Immunomonitoring of human responses to the rVSV-ZEBOV Ebola vaccine

Immunomonitoring

of

human

responses

to

the

rVSV-ZEBOV

Ebola

vaccine

Donata

Medaglini

and

Claire-Anne

Siegrist

TherVSV-ZEBOVvaccineiscurrentlytheonlyEbolavaccine withdemonstratedclinicalefficacyinaring-vaccinationclinical trial.Ithasbeenshowntobereactogenicbutimmunogenicand safeinseveralPhaseIclinicalstudies.However,its

mechanismsofprotectionareunknownandavailable immunogenicitydataaremostlylimitedtoclassicalserological analysis;itisnowofparamountimportancetoapply cutting-edgetechnologies,includingtranscriptomicandmetabolomic analyses,andtoperformintegrativeanalyseswithstandard serologyandclinicaldatatocomprehensivelyprofilethe rVSV-ZEBOVimmunesignature.

Addresses

1

LaboratoryofMolecularMicrobiologyandBiotechnology,Department

ofMedicalBiotechnologies,UniversityofSiena,Siena,Italy

2_{WorldHealthOrganizationCollaboratingCenterforVaccine}

Immunology,DepartmentsofPathology-Immunology,Universityof

Geneva,1211Geneva,Switzerland

Correspondingauthor:Siegrist,Claire-Anne(claire-anne.siegrist@unige.

ch)

CurrentOpinioninVirology2017,23:88–94

ThisreviewcomesfromathemedissueonPreventiveand

therapeuticvaccines

EditedbyRinoRappuoliandGerdSutter

ForacompleteoverviewseetheIssueandtheEditorial

Availableonline28thApril2017

http://dx.doi.org/10.1016/j.coviro.2017.03.008

1879-6257/_ã2017TheAuthors.PublishedbyElsevierB.V.Thisisan

openaccessarticleundertheCCBY-NC-NDlicense(

http://creative-commons.org/licenses/by-nc-nd/4.0/).

Need

for

an

Ebola

vaccine

Ebola Virus Disease (EVD) has occurred in numerous

sporadicoutbreakssinceitsidentification.In2014,West Africa experienced the largest outbreak of Ebola Virus Disease (EVD) in history, with six countries affected: Guinea,Liberia,Nigeria,Senegal,MaliandSierraLeone; withover28652confirmedorprobablecasesandmorethan 11325 deaths [URL://www.cdc.gov/vhf/ebola/outbreaks/

2014-west-africa/; [1,2]. The extremely high mortality

and therisks of exportationhave triggereda high level ofglobalconcern.Furthermore,Ebolavirusesmaybecome endemicincountrieswithpoorhealthcareinfrastructures, thusrepresentingasignificantlong-termthreat.

Currently, no effective therapies or licensed vaccines

exist for Ebola virus and for any member of the

Filoviridae family. The availability of effective Ebola vaccines could be of critical importancefor preventing the disease in high risk areas, using ring vaccination

strategies, and would maximize safety for front line

workers at greatest risk during outbreaks [3]. An

immunization capable of rapidly eliciting sustained

protectiveimmunityinmostrecipientswithasingledose wouldbehighlydesirable.

The 2014 EVD outbreak promoted a strong

unprece-dentedeffortforthedevelopmentofEbolavaccinesand several vaccine candidates were advanced to the clinic

including: (i) the ChAd3-EBO non-replicating vaccine

vector based on the chimpanzee adenovirus type 3

(ChAd3) [4,5]; (ii) the heterologous prime-boost Ebola

vaccineregimen based onrecombinantadenovirustype

26vector (Ad26.ZEBOV)priming followedbyboosting

with modified vaccinia Ankara vector (MVA-BN-Filoã) [6];(iii)the adenovirus5-vectored Ebola vaccine [7–9], (iv)theDNAEbolavaccine[10,11]and(v)the recombi-nantVesicularStomatitisVirus(VSV)vector-basedEbola

vaccine(rVSV-ZEBOV),themostadvancedcandidateto

haveshown immunogenicity,safetyand protective effi-cacyin humans [12,13,14,15,16] on which this reviewfocuses.

The

rVSV-ZEBOV

vaccine

candidate

ThevectorisasinglerecombinantVSVwildtypeisolate

in which the VSV envelope glycoprotein was replaced

withtheZairestrainEbolavirus(ZEBOV)glycoprotein

(GP), giving rise to rVSVDG-ZEBOV-GP

(rVSV-ZEBOV) (Figure 1) [17,18]. This Ebola vaccine

candi-date was developed by Public Health Canada

(BPSC1001), licensed to NewLink Genetics and

subsequently transferred to Merck (renamed as V920).

ThecharacteristicsofrVSV-basedvectorsarereviewedin Ref. [17].Previous experience showed that rVSV lacks the potential for reassorting with viruses and cannot integrateintohost cellDNA.Theinvivoreplication of rVSV-ZEBOVissignificantlyattenuatedbytheexchange

of the rVSV glycoprotein for the EBOV-GP. The

VSV-ZEBOVvaccinewasidentifiedbytheinternational consultationledbyWHOinSeptember2014asoneofthe

only two Ebola vaccine candidates with 100%

demon-strated protection in non-human primates (NHP) and

clinicalgradematerialreadyfor clinicaltesting[3]. ProtectionagainstEVDwasfirstreportedin nonhuman primates(NHP)with asingleintramuscular(IM) injec-tion of rVSV-ZEBOV [19,20]. Notably, vaccine vector

shedding was not detectablein NHP and none of the animalsdevelopedovertfeverorotherdetectableadverse events [19,21].rVSV-ZEBOVinduced humoraland cel-lularimmuneresponsesinallvaccinatedmonkeysandno evidence of Ebola virus replication was detected after intravenouschallengewithahighlethaldoseofZEBOV. Completeandpartialprotectionwasevenachievedwitha single injection seven or three days before challenge,

respectively, showing that VSV-ZEBOV may rapidly

confer protection further supporting its use for rapid responses duringoutbreaks[22].Safetyin

immunocom-promised hosts, an important consideration given the

potential presence of HIV infected individuals in the targetvaccinepopulation,wasevaluatedinafewrhesus

macaques infected with simian-human

immunodefi-ciencyvirus(SHIV),noneoftheinfectedanimalsshowed

evidence of illness following rVSV-ZEBOV

immuniza-tion and four out of six were protected from EBOV

challenge [23].The rVSV-ZEBOVvaccine also showed

anefficacyof33–67%followinga24hourspost-exposure injection of Resus macaques infected with Ebola virus Makona [24].Thepotentialforprotectionagainstnewly emerging, phylogenetically related strainswas assessed

by immunizing macaques with rVSV-ZEBOV prior to

challengewithBundibugyoebolavirus(BEBOV),anewly emerged EBOV species [25].A single vaccination with rVSV-ZEBOVprovidedsignificantcross-protection(75% survival), suggesting that monovalent rVSV-based vac-cinesmightbealsousefulagainstnewlyemergingspecies [23].

TherVSV-ZEBOVvaccinewastestedinseveralclinical

trials (Table 1) in over 1000 subjects, showing

high immunogenicity, safety and protective efficacy

[12,13,14,15,16].PhaseIVEBCONclinicaltrials

havebeenorganizedinAfricaandEuropeas investigator-driven andinvestigator-sponsored trialsunder the coor-dinationofWHO,usingvaccinevialsdonatedtoWHOby

Public Health Canada and financially supported by a

grant of theWellcome TrustFoundation to the WHO.

AdditionalfundswereprovidedbytheBillandMelinda

GatesFoundationandtheGermanCenterforInfection

Research.Thefirstresultsof thesafetyand

immunoge-nicityofrVSV-ZEBOVwerereportedonlyafewmonths

aftertheinitiationofthefirstclinicaltrials[12,13].At high doses, ranging between 3106and 5108PFU, mild-to-moderate reactogenicity (fever, myalgia, chills, fatigue, headaches, etc.) affected most subjects. Symp-toms occurredearly(onset day 1 and 2) andwere tran-sient, associated to viremiaandhaematological changes reflecting vaccinereplication[13].The rVSV-ZEBOV

vaccine generated glycoprotein-binding antibodies in

almost all participants at any dose, showing its high immunogenicityinhumans.Unexpectedly,oligoarthritis wasidentifiedinthesecondweekaftervaccinationwith 107

PFUin11/51(22%)ofGenevaclinicaltrialsubjects. Itlastedanaverageof8days,occasionallyassociatedwith maculopapularorvesiculardermatitis.Theidentification of rVSV-ZEBOVinsynovialfluidandskinvesicles con-firmed viraldissemination and replicationin peripheral tissues [14]. Only two similar cases of arthritis were

observed at other VEBCON trial sites, suggesting an

influenceofthevaccinedose,hostfactors,and/or report-ingandinvestigativeapproaches.

Afterasafety-drivenstudyhold,theGenevarandomized clinical trial was restarted with a lowerdose of 3105 PFUandtheGabon/Germantrialswereconductedatthe sameorlowerdoses,providingtheopportunitytoassess therelative influence of vaccinedoseonvaccine safety

andimmunogenicityinthesamepopulations.Reducing

thevaccinedosemarkedlyreducedacutereactogenicity, butdidnotavoidarthritis,dermatitisorcutaneous vascu-litis, and limited the magnitude of antibody responses [14].Novaccine-associatedsevereadverseeventswere

reported in any of the rVSV-ZEBOV phase 1 trials.

Hence, the vaccine was selected for use at a dose of

2107 PFU in further phase 2/3 trials sponsored by

WHO (Guinea), the U.S. Centers for Disease Control

and Prevention (CDC) (Sierra Leone) and the U.S.

NationalInstituteofHealth(NIH)(Liberia),alongwith theNationalHealthAuthoritiesofthehostcountries. Protective efficacy was demonstrated in an open-label, cluster-randomised ringvaccinationtrialin the commu-nitiesofConakry,Guinea,andSierraLeone [15,16]. The analysisincludedatotalpopulation of11841 indi-viduals,assignedto117clusters(rings)vaccinatedeither

immediately or after a 21-day delay. rVSV-ZEBOV

showed up to 100% efficacy, with no cases of EVD as

of10daysafterimmunization,comparedto23casesinyet unvaccinatedsubjects [16].Infact,new casesofEVD

ImmunomonitoringofhumanresponsestotherVSV-ZEBOVEbolavaccineMedagliniandSiegrist 89

Figure1

V

VSV ZEBOV

rVSV- ZEBOV

Current Opinion in Virology

SchematicrepresentationofrVSV-ZEBOV.TherVSV-ZEBOVvaccine

isarecombinantvirusinwhichtheVSVenvelopeglycoprotein(inblue)

isreplacedwiththeZairestrainEbolavirus(ZEBOV)glycoprotein

(inred)givingrisetothechimericvirusrVSV_DG-ZEBOV-GP

(rVSV-ZEBOV).

ve

and

therapeutic

vaccines

Phase Dose (pfu) Subjects n. Age (years) Status Sponsor Country Clinical trial n. Ref. 1 – Safety, and immunogenicity 2.5 104

2.5 105

2.0_{ 10}6

38 18–65 Completed Profectus Bioscience Inc. USA NCT02718469a

1 – Safety, and immunogenicity 3.0_{ 10}6

2.0 107 1.0 108

39 18–65 Completed Merck Sharp & Dohme Corp USA NCT02280408a _[13_]

1 – Safety, tolerability and immunogenicity 3.0 105

3.0_{ 10}6

2.0_{ 10}7

30 18–55 Completed University of Hamburg-Eppendorf Germany NCT02283099 [12]

1 – Safety and immunogenicity 3.0 103 3.0 104 3.0 105 3.0_{ 10}6 9.0_{ 10}6 2.0 107 1.0 108

512 18–60 Completed Merck Sharp & Dohme Corp. USA NCT02314923

1 – Safety, tolerability and immunogenicity 1.0 105

5.0_{ 10}5

3.0_{ 10}6

40 18–65 Completed Dalhousie University Canada NCT02374385

1 – Safety, tolerability and immunogenicity 3.0 106 1.0 107

40 18–55 Ongoing University of Oxford Kenya NCT02296983 [12] 1 – Safety and immunogenicity 3.0 103

3.0_{ 10}4

3.0_{ 10}5

3.0 106 2.0 107

155 6–50 Ongoing University of Tuebingen Gabon PACTR201411000919191 [12]

1 – Immune durability 3.0 105

1.0 107

5.0_{ 10}7

100 18–68 Ongoing University Hospital Geneva

Switzerland NCT02933931

1 – Safety and immunogenicity 3.0 106 2.0 107 1.0 108

39 18–50 Completed Merck Sharp & Dohme Corp. USA NCT02269423 [13]

1/2 – Safety and tolerability 3.0 105

1.0_{ 10}7b

5.0 107b

115 18–65 Completed University Hospital Geneva Switzerland NCT02287480 [12,14]

2 – Immunogenicity and durability 2.0 107 300 >18 Ongoing NIAID USA NCT02788227 2 – Safety and efficacy 2.0 107 _{900 >18 Ongoing NIAID Liberia NCT02344407}

2/3 – Safety immunogenicity 2.0 107 _{8000 >18 Ongoing CDC Sierra Leone NCT02378753}

3 – Safety, immunogenicity and efficacy 2.0_{ 10}7 _{8851 6–90 Completed WHO Guinea (Conakry) PACTR201503001057193 [15}_,16_]

3 – Safety, immunogenicity and efficacy 3 consistency lots and a high-dose lot

1198 18–65 Ongoing Merck Sharp & Dohme USA NCT02503202

a_{Prime-boost schedule.} in Virolo gy 2017, 23 :88–94 www.sci encedirect.com

werenotdiagnosedfromsixdaysaftervaccination, indi-cating the rapid onset of protective immune responses

considering that exposure may have occurred prior to

immunization [15]. Adverse effects, mostly mild and self-limiting (headache,fatigue and muscle pain), were reported in more than half of vaccinees.These results demonstratedthefeasibilityof usingrVSV-ZEBOVina

ring-vaccination design to help control outbreaks. A

Merck-sponsored phase III clinical trial is ongoing to evaluatethesafetyandimmunogenicityofthreedifferent

lots ofrVSV-ZEBOV inabout1200healthyadults.The

primary purposeof thestudy is to demonstrate consis-tencyintheimmuneresponsesof participantsreceiving threeseparatelotsofV920through28days post-vaccina-tion. Asubset of participants will continueto be

moni-tored through 24 months post-vaccination for the

evaluationof safety,anddurabilityofimmuneresponse (NCT02503202).

Yet,manyquestionsarestillopen.Theymainlyrelateto

thesafetyandimmunogenicityof rVSV-ZEBOVin

vul-nerable populationssuch asyoung childrenor pregnant womenandtothedurationofprotection.Intheabsence

of current outbreaks, these may only be assessed by

immunomonitoring.

Immunomonitoring

of

rVSV-ZEBOV

Conventionalserologyandcellularimmunologyprovide

significant insights into vaccine induced protective

immunity. However the integration of multiple layers

ofinformationderivedfromtheintegrationofclinicaland state-of-the-art immunological read outs with distinct ‘Omics’analyses, suchas transcriptomicsand metabolo-mics,mayprovideabetterunderstandingofthecomplex mechanismsofvaccine-inducedprotectivereactogenicity and immunity (Figure 2). Thismayhelp in identifying

early signatures/biomarkers predictive of magnitude,

quality and duration of vaccine-induced adaptive

immuneresponsesaswellassurrogatemarkersofvaccine

induced adverse events. Recently, systems biology

approaches havebeen employed to pinpoint signatures

of immunogenicity for few human vaccines [26].Using thelatestsystemsbiologytechniquesandmodels (includ-ing integrated and validated transcriptomicsand meta-bolomicsanalyses)todissectthecomplexityofthe

inter-actions between the various components of the human

immune system in responseto vaccinationis of critical importancetofullydeciphertheimmunologicalsignature of rVSV-ZEBOV[27,28].

Serological analyses conducted in Phase 1 clinical trials

usingescalatingvaccine doseshaveshown thatEBOV–

glycoprotein(GP)–specificIgGantibodyresponseswere detected in almost all participants, with significantly higher titresof EBOV neutralizing antibodiesathigher vaccinedoses[14].Theimportanceofapredominantly

anti-GP IgM response for EBOV neutralization was

revealedaswellasanindependentevolutionofantibody immuneresponses–intermsofantibodyepitope reper-toirediversity,affinitymaturation,durabilityandisotype switching–aftervaccinationwithrVSV-ZEBOVinthree dosegroups(3million,20millionand100millionPFUs) [29].Astrong correlationbetweeninvitro EBOV

neu-tralization and serum GP-binding antibody titres was

observed. Interestingly, a second dose did not boost

antibodyorvirusneutralizationtitersandelicitedlimited antibodyaffinitymaturation[27].Ithasalsobeenrecently

shown that ZEBOV-specific circulating follicular T

helpercells(cTfh)correlatewithantibodytitersandwith theTfh17subset[30].

In arecentprospectivederivationand validationcohort

study nested within the phase I randomized,

placebo-ImmunomonitoringofhumanresponsestotherVSV-ZEBOVEbolavaccineMedagliniandSiegrist 91

Figure2

Immuno

moni

torin

g

in

clinical trials

Cli

nica

l

trials

Integrative

analysis

Current Opinion in Virology

IntegrativeimmunologicalanalysisofrVSV-ZEBOV.Immunomonitoring

inclinicaltrialsisconductedtoidentifymolecularsignaturesofthe

rVSV-ZEBOVvaccineintegratingstandardanalysisofinnateand

adaptiveimmuneresponsesandclinicaldatawithtranscriptomicand

matabolomicanalysis.

controlled (Geneva, Switzerland) and dose-escalation (Lambare´ne´, Gabon) trials, we observed an early (day

1) dose-dependent plasma signature of the safety and

immunogenicityoftherVSV-Ebolavaccine.Thesedata

highlight that monocytes play a critical role in

rVSV-ZEBOV-linked reactogenicity and adverse events,

includingarthritis [31].Preliminary analysisof whole-bloodtranscriptomicdata,obtainedbytargeted

transcrip-tome sequencing on Ion Proton platform, revealed an

activationofgenesinvolvedininnateimmuneresponses, whichstartsatday1aftervaccinationandlastsuntilday7 (manuscriptin preparation).

ResultsobtainedfromtherVSV-ZEBOVclinicaltrialsso farhaveshownthatthereare differencesamong volun-teersreceivingdifferentdosesofthevaccineaspertainto the magnitude of the antibody response as well as the magnitude and duration of adverse events. Hence, this stratifiedresponsetothevaccine offersaunique

oppor-tunityforbiomarkerdiscovery.TherVSV-ZEBOVphase

ItrialsperformedinGeneva,Lambare´ne´ andKilifihave

generated a unique set of immunological and vaccine

safetyobservations,thebiologicalbasesofwhicharenow beingstudied throughthousandsof collectedbiological samples.Asignificantstepbeyondthestate-of-the-artis

now required, through the harmonisation and in depth

integrated analyses of data generated by all different

clinical and immunological/molecular read outs. This

includes safety, immunogenicity, innate and adaptive

immunity, immunological memory, transcriptomics and

metabolomics(Figure2).Thiswillensuregaining maxi-mumleverageofthePhaseIEbolavaccinetrials,through

a comprehensive characterization of the immune

responses induced by rVSV-ZEBOV and ensuring that

all information resulted from clinical studies is fully exploitedandshared.Indeed,thefieldprotectiveefficacy trialsdidnotincludeharvestingsamplesfor immunomo-nitoring[15,16].

Joining

efforts

to

profile

immune

and

molecular

signatures

of

rVSV-ZEBOV

The VSV-EBOVAC project (www.vsv-ebovac.eu)

sup-portedbyInnovativeMedicinesInitiative2Joint

Under-taking (IMI2 JU, http://www.imi.europa.eu/), was

launched to comprehensively characterize the immune

andmolecularsignaturesinducedinhumansbythe rVSV-ZEBOVvaccine[32].VSV-EBOVACseekstocarryout in-depth transcriptomics and metabolomics analyses of bloodsamplesobtainedatdifferenttimepointsfollowing

immunizationwithrVSV-ZEBOVinSwitzerland,Kenya

andGabon,harnessingstate-of-the-artandcuttingedge

technologies to carry out systems analysis of human

innateandadaptiveimmuneresponsestorVSV-ZEBOV.

Transcriptomic response is assessed in the RNA

extracted from blood stored in PAXgene tubes and

RNAsequencingisperformedusingtheIonAmpliSeqTM

Transcriptome Human Gene Expression Kit (Thermo

Fisher)onanIonProtoninstrument.Thisprotocolallows

the simultaneous quantification of transcripts from

20802humangenes [28].Geneexpression dataare cor-relatedwithclinicalandimmunologicdataandanalyzed usingthebloodtranscriptionmodulesidentified in pre-viousvaccinologystudies[27].Metabolomicanalysesare

performed onplasma samplesusingthe qTOFLC/MS

system(Agilent)which combinesaccuracy and

sensitiv-ity.TargetedmetabolomicsusingISQGC–MSplatform

(ThermoFisher)iscarriedoutforabsolutequantification ofalimitednumberofkeymetabolites.Integrativedata

analysis using a systems biology approach are being

employedto pinpointmolecularpatterns andpathways. Theunderlyingpremiseisthatresultsobtainedfromsuch integrated omics approach, combined with clinical and immunologicalread outs,couldlay afoundationforthe

discoveryofmolecularbiomarkersofrVSV-ZEBOV

vac-cinesafety andimmunogenicity.

Synergyand complementaritywithother Ebolavaccine

projectsas wellas with otherEU projectsactive in the fieldofsystemsvaccinologysuchastheHighImpactFP7

ProjectonAdvancedImmunizationTechnologies

(ADI-TEC, www.aditecproject.eu), aiming to accelerate the

developmentofnovel,powerfulimmunisation technolo-giesfornext-generationvaccines[33],arealsopromoted byVSV-EBOVAC.Itisexpectedthattheresultsobtained from this joint efforts will enhance and accelerate the

developmentof rVSV-ZEBOV as asafe andefficacious

vaccineto counterEbolainfection inhumans.

Concluding

remarks

rVSV-ZEBOVis currently the only Ebola vaccine with

demonstratedefficacyinaringvaccinationclinicaltrialas

well as excellent immunogenicity and safety. Current

immunogenicitydataare limitedtoadultresponses and theuseofserology, showing theimportantrole of anti-bodiesinprotectionwithemergingevidencesindicating thekeyinvolvementofinnateimmuneresponsesinthe

shaping of both immunogenicity and safety. Applying

cutting-edge technologies including transcriptomic and metabolomic analyses,and integratingwith clinical and

serological data is needed to fully decipher the

rVSV-ZEBOVvaccine immunesignature.

Acknowledgement

ThisworkwassupportedbytheInnovativeMedicinesInitiative2Joint

Undertaking(IMI2JU)undertheVSV-EBOVACproject[grantnumber

115842].IMI2receivessupportfromtheEuropeanUnion’sHorizon

2020ResearchandInnovationProgrammeandEFPIA.

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