Studiesofpathogenesis,innateimmunityandtherapeuticsofhumanentericvirusesingnotobioticpigs

TammyBuiCastellucci

DissertationsubmittedtothefacultyoftheVirginiaPolytechnicInstituteandStateUniversityin

partialfulfillmentoftherequirementsforthedegreeof

DoctorofPhilosophy

In

BiomedicalandVeterinarySciences

LijuanYuan,Chair

Xiang-JinMeng

XinM.Luo

LiwuLi

April24th,2017

BlacksburgVA

Keywords:humanrotavirus,humannorovirus,gnotobioticpigs,pathogenesis,dendriticcells,

anti-diarrhealdrugs

CopyrightÓ2017TammyBuiCastellucci

Studiesofpathogenesis,innateimmunityandtherapeuticsofhumanentericvirusesingnotobioticpigs

TammyBuiCastellucci

ACADEMICABSTRACT

Norovirusandrotavirusarethemostcommonviralcausesofacutegastroenteritis

amongallagegroupsandinchildrenunder5yearsofage,respectively.Understandingthe

pathogenesisofthevirusandcorrelatesofprotectiveimmunityisfundamentaltodeveloping

effectivepreventionandtreatmentstrategies.Gnotobiotic(Gn)pigsareanattractiveanimal

modelforstudyingentericvirusesduetotheirsimilaritiestohumans,particularlyinregardsto

theimmunesystemandgastrointestinalanatomyandphysiology.Here,toestablishareliable

Gnpigmodelofhumannorovirus(HuNoV)infectionanddisease,wedeterminedthemedian

infectiousdose(ID50)ofaGII.42006bvariantinpigs.Wealsoevaluatedtheeffectsofageand

administrationofthecholesterol-loweringdrugsimvastatinonsusceptibilitytoNoVinfection.

Inneonatalpigs(4-5daysofage,theID50wasdeterminedtobe£2.74x103viralRNAcopies.

TheID50wasincreasedin33-34dayoldpigs(6.43x104),butdecreasedto

weightcomparedtocontrolsduringthecourseofthestudy.Wealsodeterminedthatahigh

doseoftheprobioticLactobacillusacidophilusNCFM(LA)wasabletoreduceRVdiarrhea

severityanddurationcomparedtoalowdose.Thedifferenceintherapeuticpotentialwas

attributedtodivergenteffectsininnateimmunitypre-andpost-challenge.HighdoseofLA

(HiLA)inducedananti-inflammatorydendriticcell(DC)profile,characterizedprimarilyby

upregulationofTLR2expressionandproductionofcytokineIL-10.Conversely,lowdoseofLA

(LoLA)upregulatedTLR3andTLR9andincreasedsecretionofcytokineIL-6.Additionally,HiLA

inducedbothIFN-aandTNF-aresponsesinDCs,butLoLAwasonlyabletoincreasethe

frequencyofTNF-a-producingDCs.TheseresultsprovidefurthersupportofGnpigsasahighly

applicableanimalmodelforstudyingpathogenesis,innateimmunityandtherapeuticsof

humanentericviruses.

Studiesofpathogenesis,innateimmunityandtherapeuticsofhumanentericvirusesin

gnotobioticpigs

TammyBuiCastellucci

GENERALAUDIENCEABSTRACT

Norovirusandrotavirusarethemostcommonviralcausesofacutegastroenteritis

amongallagegroupsandinchildrenunder5yearsofage,respectively.Understandingthe

pathogenesisofthevirusandcorrelatesofprotectiveimmunityisfundamentaltodeveloping

effectivepreventionandtreatmentstrategies.Gnotobiotic(Gn)pigsareanattractiveanimal

modelforstudyingentericvirusesduetotheirsimilaritiestohumans,particularlyinregardsto

theimmunesystemandgastrointestinalanatomyandphysiology.Here,weestablisheda

reliableGnpigmodelofhumannorovirus(HuNoV)infectionanddisease.Overall,the

developmentofdiarrhea,fecalvirussheddingandsmallintestinalcytopathologicalchanges

confirmedtheusefulnessoftheGnpigasanappropriateanimalmodelforstudyingHuNoVs.

Wealsoutilizedthewell-establishedGnpigmodelofhumanrotavirus(HRV)infectionand

diseasetoevaluateadjunctivetreatmentoptionsforHRV-induceddiarrhea.Wedemonstrated

thattheanti-secretorydrugracecadotrilwascapableofdiminishingclinicalsignsofHRV

infectionandshorteningdurationofillness.Wealsodeterminedthatahighdoseofthe

probioticLactobacillusacidophilusNCFM(LA)wasabletoreduceRVdiarrheaseverityand

durationcomparedtoalowdose.TheseresultsprovidefurthersupportofGnpigsasahighly

applicableanimalmodelforstudyingpathogenesis,innateimmunityandtherapeuticsof

humanentericviruses.

v

DedicatedtoErnestandtoallthosewholovedhim,becausethatprettymuchcoverseveryoneI

careaboutaswell.

vi

ACKNOWLEDGEMENTS

Mygraduatecareerbeganinthefallof2008andhasprovidedmetheopportunitytomeetand

interactwithnumerousintelligent,genuinelyhelpfulandabsolutelywonderfulindividuals.

Completingthisdegreewouldnothavebeenpossiblewithouttheirassistance.Wordscannot

fullyexpressmyappreciation,butIwillcertainlytry.

Iowemuchthankstomyamazingadvisor,Dr.LijuanYuan.Shetookmeinasafairlynaïve

graduatestudentandhasprovidedsomanylessons,bothinthelabandinlife.Herguidance

andencouragementhavebeenintegraltomydevelopmentasascientist.Amorebrilliant,

dependable,caringandsupportivementorisdifficulttoimagine.Thankyoufortheopportunity

tolearnfromyouandtoconductresearchinyourlab.

IhavethoroughlyenjoyedworkingwitheveryindividualwhohaspassedthroughtheYuanLab

throughtheyears.IwouldespeciallyliketooffersincereappreciationtoDr.GuohuaLi,Dr.Ke

Wen,Dr.FangningLiu,Dr.JacobKocher,Dr.YanruLi,Dr.XingdongYang,Dr.EricaTwitchell,

MariahWeiss,AshwinRamesh,ShaohuaLei,ChristineTinandRichardKevorkian.Thankyouall

foryouradvice,assistanceandfriendship.IalsobelieveIhaveaskedeachoneofyouatone

timeoranothertocheckifIhadleftafridge/freezerdooropen.Thankyouforacceptingmy

absentmindednessandhumoringmyparanoia.

GnotobioticpigworkwouldnotberemotelyfeasiblewithouttheTRACSSstaff,includingPete

Jobst,KarenHall,MariahWeiss,KimAllen,AndreaPulliam,MelissaSquib,LauraThomas,Cassi

vii

BashamandJillStafford.Iwouldalsoliketothanktheveterinarystaff,includingDrs.Sherrie

Clark-Deener,KevinPelzer,NicoleLindstromandMarliceVonck.Thankyoufortheexcellent

animalcareandforallyoureffortsinmakingourpigprojectssuccessful.

Muchgratitudeisalsoduetomyadvisorycommitteememberspastandpresent,includingDrs.

Meng,Subbiah,Myles,LuoandLi.Yourmentorship,insightfuldiscussionsandsupporthave

beeninvaluable.

IwouldalsoliketothankthefacultyandstaffoftheVirginia-MarylandCollegeofVeterinary

MedicineandtheBMVSprogram,includingDr.AnsarAhmed,Dr.RogerAvery,BeckyJones,

CindyBoothandSusanRosebrough.Thankyouforprovidingthefoundationforsucha

wonderfulgraduateprogram.

IwouldliketothankourcollaboratorsatCincinnatiChildren’sHospitalMedicalCenterand

PATH,includingXiJiang,MingTan,MingXia,WeimingZhongandRobertChoy.These

individualsgraciouslyprovidedtheirtime,scientificexpertiseandreagents.Iwouldalsoliketo

acknowledgethefollowingfundingsourcesfortheirsupport:NationalInstituteofAllergyand

InfectiousDiseases,NIH(R01AI089634-01);PATH;NationalCenterofComplementaryand

AlternativeMedicine,NIH(R01AT004789).

Myparents,sisterandfriendshavebeenamazingintheirsupportandformaintainingmy

humorandsanity.Specialthanksarereservedformylovingandimmenselypatienthusband,

viii

Matt.He’slistenedtomevent,helpedmetroubleshootvariouslabequipment-related

emergencies,fedmechocolateandallowedmetoadoptsixanimalsinthespanof8years.

There’snooneelseI’dwanttoshareabathroomwithwhilesufferingfromgastroenteritis.

ix

ATTRIBUTIONS

Severalcolleaguescontributedtotheprojects,research,writing,andeditingofeachchapterof

thisdissertation.

Chapters2through4:

LijuanYuan,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)iscurrentlyan

AssociateProfessoratVMCVMandisthecorrespondingauthoronthesemanuscripts.She

aidedinprojectdevelopment,writingandeditingofthemanuscripts.

GuohuaLi,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthe

manuscriptsandiscurrentlyaResearchScientistatCincinnatiChildren’sHospitalMedical

Center.Hecontributedtoprojectdevelopment,samplecollection,sampleprocessing,flow

cytometricanalysis,datainterpretationandeditingofthemanuscripts.

KeWen,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthe

manuscriptsandiscurrentlyaPost-doctoralResearchAssociateatTexasTechUniversityHealth

SciencesCenter.Hecontributedtosamplecollection,sampleprocessingandeditingofthe

manuscripts.

XingdongYang,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoron

themanuscriptsandiscurrentlyaPost-doctoralResearchAssociateattheNationalInstitutesof

Health.Hecontributedtosamplecollectionandeditingofthemanuscripts.

Chapters2and4:

JacobKocher,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoron

thesemanuscriptsandiscurrentlyaPost-doctoralResearchAssociateattheUniversityofNorth

CarolinaatChapelHill.Hecontributedtoprojectdevelopment,samplecollectionandeditingof

themanuscripts.

FangningLiu,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoron

thesemanuscriptsandcontributedtosamplecollectionandeditingofthemanuscripts.

Chapters3and4:

MariahWeiss(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthese

manuscriptsandiscurrentlytheProjectandLabManagerwiththeTeaching&ResearchAnimal

CareSupportServiceatVMCVM.Shecontributedtosamplecollectionandeditingofthe

manuscripts.

Chapter2:

x

YanruLi,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthis

manuscriptandcontributedtosamplecollection,histopathologicalreviewandeditingofthe

manuscript.

TanyaLeRoith,DVM,Ph.D(DepartmentofBiomedicalSciencesandPathobiology)isaco-author

onthismanuscriptandiscurrentlyaClinicalAssociateProfessoratVMCVM.Shecontributedto

samplecollection,histopathologicalreviewandeditingofthemanuscript.

MingTan,Ph.D.(CincinnatiChildren’sHospitalMedicalcenter)isaco-authoronthis

manuscriptandprovidedmaterialsandcontributedtoeditingofthemanuscript.

MingXia,Ph.D.(CincinnatiChildren’sHospitalMedicalcenter)isaco-authoronthismanuscript

andprovidedmaterialsandcontributedtoeditingofthemanuscript.

WeimingZhong(CincinnatiChildren’sHospitalMedicalcenter)isaco-authoronthismanuscript

andprovidedmaterialsandcontributedtoeditingofthemanuscript.

XiJiang,Ph.D.(CincinnatiChildren’sHospitalMedicalcenter)isaco-authoronthismanuscript

andaidedinprojectdevelopment,providingmaterialsandeditingofthemanuscript.

Chapter3:

InyoungKim,Ph.D.(DepartmentofStatistics)isaco-authoronthismanuscriptandiscurrently

anAssociateProfessoratVirginiaTech.Shecontributedtostatisticalanalysisandeditingofthe

manuscript.

EricaTwitchell,DVM(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoron

thismanuscriptandiscurrentlyaGraduateResearchAssociateatVirginiaTech.She

contributedtosamplecollectionandeditingofthemanuscripts.

ShaohuaLei(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthis

manuscriptandiscurrentlyaGraduateResearchAssociateatVirginiaTech.Hecontributedto

samplecollectionandeditingofthemanuscripts.

AshwinRamesh(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthis

manuscriptandiscurrentlyaGraduateResearchAssociateatVirginiaTech.Hecontributedto

samplecollectionandeditingofthemanuscripts.

SherrieClark-Deener,DVM,Ph.D.(DepartmentofLargeAnimalClinicalSciences)isaco-author

onthismanuscriptandiscurrentlyanAssociateProfessoratVMCVM.Shecontributedwith

animalderivationandeditingofthemanuscript.

xi

RobertK.M.Choy,Ph.D.(PATH)isaco-authoronthismanuscript.Hecontributedwithproject

development,datainterpretationandeditingofthemanuscript.

Chapter4:

HaifengWang,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoron

thismanuscriptandcontributedtosamplecollectionandeditingofthemanuscript.

KevinPelzer,DVM,MPVM(DepartmentofLargeAnimalClinicalSciences)isaco-authoronthis

manuscriptandiscurrentlyaProfessoratVMCVM.Hecontributedwithanimalderivationand

editingofthemanuscript.

xii

TABLEOFCONTENTSAbstract...................................................................................................................................................iiDedication...............................................................................................................................................vAcknowledgements.................................................................................................................................viAttributions.............................................................................................................................................ixListofTables............................................................................................................................................xviListofFigures..........................................................................................................................................xvii

Chapter1:Literaturereview

Acuteviralgastroenteritisduetonorovirusandrotavirusinfections:Studiesofpathogenesis,

innateimmunity,andtherapeuticsinagnotobioticpigmodel

1.1INTRODUCTION:ACUTEVIRALGASTROENTERITIS 2

1.2NOROVIRUS(NOV) 2

1.2.1CLINICALSIGNSOFNOVINFECTION 31.2.2PATHOGENESISOFNOVINFECTION 5

1.3ROTAVIRUS(RV) 11

1.3.1CLINICALSIGNSOFRVINFECTION 111.3.2PATHOGENESISOFRVINFECTION 13

1.4GNOTOBIOTIC(GN)PIGMODELSFORACUTEVIRALGASTROENTERITIS 17

1.4.1GENERALOVERVIEW 171.4.2GNPIGMODELOFHUNOVINFECTION 191.4.3GNPIGMODELOFHRVINFECTION 21

1.5ROLEOFDENDRITICCELLSININNATEIMMUNITYAGAINSTACUTEVIRALGASTROENTERITIS 22

1.5.1GENERALOVERVIEW 221.5.2DENDRITICCELLRESPONSESTONOVINFECTION 241.5.3DENDRITICCELLRESPONSESTORVINFECTION 24

1.6PREVENTIONOFACUTEVIRALGASTROENTERITIS 25

1.6.1DISINFECTIONMETHODS 251.6.2NOVVACCINEDEVELOPMENT 261.6.3RVVACCINEDEVELOPMENT 29

1.7TREATMENTOFACUTEVIRALGASTROENTERITIS 33

1.7.1ORALREHYDRATIONSOLUTIONS 331.7.2GENERALANTI-DIARRHEALANDANTI-VIRALAGENTS 341.7.3RV-SPECIFICADJUNCTIVETREATMENTS 361.7.4NOV-SPECIFICADJUNCTIVETREATMENTS 36

xiii

1.8CONCLUDINGREMARKS 37

1.9REFERENCES 39

Chapter2

MedianinfectiousdoseofhumannorovirusGII.4ingnotobioticpigsisdecreasedbysimvastatin

treatmentandincreasedbyage

2.1ABSTRACT 69

2.2INTRODUCTION 70

2.3MATERIALSANDMETHODS 71

2.3.1VIRUS 712.3.2INOCULATIONOFGNPIGS 722.3.3SIMVASTATIN 722.3.4BLOODTYPINGOFGNPIGSBYPCRANDIMMUNOFLUORESCENCEASSAY 732.3.5ASSESSMENTOFNOVDIARRHEA 742.3.6DETECTIONOFNOVSHEDDINGBYRT-PCR 742.3.7DETERMINATIONOFNOVSHEDDINGTITERSBYTAQMANREAL-TIMERT-PCR 752.3.8DETERMINATIONOFID50FORNOVGII.4VARIANT092895 762.3.9DETECTIONOFNOVANTIGENININTESTINALTISSUESBYINDIRECTIMMUNOFLUORESCENCE 762.3.10EVALUATIONOFCYTOPATHOLOGICALCHANGESBYTRANSMISSIONELECTRONMICROSCOPY 772.3.11STATISTICALANALYSIS 77

2.4RESULTS 77

2.4.1SIMVASTATINFEEDINGLOWERSTHESERUMCHOLESTEROLLEVELS 772.4.2OBSERVATIONOFDIARRHEAINBOTHAGEGROUPSOFGNPIGSFOLLOWINGINOCULATIONWITHNOV 792.4.3GNPIGSSHEDVIRUSINFECESASDETERMINEDBYRT-PCRANDQUANTIFIEDBYTAQMANREAL-TIMERT-PCR 792.4.4VIRUSAMPLIFICATIONINGNPIGS 842.4.5SIMVASTATININCREASESSUSCEPTIBILITYOFNOVINFECTIONANDTHEINCIDENCEOFDIARRHEAINGNPIGS 842.4.6DETERMINATIONOFID50FORTHEGII.4092895VARIANTINNEONATALANDOLDERPIGS,ANDLOWERINGOFTHEID50INTHEOLDERAGEGROUPBYSIMVASTATIN 862.4.7DETECTIONOFNOVANTIGENINTHEDUODENALVILLIOFINFECTEDPIGS 862.4.8CYTOPATHOLOGICALCHANGESINTHEDUODENUMANDJEJUNUMININFECTEDPIGS 88

2.5DISCUSSION 88

2.6REFERENCES 96

xiv

Chapter3

Effectsofracecadotrilonweightlossanddiarrheaduetohumanrotavirusinneonatal

gnotobioticpigs(Susscrofadomesticus)

3.1ABSTRACT 101

3.2INTRODUCTION 101

3.3MATERIALSANDMETHODS 103

3.3.1ANIMALS,DIETS,HOUSINGCONDITIONS,ANDHEALTHMONITORING 1033.3.2STERILITY 1053.3.3VIRUS 1053.3.4DRUGTREATMENTGROUPSANDINOCULATIONOFGNOTOBIOTICPIGS 1053.3.5SAMPLECOLLECTION 1063.3.6ASSESSMENTOFROTAVIRUSDIARRHEAANDVIRUSSHEDDING 1073.3.7STATISTICALANALYSIS 109

3.4RESULTS 110

3.4.1DETECTIONOFTHIORPHANINPLASMAAFTERRACECADOTRILADMINISTRATION 1103.4.2EFFECTSOFRACECADOTRILONTHEDURATIONANDSEVERITYOFDIARRHEAINGNOTOBIOTICPIGSINFECTEDWITHHRV 1103.4.3EFFICACYOFRACECADOTRILINREDUCINGDEHYDRATIONINGNOTOBIOTICPIGSINFECTEDWITHHRV 113

3.5DISCUSSION 118

3.6REFERENCES 124

Chapter4

DivergentimpactsofLactobacillusacidophilusdosageonintestinalandsystemicdendriticcellsingnotobioticpigsinfectedwithhumanrotavirus

4.1ABSTRACT 128

4.2INTRODUCTION 129

4.3MATERIALSANDMETHODS 131

4.3.1VIRUSANDPROBIOTICS 1314.3.2TREATMENTGROUPSANDINOCULATIONOFGNPIGS 1314.3.3ASSESSMENTOFSTERILITY 1334.3.4ASSESSMENTOFLACOLONIZATION 1334.3.5ASSESSMENTOFROTAVIRUSDIARRHEAANDVIRUSSHEDDING 1344.3.6ASSESSMENTOFFREQUENCIESOFCYTOKINEANDTLREXPRESSIONINPDCSANDCDCSBYFLOWCYTOMETRY 1344.3.7STATISTICALANALYSIS 138

xv

4.4RESULTS 138

4.4.1LACOLONIZATION 1384.4.2ROTAVIRUSSHEDDINGANDDIARRHEA 1404.4.3HIGHEROVERALLTLR2RESPONSESWEREOBSERVEDINHILAPIGS,WHILELOLAINDUCEDHIGHERTLR3ANDTLR9RESPONSESINILEUMANDSPLEEN 1404.4.4HIGHERIL-6RESPONSESWEREOBSERVEDINLOLAPIGSPRE-CHALLENGEANDINHILAPIGSPOST-CHALLENGE,WHILEIL-10WASMORECONSISTENTLYUPREGULATEDINDCSOFTHEHILAGROUP 1454.4.5INCREASEDPRODUCTIONOFIFN-ΑBYDCPOPULATIONSOCCURREDMOREFREQUENTLYINHILAPIGSPOST-CHALLENGE,WHILETNF-ΑPRODUCTIONWASOBSERVEDINBOTHLOLAANDHILAPIGSPRE-ANDPOST-CHALLENGE 148

4.5DISCUSSION 148

4.6REFERENCES 158

Chapter5

Generalconclusions

5.1GENERALCONCLUSIONS 163

5.2FUTUREDIRECTIONS 166

5.3REFERENCES 168

xvi

LISTOFTABLES

Chapter2

MedianInfectiousdoseofhumannorovirusGII.4ingnotobioticpigsisdecreasedbysimvastatintreatmentandincreasedbyageTable1:Cholesterollevels(mg/dL)after11daysofsimvastatinfeeding(8mg/day)............78 Table2:SummaryofdiarrheaandnorovirussheddinginGnpigschallengedwithGII.42006b

variant(inoculum092895)at4-5daysofage.............................................................80Table3:SummaryofnorovirussheddinginGnpigschallengedwithGII.42006bvariant

(inoculum092895)at33-34daysofagewithandwithoutsimvastatinfeeding........81Table4:VirusamplificationinolderGnpigswithandwithoutsimvastatinfeeding.............85

Chapter3

EffectsofracecadotrilonweightlossanddiarrheaduetohumanrotavirusinneonatalgnotobioticpigsTable1:PlasmathiorphanconcentrationsinGnpigstreatedwithracecadotril(dose80mg/kg)

...................................................................................................................................111Table2:Percentageofdayswithdiarrhea(relativetoPID1;mean±SEM)ingnotobioticpigs

infectedwithhumanrotavirusandtreatedwithPBS(mock),racecadotril,or

chlorpromazine...........................................................................................................112Table3:Summaryofdiarrheadataanalysisusinglinearmixed-effectsmodel......................112Table4:Bodyweights(%relativetoPID0,mean±SEM)ingnotobioticpigsinfectedwithhuman

rotavirusandtreatedwithPBS(mock),racecadotril,orchlorpromazine...................117

Chapter4

DivergentimpactsofLactobacillusacidophilusdosageonintestinalandsystemicdendriticcellsingnotobioticpigsTable1:ProbioticdoseandfeedingregimensandVirHRVchallenge.....................................132Table2:ProtectionagainstrotavirusdiarrheaafterVirHRVchallenge...................................141

xvii

LISTOFFIGURES

Chapter2

MedianInfectiousdoseofhumannorovirusGII.4ingnotobioticpigsisdecreasedbysimvastatintreatmentandincreasedbyageFigure1:RT-PCRtodetectNoVsheddinginlargeintestinalcontents(LIC)...........................83Figure2:DetectionofNoVantigeninintestinaltissuebyimmunohistochemistry................87Figure3:CytopathologicalchangesintheduodenumandjejunumofGnpigsinfectedwithNoV

092895......................................................................................................................89

Chapter3

EffectsofracecadotrilonweightlossanddiarrheaduetohumanrotavirusinneonatalgnotobioticpigsFigure1:MeandiarrheascoreafterinfectionwithhumanrotavirusandtreatmentwithPBS

(mock),racecadotril,orchlorpromazine.....................................................................114Figure2:Changesinbodyweight(kg)afterinfectionwithhumanrotavirusandtreatmentwith

PBS(mock),racecadotril,orchlorpromazine..............................................................115

Chapter4

DivergentimpactsofLactobacillusacidophilusdosageonintestinalandsystemicdendriticcellsingnotobioticpigsFigure1:RepresentativedotplotsoffrequenciesofTLRs,cytokines,CD80/86andMHCIIamong

SWC3a+CD4+(pDC)andSWC3a+CD11R1+(cDC)cells................................................135Figure2:LAcolonizationinLoLAandHiLAgroups..................................................................139Figure3:TLRexpressionprofileofDCsfromileum,spleenandPBLofGnpigspre-(PCD0)and

post-challenge(PCD7)withVirHRV............................................................................142Figure4:IL-6andIL-10expressionprofileofDCsfromileum,spleenandPBLofGnpigspre-

(PCD0)andpost-challenge(PCD7)withVirHRV........................................................146Figure5:IFN-aandTNF-aexpressionprofileofDCsfromileum,spleenandPBLofGnpigspre-

(PCD0)andpost-challenge(PCD7)withVirHRV........................................................149

1

Chapter1

Acuteviralgastroenteritisduetonorovirusandrotavirusinfections:Studiesof

pathogenesis,innateimmunity,andtherapeuticsinagnotobioticpigmodel

T.Castellucci1

1DepartmentofBiomedicalSciencesandPathobiology,Virginia-MarylandRegionalCollegeof

VeterinaryMedicine,VirginiaTech,IntegratedLifeScienceBuilding,1981KraftDr,Blacksburg,

VA24061-0913,USA.

2

1.1Introduction:Acuteviralgastroenteritis

Acutegastroenteritis(AGE)presentsasasuddenonsetofdiarrheawithorwithout

vomiting,andcanincludesymptomsofnauseaandabdominalpainaswell(1).Whiletypically

self-limitinginduration,casesofAGEstillhaveasignificantimpactonhealthcareand

economiccosts.Furthermore,diarrheaiscurrentlythefourthleadingcauseofmortalityamong

allagesglobally,causingapproximately1.3milliondeathsin2015(2,3).Youngchildrenare

especiallyaffectedbyinfectiouscausesofAGE;diarrheaisthesecondmostcommoncauseof

deathduetocommunicablediseaseworldwideinindividualsunder5yearsofage,withan

estimated578,000deathsoccurringin2013(4,5).Amonginfectiouscausesofdiarrhea,

norovirus(NoV)androtavirus(RV)arethepredominantviralagents.NoVsareimplicatedinthe

majorityofAGEcasesinallagegroupsworldwide,whileRVsarespecificallythemostcommon

causeinchildrenunder5yearsofage(6,7).

1.2Norovirus(NoV)

NoVsbelongtotheCaliciviridaefamilyandaresmall,non-enveloped,positive-stranded

RNAviruses(8-12).Theprototypeofthevirus,Norwalkvirus(GI.1),wasdiscoveredfollowing

anoutbreakofgastroenteritisinNorwalk,Ohioin1968(13,14).Currently,sixgenogroupsand

>40genotypeshavebeenidentified,withGII.4strainsbeingresponsibleforthemajorityof

humanNoV(HuNoV)infections(9).MorerecentoutbreakshavealsoincludedGII.17strains

(15-18).AsidefromGII,othergenogroupsthatareknowntoinfecthumansincludeGIandGIV

(12).OtheranimalNoVsincludethosethatinfectmice(GV),pigs(GII),cattle(GIII)anddogs

(GIV,GVI)(19,20).

3

1.2.1ClinicalsignsofNoVinfection

TransmissionoftheNoVprimarilyoccursthroughafecal-oralroutefromcontaminated

food,waterandpersons(21,22).However,NoVinvomitusmaybeacontributingfactorin

spreadingofthevirusaswell,eitherbyaerosolizationorcontaminationoffomites(23,24).

Additionally,detectionofNoVinnasopharyngealswabsraisesthequestionofwhether

respiratorytransmissionispossible(25).Overall,theinfectiousdosehasbeenreportedtobe

fairlylowat10-100viralparticles(26).

Afterashortincubationperiodof12-48hours(8,9,11,21,27),symptomsmanifestas

vomitingandnon-bloodydiarrheawithsomeindividualsalsoexperiencing,nausea,abdominal

pain,mildfeverandmyalgia(8-10,12,21,27,28).Mostcasesofnorovirusinhealthy,

immunocompetentindividualsareself-limitingandsymptomsceaseafteratypicalperiodof2-3

days,butcanlastforupto6days(8-11).Virussheddinginfecescanstartbeforeclinical

presentation(asearlyas18hoursafterinfection)andtendstopersistformuchlonger,though

peaksheddinghasbeenreportedatfourdayspost-infection(28).Ingeneral,virussheddingcan

continueforthreetoeightweeks(8,11,27,28).Asymptomaticsheddingisalsopossibleand

hasbeenreportedinapproximately1/3ofcases(8,29).Inbothsymptomaticand

asymptomaticinfections,highsheddingtitersbetween105-10

11viralcopiespergramoffeces

canbeexpected(29).Inarecentstudy,NoVtitersinvomituswerereportedas8.0x105and3.9

x104viralcopiespermilliliterforGIandGIIviruses,respectively(24).Thecombinationof

sheddinginhightiters,oflongdurationandinasymptomaticindividuals,alongwithlow

infectiousdosefacilitateseaseofNoVtransmission.

4

WhileNoVsarecapableofinfectingallagegroups,moreseverepresentationsandeven

mortalitiescanoccurintheyoung(65yearsofage)or

immunocompromised(9,30,31).Aswithmostcausesofdiarrhea,severedehydrationisa

commoncomplicatingfactor.IntheUS,childrenunder5yearsofagehavehigherratesof

clinicalvisitspertainingtoNoV-relatedillnesses(32).Longerdurationofsymptoms(7versus3.5

days)andgreaterseverityofdiarrheahasbeenreportedinchildren

5

(44,54).Inadditiontoseveredehydration,complicationsarisingfromchronicdiarrheacan

includedisruptionoftheintestinalbarrier,malnutrition,acutekidneyinjuryand

transplant/graftfailure(48,55).Anincreasedoccurrenceofpneumatosisintestinalis,orgas

withintheintestinalwall,hasbeenreportedaswell.Fouroutoffiveimmunocompromised

individualswithpneumatosisintestinaliswereNoV-positiveinoneretrospectivestudy(56).

Virussheddingisprolongedcomparedtohealthy,immunocompetentpatientsandhas

continuedforoverayearincertaincases(44,57).Suchlongperiodsofsheddingcanpossibly

actasareservoirforinfectionandallowfortheemergenceofnewvariants(55,58).

NoVinfectionshavealsobeenrarelyassociatedwithvariousotherunique

presentations.Asidefromnecrotizingenterocolitisandneurologicalmanifestationsinyoung

children,theseincludehemolytic-uremicsyndrome(59),ischemiccolitis(60),flare-upsof

inflammatoryboweldisease(61),post-infectiousirritablebowelsyndrome(62)and

spontaneousbowelperforation(63).ViremiaisnotacommonoccurrencewithNoVinfections

andmayexplainthepredominanceofintestinaldisease.Viremiahasnotbeenreportedin

healthyadults;onlyinchildren(64),immunocompromisedindividuals(65)andgnotobioticpigs

(66,67).

1.2.2PathogenesisofNoVinfection

Historically,thelackofanefficientcellculturesystemandsmallanimalmodelfor

humanNoVs(HuNoVs)haslimitedtheunderstandingofpathogenesis.Themostwell-studied

aspectislikelythebindingofNoVstohisto-bloodgroupantigens(HBGAs).HBGAsarecomplex

carbohydratesthatcanbefoundonerythrocytesandvariousepithelialtissues,aswellas

6

secretedfreelyinbiologicalfluidsofsecretors(seebelow)(68).Thespecificphenotypic

expressionofHBGAsinanindividualisdependentondifferentfunctioningglycosyltransferases.

Inhumans,ABO,secretorandLewisfamiliesareinvolvedinthebiosynthesispathwaysthat

producethemajorHBGAs(69).TheFUT1geneencodesana1,2fucosyltransferasethat

synthesizesH(O)antigenonerythrocytes,whiletheFUT2geneisresponsibleforthea1,2

fucosyltransferasethatresultsinHantigenexpressiononmucosalepithelialsurfacesandas

freeoligosaccharidesinsaliva,milkandintestinalcontents(70,71).Individualswithatleastone

functionalFUT2allelearedesignatedas‘secretors’.AandBantigensareproducedthrough

subsequentmodificationoftheHantigenbyAandBglycosyltransferases(ABOgene),

respectively(71).FurtheradditionofcarbohydratemoietiesbytheFUT3gene-encodeda1,3/4

fucosyltransferaseleadstoexpressionofLewisantigensinsecretors(Leb,Le

y)andnon-

secretors(Lea,Le

x)(68,69,72).Altogether,individualscanbeclassifiedbasedontheirABO

blood-type,aswellaswhethertheyaresecretorsornon-secretorsandLewis-positiveor

negative.

Basedontheknowledgethatrabbithemorrhagicdiseasevirus,whichisacalicivirus,

bindstoaHGBA(73,74),itwaspostulatedthatNoVsmayusesimilarantigensascellular

receptors.Indeed,studiesdemonstratedHuNoVvirus-likeparticles(VLPs)hemagglutinating

andbindingtoHBGASinsalivaandonintestinalepithelialcellsofsecretors(75-78).CanineNoV

VLPshavealsobeenshowntobindHBGAsinvitro(20).TheuseofHBGAsascellularligandsfor

NoVbindingwasfirstsupportedinvivobyastudyreportingassociationofNorwalkvirus(GI.1)

infectioninhumanswithABOhisto-bloodtype.Thestudyrevealedanincreasedriskof

infectioninthosewithOblood-typecomparedtoBblood-type(79).Furthercharacterizationof

7

susceptibilityconfirmedthat,inadditiontoblood-type,secretorstatuswasanequally

importantdeterminantofsymptomaticinfectionsasnon-secretorswereresistanttoinfection

withaGIINoVinonestudy(80).Manyotherinvestigationssincethenhavealsosupported

increasedsusceptibilitytoNoVinfectioninpersonswhoaresecretors(17,72,81-86).

Resistanceinsomesecretorindividuals,aswellassymptomaticinfectioninnon-secretors(87-

89),hasbeenexplainedbyrecognitionofseveralstrain-specificbindingprofilestoABOand

Lewisantigensthroughhumanstudies,invitroassaysandstructuralanalysisoftheNoVcapsid

protein(71,90-94).Ingeneral,twomajorclassificationsofHBGAbindinghavebeenproposed

andincludethosestrainsthatbindA/BantigensandthosethatbindLewisantigens(90).

ThroughX-raycrystallography,ithasbeendeterminedthattheP2subdomainofthe

NoVcapsidprotein(VP1)istheprimarysiteofbindinginteractionswithHBGAs.TheNoVcapsid

proteiniscomprisedofshell(S)andprotruding(P)domains,withthePdomainbeingfurther

subdividedintoP1andP2.TheP2subdomainisfoundontheoutermostsurfaceofVP1,making

itanidealtargetasareceptorinterface.IthasbeensuggestedthatthepolymorphismofHBGA

phenotypesinthehumanpopulationandmutationsoftheP2domaininfluenceNoVevolution

(95-97)

OtherpotentialreceptorcandidatesforHuNoVinfectionhaveincludedheparinsulfate

(98),sialicacid(99)andb-galactosylceramide(100).FormurineNoVs(MNV),inadditionto

sialicacid(101),glycolipidandglycoproteinreceptors(102),recentevidenceforproteinaceous

cellreceptors,CD300lfandCD300ld,hasbeendemonstrated(103,104).

ThecellulartropismforNoVsremainsincompletelyunderstood,withbothintestinal

epithelialcells(IECs)orenterocytesandimmunecellsbeingviablecandidates(105-109).

8

Initially,basedonthemultitudeofevidenceforHBGAsascellularligandsandthenatureof

NoVsasentericviruses,itwasspeculatedthatIECsexpressingHBGAsweretheprimarytargets

ofNoVinfection.DetectionofNoVantigeninIECshasbeendemonstratedinchronically

infectedhumantransplantpatients(110),Gnpigs(66),Gncalves(111)andSTAT1-/-mice(112),

indicatingthatthesecellscanatleastbeinfectedinimmunocompromisedindividuals.Yet,until

recently,multipleattemptstocultureHuNoVinepithelialcelllineshavefailed(113-116),even

withthepresenceofHBGAs(117).SuccessfulHuNoVreplicationhasnowbeenestablished

utilizingstemcell-derivedhumanintestinalenteroidswithandwithouttheinclusionofbileacid

supplementation(118),thoughroutineuseofthismodelremainstobeimplemented.Still,the

enteroidcellculturesystemprovidesstrongevidenceforthecontinuednotionthat,similarto

otherentericviruses,NoVscaninfectenterocytes.

Meanwhile,MNVshavebeenreadilyculturedindendriticcells,macrophagesandBcells

(119,120).Invivo,MNVantigenhasalsobeendetectedintheaforementionedimmunecells

(112,119-121)andintestinalviraltiterswerelowerinBcell-deficientmice(122),further

suggestingtheimportanceofimmunecellsinthepathogenesisofMNVinfection.Ithasbeen

proposedthat,inordertotraversetheintestinalepithelialbarrierandaccessimmunecells,

virusistrancytosedbymicrofold(M)cells(106,107,123).Mcellsarespecializedintestinalcells

thatareheavilyinvolvedinimmunosurveillancethroughtheengulfmentofmicroscopic

materialfromthelumen,whichalsomakesthemakeyentryportalforpathogens(124).In

micethatweredepletedofMcells,reducedviraltiterswereobservedcomparedtocontrols

(125).

EvidenceofHuNoVsinfectingimmunecellsalsoexists,thoughismoreconflicting.Inthe

9

previouslymentionedstudydemonstratingviralantigendetectioninenterocytesof

immunocompromisedhumantransplantpatients(110),antigenwasalsodetectedin

macrophages,dendriticcellsandTcellsinintestinalbiopsies.Yet,Norwalkvirus(GI.1)was

incapableofreplicatinginhumanmonocyte-derivedmacrophagesanddendriticcells(126).In

termsofBcellinfection,onlytheGII.4SydneystrainamongHuNoVshasbeenshownto

replicateinBcellsinvitro(120,127).Thesituationinvivoislikelyevenmorecomplicated,as

similarincidenceofNoVinfectionoccurredinBcellcompetent(60%)anddeficient(63%)

childrenwithseverecombinedimmunodeficiency(SCID)(128).However,theviraltiterinBcell

deficientindividuals,whilestillonthescaleofmillionsofgenomiccopiespermilliliter,was

significantlyreducedcomparedtothosepatientswithBcells.Incontrast,infectionwithaGII.4

HuNoVinRAG2/IL2RGdeficientGnpigs,whichlackBcells,Tcellsandnaturalkillercells,

provedtobeofhighertiterandlongerdurationversuswildtypecontrols(67).Aswithprevious

studiesinGnpigs,detectionofviralantigenoccurredinenteroctyesofthesmallintestine(66,

129).Overall,itappearsthatregulationofNoVcelltropismiscomplex,possiblydependingon

virusstrain,hostspecies,immunestatusandotherpotentiallyundeterminedinnateor

environmentalfactors.

Interestingly,theabilityofNoVtoreplicateintheBJABhumanBcelllinewasdependent

onthepresenceofHBGA-expressingentericbacteriaasuseoffilteredstoolsamplesforculture

resultedindecreasedtiters(120).AdditionofHtype-positiveEnterobactercloacaetocultures

subsequentlyreestablishedNoVinfectivity.Inthesamestudy,antibiotictreatmentofmice

priortoMNVinfectionsignificantlyreducedviraltiters.Anotherrecentstudyalsorecognized

GII.4andGI.6NoVbindingtoentericbacteria,againpossiblyduetointeractionswithHBGA-like

10

moieties(130).Thus,HBGAsinthecontextofintestinalmicrobiotamaybeinvolvedin

regulatingNoVpathogenesisaswell.However,theexactinteractionbetweenvirusand

bacteriaisstilluncertain.ArecentstudyinGnpigsdemonstratedreducedviraltitersinpigs

colonizedwithE.cloacaeandsubsequentlyinfectedwithaGII.4humannorovirus(131).The

authorssuggestedthatHBGA-expressingbacteriamayfunctiontoactasdecoyreceptors,thus

blockingratherthanenablingbindingtoviralreceptors.Furtherstudieswillneedtobe

conductedtoclarifytheroleofHBGA-expressingbacteriainNoVinfection.

AnotherunansweredquestionregardingNoVpathogenesisinvolvesreconcilingthe

relativelackofintestinalpathologyandinflammationwiththeseverityofdiarrheathatcan

occur(108,109).Moststudiesinbothhumans(132-134)andanimalmodels(66,111,135)

reportfairlymilddisruptionoftheintestinalepithelium,withbluntingofvillibeingmost

common.Otherchangesobservedincludemicrovillidestruction,mitochondrialdistension,

cytoplasmicvacuolization,intercellularedemaandcellapoptosis(129).Therefore,itis

reasonabletoassumethatothermechanismsofdiarrheamaybeinvolved.Onestudyofhuman

duodenalbiopsiesreportedadecreaseintransepithelialresistancefollowingNoVinfection,

likelysecondarytoareductionintightjunctionproteinsoccludin,claudin-4andclaudin-5(133).

SuchfunctionalimpairmentsmaycontributemoretoNoVdiarrheathanstructuraldamageof

enterocytes.FurtherstudiesareneededtounderstandthepathophysiologyofNoVdiarrhea.

11

1.3Rotavirus(RV)

RVsbelongtotheReoviridaefamilyandarenon-enveloped,segmented,double-

strandedRNAviruses(136,137).BasedonthestructuralproteinVP6,RVsareclassifiedintoat

least8differentgroups,denotedwithlettersA-H(136,138).RVA,RVBandRVCcauseinfection

inhumansandcertainanimals,whileRVD-RVHareexclusivelyassociatedwithanimal

infections.Recently,newcanineandbatspeciesofRVhavebeensuggestedtocompriseRVI

andRVJgroups,respectively(139,140).Withingroups,RVsarefurtherclassifiedbasedon

capsidproteinsVP7(glycoprotein-Gtype)andVP4(proteasesensitiveprotein-Ptype)(137).

AmongtheRVAgroup,whichisthemostcommonlyinvolvedinhumaninfections,G1P[8],

G2P[4],G3P[8],G4P[8],G9P[8]andG12P[8]arethemostprevalentstrains(141).

1.3.1ClinicalsignsofRVinfection

RVinfectionismostprevalentininfantsandyoungchildrenandischaracterizedbynon-

specificsymptomsofacutegastroenteritis.Vomitingfollowedbywatery,non-bloodydiarrhea

typicallyoccurswithin48-72hoursofinfectionviathefecal-oralrouteandcanbeaccompanied

byfever,abdominalcramps,nauseaandheadaches(136,142-146).Severityofsymptomscan

varyfrommildtodebilitatinganddiarrheacanpersistforuptooneweek(146,147).

Duetotheonsetofbothvomitinganddiarrhea,deathcanoccursubsequenttoextreme

dehydration,electrolyteimbalancesandcardiovascularfailureifmedicalinterventionisnot

available(136,142).DehydrationismorecommonwithRVinfectioncomparedtoother

bacterialorviralagentsofgastroenteritisinchildren(143).Casesthatrequirehospitalization

andeitheroralorintravenousrehydrationtherapyareintheminoritythough,andRVinfection

12

ismoreoftenself-limiting(147).Childrenbetweenthreemonthstothreeyearsofagearemore

likelytopresentwithsevereillness(145).Mortalityratesarehighestinunderdeveloped

regions,suchasinSaharanAfricaandsoutheastAsia,whereaccesstoappropriatemedicalcare

canbelimitedandmalnutritionorcoinfectionswithotherenteropathogensexacerbateclinical

symptoms(136,147-149).

Virussheddingoftenprecedesonsetofclinicalsymptomsandcanusuallybeexpected

tocontinueforupto10days,withanaverageoffourdaysduration(145,150).Peaklevelsof

sheddinginyoungchildrencanproduceasmuchas1010-11

viralparticlespergramoffeces

(142).Prolongedsheddinghasbeenreportedwithsevereinfections(150)andin

immunocompromisedindividuals(145,150,151).

AsymptomaticRVinfectionsareknowntooccur,bothinyoungchildrenandinadults.

Thoughsevereinfectionscanoccurinneonates,infectionsatthisagearetypically

asymptomaticduetomaternalantibodyprotectionviaplacentaltransferorbreastfeeding(145,

152,153).Furthermore,individualsencountermultipleRVinfectionswithageandhaveless

severesymptomswithsuccessiveinfectionsduetopartialimmunity(145,154-156).Fecalvirus

sheddingcontinuestooccurintheseinfections,butoftenatmuchlowertitersthanwith

symptomaticcases(142,157).

However,notallRVre-infectionsintheadultpopulationareasymptomatic(158-160).In

arecentstudyofadultsrequiringhospitalizationforacutegastroenteritisinDenmark,RVwas

determinedtobethesecond-mostcommon(25/265patientsor9.4%)enteropathogen

implicated(161).Similarly,intheUS,RVwasdetectedasthecauseofdiarrheain18%ofcases

(19/106)fromseveralemergencycenters(162).Althoughelderlyandimmunocompromised

13

individualsareunderstandablymorepredisposedtoinfection(163,164),outbreakshavebeen

reportedinyoungadultsaswell(159).Thesefindingssuggestthatrotavirusshouldstillbe

consideredasacausativeagentinpresentationsofacutegastroenteritisinadults.Symptomsin

adultsaresimilartothoseobservedinyoungchildren(142).

RVreplicatesinthesmallintestineandexpectedlyresultsinlocaldisease,butthereare

severalreportedinstancesofextraintestinalmanifestations.Seizuresarethemostcommonly

observedpresentationofsystemicinfection(165-167),withbothfebrileandafebrileseizures

beingpossibleandgenerallyoccurringwithoutlastingconsequences.Meningitisand

encephalitisareamongtheotherprevalentneurologicaldiseasesoccurringwithRVinfection

(165,168,169).Thoughevenrarer,tentativeassociationshavealsobeenproposedforcasesof

pneumonia(170),disseminatedintravascularcoagulation(171),exanthema(172),

haemophagocyticlymphohistiocytosis(173)andautoimmunediseasessuchdiabetesmellitus

amongothers(174).

Ingeneral,ithasbecomeacceptedthatantigenemiaandviremiafrequentlyoccurwith

acuteRVinfections,eitherwithorwithoutdiarrhea(168,175-178).Althoughusuallyclinically

insignificantinthecourseofillness,thisevidencemayexplaintheabilityofRVtocausedisease

outsideofthegastrointestinaltract.

1.3.2PathogenesisofRVinfection

Rotavirusiswidelyknowntoinfectandreplicateinmatureenterocytesofsmall

intestinalvilli(136,179,180).HowexactlyRVbindstothesecellsandinduceswaterydiarrhea

14

andvomitinghasbeenthesubjectofmultiplestudies.Fromtheseobservations,itisclearthat

thepathogenesisofRVinfectionismultifactorialandpossiblyincompletelyunderstood.

ThemaincapsidproteinsofRVareVP4andVP7,withVP7comprisingtheoutershellof

thecapsidandVP4formingthespikeprotein(137).Assuch,VP4,isprimarilyresponsibleforRV

attachmenttoenterocytes.VP4isenzymaticallycleavedbytrypsinintoVP5*andVP8*,with

VP5*formingthemainstemandVP8*formingtheheadofthespikethatdirectlyencounters

receptorsonsmallintestinalcells(136,181).Initially,sialicacidmoietieswerethoughttobe

involvedinRVbinding,sinceinteractionofsomeanimalRVstrainswithcellsurfacesis

interruptedbysialidasetreatment(182).However,itwaseventuallydeterminedthathuman

andmostanimalRVstrainsweresialidase-resistantandlikelynotdependentonsialicacidfor

cellularentry(181,183).Basedonmultidisciplinarystudiesinvolvingstructuralbiologyand

glycobiolity,itbecameevidentthatRVscanuseHBGAsascellularreceptors(181,184-188).

Asmentioned,theseligandshavepreviouslybeendeterminedtoberesponsibleforthe

cellularbindingofNoV(76,92).SinceNoVcaninfectenterocytes,itwasspeculatedthatRV

mayproceedsimilarly.Indeed,frominvitroassaysusinghumansaliva,milksynthetic

oligosaccharides,rotavirusVP8*wasdemonstratedtobindtoHBGAs(186).Currentknowledge

indicatesRVstrain-specificbindingtoA,Htype1and/orLewisbantigens(185,186).Most

studieshavealsodeterminedincreasedsusceptibilitytosymptomaticinfectioninsecretor-

positiveindividuals(82,189-191),thoughconflictingevidenceofinfectioninnon-secretors

doesexist(192).Yet,arecentstudydiddemonstrateincreasedRV-specificsalivaryIgAtitersin

secretors(193).

15

HBGAsarenottheonlyglycansthathavebeendemonstratedtointeractwithRVs.VP8*

alsointeractswithgangliosidesinastrain-dependentmanner(194).Asstatedpreviously,some

animalstrainsdobindsialicacidasconfirmedbytheirsensitivitytosialidases.Thesestrainsare

abletobindthesialicacidfoundasaterminalmoietyonthegangliosideGM3(195,196)andas

terminalandinternalmoietiesongangliosideGD1a(197).Incontrast,sialidase-insensitiveRVs,

whichincludemosthumanstrains,appeartointeractwiththegangliosideGM1athatlacksa

terminalsialicacid(197).Ofnote,GM1acontainsaninternalsialicacidmoietythatcaninteract

withVP8*anditisnotaffectedbysialidases.

InadditiontoVP8*,VP5*andVP7havealsobeenimplicatedfortheirroleincellular

binding.VP5*hasbeendemonstratedtobindintegrina2b1(198),whileVP7bindsintegrins

axb2andavb3(198,199).Heatshockcognateproteinhsc70(200)andtightjunctionprotein

JAM-A(201)arealsoinvolvedinRV-hostcellinteraction.Onceboundtoenterocytes,RVhas

beenshowntoenterthecellviaendocytosis,thoughtheexactmechanismappearstobestrain

dependent(202-204).

MalabsorptionisoneofthefundamentalmechanismsofRV-induceddiarrhea.Inthe

smallintestine,maturevillusenterocytesarenon-replicatingcellswithnumerousmicrovillithat

areinvolvedinbothactiveandpassiveabsorptionoffluidandelectrolytes,aswellasenzymatic

digestionoffood.Conversely,intestinalcryptcellsproliferateinself-renewalofintestinal

epitheliumandmaintainasecretorycapacity,releasingchlorideionsintotheintestinallumen

(179).WithRVinfection,structuraldamagetotheabsorptivematureenterocyteshaslongbeen

acceptedasamajorcontributortothepresenceofwaterydiarrhea.Earlyhistologicalstudyof

duodenumsamplesformchildrendiagnosedwithRVinfectionidentifiedbluntingofvilli,

16

vacuolizationofepithelialcells,increaseincuboidalversuscolumnarepithelium,intestinal

crypthyperplasiaandamildinfluxofinflammatorycells(136,179,205-208).Intestinalvilli

damagehasalsobeenreportedduetoischemiainmice,thoughtherelevancetohumansis

unclear(209).Combined,thereducedabsorptivecapacityduetoenterocytelossresultsin

increasedintestinalfluid.

However,theselesionsarenotalwaysconsistentlyobservedandoftendonot

necessarilycorrelatewithclinicalsymptoms(146,179).Assuch,amaldigestioncomponenthas

alsobeenimplicatedasacauseofRVdiarrhea.Adecreaseindigestivefunctionhasbeen

reportedsubsequenttoalossofdisaccharidasesthatarelocatedatvillitips(205,208,210,

211).Thisproducesanabundanceofcarbohydrates,whichactasosmoticallyactiveparticles

andfurtheraddstothewaterbeingdrawnintotheintestinallumen.

AuniqueaspectofRV-induceddiarrheaistheroleofnonstructuralprotein,NSP4,which

actsasanenterotoxin.NSP4isproducedbyinfectedenterocytes(146,179,208,212,213)and

peptidesaresecretedintotheintestinallumentofurtheraffectneighboringcells(214).NSP4

appearstohavemultipleeffectsonenterocytesthatcollectivelycontributetointestinalfluid

lossandsecretion.ItactstoreleaseintracellularCa2+fromcells,whichcaninducechangesin

cellulariontransportandpermeability(208,215,216).IthasbeensuggestedthattheCa2+

signaltransductionproducesamild,netchloridesecretionthroughacysticfibrosis

transmembraneconductanceregulator(CFTR)-independentprocess(208,212,217).NSP4has

alsobeendemonstratedtoinhibitthesodium-glucosetransporterSGLT1,reducingwater

reabsorption(218).Additionally,thereissomeevidencethatthissignalingcanmodifyactin

17

filamentsofmicrovillianddisruptintestinaltightjunctions,possiblyincreasingintestinal

permeability(216,219,220).

Furthermore,Ca2+signalingcaninciteproductionofcytokines,prostaglandinsand

reactiveoxygenspeciesthathavebeenpostulatedtostimulatetheentericnervoussystem

(ENS)(208).Locatedintheintestinalwall,theENSisconvenientlyabletosensechangesinthe

intestinalenvironmentandaltersecretionoffluidandelectrolytesinresponse(221).Serotonin

(5-hydroxytryptamineor5-HT)isanimportantneurotransmitteroftheENSandissecreted

exclusivelyinthesmallintestinebyenterochromaffin(EC)cells(207).Intermsof

gastrointestinaleffects,itisresponsibleforgutmotility,intestinalsecretion,bloodflowand

vagally-mediatedvomiting(207,222).Recently,RVhasdemonstratedcapabilityofinfectingEC

cellsandsubsequentNSP4releaseactivatesserotoninsynthesistoinfluencediarrhea,nausea

andvomiting(207,222).Additionally,serotoninantagonistshavebeenshowntoreduceRV

diarrheainmice,furtherconfirmingtheroleofENSinRVpathogenesis(207,223).Intermsof

ENSinvolvementinintestinalmotility,thedrugsloperamideandatropine,whichfunctionto

increaseintestinaltransittimeandallowforagreaterperiodoffluidabsorption,havebeen

showntoattenuatediarrheainducedbyRVinfection(224).

1.4Gnotobiotic(Gn)pigmodelsforacuteviralgastroenteritis

1.4.1Generaloverview

Domesticpigs(Susscrofadomesticus)arebeingincreasinglyutilizedasananimalmodel

inbiomedicalresearch.Whilepigs,evenminipigbreeds,representagreaterhusbandry

18

challengeduetotheirsize,thereareseveralfactorsthatmakethemmoreidealthanrodentor

otheranimalmodelsforstudyinghumandiseases.Inparticular,pigsareanatomically,

physiologicallyandimmunologicallyfairlyequivalenttohumans(225-227).Manystudiesthus

haveexploitedthesimilaritieswiththeintegumentary,cardiovascular,urinaryanddigestive

systems(225).Additionally,thereproductivecharacteristics(shortgenerationinterval,large

littersize),easeofadequatesamplecollectionandavailabilityofnumerousoutbredandinbred

linesarealsoadvantageousqualities(227).

Intermsofutilizingpigsasananimalmodelforstudyingentericviruses,the

resemblancewiththehumangastrointestinaltractandimmunesystemareespecially

appropriate.Therearegrossanatomicaldifferencesinthestomach,smallandlargeintestine

(225),butthedigestivephysiology,aswellassusceptibilityandresponsetodiseaseare

comparable(227-229).Furthermore,pigshavesimilarintestinalvillistructureandcell

classification(228)(230),enablingstudiesofdiseasepathophysiology.Forimmunological

evaluation,itisespeciallybeneficialthat>80%oftheimmuneparametersassessedare

analogoustohumans(231)andthatallimmunecellpopulationsaresimilarlypresentinpigs

(227).

Germ-freeanimalsarethosethataredeemedmicrobiologicallysterilebyspecific

testing,whilegnotobiotic(Gn)animalsarethosethatareassociatedwithknownand

identifiableorganisms(232).Technically,germ-freeanimalscanalsobecalledGnbecausethe

statusofmicroorganismsisknown.Gnmammalsarederivedbyaseptichysterectomyinto

sterileisolators,wheretheyarefedasterilized,commercialmilkdiet.Gnpigshavebeen

availableforbiomedicalresearchsincethe1960’sandpresentauniqueopportunitytoevaluate

19

host-pathogeninteractionsandrelatedimmunity.Unlikehumansandmice,pigshaveadiffuse

epitheliochorialplacentation,whichpreventsthetransferofmaternalantibodiesduring

intrauterinedevelopment(225,233).Neonatalpigsacquirepassiveimmunityviaingestionof

colostrumwithinthefirst36hoursafterbirth(234)toaidinprotectionagainstpathogenswhile

theirimmunesystemsarestilldeveloping.Thelackofmaternalantibodyinterference,

intestinalmicrobiotaandunknownpathogensallowsforspecificevaluationofprimaryimmune

responsestochosenentericmicrobesandvaccines.

Still,recentstudieshavehighlightedtheimportanceoftheintestinalmicrobiotain

influencingentericimmunityandresponsetoinfections(235,236).Tobetterelucidatethese

relationships,Gnpigshavebeensuccessfullytransplantedwithhumangutmicrobiota(235-

237).Additionally,withthedevelopmentoftheCRISPR/Cas9systemforgeneediting,the

manipulabilityoftheGnpigmodelhasbroadenedevenfurther(67).

Currently,inadditiontoseveralotherGnpigmodelsofentericpathogens(238-240),

modelsforHuNoVandHRVhavebeenwell-established.

1.4.2GnpigmodelofHuNoVinfection

Asstated,efficientcellculturesystemsforHuNoVarelacking,makinganimalmodels

evenmorevitalforstudyingpathogenesis,immunityandresponsetovaccinesand

therapeutics.AsidefromGnpigs,otheranimalmodelsincludemice(241),Gncalves(111),and

non-humanprimates(135,242).However,Gnpigshavebeensuggestedasamoresuitable

modelduetopresenceofclinicalsigns,i.e.diarrhea,comparedtomiceandnon-human

20

primates.Additionally,pigshavealsobeendemonstratedtohavesimilarintestinalHBGA

expressionandNoVbindingashumans(243).

TheGnpigmodelofHuNoVinfectionanddiseasewasfirstdescribedin2006usinga

GII.4strain(HS66)(66).Theauthorsreportedmilddiarrheain74%ofinfectedpigs,aswellas

fecalvirussheddingasdeterminedbyRT-PCRandELISA.NoVcapsidantigenwasdetectedin

enterocytesofboththeduodenumandjejunum,supportingoccurrenceofviralreplication.

Similartofindingsinhumans,onlymildhistopathologicallesionsoftheintestineswere

observed,mainlyconsistingofduodenalvillousatrophyandedemaofthelaminapropria.Ina

follow-upstudywiththesameGII.4strain,thegroupevaluatedcytokineandantibody

responsespost-infection(244).Itwasnotedthatpigswithgreaterseverityofdiarrheawere

morelikelytoseroconvertandhadhigherintestinalIgAandIgGtiters.Intermsofcytokine

secretion,HuNoVinfectioninducedabalancedTh1/Th2(IFN-g,IL-12,IL-4,IL-10)anddelayed

IFN-aresponsesystemically,whileonlyIFN-aandIL-12weredetectedlocallyinintestinal

contents.

GnpigscontinuetobeusedtoevaluateNoVvaccinecandidates,includingbothVLPand

Pparticle-basedvaccines(245,246).Otherstudieshaveutilizedthemodeltoassessefficacyof

immunomodulatorsandtherapeutics,suchasIFN-a,probioticsandricebran(247,248).As

previouslymentioned,recentpathogenesisstudiesalsodemonstratedthat,incontrasttoMNV,

HuNoVdoesnothaveatropismforBcellsinGnpigsandthatE.cloacaeinhibitsinfectivity

instead(67,131,249).

21

1.4.3GnpigmodelofHRVinfection

IncontrasttoHuNoVs,HRVscanbegrownefficientlyincellcultureandcommercial

vaccinesarereadilyavailable.However,thesevaccineshaveloweredefficacyindeveloping

countries,thustheGnpigmodelisstillfairlyrelevantforstudyingRVs.SimilartoHuNoVs,

infectionwithHRViscapableofcausingclinicaldiseaseinGnpigs.Additionally,unlikemiceand

rabbitmodels,GnpigscontinuetobesusceptibletoRV-induceddiarrheauntil6weeksofage

(250).

GnpigswerefirstutilizedtostudyHRVsstartinginthe1970’s(251,252),demonstrating

developmentofdiarrheaandfecalvirusshedding.Yet,itwasnotuntil1996whentheGnpig

modelofHRVinfectionanddiseasewasfullyestablished(253).Thismodelwasdevelopedusing

apig-adaptedHRVWastrain(G1P1A[P8]),whichinduceddiarrheainallpigsatadoseof~105

focus-formingunits(FFU)approximately13hourspost-inoculationandlastingforanaverageof

fourdays.Thediarrheawasassociatedwithhistopathologicallesionsintheduodenumand

jejunum(villousatrophy),seroconversionandfecalvirussheddingupto2x107FFU/ml.

Sincethattime,themodelhasbeenutilizedtodetermineimmuneresponsestoHRV

infectionandevaluationofvaccineandtherapeuticcandidates.AstudyconductedinGnpigs

wasthefirsttodemonstrateintestinalIgAantibody-secretingcells(ASCs)asacorrelateof

protectiveimmunityinanHRVanimalmodel(254).TwootherGnpigstudiessuggestedthat

vaccinecandidatesforregionswithinfantshavinghighmaternalantibodylevels(developing

countries)mayrequirespecializedformulationsand/ordosingregimenstocircumvent

maternalantibodyinterferenceonthedevelopmentofprotectiveASCresponses(255,256).An

additionalstudyhighlightedtheimportanceofabalancedTh1/Th2responseinducedbyWa

22

HRVinprovidingprotectionagainstsubsequentre-challenge(257).PotentialRVvaccinesand

therapeuticsevaluatedinGnpigsincludeVLP-basedvaccines(258,259),chickeneggyolkIgY

antibodies(260),llama-derivedantibodies(261),probiotics(262-265)andricebran(266,267).

Recently,aGnpigmodelofentericdysbiosiswasestablishedtoelucidatetheeffectsofhuman

gutmicrobiotaonimmuneresponsesinducedbyRVvaccination(235).

1.5Roleofdendriticcellsininnateimmunityagainstacuteviral

gastroenteritis

1.5.1Generaloverview

Dendriticcells(DCs)arespecializedcellsoftheinnateimmunesystemthatare

particularlyadaptedforantigencaptureintheirimmaturestate(268,269).Recognitionof

antigenicstimulibyDCsarecontrolledbypatternrecognitionreceptors(PRRs)thatare

associatedwithconservedpathogenassociatedmolecularpatterns(PAMPs)ofmicrobes(268,

270).Thiscontactpromptsthesecretionofcytokinesandchemokinesthatcanattractother

innateimmunecells(268).Uponmaturation,theactivatedDCsmigratewiththecaptured

antigenstolymphoidtissuesandinteractwithT,BandNKcells(271).Significantly,DC

presentationofantigenstoTcellsresultsinguidanceoftheadaptiveimmuneresponseto

pathogens,inducingeitheranimmunogenicortolerogenicstate(270).DCsaregenerally

classifiedaseithermyeloid/conventionalDCs(cDCs)orplasmacytoidDCs(pDCs)(272).cDCsare

recognizedprimarilyforantigenpresentation,whilepDCsareknownaspotentsecretorsof

typeIinterferons(IFN-aandIFN-b)(273).Inmice,cDCsarecomprisedoftwomajorsubsets,

23

CD103+CD11b

-andCD11b

+cells(274,275).TwocDCsubsetsarealsopresentinhumansandare

mainlycharacterizedaseitherCD1c+orCD141

+(275,276).AsforpDCs,thoseinhumansare

knowntoexpressCD303(CLEC4C),CD304(neuropilin-1),andCD123(IL3RA),whilethosein

miceexpressB220,sialicacid-bindingimmunoglobulin-likelectinH(SiglecH),andintermediate

levelsofCD11c(275,277).cDCandpDCpopulationsinpigshavebeendefinedas

CD172a(SWC3a)+CD4

-CD11R1

+andCD172a(SWC3a)

+CD4

+CD11R1

-,respectively(278).Itis

importanttonotethatCD11R1inpigs,whichisrecognizedbymouseanti-pigCD11R1antibody

cloneMIL4,isanalogoustoCD11binhumansandthusalsorecognizedbythecross-reactive

anti-humanCD11bcloneTMG6-5.

DCsarefoundinseverallocationsthroughoutthebodywherethereisahigher

incidenceofencounteringpathogens.Specifically,inthegastrointestinaltract,DCsarelocated

inPeyer’spatchesofthesmallintestineandthroughoutthelaminapropria(279,280).Inmice,

themajorityoftheDCsinthelaminapropriaofthesmallintestinearecharacterizedas

CD103+CD11b

+,whilethoseinthecolonarecomprisedofCD103

+CD11b

-andCD103

-CD11b

+

populations(281).StudiesofintestinalDCpopulationsinhumanshavedemonstratedthat

CD103+CD172a

+DCsarecloselyrelatedtomurineCD103

+CD11b

+DCs.Similarly,CD103

+CD172a

-

DCsinhumansarehomologoustoCD103+CD11b

-DCsinmice(282).Inpigs,DCsinthelamina

propriaaredefinedasCD11b+CD172a

+,whileDCsinPeyer’spatchesaredividedintoCD11b

-

CD172a+insubepithelialdomesandCD11b

-CD172a

-ininterfollicularregions.Afourthsubsetin

pigsisrepresentedbyCD11b+CD172a

-DCsinmesentericlymphnodes(283).Asthe

gastrointestinaltractisaprimesiteforinteractionswithentericpathogens,recentstudieshave

beenconductedtounderstandtheroleofDCsinNoV-andRV-inducedimmunity.

24

1.5.2DendriticcellresponsestoNoVinfection

StudiesofDCresponsestoNoVinfectionarelimitedandhaveprimarilybeenconducted

inmicethusfar.InadditiontotheirpermissivenesstoMNVinfection,DCshavealsobeen

implicatedinclearanceofvirusandinfluenceofantibodyresponses.OnestudydepletedcDCs

frommicepriortoMNVinfectionandreportedincreasedvirussheddinganddecreasedMNV-

specificIgGtiters(279).AmorerecentstudyassociatedtheabilityofDCstocontrolMNV

replicationwiththesecretionofTypeIIFN(284).MicewithDCsthatwereunabletorespondto

TypeIIFNduetoareceptordeficiencydemonstratedpersistenceofMNVinfection.This

persistenceoccurreddespitecompensatoryincreasedhumoralresponses.

1.5.3DendriticcellresponsestoRVinfection

WhilenumerousstudieshaveadvancedunderstandingofadaptiveimmunityagainstRV

infection,knowledgeofinnateimmunityintermsofDCresponsesisstillincomplete.Two

featuresseemtobewell-acceptedfromrecentstudiesthough.First,DCsareclearlycapableof

beingactivatedbyRVinfectionasdemonstratedbyup-regulationofCD40,CD86,MHCII,TLR3

andTLR4,cytokinesecretion,andreducedbindingtoVLPs(285,286).Second,TypeIinterferon

secretionbypDCs(272)isimportantforimmunedefensesagainstRVinfection.pDCsecretion

ofIFN-ahasbeenassociatedwithstimulationofIFN-g-producingTcellresponsesinvitro(287).

AnotherinvitrostudyrevealedthenecessityofTypeIIFNproductionbypDCsinregardstoB

cellactivation(288).ThesamestudyalsodemonstratedthatmicewithoutfunctionalpDCs

25

wereunabletomountsufficientRV-specificantibodyresponsestocontrolinfectionasthese

micehadincreasedvirusshedding.

1.6Preventionofacuteviralgastroenteritis

1.6.1Disinfectionmethods

BothNoVandRVarespreadthroughthefecal-oralrouteandarefairlyeasily

transmittedfrompersontopersonviacontaminatedfood,waterandfomitesifproperhygiene

anddisinfectionarenotemployed.Numerousstudieshavebeenconductedtodeterminethe

bestsanitationpracticestoreducespreadofeachvirus.

DuetothelackofanefficientcellculturesystemforHuNoVs,moststudiesevaluating

disinfectionmethodseitherutilizeothercalicviruses(MNV,FCV)assurrogates(289,290)or

measurereductionofHuNoVsbyPCRmethods(291,292).NoVappearstobemostsuccessfully

removedfromhandsviawashingwithregularsoapandwater(291-293).Different

concentrationsofalcohol-basedhandsanitizershaveproveneitherineffective(291,292)or

possiblyevendetrimental.Asurveyoflong-termcarefacilitiesfoundanassociationwiththe

useofalcohol-basedhandsanitizersandanincreasedriskofNoVoutbreaks(294).

ChlorhexidineisalsoconsideredineffectualasahandsanitizeragainstNoVs(295).Intermsof

surfacedisinfectants,itseemsthatNoVsarefairlyresistanttoquaternaryammonium

compounds(293).Sodiumhypochloriteorbleachataminimumconcentrationof500-1000

ppmhasproventobeeffectiveinNoVdecontamination(296-298).

26

Incontrast,simplehandwashingwithregularsoapisnotnecessarilyeffectiveat

removingRV.Studieshaveshownthatalcohol-basedhandsanitizersaremorereliableat

reducingRVloadsfromhumanhands(145,299,300)andshouldbeutilizedinfoodpreparation

andwhencontactwithinfectedindividualsorcontaminatedfomitesissuspected.Asfaras

surfacedisinfectantsareconcerned,theseshouldalsocontainalcoholinhighconcentrationas

alcoholhasbeenshowntodamagetheoutercapsidofRV(291,301).Chlorhexidinegluconate

0.5%in70%ethanol,quaternaryammoniumcompoundscontaining>40%isopropylalcohol,

0.1%o-phenylphenolwith79%ethanolor95%ethanolalonearecapableofinactivatingRV

(145,302).DomesticbleachalsohasacceptableactivityagainstRV(302).

1.6.2NoVvaccinedevelopment

ThereiscurrentlynolicensedNoVvaccineavailable.Thedifficultyindevelopinga

suitableNoVvaccineismultifactorialandincludestheinabilitytoefficientlycultureHuNoVs

efficientlyandcost-effectively,thelackofappropriateanimalmodelsandthesignificant

antigenicdiversityofNoVs(2,303,304).Additionally,thediversitybetweengenogroupsand

thecontinualevolutionofthemoreprevalentGII.4genotypehascloudedunderstandingof

howpreviousexposuretomultipleNoVstrainsmayinfluencetheresponsetovaccinesand

whetherlong-lastingimmunityexists(303,305).Studieshavesuggestedimmuneprotection

maylapseafter2monthsto2years(306)ormaypersistforupto8years(307).

Progresshassteadilybeencontinuingtoaddresstheseissuesthoughandseveral

candidatesarebeingevaluatedinpre-clinicalandclinicaltrials(304,305,308,309).Since

HuNoVcellculturesystemsarenotwell-established,recombinantNoVvaccinesarebeing

27

developedatthistime.ParticularinteresthasbeenaffordedtoVLPvaccines.VLPsare

producedbyexpressionofthemajorcapsidproteinVP1inanappropriatesystem(304,309),

includinginsectcells(310),mammaliancelllines(311),E.coli(312),yeast(313),potatoes(314),

tobacco(315)andtomatoes(316).Allleadtoformationofparticlesthataremorphologically

andantigenicallycomparabletotheactualviruscapsiditself(304,317).Whenadministered

eitherorally(318,319),intranasally(320)orintramuscularly(321),VLPvaccineshavebeen

capableofinducinghomotypicandheterotypichumoral,mucosalandcellularimmunitiestoa

varyingdegree(309).Variousadjuvants,includingLT(R192G)toxinfromE.coli(320),

monophosphoryllipidA(MPL)andchitosan(322),havethusbeenutilizedtoincreaseimmune

responsestoVLP-basedvaccines.Severalpreclinicalstudieshavealsoconsideredthedelivery

ofVLPvaccinesviaaviralvectortoinducegreaterimmunity(323-325),thoughconcerns

regardingbiosafetyandefficacyrelatedtoprevioushostexposurehavereducedtherelevance

ofsuchvaccines(309,326).

OfthecurrentNoVvaccinecandidates,aVLP-basedvaccinefromTakeda

Pharmaceuticalsisthemostadvancedinclinicaltrialprogress.Toovercomethehurdleof

antigenicdiversityamongNoVs,designofmultivalentvaccinesisconsideredimperative(327).

Thephase2clinicaltrialcandidateisabivalentGI.1/GII.4vaccinethatisadjuvantedwithMPL

andaluminumhydroxide;itisadministeredintramuscularlyintwodoses,28daysapart(328-

332).ThevaccineiscomprisedoftheNorwalkGI.1strainVLPandaGII.4VLPdevelopedfroma

consensussequenceofthreeGII.4variants.Variousdosageformulationshavebeenevaluated,

withmorerecentresultsindicatinga15/50(GI.1/GII.4)formulationproducingrobust

serologicalresponsesfollowingjustonedoseofvaccine,whilemaintainingtolerability(331).

28

Aftervaccinationofhumanadults,IgAandIgGASCresponsespeakedatday7post1stdoseof

vaccine.Allvaccinerecipientsshowed>4-foldrisesinASCsfrompre-vaccinationlevelsatthis

timepointandminimalincreasesinASCswereobservedfollowingasecondvaccinedose,

indicatingtheimmuneresponsesareanamnesticinnature.GI.1-andGII.4-specificIgGmemory

Bcellsweredetected180dayspost-vaccination.Butitremainstobeevaluatedifthese

responsesfunctionasvaccine-inducedcorrelatesofprotection(332).Overall,thehighestGI.1

antibodylevels(pan-Ig,IgAandHBGA-blockingtiters)wereseenwitha50/50formulation,but

the15/50formulationunexpectedlyproducedbothgoodGI.1responsesandevenbetterGII.4

responses.AsGII.4NoVsarethepredominantcirculatingstrains,thiswillmostlikelybethe

formulationmovingforwardwithfuturestudies.Currentplansincludedeterminingwhether

theMPLadjuvantisabsolutelyessential.

The other recombinant protein utilized inNoV vaccine development is the P particle,

whichisasubviralparticleformedfromexpressionoftheVP1PdomaininE.colioryeast(333).

Asmentionedpreviously,thePdomain,specificallytheP2subdomain, isresponsibleforhost

bindinginteractions(334).AnadvantageofthePparticleistheeaseofproduction,makingthem

likelyamoreeconomicalvaccineoptionthanVLPs(334).StudiesindicatePparticlesretainHBGA-

binding properties (333) and are immunogenic in mice (335) and Gn pigs (246) following

intranasal administration. The study conducted in pigs demonstrated 47% cross-variant

protectionagainstdiarrheacompared to60%with thesameGII.4strain-derivedVLPvaccine.

However,theauthorsnotedthatimmunizationwithPparticlesinducedgreaterTcellresponses

thanwithVLPs.Conversely,anotherstudyrevealedthatVLPs,butnotPparticles,werecapable

ofprimingTcellsforIFN-gproductionandinducingcross-reactiveTandBcellresponsesinmice

29

(321).QuestionableformationofthePparticlesusedinthemousestudymayhaveaffectedthe

immunological responses though (326, 336). Further studies are likely required to better

characterizetheimmunityinducedbyPparticlevaccination.

Asmultipleentericpathogenscancauseacutegastroenteritis,therehasbeen

considerableinterestindevelopingcombinationvaccinesthatincludeNoV.Inparticular,

inclusionofRVrecombinantVP6proteinwithNoVVLPshasbeenevaluatedinseveralstudies

(337-339).Additionally,thePparticlehasproventobeanexcellentplatformforpresentation

ofadditionalantigens(340);PparticleshavebeendevelopedwithadditionofRV(340),

hepatitisEvirus(341),influenzavirus(342)andastrovirus(343)antigens.

ItremainstobeseenwhetherNoVvaccineswillbecapableofproducingdurableand

adequatehomotypicandheterotypicimmuneresponses.ItispossiblethatNoVvaccineswill

needtobereformulatedasemergentstrainsappear.

1.6.3RVvaccinedevelopment

RVvaccinedevelopmenthasbeenanimportantcontributortoreducingdiseaseburden.

ThefirstlicensedoralRVvaccinewasatetravalentreassortantofarhesusRVstrain(G3)with

VP7fromhumanG1,G2,andG4(137,344).RotaShieldorRRV-TVwaslicensedin1998after

provingtobesafeandefficaciousinstudiesconductedintheUS,FinlandandVenezuela(137,

344-347).Unfortunately,itwasbelievedtobeassociatedwithanincreasedriskof

intussusception,aconditionwhenapartoftheintestinallumenfoldsintoitselfandmayresult

inobstruction,ischemiaandperforation.Theincidencewasgreatestinthe3-7daysfollowing

thefirstdoseandmoreoftenwhenthevaccinewasadministeredtochildrenbetween3and9

30

monthsofage(344,348,349).Duetosafetyconcerns,RotaShieldwasvoluntarilywithdrawn

fromthemarketin1999,lessthanayearafterlicensure.

Since2006,twonewlive,attenuatedoralRVvaccineshavebeencommerciallyavailable

–RotaTeq(approved2006)andRotarix(approved2008).Rotarix(GlaxoSmithKlineBiologicals)

isamonovalenthumanG1P1A[8]strainvaccinethatwasattenuatedthroughserialcellculture

passage(136,137,344).Upondevelopment,thesafetyandefficacywastestedin60,000

infantsinEuropeandLatinAmerica(147,350,351).Thevaccineisadministeredin2dosesat2

and4monthsofage.Duetothehumanstrainoriginofthevaccine,intestinalreplicationdoes

occurandsheddingfollowingthefirstdoseiswell-observed.Immunityshouldbehighenough

bytheseconddosethatsheddingshouldbeveryminimal(147,344).

RotaTeq(Merck)isapentavalent,reassortantvaccinecontainingabovineRVstrain

(WC-3,G6P[5])backbonewithVP7ofhumanG1,G2,G3andG4strainsandVP4ofaP1A[8]

strain(344).SimilartoclinicaltrialsofRotarix,RotaTeqwasevaluatedin>60,000infantsinthe

USandEurope(147,352).MostlikelyduetolessintestinalreplicationthanRotarix,RotaTeq

requiresa3-dosescheduleofadministrationat2,4and6monthsofage.Inaccordancewith

thelimitedreplication,muchlowerincidencesofsheddingofthevaccinestrainoccuraswell

(137,147).

RegionalRVvaccinesincludetheLanzhoulambRVvaccine(LLR)licensedinChina,a

monovalenthuman-bovinevaccine(Rotavac)inIndiaandanattenuatedG1P[8]strainvaccine

(Rotavin)inVietnam(353-356).Morerecently,anoralbovine-humanreassortantpentavalent

vaccine(Rotasiil)demonstrateda66.7%efficacyagainstsevereRVgastroenteritiswith3doses

inarandomized,placebo-controlledtrial(357).ThevaccinecontainshumanRVserotypesG1,

31

G2,G3,G4,andG9onabovineRVG6P[7]backbone(BRV-PV).Ofimportanceforusein

resource-limitedcountries,thevaccineisheat-stablefor2yearsatatemperatureof37°Cand

for6monthsat40°C.

BothRotaTeqandRotarixweredemonstratedtoinducegoodimmunityinlicensing

trials,withefficacyagainstsevereRVgastroenteritisrangingfrom85-98%thatseemstopersist

forafewyearsfollowingimmunization,atleastindevelopedcountries(350,352,358-362).As

countriescontinuetoincludeRVvaccinationinstandardimmunizationprotocolsas

recommendedbytheWorldHealthOrganization(WHO),thetrueclinicalimpactisbecoming

betterunderstood.AsofMay2016,81countrieshaveincorporatedRVvaccinationintotheir

nationalimmunizationprograms(363).

DeclineincasesofRVdiarrheaandrelatedhospitalizationhasbeenobservedinthe

majorityofvaccinestudiesinbothdevelopedanddevelopingcountries.OnestudyintheUS

reporteda74-90%decreaseinthenumberofpositiveRVtestsbetween2010-2012compared

toprevaccinebaselines(2000-2006)(364).Additionally,RV-associatedhospitalizationsinthe

USweredecreasedbetween60-94%from2007-2011whencomparedwithratesobserved

from2001-2006(365).Similarly,hospitalizationratesinEuropewereestimatedtodeclineby

65-84%whenreviewingdatafrom2006-2012(366).Furthermore,evidenceofherdimmunity

hasemergedasdeclineinall-causediarrheahospitalizationshaveoccurredinolderchildren

andadultssincereleaseofcurrentRVvaccines(367,368).RegardingreductionofRVburdenin

developingcountries,arecentreportfromRwandaindicatedthatintroductionofRotaTeq

vaccinationreducedRV-relatedhospitalizationsby61-70%between2011to2014(369).

32

Likewise,astudyfromGhanaconductedduringthefirstthreeyearsfollowingRotarixvaccine

introductiondemonstrateda49%reductioninhospitalizationsduetoRV(370).

DespitethepromisingresultsfromimplementationofRVvaccineusageindeveloping

countries,overallvaccineefficacy(50-64%)stillremainslowerthanthatofdevelopedregions

(354,363).Reasonsforthisobservationappeartobemultifactorialandencompass

interferencebymaternalantibodies,concurrentadministrationoforalpoliovaccine,

malnutrition,coinfectionswithotherentericpathogensanddifferencesingutmicrobiome

(147,353,371).Addressingthisissuemayrequirealterationsofvaccineadministration

schedulesordevelopmentofparenteralvaccinecandidates(147).

SincetheintroductionofRotaTeqandRotarix,acoupleofsafetyconcernshavearisen,

thoughneitherhavebeenseriousenoughtopermanentlyremoveeithervaccinefromthe

market.Despitecarefulsafetyevaluationinclinicaltrials,bothvaccineshavepossiblybeen

associatedwithaslightincreaseinintussusceptionriskinpostlicensurestudiesinseveral

countries,includingtheUS,Australia,andMexico(372-375).SimilartoRotashield,the

incidenceismostlikelytooccurinthefirstweekfollowinginitiationofthevaccineseries(376).

However,theoverallincidenceismuchlowerthanobservedwithRotashield,equaling1-5

excesscasesforevery100,000childrenvaccinated(147,376).

Another,thoughtemporary,concernwasthediscoveryofporcinecircovirusI(PCV-1)

DNAinRotarixandPCV-1andPCV-2DNAfragmentsinRotaTeqin2010.PCV-1DNAwasfound

originatinginthemasterseedvirusofRotarix,whilecontaminatedtrypsinusedinthe

productionofRotaTeqwasimplicated(344).Sinceporcinecircovirusdoesnotinfecthumans,

useofbothvaccineswasallowedtocontinuewhileproductionofPCV-freevaccineswas

33

initiated.Overallthough,thebenefitsofRVvaccinationinreductionofmorbidityandmortality

outweighanyperceivedrisks.

1.7Treatmentofacuteviralgastroenteritis

1.7.1Oralrehydrationsolutions

DuetothelackofavailabilityofNoVvaccinesandthestillunder-utilizedRVvaccines,

adjunctivetreatmentsremainsignificant.MosttreatmentmodalitiesforNoVandRVareaimed

atreducingclinicalsymptomsofdiseaseandarethussimilarbetweenthetwo.Themost

importantandbeneficialaspectoftreatmentisappropriaterehydrationtherapy.Fatalities

occurwhenintestinalwaterlossissevereenoughtocauseelectrolyteimbalancesand

cardiovascularfailure.WHOrecommendstheuseoforalrehydrationsolution(ORS)in

treatmentofacutewaterydiarrheaofallcauses.UseofORShelpspreventlife-threatening

dehydration,thoughitdoesnotalleviatesymptomsofdiarrheaorshortenduration.ORS

containsglucose,electrolytesandcitrateinanhypo-osmolarformulationtoreplenishlosses

andreduceacidosisassociatedwithdiarrheaandvomiting(377).ThebasisofORStherapyis

thatglucosestimulateswaterandsaltabsorptioninthesmallintestineviatheNa+/glucose

cotransporterSGLT1(378).Insituationswhereoralrehydrationisnotadequateenoughto

counteractdehydration,thenintravenousfluidtherapyisnecessary(379).

Since2004,WHOhasalsorecommendedtheuseofzincsupplementationfortwoweeks

inconjunctionwithORS(380,381).Zinchasbeenshowntobeimportantformucosalintegrity

oftheintestineandinhibitionofpotassiumchannelsinvolvedincyclicadenosine

34

monophosphate(cAMP)mediatedchloridesecretion(146,382,383).Consequently,low

plasmazinclevelshavereportedlybeenlinkedwithincreasedriskofmorbidityduetosevere

diarrhea(384)andsupplementationwithORScanreducediarrheavolumeandduration(385).

1.7.2Generalanti-diarrhealandanti-viralagents

SinceORSonlyservestooffsetdehydration,non-specificadjunctivetreatments

continuetobeproposedandstudiedwiththeaimofeitherreducingdiarrheaorlimitingviral

replication.Probioticsarewell-knownfortheirgastrointestinalhealthybenefit.Multiple

probioticstrainshavebeenevaluatedfortheirabilitytodecreasedurationofdiarrheawith

generallypositive,thoughvariablestrainanddose-dependentresponses(263,386,387).The

mostcommonlyadministratedstrainsincludeLactobacillusandBifidobacteriumspecies(388).

Themechanismofactionofprobioticsisnotfullydefined,butmayinvolveimprovinggut

barrierfunction,regulatingtheintestinalenvironmentandotherentericmicrobesand/or

influencingthehostimmunesystem(389,390).

Inparticular,LactobacillusrhamnosusGG(LGG)hasgarneredthemostsupportive

evidenceforlesseningclinicalsymptomsassociatedwithacutegastroenteritis(391).Inaclinical

trialinPakistan,LGGsupplementationfollowingrehydrationelicitedaresponsebythesecond

dayoftreatmentinchildrenwithacute,non-bloodygastroenteritis(392).Bythistime,31%of

childrenintheLGGgrouphadcontinueddiarrheacomparedtothe75%intheplacebogroup.

Similarly,inaclinicalstudyconductedinEurope,LGGadministrationinconjunctionwithORS

reducedthedurationofdiarrheainchildrenwithassumedviralgastroenteritis(noidentifiable

bacterialpathogensdetected)(393).LGGhasalsodemonstratedefficacyinpreventing

35

nosocomialdiarrheainyoungchildren(394).StudiesinGnpigshavealsoconfirmedspecific

efficacyofLGGagainstbothHRV(265,395)andNoV-induceddiarrhea(248).

Inrelationtoprobioticadministration,theprebioticricebranhasalsobeenreportedto

ameliorateacute,waterydiarrhea.Comprisingtheouterlayerofrice,ricebranhasbeen

identifiedasadietarysourceofbioactivecompoundsandhasbeensuggestedtobebeneficial

inintestinalhealthaswell(396,397).Themechanismofactionincludespromotingthegrowth

ofdiarrhea-reducingprobioticspecies,maintaininggutbarrierintegrityandimprovinginnate

immunity(266).InGnpigsinfectedwithHuNoVandcolonizedwithLGGandE.coliNissle1917,

ricebranfeedingreducedtheincidenceanddurationofdiarrhea,aswellasvirusshedding

(248).SimilarlyinHRV-infectedGnpigs,ricebranfeedingalonewasabletodecreasethe

incidenceandseverityofdiarrhea,thoughdidnotaffectvirusshedding(266).

Commercialdrugsarealsoavailableforthetreatmentofacutegastroenteritis.Theanti-

secretorydrugracecadotrilhasproventobeeffectiveindecreasingintestinalwaterlossand

hasbeenlicensedforuseinEurope(398,399).Racecadotrilactstoinhibittheenzymeneutral

endopeptidaseand,indoingso,increasestheintestinalconcentrationofenkephalins,which

havenaturalanti-secretoryproperties(400).Anotherdrugthathasbeenevaluatedisthe

broad-spectrumanti-infectivenitazoxanide(401,402).Nitazoxanidewasinitiallydevelopedas

ananti-parasiticagent,buthasalsoshownactivityagainstanaerobicbacteria(403)andviruses,

includingNoV(401,404)andRV(401,402,405).ItsuseisparticularlyapplicableincasesofNoV

infectioninimmunocompromisedindividualswherereductioninimmunosuppressivetherapy

oradministrationofintravenousimmunoglobulinisnotpossible(404).InRVinfectionatleast,

36

itappearsthatnitazoxanideinhibitsVP7maturation(406);themodeofactionagainstNoVis

unknown(407).

Recombinantllama-derivedsinglechainantibodyfragments(VHH)areanadditional

therapeuticagentthathavebeenproposedforbothNoVandRV-induceddiarrhea.Theseso-

callednanoantibodiesareabletobeproducedagainstaspecificantigen,includingVP6ofRV

(261)andVP1ofNoV(408,409),andrepresentapotentialimmunotherapy.

1.7.3RV-specificadjunctivetreatments

ForRVinfection,othernovelapproachestoinducepassiveimmunityincludetheuseof

hyperimmunebovinecolostrum(410)andhyperimmunechickeneggyolkimmunoglobulin

(260).AdditionalpotentiallyeffectivetreatmentsincludeN-acetyl-cysteineandergoferon.The

antioxidantN-acetyl-cysteinehasbeendemonstratedtoreduceRVinfectivityincellculture

(411)andresolveRVdiarrheainchildrenafterjusttwodaysoforaladministrationat60

mg/kg/day(412).Ergoferon,whichiscomposedofrelease-activepolyclonalantibodiesagainst

IFN-g,CD4andhistamine,wasrecentlytestedforantiviralactivityagainstRVincellculturewith

promisingresults(413).InvivostudiesevaluatingErgoferonhaveyettobeconducted.

1.7.4NoV-specificadjunctivetreatments

VariouscompoundshavebeenevaluatedinattempttodisrupttheNoVreplicationcycle

(407).Inresponsetothewell-establisheduseofHBGAsascellularreceptorsbyNoVs,therehas

beenconsiderableinterestinblockingthisbindinginteractiontolimitinfection(414-417).

37

Otheranti-NoVdrugsaimtoreducereplicationbytargetingtheviralprotease(418-420)or

RNA-dependentRNApolymerase(421-423).

1.8Concludingremarks

Mydissertationresearchinvolvesinvestigationsofpathogenesis,innateimmunityand

therapeuticsinGnpigmodelsofNoVandRVinfectionanddiarrhea.Asdiscussed,pigsarea

particularlysuitableanimalmodelforstudyingthepathogenesisofentericvirusesduetotheir

similaritiesinanatomy,physiologyandimmunefunctionwithhumans(225,227-229).

UtilizationofGnpigsenablesevaluationofprimaryimmuneresponsestovirus,vaccination

and/ortherapeuticswithoutconfoundingvariablessuchasmaternalantibodyinterference,

priorantigenicexposureandpossibleco-infectionswithotherentericpathogens.

Thefirstphaseofmydissertationresearchwasfocusedontheestablishmentofa

reliableGnpigmodelofNoVinfectionanddisease.Ithaspreviouslybeendemonstratedthat

GnpigsareindeedsusceptibletoinfectionwithHuNoVsusingGII.4andGII.12strains(66,424).

However,thesepriormodelsfailedtodeterminethemedianinfectiousdose(ID50)ofthe

strainsused.Thischaracterizationenablesmoreconsistentincidenceofinfectionfor

appropriatelydeterminingprotectiveefficacyinvaccineandtherapeutictrials.Basedonthe

predominanceofGII.4strainsinoutbreaksofNoVgastroenteritis(8),aGII.42006bvariantwas

selectedasthechallengestrain.TodeterminetheeffectofageontheID50,bothneonatal(4-5

daysofage)andolder(33-34daysofage)wereinfected.Wealsoconfirmedwhether

38

administrationofacholesterol-loweringdrug,simvastatin,wasabletoincreasesusceptibilityto

NoVinfectionasreportedpreviously(247,425).

Thesecondphaseofmydissertationresearchinvolvedutilizingthewell-establishedGn

pigmodelofRVinfectionanddiseasetoevaluateadjunctivetreatmentsforRV-induced

diarrhea.WhileRVvaccinationhasreducedmorbidityandmortality,theefficacyofRotarixand

RotaTeqisstillunderwhelmingindevelopingcountries(354).Aneffectivetherapeuticto

reducediarrheaandshortendurationofillnesswouldthusbeespeciallybeneficial.

Racecadotrilisanattractiveanti-diarrhealdrugcandidateduetoitsefficacyand

tolerability(399,400).Asitdecreasesintestinalsecretion,itaddressesoneofthepossible

mechanismsofdiarrheaduetoRVinfection(208).Previousstudieshavereportedgenerally

favorableresultsinall-causeandRV-induceddiarrhea,thoughconflictingevidenceexists(426,

427),possiblyduetoconfoundingvariablessuchaspatientcomplianceandco-infectionswith

otherentericpathogens.Subsequently,theGnpigmodelpresentsanidealopportunityto

determinethespecificefficacyofracecadotrilagainstRV-induceddiarrhea.Suchevaluationis

prudentbeforestandardizationofitsuseintreatmentprotocols.

Probioticshavealsobeenhighlystudiedfortheiranti-diarrhealeffects.However,

numerousstrainsareavailableanditiswell-acceptedthatresultsmaybebothstrain-and

dose-dependent(263).Furthermore,theexactmechanismofactionisill-defined,though

immunomodulationislikelyinvolved(389).Again,theimmunologicallynaïvebackgroundofGn

pigsfacilitateselucidationofimmuneresponseselicitedbyprobioticadministrationandthe

possibleassociationwithdiarrheareduction.Ourpreviousstudiesevaluatedthedose-

dependenteffectsofLactobacillusacidophilusNCFM(LA)onTcellresponsesinGnpigs

39

receivinganoralHRVvaccine(428).Currently,limitedstudieshavebeenconductedregarding

theroleofDCsininnateimmunitytoRVinfection.AsDCsinthegastrointestinaltractwould

expectedlyencounterbothprobioticsandRVs,weelectedtoexplorethisinteractioninGnpigs

administeredeitheraloworhighdoseofLA,bothbeforeandafterHRVinfection.

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