UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)

UvA-DARE (Digital Academic Repository)

The role of the intestinal microbiome in rotavirus vaccine immunogenicity

Harris, V.C.

Link to publication

Citation for published version (APA):Harris, V. C. (2018). The role of the intestinal microbiome in rotavirus vaccine immunogenicity: An explorationfrom correlation to causation

General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.

Download date: 11 Jun 2018

9

chapter oneGeneral introduction

10

11

General introduction

Cha

pter

1Rotavirus disease Over the past 5 decades anywhere on earth, the risk of a child dying before they reach their 5th birthday has decreased dramatically (Wang et al., 2017). Despite this monumental public health achievement, the global under-fi ve mortality burden is borne unequally across countries. In 2016 only 3.8 children per 1000 livebirths died in the Netherlands before reaching the age of 4 whereas in Ghana this number was 52.3, in Pakistan 53.4 and in the Central African Republic as high as 130.5 deaths (Wang et al., 2017). After the fi rst month of life, the predominant diseases causing these childhood deaths are diarrhea and pneumonia, collectively responsible for up to 28% of under-5 deaths globally (Black et al., 2010).

Global health research has made it possible to describe in increasing detail which infectious diseases are driving childhood pneumonia and diarrheal mortality in developing countries (Walker et al., 2013). Rotavirus is the most common cause of severe and life-threatening gastroenteritis in young children in developing countries (Platts-Mills et al., 2016). Prior to rotavirus vaccine introduction, di-arrhea attributable to rotavirus caused an estimated 453,000 deaths world-wide (37% of diarrheal deaths and 5% of all deaths) in children under fi ve (Tate et al., 2012). By 2013, this number was still 215,000 per year and the vast majority of these deaths were in sub Saharan African and Asia (Tate et al., 2016) (Figure 1).

figure 1: rotavirus-associated mortality in children < 5 years of age in 2013. as of april 2016, the Who estimated that worldwide, 215,000 children < 5 years of age died because of rotavirus infec-tion in 2013. (figure reprinted with permission from the World health organization. http://www.who.int/immunization/monitoring_surveillance/burden/estimates/rotavirus/rotavirus_deaths_map_b.jpg?ue=1. accessed february 28, 2018.)

chapter one

12

Rotavirus pathophysiologyRotavirus is a double-stranded RNA virus of the family Reoviridae that infects enterocytes and enteroendocrine cells such as enterochromaffi n cells in the small intestine (Saxena et al., 2015). The virus is a sphere with outer spikes and de-rives its name from the Latin root rota, or wheel. (Figure 2) In the sphere, three concentric layers surround 11 segments of dsRNA. The RNA segments encode six structural viral proteins (VP1, VP2, VP3, VP4, VP6 and VP7) and six non-structural proteins (NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6). The structural proteins determine the architecture of the virus: the inner capsid and virion core are made up of VP2 with the enzymes VP1 and VP3; the middle capsid is VP6, which determines host specifi city; the outer capsid displays the epitopes that elicit host immune responses - VP7, a smooth protein, and VP4, a protruding spike protein that is cleaved into VP8* and VP5* (Figure 2). The non-structural proteins are needed for rotavirus replication, contain the enterotoxin NSP4, and can antagonize host innate immune responses (via NSP1)(Crawford et al., 2017).

Rotavirus causes particularly severe gastroenteritis in young children. Transmitted by fecal-oral contamination, the virus transits to the small intestine and enters host enterocytes through binding of the outer spike protein, VP4, to sialoglycans

figure 2: schematic representation of a rotavirus virion. the virus is composed of three protein shells, an outer capsid, an inner capsid, and an internal core, that surround the 11 segments of dou-ble-stranded RNA. The outer capsid proteins VP4 and VP7 are neutralization antigens and defi ne the P and G serotypes, respectively. vP6, the inner capsid structural protein, is the subgroup antigen. (reprinted with permission from J. angel, m.a. franco, and h.b. Greenberg. 2007. rotavirus vac-cines: recent developments and future considerations. nat. rev. microbiol. 5:529– 539.)

13

General introduction

Cha

pter

1and perhaps histo-blood group antigens (HBGA) in a strain-dependent manner (Arias et al., 2015). The virus is then internalized by endocytosis.

Clinical manifestations of rotavirus disease include diarrhea, vomiting, and fever. The diarrhea is primarily secretory through increased intracellular calcium up-regulating calcium-dependent chloride channels and chloride secretion following NSP4 enterotoxin signaling (Ball et al., 1996; Ko et al., 2014). Malabsorption secondary to enterocyte damage (Crawford et al., 2017) and increased intestinal motility due to serotonin release from enterochromaffin cells also play a role in diarrhea (Bialowas et al., 2016). Vomiting resulting in dehydration, an important clinical manifestation that exacerbates the severity of rotavirus disease, is caused by NSP4-induced serotonin release activating vagal afferent nerves (Hagbom et al., 2011).

Immunity to rotavirus is incompletely understood partially due to differences in in vitro and animal findings and clinical studies. Rotavirus is able to evade the innate immune system. Rotavirus is recognized by several pattern recogni-tion receptors (PRRs) but it has evolved mechanisms to inhibit PRR signaling through NSP1 interference (Arnold et al., 2013). Adults and children are subject to recurrent infections (Gladstone et al., 2011; Ward et al., 1989) suggesting that adaptive immunity to RV is poor, potentially a result of NSP1 alteration of innate immunity. Currently, serum anti RV IgA is the best correlate of protection for natural and vaccine-induced immunity (Angel et al., 2012; Patel et al., 2013) but is not a perfect correlate. Both antibodies and T cells are likely important in protec-tion against disease, as infants with SCID develop chronic rotavirus infection (Klinkenberg et al., 2015).

Rotavirus vaccinesIn 1998, Rotashield was the first licensed vaccine to prevent severe rotavirus gas-troenteritis in infants. The vaccine was withdrawn from the market as a result of post-licensure reporting of an increased risk of intussusception. It took till 2006 for Merck to license RotaTeq (RV5), a three-dose pentavalent, bovine re-assortant live oral rotavirus vaccine, and for Glaxosmithkline to license Rotarix (RV1), a two-dose live attenuated G1[P8] human strain vaccine. These vaccines have been broadly introduced in the US and countries in Europe, South America, and Africa and Asia, often with GAVI support. India approved licensure of an inexpensive human-bovine monovalent rotavirus vaccine, ROTAVAC, in 2016 for national use, and several other rotavirus vaccines are licensed or in the development pipe-line (Kirkwood et al., 2017). (Table 1) Countries in Asia and the Western Pacific

chapter one

14

have been particularly slow to introduce rotavirus vaccines and currently 67% of children globally still lack access to rotavirus vaccines (International Vaccine Access Center (IVAC)).

Although many children are not protected against severe rotavirus gastroenteritis because they are not getting vaccinated, the currently licensed RV vaccines also have significantly impaired protection when administered in developing coun-tries as compared to developed countries(Fischer Walker and Black, 2011). This characteristic is shared with several other orally administered vaccines, including live-attenuated and heat-killed vaccines against viral and bacterial pathogens such as polio, cholera, shigella, and typhoid (Qadri et al., 2013). Prior to deliberating on a global recommendation for rotavirus vaccine introduction, the WHO requested data on RV1 and RV5 vaccine efficacy in developing country settings. While trials of RV1 and RV5 demonstrated very high RVV efficacy rates against severe gas-troenteritis in developed countries – 85% and 98%, respectively, data from devel-oping countries was much lower (Ruiz-Palacios et al., 2006; Vesikari et al., 2006). RV1 trials in Malawi and South Africa demonstrated a pooled vaccine efficacy of 61.2% against severe RV gastroenteritis in the first year of life (Madhi et al., 2010) with waning immunity in the second year of life. A parallel RV5 trial showed a vaccine efficacy of 39.3% in Ghana, Kenya and Mali (Armah et al., 2010) and 48.3% in Bangladesh and Vietnam (Zaman et al., 2010). ROTAVAC (introduced later using a neonatal reassortant G9P[11] strain) had an efficacy of 53.6% in

table 1: overview of currently licensed vaccines with manufacturer and strain

Vaccine Manufacturer Strain Licensed

RotaTeqR Merck & Co, USA a pentavalent human-bovine reassortant vaccine

Globally, WHO pre-approved

RotarixTM GlaxoSmithKline Biologicals, Belgium

a human monovalent (G1P[8])

Globally, WHO pre-approved

ROTAVAC Bharat Pharmaceuticals, India

116E, naturally-occurring human reassortant strain (G9P[11])

India

Rotavin-M1 The Center for Research and Production of Vaccines and Biologicals (POLYVAC), Vietnam

KH0118-2003, naturally-occuring attenuated human strain (G1P[8])

Vietnam

Lanzhou Lamb virus vaccine

Lanzhou Institute of Biological Products (LIBP), China

monovalent lamb rotavirus strain (G10P[12])

China, private market

15

General introduction

Cha

pter

1India (Bhandari et al., 2014). RVV has a lower efficacy in sub Saharan Africa and Asia, but the rotavirus attack rate and mortality burden in developing country settings is also much higher. This means that despite having a reduced vaccine efficacy, significantly more child deaths are prevented upon RVV introduction in a developing country setting than in a developed country setting (Lopman et al., 2012). This rationale led the WHO to recommend that rotavirus vaccines be included in all national immunization programs globally in 2009 (World Health Organization (WHO).

Introduction of rotavirus vaccines into countries’ national vaccination programs have dramatically reduced rotavirus gastroenteritis (Desai et al., 2012; Bar Zeev et al., 2015) as well as diarrheal emergency department visits, hospitalizations (Cortes et al., 2011; Desai et al., 2012; Platts-Mills et al., 2017; Shah et al., 2016) and childhood diarrheal deaths (Enane et al., 2016; Richardson et al., 2011) across numerous countries. The path from RVV development to global RVV roll-outs and confirmation of this magnitude of effectiveness is a public health triumph. However, RVV impact could be even higher. Post-licensure effectiveness data have confirmed the gap in vaccine performance between developing and devel-oped country settings. A meta-analysis evaluated RVV post-licensure vaccine effectiveness data from 24 countries. When countries were segregated by under-5 mortality rates, mean RV1 effectiveness was 84%, 75%, and 57% for countries with low, middle, and high child mortality, respectively (Jonesteller et al., 2017). Therefore, while currently licensed rotavirus vaccines have significantly reduced childhood gastroenteritis morbidity and mortality, the vaccine is not adequately protecting those children most at risk of dying. Further improvements in rotavi-rus vaccine efficacy could likely prevent tens of thousands of additional deaths per year in developing countries with high under-five mortality rates (Patel et al., 2012).

Known determinants of rotavirus vaccine underperformanceImproving RVV impact requires urgent identification of modifiable etiologies of the poor RVV performance in developing country settings. Numerous factors are already known to correlate with diminished RVV performance and causality is very likely multi-factorial. Here follows a brief overview of the major factors hypothesized to alter RVV performance in developing country settings, followed by a rationale for the hypothesis underpinning this thesis – namely that the infant intestinal microbiome composition is a major determinant of the reduced RVV efficacy observed in developing countries.

chapter one

16

High maternal antibody titers to rotavirus in developing countries may diminish rotavirus vaccine take by interfering with vaccine strain replication. Higher pre-vaccination titers of both breast milk anti-RV IgA (Chilengi et al., 2016; Moon et al., 2010) and trans-placentally acquired anti-RV IgG (Appaiahgari et al., 2014; Moon et al., 2016) correlate with diminished RVV immunogenicity in several countries. Three randomized intervention studies built on these findings and tested if transiently withholding breast milk directly prior to vaccination could improve RVV seroconversion(Ali et al., 2015; Groome et al., 2014; Rongsen-Chandola et al., 2014). None of the studies demonstrated an improvement in RVV response. (Table 3) Hypothesizing that the effect of maternal antibodies might wane at older ages, additional studies have compared delayed or additive RVV dosing schedules. Results depended on location – with no change in im-munogenicity in Pakistan and a 14% gain in seroconversion in Ghana when three RV1 doses were used instead of two (Ali et al., 2014; Armah et al., 2016; Colgate et al., 2016) (Table 3).

Additional research supports a correlation between RVV and secretor and histo-blood group antigen (HBGA) expression. Rotavirus has two major surface pro-teins – a spiked protease-sensitive P protein (VP4) and a glycoprotein G (VP7). Some rotaviruses may recognize HBGAs in a P-genotype dependent manner. An association between salivary HBGA expression and RV1 (a G1P[8] rotavirus strain) vaccine seroconversion were tested in an infant cohort in Pakistan and showed that RVV seroconversion rate varied significantly by salivary HBGA phenotype. Non-secretors (not expressing carbohydrates synthesized by FUT2) had the lowest seroconversion, while secretors with O blood group had the high-est seroconversion (Kazi et al., 2017). This suggests that vaccine P[8] rotavirus strains may, for example, interact with H type 1 and Lewis b oligosaccharides, although in vitro data supporting such a mechanism has not been consistent (Sun et al., 2016). HBGA expression is likely only one of several factors affecting RVV immunogenicity, as developed countries have close to 95% RVV efficacy, despite having heterogeneous HBGA phenotypes in the population. Additionally, HBGA phenotype is an intrinsic genetic characteristic, and therefore not easily amenable to intervention.

Numerous studies have evaluated if nutrient and micronutrient deficiencies, malnutrition or HIV correlate with reduced RVV immunogenicity in developing countries (Colgate et al., 2016; Gastañaduy et al., 2016; Levin et al., 2017; Perez-Schael et al., 2007). These studies having conflicting results, and show no strongly

17

General introduction

Cha

pter

1significant correlations with RVV immunogenicity. Serum zinc has been the most promising, correlating significantly with both RVV seroconversion and efficacy in Bangladesh. However, an intervention study evaluating the effect of zinc supple-mentation alone prior to vaccination in Indian infants was unable to demonstrate an increase in RVV seroconversion (Lazarus et al., 2017). Probiotics prior to RVV administration in combination with zinc or alone show modest improvements in RVV seroconversion (Isolauri et al, 2003; Lazarus et al. 2017) (Table 3).

Finally, co-administration of both bi and tri-valent oral polio vaccine (OPV) and RVV has been shown to modestly reduce RVV seroconversion by 5-15% (Em-perador et al., 2016; Ramani et al., 2016; Steele et al., 2010), (Table 3), possibly through viral competition (Wang et al., 2012). Ameliorating this interaction is difficult as OPV use will depend upon the timing of the Global Polio Eradica-tion Campaign. Currently inactivated polio vaccine (IPV) is being introduced and trivalent OPV is being replaced by bivalent OPV.

While all these factors may explain a component of the diminished RVV efficacy in developing countries, they in aggregate fail to adequately account for how RVV efficacy and effectiveness can be as low as 30-40% in some settings (Ali et al., 2014). An underexplored hypothesis explaining rotavirus vaccine performance is that the intestinal microbiome composition may be modulating RVV immunoge-nicity. Because manipulation of the microbiome is becoming increasingly feasible, identifying robust correlations between the microbiome composition and RVV response opens avenues to improving RVV performance in those settings where children are disproportionately burdened with rotavirus disease. Testing this hypothesis is the main aim of this thesis.

The intestinal microbiome and rotavirus vaccine underperformanceThe term microbiome was first used by the Nobel Laureate Joshua Lederberg, who described it as the ‘collective genome of our indigenous microbes’ (Hooper and Gordon, 2001). In the last 15 years, application of next-generation sequenc-ing techniques derived from the environmental sciences has revolutionized the understanding of these indigenous microbes. The human organism contains approximately 1014 cells, of which only 10% are animal – the rest are bacteria, archaea, fungi, and viruses and largely reside in the human intestinal tract (Harris et al., 2017; Savage, 1977). These microbes have co-evolved with humans and their mammalian ancestors and there is rapidly expanding evidence of their roles in human health and disease. (See list of definitions, Table 2). Chapter Two of this

chapter one

18

thesis provides an overview of the known roles that the intestinal microbiome plays in clinical infectious diseases.

A set of four key findings support a role for the intestinal microbiome in deter-mining diminished RVV immunogenicity in developing countries: (1) Across all ages, the intestinal microbiome differs by geography(2) The intestinal microbiome shapes and regulates infant immunity(3) Enteric viruses are known to interact with the bacterial microbiome(4) The intestinal microbiome can alter vaccine responses in mouse models

(1) The intestinal microbiome differs by geography across all ages:In 2012, a key article (Yatsunenko et al., 2012) described differences in bacte-rial microbiome composition between healthy children and adults in the United States, rural Malawi, and Amazonas in Venezuela. At all ages the microbiome composition and functional gene reservoirs differed significantly between the US and both developing country populations. The study also described the devel-opment of the infant microbiome over time: regardless of geography –infants begin with low intra- and high inter-individual variability and move progressively towards higher intra-individual diversity with lower inter-individual differences or an ‘adult’ state over time. All factors driving microbiome maturity and geographic differences are not known, but an infant’s microbiome is shaped early by genetics, a mother’s own microbiota, mode of delivery (vaginal or caesarian), antibiotic use, and feeding practices (breast-feeding, formula, type and introduction of solid foods)(De Filippo et al., 2010; Duranti et al., 2017; Milani et al., 2017). In poor countries, undernutrition and repeated exposure to fecally-contaminated foods in combination with a high burden of eukaryotic disease likely significantly impact the microbiome over time (Blanton et al., 2016; Subramanian et al., 2015). There

Table 2: List of definitions

Term Definition

Microbiome All microorganisms, commensal, symbiotic or pathogenic that share our body space

Bacterial Microbiome All bacteria that share our body space

Virome All viruses that share our body space, both those that can infect bacterial cells and those that can infect eukaryotic cells

Mycobiome All fungal communities that share our body space

19

General introduction

Cha

pter

1is therefore a parallel in the differences in microbiome composition and RVV performance by geography.

(2) The intestinal microbiome shapes and regulates infant immunityIt has long been known that presence of an intestinal microbiome is essential for normal immune development. The clearest example of this is the aberrant immune development observed in germ-free mice, who do not have normal gut-associated lymphoid tissue (GALT) and IgA B cell maturation (Bouskra et al., 2008; Kunisawa et al., 2013). The resident intestinal bacteria interact with the innate and adaptive immune system and are important regulators of the immune steady state and readiness for infection (Collins and Belkaid, 2017; Honda and Littman, 2016; Kamada and Núñez, 2014). Therefore, geographic differences in key microbial taxa could potentially influence the maturation and regulation of infant immunity and responses to the live RV vaccine strain.

(3) Enteric viruses are known to interact with the bacterial microbiomeEnteric viruses have been shown to engage in a unique triangulation with the bac-terial intestinal microbiome and the immune system once they enter the human intestinal tract (Pfeiffer and Virgin, 2016). Poliovirus, mouse mammary tumor virus, and norovirus as well as murine rotavirus have all been shown to interact with the intestinal microbiome(Baldridge et al., 2014; Kane et al., 2011; Kuss et al., 2011; Robinson et al., 2014; Uchiyama et al., 2014). Specifically, study of murine rotavirus showed that microbiome depletion (either in gnotobiotic mice or through antibiotics) significantly delayed RV infection and reduced RV infec-tivity (Uchiyama et al., 2014). Enteric viral - virome interactions have also been described for rotavirus. In Bangladesh, the presence of non-polio enteroviruses in stool prior to vaccination correlated with a diminished RVV seroconversion, suggesting competitive viral replication (Taniuchi et al., 2016; Wang et al., 2012). These findings are not surprising when considering the millions of years of co-evolution between enteric viruses, resident bacteria, and human immunity. There is therefore an experimental precedent for viral-bacterial-immune responses underpinning a RVV-microbiome-immune response interaction.

(4) The intestinal microbiome can alter vaccine responses in mouse modelsFinally, one set of experimental evidence supports a role for the microbiome in systemic vaccination (Collins and Belkaid, 2017; Littman, 2017; Valdez et al., 2014). In a mouse model, normal antibody response to the seasonal influenza vaccine required the presence of resident bacteria or flagellated E. coli, which directly stimulated plasma cell differentiation (Oh et al., 2014). These results sug-

chapter one

20 21

General introduction

Cha

pter

1

Tabl

e 3:

Exa

mpl

es o

f int

erve

ntio

ns to

impr

ove

RVV

imm

unog

enic

ity

and

thei

r effe

ct o

n an

ti-R

V Ig

A se

roco

nver

sion

.

Inte

rven

tion

Vacc

ine

Cou

ntry

Inte

rven

tion

anti

-RV

IgA

sero

conv

ersi

onR

ef

Prob

iotic

DxR

RVFi

nlan

dLa

ctob

acill

us c

asei

i GG

74

% w

/o v

s 93%

with

p=0

.05

(Iso

laur

i, 20

03)

Prob

iotic

and

Zin

cRV

1In

dia

Prob

iotic

alo

neZi

nc a

lone

Prob

iotic

and

zinc

NS

NS

12.0

% in

crea

se (C

I,0.8

, 22.

8), p

=0.0

4

(Laz

arus

, 201

8)

Del

ayed

or a

dded

do

sing

RV1

Bang

lade

sh10

and

17

wee

ks, n

o co

ntro

l73

.5%

(CI,4

5-87

%)

(no

com

para

tor)

(Col

gate

., 20

16)

RV1

Gha

na6/

10 v

s 6/1

0/14

6/10

vs 1

0/14

wks

28.9

% v

s 37.

4%, p

=0.0

14

28.9

% v

s 43.

4% p

=0.1

63

(Arm

ah, 2

016)

RV1

Paki

stan

6/10

wks

6/10

/14

wks

10/1

4 w

ks

36.1

% (C

I, 29

.0, 4

3.9)

36.7

% (C

I, 29

.8, 4

4.2)

38.5

% (C

I, 31

.2, 4

6.3)

All c

ompa

rison

s NS

(Ali,

201

4)

RV1

Indi

a,

6/10

/14

wks

6/10

/14/

18 w

ksN

S(K

ompi

thra

,201

4)

With

hold

br

eastf

eedi

ng (B

F)RV

1In

dia

With

hold

30

min

pre

/pos

t RV

V

dose

NS

(Ron

gsen

-Cha

ndol

a,

2014

)

RV1

Paki

stan

With

hold

1 h

our p

re/p

ost R

VV

dos

eIn

crea

sed

in im

med

iate

BF

arm

: △-

12.5

% (C

I,-21

.2,-3

.8) p

=0.0

05

(Ali,

201

5)

RV1

Sout

h Af

rica

With

hold

1 h

our p

re/p

ost R

VV

dos

eN

S(G

room

e, 2

014)

chapter one

20 21

General introduction

Cha

pter

1

Tabl

e 3:

Exa

mpl

es o

f int

erve

ntio

ns to

impr

ove

RVV

imm

unog

enic

ity

and

thei

r effe

ct o

n an

ti-R

V Ig

A se

roco

nver

sion

. (co

ntin

ued)

Inte

rven

tion

Vacc

ine

Cou

ntry

Inte

rven

tion

anti

-RV

IgA

sero

conv

ersi

onR

ef

Stag

gere

d O

PV -

RRV

ad

min

istra

tion

RV1

Bang

lade

shR

RV w

/OPV

RRV

w/o

OPV

56.5

%(C

I,44%

,68%

)66

.7%

(CI,5

4%,7

8%)

NS

(Zam

an, 2

009)

RV1

Chi

leR

RV w

/bO

PVR

RV w

/IPV

50%

(CI,4

2%,5

8%)

65%

(CI,

60%

,71%

)p=

0.00

4

(Ram

ani,

2016

)

RV1

Bang

lade

shR

RV w

/OPV

RRV

>1

d sta

gger

ed w

/OPV

47%

(CI,3

9%,5

4%)

63%

(CI,5

7%,7

0%)

p=0.

001

(Em

pera

dor,2

016)

Abb

revi

atio

ns: B

F, b

reas

tfeed

ing;

RV

1, R

otar

ix; D

xRRV

, rhe

sus h

uman

reas

sort

ant v

acci

ne; O

PV, o

ral p

olio

vac

cine

; bO

PV, b

ival

ent O

PV; I

PV, i

nact

ivat

ed p

olio

vac

cine

wks

, wee

ks;

NS,

not

sign

ifica

nt; C

I, 95

% C

onfid

ence

Inte

rval

; Ref

, ref

eren

ce.

chapter one

22

gest that resident microbial taxa can stimulate vaccine-induced immunity, acting similarly to an adjuvant for a systemic vaccine.

These four lines of evidence formed the rationale for embarking upon the work presented in this thesis.

Aims and Outline of this thesisThe central aim of this thesis is to test the hypothesis that the intestinal microbi-ome modulates RVV immunogenicity and thereby contributes to the diminished RVV efficacy observed in developing country settings.

Part I – BackgroundChapter Two aims to give an overview of the known roles for the microbiome in clinical infectious diseases. This provides a background for understanding the microbiome’s relevance to early immune development as well as enteric infections.

Part II – Correlations of microbiome composition and RVV immunogenicityIf the microbiome modulates RVV immunogenicity, then there must be dif-ferences in microbiome composition between infants who do and who do not seroconvert to RVV in developing country settings.

Chapter Three tests whether there are bacterial microbiome differences prior to vaccination between RVV responders and non-responders in an urban slum in Karachi, Pakistan.

Chapter Four tests whether there are bacterial microbiome differences prior to vaccination between RVV responders and non-responders in rural Navrongo in northern Ghana.

Chapter Five provides a more extensive and temporal analysis of microbiome and RVV correlations, testing whether there are differences not only in bacterial microbiome, but also virome and mycobiome composition over time between RVV responders and non-responders in northern Ghana.

Part III – From correlation to causationIf the microbiome modulates RVV immunogenicity, then modulating the micro-biome should alter RVV immunogenicity

23

General introduction

Cha

pter

1Chapter Six describes establishment of a novel mouse model to test the effect of modulation of the microbiome on RVV immunogenicity in vivo.

Chapter Seven describes a human volunteer study whose aim was to evaluate the effect of antibiotic modulation of the intestinal microbiome on RVV im-munogenicity

Part IV – From causation to interventionChapter Eight provides a synthesis of the data presented in this thesis with sug-gestions for future work to establish if a microbiome-based intervention can improve RVV performance and decrease rotavirus-related deaths in developing country settings.

chapter one

24

references

Ali, A., Kazi, A.M., Cortese, M.M., Fleming, J.A., Moon, S., Parashar, U.D., Jiang, B., Mcneal, M.M., Steele, D., Bhutta, Z., et al. (2015). Impact of Withholding Breastfeeding at the Time of Vaccination on the Immunogenicity of Oral Rotavirus Vaccine—A Randomized Trial. PLoS ONE 10, e0127622.

Ali, S.A., Kazi, A.M., Cortese, M.M., Fleming, J.A., Parashar, U.D., Jiang, B., McNeal, M.M., Steele, D., Bhutta, Z., and Zaidi, A. (2014). Impact of different dosing schedules on the immunogenicity of the human rotavirus vaccine in infants in Pakistan: a randomized trial. J Infect Diseas 210, 1772–1779.

Angel, J., Franco, M.A., and Greenberg, H.B. (2012). Rotavirus immune responses and correlates of protection. Curr Opin Virol 2, 419–425.

Appaiahgari, M.B., Glass, R., Singh, S., Taneja, S., Rongsen-Chandola, T., Bhandari, N., Mishra, S., and Vrati, S. (2014). Transplacental rotavirus IgG interferes with immune response to live oral rotavirus vaccine ORV-116E in Indian infants. Vaccine 32, 651–656.

Arias, C.F., Silva-Ayala, D., and López, S. (2015). Rotavirus Entry: A Deep Journey into the Cell with Several Exits. J Virol 89, 890–893.

Armah, G.E., Sow, S.O., Breiman, R.F., Dallas, M.J., Tapia, M.D., Feikin, D.R., Binka, F.N., Steele, A.D., Laser-son, K.F., Ansah, N.A., et al. (2010). Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in sub-Saharan Africa: a randomised, double-blind, placebo-controlled trial. Lancet 376, 606–614.

Armah, G., Lewis, K.D.C., Cortese, M.M., Parashar, U.D., Ansah, A., Gazley, L., Victor, J.C., Mcneal, M.M., Binka, F., and Steele, A.D. (2016). A Randomized, Controlled Trial of the Impact of Alternative Dosing Schedules on the Immune Response to Human Rotavirus Vaccine in Rural Ghanaian Infants. J Infect Dis 213, 1678–1685.

Arnold, M.M., Sen, A., Greenberg, H.B., and Patton, J.T. (2013). The battle between rotavirus and its host for control of the interferon signaling pathway. PLoS Pathog 9, e1003064.

Baldridge, M.T., Nice, T.J., McCune, B.T., Yokoyama, C.C., Kambal, A., Wheadon, M., Diamond, M.S., Ivanova, Y., Artyomov, M., and Virgin, H.W. (2014). Commensal microbes and interferon- determine persistence of enteric murine norovirus infection. Science 347, 266–269.

Ball, J.M., Tian, P., Zeng, C.Q., Morris, A.P., and Estes, M.K. (1996). Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272, 101–104.

Bar-Zeev N, Kapanda L, Tate JE, et al. (2015). Effectiveness of a monovalent rotavirus vaccine in infants in Malawi after programmatic roll-out: an observational and case-control study. Lancet Infect Dis 15, 422–428.

Bhandari, N., Rongsen-Chandola, T., Bavdekar, A., John, J., Antony, K., Taneja, S., Goyal, N., Kawade, A., Kang, G., Rathore, S.S., et al. (2014). Efficacy of a monovalent human-bovine (116E) rotavirus vaccine in Indian infants: a randomised, double-blind, placebo-controlled trial. Lancet 383, 2136–2143.

Bialowas, S., Hagbom, M., Nordgren, J., Karlsson, T., Sharma, S., Magnusson, K.-E., and Svensson, L. (2016). Rotavirus and Serotonin Cross-Talk in Diarrhoea. PLoS ONE 11, e0159660.

Black, R.E., Cousens, S., Johnson, H.L., Lawn, J.E., Rudan, I., Bassani, D.G., Jha, P., Campbell, H., Walker, C.F., Cibulskis, R., et al. (2010). Global, regional, and national causes of child mortality in 2008: a systematic analysis. Lancet 375, 1969–1987.

25

General introduction

Cha

pter

1Blanton, L.V., Barratt, M.J., Charbonneau, M.R., Ahmed, T., and Gordon, J.I. (2016). Childhood undernutrition, the gut microbiota, and microbiota-directed therapeutics. Science 352, 1533–1533.

Bouskra, D., Brézillon, C., Bérard, M., Werts, C., Varona, R., Boneca, I.G., and Eberl, G. (2008). Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 456, 507–510.

Chilengi, R., Simuyandi, M., Beach, L., Mwila, K., Becker-Dreps, S., Emperador, D.M., Velasquez, D.E., Bosom-prah, S., and Jiang, B. (2016). Association of Maternal Immunity with Rotavirus Vaccine Immunogenic-ity in Zambian Infants. PLoS ONE 11, e0150100.

Colgate, E.R., Haque, R., Dickson, D.M., Carmolli, M.P., Mychaleckyj, J.C., Nayak, U., Qadri, F., Alam, M., Walsh, M.C., Diehl, S.A., et al. (2016). Delayed Dosing of Oral Rotavirus Vaccine Demonstrates De-creased Risk of Rotavirus Gastroenteritis Associated With Serum Zinc: A Randomized Controlled Trial. Clin Infect Dis 63, 634–641.

Collins, N., and Belkaid, Y. (2017). Do the Microbiota Influence Vaccines and Protective Immunity to Patho-gens? Cold Spring Harb Perspect Biol a028860–a028869.

Cortes, J.E., Curns, A.T., Tate, J.E., Cortese, M.M., Patel, M.M., Zhou, F., and Parashar, U.D. (2011). Rotavirus vaccine and health care utilization for diarrhea in U.S. children. N Engl J Med 365, 1108–1117.

Crawford, S.E., Ramani, S., Tate, J.E., Parashar, U.D., Svensson, L., Hagbom, M., Franco, M.A., Greenberg, H.B., O’Ryan, M., Kang, G., et al. (2017). Rotavirus infection. Nat Rev Dis Primers 3, 17083–16.

De Filippo, C., Cavalieri, D., Di Paola, M., Ramazzotti, M., Poullet, J.B., Massart, S., Collini, S., Pieraccini, G., and Lionetti, P. (2010). Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci U S A 107, 14691–14696.

Desai, R., Curns, A.T., Steiner, C.A., Tate, J.E., Patel, M.M., and Parashar, U.D. (2012). All-Cause Gastroenteritis and Rotavirus-Coded Hospitalizations Among US Children, 2000–2009. Clin Infect Dis 55, e28–e34.

Duranti, S., Lugli, G.A., Mancabelli, L., Armanini, F., Turroni, F., James, K., Ferretti, P., Gorfer, V., Ferrario, C., Milani, C., et al. (2017). Maternal inheritance of bifidobacterial communities and bifidophages in infants through vertical transmission. Microbiome 5, 1–13.

Emperador, D.M., Velasquez, D.E., Estivariz, C.F., Ben Lopman, Jiang, B., Parashar, U., Anand, A., and Zaman, K. (2016). Interference of Monovalent, Bivalent, and Trivalent Oral Poliovirus Vaccines on Monovalent Rotavirus Vaccine Immunogenicity in Rural Bangladesh. Clin Infect Dis 62, 150–156.

Enane, L.A., Gastañaduy, P.A., Goldfarb, D.M., Pernica, J.M., Mokomane, M., Moorad, B., Masole, L., Tate, J.E., Parashar, U.D., and Steenhoff, A.P. (2016). Impact of Rotavirus Vaccination on Hospitalizations and Deaths From Childhood Gastroenteritis in Botswana. Clin Infect Dis 62, S168–S174.

Fischer Walker, C.L., and Black, R.E. (2011). Rotavirus vaccine and diarrhea mortality: quantifying regional variation in effect size. BMC Public Health 11 Suppl 3, S16.

Gastañaduy, P.A., Steenhoff, A.P., Mokomane, M., Esona, M.D., Bowen, M.D., Jibril, H., Pernica, J.M., Mazhani, L., Smieja, M., Tate, J.E., et al. (2016). Effectiveness of Monovalent Rotavirus Vaccine After Program-matic Implementation in Botswana: A Multisite Prospective Case-Control Study. Clin Infect Dis 62, S161–S167.

Gladstone, B.P., Ramani, S., Mukhopadhya, I., Muliyil, J., Sarkar, R., Rehman, A.M., Jaffar, S., Gomara, M.I., Gray, J.J., Brown, D.W.G., et al. (2011). Protective effect of natural rotavirus infection in an Indian birth cohort. N Engl J Med 365, 337–346.

Groome, M.J., Moon, S.-S., Velasquez, D., Jones, S., Koen, A., van Niekerk, N., Jiang, B., Parashar, U.D., and Madhi, S.A. (2014). Effect of breastfeeding on immunogenicity of oral live-attenuated human rotavirus

chapter one

26

vaccine: a randomized trial in HIV-uninfected infants in Soweto, South Africa. Bull World Health Organ 92, 238–245.

Hagbom, M., Istrate, C., Engblom, D., Karlsson, T., Rodríguez-Díaz, J., Buesa, J., Taylor, J.A., Loitto, V.-M., Magnusson, K.-E., Ahlman, H., et al. (2011). Rotavirus Stimulates Release of Serotonin (5-HT) from Human Enterochromaffin Cells and Activates Brain Structures Involved in Nausea and Vomiting. PLoS Pathog 7, e1002115–10.

Harris, V.C., Haak, B.W., Boele van Hensbroek, M., and Wiersinga, W.J. (2017). The Intestinal Microbiome in Infectious Diseases: The Clinical Relevance of a Rapidly Emerging Field. Open Forum Infect Dis 4, ofx144.

Honda, K., and Littman, D.R. (2016). The microbiota in adaptive immune homeostasis and disease. Nature 535, 75–84.

Hooper, L.V., and Gordon, J.I. (2001). Commensal host-bacterial relationships in the gut. Science 292, 1115–1118.

International Vaccine Access Center (IVAC) View-hub.org. Www.View-Hub.org.

Isolauri, E., Joensuu, J., Suomalainen, H., Luomala, M., and Vesikari, T. (2003). Improved immunogenicity of oral D x RRV reassortant rotavirus vaccine by Lactobacillus casei GG. Vaccine 13, 310–312.

Jonesteller, C.L., Burnett, E., Yen, C., Tate, J.E., and Parashar, U.D. (2017). Effectiveness of Rotavirus Vaccina-tion: A Systematic Review of the First Decade of Global Postlicensure Data, 2006–2016. Clin Infect Dis 65, 840–850.

Kamada, N., and Núñez, G. (2014). Regulation of the Immune System by the Resident Intestinal Bacteria. Gastroenterology 146, 1477–1488.

Kane, M., Case, L.K., Kopaskie, K., Kozlova, A., MacDearmid, C., Chervonsky, A.V., and Golovkina, T.V. (2011). Successful transmission of a retrovirus depends on the commensal microbiota. Science 334, 245–249.

Kazi, A.M., Cortese, M.M., Yu, Y., Lopman, B., Morrow, A.L., Fleming, J.A., Mcneal, M.M., Steele, A.D., Para-shar, U.D., Zaidi, A.K.M., et al. (2017). Secretor and Salivary ABO Blood Group Antigen Status Predict Rotavirus Vaccine Take in Infants. J Infect Dis 215, 786–789.

Kirkwood, C.D., Ma, L.-F., Carey, M.E., and Steele, A.D. (2017). The rotavirus vaccine development pipeline. Vaccine 1–8.

Klinkenberg, D., Blohm, M., Hoehne, M., Mas Marques, A., Malecki, M., Schildgen, V., Schneppenheim, R., Müller, I., Schildgen, O., and Kobbe, R. (2015). Risk of Rotavirus Vaccination for Children with SCID. Pediatr Infect Dis J 34, 114–115.

Ko, E.-A., Jin, B.-J., Namkung, W., Ma, T., Thiagarajah, J.R., and Verkman, A.S. (2014). Chloride channel in-hibition by a red wine extract and a synthetic small molecule prevents rotaviral secretory diarrhoea in neonatal mice. Gut 63, 1120–1129.

Kompithra, R.Z., Paul, A., Manoharan, D., Babji, S., Sarkar, R., Mathew, L.G., and Kang, G. (2014). Immunoge-nicity of a three dose and five dose oral human rotavirus vaccine (RIX4414) schedule in south Indian infants. Vaccine 32, A129–A133.

Kunisawa, J., Gohda, M., Hashimoto, E., Ishikawa, I., Higuchi, M., Suzuki, Y., Goto, Y., Panea, C., Ivanov, I.I., Sumiya, R., et al. (2013). Microbe-dependent CD11b+ IgA+ plasma cells mediate robust early-phase intestinal IgA responses in mice. Nat Commun 4, 1772.

27

General introduction

Cha

pter

1Kuss, S.K., Best, G.T., Etheredge, C.A., Pruijssers, A.J., Frierson, J.M., Hooper, L.V., Dermody, T.S., and Pfeiffer, J.K. (2011). Intestinal microbiota promote enteric virus replication and systemic pathogenesis. Science 334, 249–252.

Lazarus, R.P., John, J., Shanmugasundaram, E., Rajan, A.K., Thiagarajan, S., Giri, S., Babji, S., Sarkar, R., Kaliap-pan, P.S., Venugopal, S., et al. (2017). The effect of probiotics and zinc supplementation on the immune response to oral rotavirus vaccine: A randomized, factorial design, placebo-controlled study among Indian infants. Vaccine 36, 273-79.

Levin, M.J., Lindsey, J.C., Kaplan, S.S., Schimana, W., Lawrence, J., Mcneal, M.M., Bwakura-Dangarembizi, M., Ogwu, A., Mpabalwani, E.M., Sato, P., et al. (2017). Safety and immunogenicity of a live attenuated pentavalent rotavirus vaccine in HIV-exposed infants with or without HIV infection in Africa. Aids 31, 49–59.

Littman, D.R. (2017). Do the Microbiota Influence Vaccines and Protective Immunity to Pathogens? Cold Spring Harb Perspect Biol a029355–a029359.

Lopman, B.A., Pitzer, V.E., Sarkar, R., Gladstone, B., Patel, M., Glasser, J., Gambhir, M., Atchison, C., Grenfell, B.T., Edmunds, W.J., et al. (2012). Understanding reduced rotavirus vaccine efficacy in low socio-economic settings. PLoS ONE 7, e41720.

Madhi, S.A., Cunliffe, N.A., Steele, D., Witte, D., Kirsten, M., Louw, C., Ngwira, B., Victor, J.C., Gillard, P.H., Cheuvart, B.B., et al. (2010). Effect of human rotavirus vaccine on severe diarrhea in African infants. N Engl J Med 362, 289–298.

Milani, C., Duranti, S., Bottacini, F., Casey, E., Turroni, F., Mahony, J., Belzer, C., Delgado Palacio, S., Arboleya Montes, S., Mancabelli, L., et al. (2017). The First Microbial Colonizers of the Human Gut: Composi-tion, Activities, and Health Implications of the Infant Gut Microbiota. Microbiol Mol Biol Rev 81, e00036–17–67.

Moon, S.-S., Groome, M.J., Velasquez, D.E., Parashar, U.D., Jones, S., Koen, A., van Niekerk, N., Jiang, B., and Madhi, S.A. (2016). Prevaccination Rotavirus Serum IgG and IgA Are Associated With Lower Immuno-genicity of Live, Oral Human Rotavirus Vaccine in South African Infants. Clin Infect Dis 62, 157–165.

Moon, S.-S., Wang, Y., Shane, A.L., Nguyen, T., Ray, P., Dennehy, P., Baek, L.J., Parashar, U., Glass, R.I., and Jiang, B. (2010). Inhibitory Effect of Breast Milk on Infectivity of Live Oral Rotavirus Vaccines. Pediatr Infect Dis J 29, 919–923.

Oh, J.Z., Ravindran, R., Chassaing, B., and Carvalho, F.A. (2014). TLR5-Mediated Sensing of Gut Microbiota Is Necessary for Antibody Responses to Seasonal Influenza Vaccination. Immunity 41, 478-92.

Patel, M., Glass, R.I., Jiang, B., Santosham, M., Lopman, B., and Parashar, U. (2013). A Systematic Review of Anti-Rotavirus Serum IgA Antibody Titer as a Potential Correlate of Rotavirus Vaccine Efficacy. J Infect Dis 208, 284–294.

Patel, M.M., Clark, A.D., Sanderson, C.F.B., Tate, J., and Parashar, U.D. (2012). Removing the Age Restrictions for Rotavirus Vaccination: A Benefit-Risk Modeling Analysis. PLoS Med 9, e1001330–13.

Perez-Schael, I., Salinas, B., Tomat, M., Linhares, A.C., Guerrero, M.L., Ruiz-Palacios, G.M., Bouckenooghe, A., and Yarzabal, J.P. (2007). Efficacy of the human rotavirus vaccine RIX4414 in malnourished children. J Infect Dis 196, 537–540.

Pfeiffer, J.K., and Virgin, H.W. (2016). Transkingdom control of viral infection and immunity in the mammalian intestine. Science 351, aad5872–aad5872.

chapter one

28

Platts-Mills, J.A., Amour, C., Gratz, J., Nshama, R., Walongo, T., Mujaga, B., Maro, A., McMurry, T.L., Liu, J., Mduma, E., et al. (2017). Impact of rotavirus vaccine introduction and post-introduction etiology of diarrhea requiring hospital admission in Haydom, Tanzania, a rural African setting. Clin Infect Dis epub ahead of print, 1-8.

Platts-Mills, J.A., Juma, J., Kabir, F., Nkeze, J., Okoi, C., Operario, D.J., Uddin, J., Ahmed, S., Alonso, P.L., Anto-nio, M., et al. (2016). Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study. Lancet 388, 1291–1301.

Qadri, F., Bhuiyan, T.R., Sack, D.A., and Svennerholm, A.-M. (2013). Immune responses and protection in children in developing countries induced by oral vaccines. Vaccine 31, 452–460.

Ramani, S., Mamani, N., Villena, R., Bandyopadhyay, A.S., Gast, C., Sato, A., Laucirica, D., Clemens, R., Estes, M.K., and O’Ryan, M.L. (2016). Rotavirus Serum IgA Immune Response in Children Receiving Rotarix Coadministered With bOPV or IPV. Pediatr Infect Dis 35, 1137–1139.

Richardson, V., Parashar, U., and Patel, M. (2011). Childhood diarrhea deaths after rotavirus vaccination in Mexico. N Engl J Med 365, 772–773.

Robinson, C.M., Jesudhasan, P.R., and Pfeiffer, J.K. (2014). Bacterial Lipopolysaccharide Binding Enhances Vi-rion Stability and Promotes Environmental Fitness of an Enteric Virus. Cell Host & Microbe 15, 36–46.

Rongsen-Chandola, T., Strand, T.A., Goyal, N., Flem, E., Rathore, S.S., Arya, A., Winje, B.A., Lazarus, R., Shanmugasundaram, E., Babji, S., et al. (2014). Effect of withholding breastfeeding on the immune response to a live oral rotavirus vaccine in North Indian infants. Vaccine 32, A134–A139.

Ruiz-Palacios, G.M., Pérez-Schael, I., Velázquez, F.R., Abate, H., Breuer, T., Clemens, S.C., Cheuvart, B., Espi-noza, F., Gillard, P., Innis, B.L., et al. (2006). Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis. N Engl J Med 354, 11–22.

Savage, D.C. (1977). Microbial ecology of the gastrointestinal tract. Annu Rev Microbiol 31, 107–133.

Saxena, K., Blutt, S.E., Ettayebi, K., Zeng, X.-L., Broughman, J.R., Crawford, S.E., Karandikar, U.C., Sastri, N.P., Conner, M.E., Opekun, A.R., et al. (2015). Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology. J Virol 90, 43–56.

Shah, M.P., Tate, J.E., Steiner, C.A., and Parashar, U.D. (2016). Decline in Emergency Department Visits for Acute Gastroenteritis Among Children in 10 US States After Implementation of Rotavirus Vaccination, 2003 to 2013. Pediatr Infect Dis 35, 782–786.

Steele, A.D., De Vos, B., Tumbo, J., Reynders, J., Scholtz, F., Bos, P., de Beer, M.C., Van der Merwe, C.F., and Delem, A. (2010). Co-administration study in South African infants of a live-attenuated oral human rotavirus vaccine (RIX4414) and poliovirus vaccines. Vaccine 28, 6542–6548.

Subramanian, S., Huq, S., Yatsunenko, T., Haque, R., Mahfuz, M., Alam, M.A., Benezra, A., DeStefano, J., Meier, M.F., Muegge, B.D., et al. (2015). Persistent gut microbiota immaturity in malnourished Bangladeshi children. Nature 510, 417–421.

Sun, X., Guo, N., Li, D., Jin, M., Zhou, Y., Xie, G., Pang, L., Zhang, Q., Cao, Y., and Duan, Z.-J. (2016). Binding specificity of P[8] VP8* proteins of rotavirus vaccine strains with histo-blood group antigens. Virology 495, 129–135.

Taniuchi, M., Platts-Mills, J.A., Begum, S., Uddin, M.J., Sobuz, S.U., Liu, J., Kirkpatrick, B.D., Colgate, E.R., Carmolli, M.P., Dickson, D.M., et al. (2016). Impact of enterovirus and other enteric pathogens on oral polio and rotavirus vaccine performance in Bangladeshi infants. Vaccine 34, 3068–3075.

29

General introduction

Cha

pter

1Tate, J.E., Burton, A.H., Boschi-Pinto, C., Parashar, U.D., World Health Organization–Coordinated Global Ro-tavirus Surveillance Network (2016). Global, Regional, and National Estimates of Rotavirus Mortality in Children. Clin Infect Dis 62 Suppl 2, S96–S105.

Tate, J.E., Burton, A.H., Boschi-Pinto, C., Steele, A.D., Duque, J., Parashar, U.D., and WHO-coordinated Global Rotavirus Surveillance Network (2012). 2008 estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis. Lancet Infect Dis 12, 136–141.

Uchiyama, R., Chassaing, B., Zhang, B., and Gewirtz, A.T. (2014). Antibiotic Treatment Suppresses Rotavirus Infection and Enhances Specific Humoral Immunity. J Infect Dis 210, 171–182.

Valdez, Y., Brown, E.M., and Finlay, B.B. (2014). Influence of the microbiota on vaccineeffectiveness. Trends Immunol 35, 526-537.

Vesikari, T., Matson, D.O., Dennehy, P., Van Damme, P., Santosham, M., Rodriguez, Z., Dallas, M.J., Heyse, J.F., Goveia, M.G., Black, S.B., et al. (2006). Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med 354, 23–33.

Walker CLF, Rudan I, Liu L, et al. (2013). Global burden of childhood pneumonia and diarrhoea. Lancet 381, 1405–1416.

Wang, H., Abajobir, A.A., Abate, K.H., Abbafati, C., Abbas, K.M., Abd-Allah, F., Abera, S.F., Abraha, H.N., Abu-Raddad, L.J., Abu-Rmeileh, N.M.E., et al. (2017). Global, regional, and national under-5 mortal-ity, adult mortality, age-specific mortality, and life expectancy, 1970-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 390, 1084–1150.

Wang, H., Moon, S., Wang, Y., and Jiang, B. (2012). Multiple virus infection alters rotavirus replication and expression of cytokines and Toll-like receptors in intestinal epithelial cells. Virus Res 167, 48–55.

Ward, R.L., Bernstein, D.I., Shukla, R., Young, E.C., Sherwood, J.R., McNeal, M.M., Walker, M.C., and Schiff, G.M. (1989). Effects of antibody to rotavirus on protection of adults challenged with a human rotavi-rus. J Infect Dis 159, 79–88.

World Health Organization (WHO) Global use of rotavirus vaccines recommended, (2009). http://www.who.int/mediacentre/news/releases/2009/rotavirus_vaccines_20090605/en/. Accessed 12/5/17.

Yatsunenko, T., Rey, F.E., Manary, M.J., Trehan, I., Dominguez-Bello, M.G., Contreras, M., Magris, M., Hidalgo, G., Baldassano, R.N., Anokhin, A.P., et al. (2012). Human gut microbiome viewed across age and geog-raphy. Nature 486, 222–227.

Zaman, K., Dang, D.A., Victor, J.C., Shin, S., Yunus, M., Dallas, M.J., Podder, G., Vu, D.T., Le, T.P.M., Luby, S.P., et al. (2010). Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in Asia: a randomised, double-blind, placebo-controlled trial. Lancet 376, 615–623.

Zaman, K., Sack, D.A., Yunus, M., Arifeen, S.E., Podder, G., Azim, T., Luby, S., Breiman, R.F., Neuzil, K., Datta, S.K., et al. (2009). Successful co-administration of a human rotavirus and oral poliovirus vaccines in Bangladeshi infants in a 2-dose schedule at 12 and 16 weeks of age. Vaccine 27, 1333–1339.