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