in absence of exogenous dietary
glycans digest mucins
Johansson, et al Nature Biotechnology, 2013
intestinal glycanstargeting and manipulation by pathogens
ETEC as pathogens must:
� compete with commensals
� traverse protective mucin
� engage the epithelial surface
� deliver toxin payloads
conservation of novel ETEC antigens
Luo et al PLoS NTD, 2015
getting a seat at the table
� ETEC secrete highly conserved proteins
� not shared with E. coli commensals� not found in other microbiota
ETEC make a sugar-binding secreted lectin
EatA dissolves MUC2 gel matrix
ETEC toxin glycoscapingLT induces MUC2 expression and promotes adhesion
ETEC glycoscapingLT modulation of intestinal glycoproteinsCEACAM6
Carlito Lebrilla, University of California, Davis
heat-labile toxin changes the glycan landscape of intestinal epithelia
Nature 2009
• all ETEC are flagellated• motility• mucin penetration
• adhesion
flagellin and ETEC vaccinology
• flagellin• most abundant secreted antigen• highly immunogenic
• protective• TLR5 agonist
FliC(H48aa_174-399)
rEtpA
Ag43volunteer 306 d7
volunteer 306 d-1
rYghJrEatAp
FliC(H48)
YghJ(1-800)
[1:2] ALS IgA ETEC microarray_EtpA
EatAp
ETEC protein microarray studies
ACE527 vaccine interrogation*
vaccine EtpA EatA YghJ LT-B FliC CFs
ACAM2022 ++
ACAM2027 ++
ACAM2025 ++
*secretome proteomics
absent present over-expressed ++
Live-attenuated ACE527 vaccinesecreted antigen production
flagellin
intestinal microbiota and enteric vaccines
microbiota� shape immune responses� are altered by infection and nutrition
� respond to changes in glycan content� can impact vaccine efficacy
� are involved in recovery from infection
enterotoxigenic E. coli ETEC � are highly diverse pathogens� secrete conserved proteins
� engage specific glycans/glycoproteins� toxin(s) glycoscape to enhance colonization
� virulence factors that impact microbiota can inform novel vaccine strategies
•Dave Rasko
•Jeticia Sistrunk
U of Maryland, Institute for Genome
Sciences
•Sachin Mani
•Dick Walker
•Heather Wentzel
PATH Enteric Vaccine
Initiative
• Lou Bourgeois
• Subhra Chakraborty
• David Sack
Johns Hopkins
Center for Immunization Research
• Arlo Randall
• Joe Campo• Xiaowu Liang
• Doug Molina
Antigen Discovery,
Inc
• Phil Felgner
University of California, Irvine
genomics/microarray development
•Sadia Afrin •Yasmin Begum•Rashed Rashu•Salma Sharmin•Firdausi Qadri
• Pardeep Kumar •Qingwei Luo•Alaullah Sheikh•Tim Vickers•Matt Kuhlmann
•Danielle Bloch
•Chase Westra
the
lab
•Scott Hultgren
•Jerry Pinkner
•Reid Townsend•Mark Miller
•Matt Ciorba
•Sri Santhanam
Göteborgs Universitet
• Gunnar Hansson
Imperial College, London,
UK
• Anne Dell• Stuart Haslam
University of British
Columbia
• Bruce Vallance• Kiran Bhullar
University of Oklahoma
• Lijun Xia
NIH/CCR
• Jeffrey Gildersleeve
glycobiology
University of California,
Davis
• Carlito Lebrilla