Antibody-guided vaccine design : identification of protective epitopes

Antibody-guided

vaccine

design:

identification

of

protective

epitopes

Antonio

Lanzavecchia

,

Alexander

Fru¨hwirth

,

Laurent

Perez

and

Davide

Corti

Inthelastdecade,progressintheanalysisofthehuman immuneresponseandintheisolationofhumanmonoclonal antibodieshaveprovidedaninnovativeapproachtothe identificationofprotectiveantigenswhicharethebasisforthe designofvaccinescapableofelicitingeffectiveB-cell immunity.Inthisreviewweillustrate,withrelevantexamples, thepowerofthisapproachthatcanrapidlyleadtothe identificationofprotectiveantigensincomplexpathogens, suchashumancytomegalovirusandPlasmodiumfalciparum, andofconservedsitesinhighlyvariableantigens,suchas influenzahemagglutininandHIV-1Env.Wewillalsodiscuss howthegenealogicalanalysisofantigen-stimulatedBcell clonesprovidesthebasistodelineatethebestsuitable prime-boostvaccinationstrategyfortheinductionofbroadly neutralizingantibodies.

Addresses

1_{InstituteforResearchinBiomedicine,ViaVincenzoVela6,6500} Bellinzona,Switzerland

2

InstituteofMicrobiology,ETHZu¨rich,Vladimir-Prelog-Weg1,8093 Zu¨rich,Switzerland

3_{HumabsBioMed,ViaMirasole1,6500Bellinzona,Switzerland}

Correspondingauthor:Lanzavecchia,Antonio(lanzavecchia@irb.usi.ch)

CurrentOpinioninImmunology2016,41:62–67 ThisreviewcomesfromathemedissueonVaccines EditedbyRinoRappuoliandEnnioDeGregorio ForacompleteoverviewseetheIssueandtheEditorial

Availableonline22ndJune2016

http://dx.doi.org/10.1016/j.coi.2016.06.001

0952-7915/#2016TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense( http://creative-commons.org/licenses/by-nc-nd/4.0/).

Introduction

Whenourimmunesystemischallengedbyaninfectious

agent,apolyclonalantibodyresponseisgeneratedagainst

multipleproteinandnon-proteinantigens.Theextentof

theresponsereflectstheimmunogenicityofthe

individ-ualcomponents,whichisdeterminedbymultiplefactors

such as their abundance, their complexity and their

capacity to bind to cellular receptor and trigger innate

immunity, notto mention the influenceof pre-existing

immunity. In general, only a fraction of theantibodies

produced exerts protective activity by binding to

moleculesthat arerequired for invasion orvirulence or

byelicitingeffectormechanisms.Forinstance,thelargest

fraction of antibodies produced in response to a virus

infectionisdirectedagainstinternalorsurfaceproteinsin

adenaturedorpost-fusionconformationandistherefore

devoidof neutralizingactivity[1,2,3].Thus,the

immu-nogenicityof the most abundant proteins offers to the

pathogen the opportunity to limit the most effective

responsebyamechanismofantigenic competition.

In this review we discuss how recent advances in the

characterizationoftheneutralizingantibodyresponseto

humanpathogens,combinedwith advancedprotein

en-gineeringmethods,haveprovidedanewwaytosolvethe

problem of antigenic competition and have led to the

production of vaccine candidates that elicit antibody

responsesofhigh magnitudeandspecificactivity.

Analytic

vaccinology:

the

cases

of

HCMV

and

malaria

Humancytomegalovirus (HCMV)is a herpesvirus that

establishesalifelonginfectioninhealthyindividuals,but

causesseriouspathologyinthefetusandin

immunosup-pressed patients and has been associated with immune

senescenceandatherosclerosis[4].HCMVusesmultiple

glycoproteincomplexesforbindingandfusiontohostcells

andhasabroadcelltropism,beingabletoinfectfibroblasts

aswellasepithelial,endothelialandmyeloidcells[5].The

fusionproteingB hasbeen consideredthemost obvious

vaccinecandidatebutclinicaltrialswithrecombinantgB

(inthepost-fusion conformation)hasshownlimited

effi-cacy[6].ToidentifythemostpotentHCMVvaccine,an

analytic vaccinology approach was used to isolate, from

memoryBcellsofnaturallyinfecteddonors,alargepanel

of monoclonal antibodies selected for their capacity to

neutralize HCMV infection of multiple cell types [7]

(Figure 1). This approach led to the identification of a

newclassof antibodiesthatwere1000foldmorepotent

thatantibodiestogBin neutralizingHCMVinfectionof

epithelial,endothelialandmyeloidcells.Theseantibodies

weremappedtoninedistinctsitesonthe

gH/gL/UL128-131Acomplex,apentamericcomplexthatwaspreviously

foundtoberequiredforinfectionofthosecelltypes[8].

Theidentificationofthepentamerasthetargetofthemost

potentantibodieswassubsequentlyconfirmedbyseveral

studies[9,10,11].Asan example,asolublepentamer

producedbystablyatransfectedCHOcelllineelicitedin

and thatwere 300-1000fold higherthan those foundin

individualsthatrecoveredfromprimaryHCMVinfection

[10].Importantly,theantibodieselicitedbythe

penta-mer vaccine prevented cell-to-cell spread and viral

dis-semination from endothelial cells to leukocytes and

neutralizedinfectionofbothepithelialcellsandfibroblasts

duetotheproductionofantibodiestothegHglycoprotein,

whichisrequiredfor fibroblastsinfection.

Thetarget-agnosticapproachcanbeparticularlyusefulto

identify targets in complex pathogens such as bacteria

andparasites.Aninterestingexampleregardsthe

identi-fication of variant surface antigens (VSAs) which are

present on the surface of Plasmodium falciparum

(Pf)-infectederythrocytesandmediateadhesionto

endothe-lia,leadingtopathology.TheVSAsareencodedbymore

that 200 genes that are polymorphic and clonally

Figure1

1

2

3

1 Analytic vaccinology

2 Structu

re-based

vaccinology

3 Epitope-focused

vaccinol

ogy

Current Opinion in Immunology

Antibody-guidedvaccinedesign.Humanmonoclonalantibodiesisolatedfromimmunedonorsareusedtoidentifyprotectiveantigensand epitopes.Theantigensdiscoveredfromcomplexpathogensareproducedasrecombinantproteins(path1;e.g.HCMVpentamer)and,when necessary,engineeredforincreasedstability(path2;e.g.stabilizedpre-fusionHRSVFprotein)ormodifiedtoexpressparticulardomainsor epitopes(path3;e.g.head-lessHAorPalivizumabepitopedisplayedonVLPs).

expressed,thus providingthepathogenwithapowerful

mechanismofescapefromtheantibodyresponse.Human

monoclonalantibodiesisolatedfrommultiparouswomen

have been used to identify VAR2CSA as the primary

targetof antibodies that protect from placental malaria

[12].In amore recentstudy, Tan etal. isolated several

antibodiesthatbroadly reactwith erythrocytes infected

with differentPf isolatesand identified the target

anti-gensas distinct RIFINs[13].Surprisingly, these

anti-bodies acquired their broad reactivity through a novel

mechanismofinsertionofalargeDNAfragmentbetween

the V and DJ segments. The insert originates from

chromosome 19 and encodes the extracellular domain

of the collagen-binding inhibitory receptor LAIR-1,

which is both necessary and sufficient for binding to

RIFINs.Importantly,theLAIR-1domaincarriessomatic

mutationsthat abolishbinding to collagenandincrease

binding to infected erythrocytes. These findings

illus-trate,withabiologicallyrelevantexample,anovel

mech-anism of antibody diversification and demonstrate the

existence of conserved epitopes as candidates for the

developmentofamalariavaccine[14].

Structure-based

vaccinology:

the

case

of

HRSV

Humanrespiratory syncytial virus (HRSV)is the most

prominentviralagentofpediatricrespiratoryinfections.

Currently, the onlytreatment availableis Palivizumab

[15],ahumanizedmonoclonalantibodythatbindstoan

epitope that is conserved in the pre-fusion and

post-fusion conformation of the HRSV F protein [16]. On

the basis of the properties of Palivizumab, the initial

effortstowards anHRSV vaccine were focusedon the

use of the post-fusion F-protein that was engineered

to increase its solubility, resulting into highly stable

immunogen forms, that were tested either as soluble

antigens or displayed onto virus-like particles (VLPs)

[17,18].

Tworecentstudiesusedthestructuralinformationonthe

Palivizumablinearepitope,a24aminoacidhelix–loop–

helix structure, to develop epitope-focused vaccines.

Correiaet al.usedcomputationalproteindesignto

gen-eratestable protein scaffolds thataccurately mimicthe

Palivizumab epitope and, when chemically linked to

VLPs,induceHRSV-neutralizingantibodiesinmacaques

[19].Inasubsequentstudy,Milichandcoworkersused

anempiricalapproachbydirectlyinsertingthe

Palivizu-mabepitopeinVLPscomposedofwoodchuck

hepadna-virus core(WHcAg) protein, which were then selected

with the antibody and found to be immunogenic in

rodents [20]. While these studies provide for the first

timeaproof-of-principleforepitope-focusedvaccinology

(Figure1),itisnotevidentwhatcouldbetheadvantage

of limiting the response to a single epitope, given the

presence of several conservedepitopes in theHRSV F

protein.

The analysis ofthe humanantibodyresponse to HRSV

[21,22]andtheisolationofhumanneutralizing

monoclo-nalantibodies [23,24] showedthatthe largemajority of

HRSVneutralizingantibodieselicitedbynaturalinfection

arespecificforthepre-fusionFproteinconformationand,

unlikePalivizumab,donotcross-reactwiththepost-fusion

conformation.Onthebasisof theseobservationsandon

thecrystalstructureofthepre-fusionFproteinincomplex

withaneutralizingantibody[25],Kwongand coworkers

usedstructure-baseddesigntoproduceastabilizedHRSV

F protein through the introduction of cysteineresidues

and the filling of hydrophobic cavities [26,27]. This

protein(dubbedDS-Cav1)maintainedbindingtopotent

neutralizingantibodiesandwasabletoelicitinmiceand

macaques levels of HRSV-neutralizing antibodies that

exceededtheprotectivethreshold.Theseresultsprovide

aclearexampleofstructure-drivenvaccinologyto

gener-atestableimmunogenscapableof inducingapolyclonal

responsetomultiple neutralizingepitopesofviralfusion

proteins,overcomingtheproblemoftheirintrinsic

insta-bility(Figure1).

Epitope-focused

vaccinology:

the

cases

of

influenza

A

InfluenzaAandHIV-1aretheprototypesofvirusesthat

evade the antibody responseby continuously mutating

surfaceglycoproteins.However,certainsitesare

relative-lyconserved since they arerequired for infectivity and

fusionandcanbethereforeexploitedtodesignvaccines

capableofinducingbroadprotection.Inthelastdecade

theisolation of broadly neutralizingantibodieshas

pro-videdapowerfulplatformtoidentifysuchepitopes,thus

speedingupvaccinedesignefforts[28,29].

Thesialicacidbindingpocketofinfluenzahemagglutinin

(HA) is a conserved site that can be targeted by

anti-bodies,suchas CH65,thatcarryatthetipof HCDR3a

distinctmotifthatmimicssialicacid[30].Importantly,

theseantibodiesarisefromdiversegermlineoriginsand

affinity maturation pathways, suggesting that such an

antibodyresponse couldberapidlygenerated by

appro-priateimmunogens.However,thebreadthofthese

anti-bodiesissomewhatlimitedduetothepresenceofhighly

variableresiduesthatflankthereceptorbindingsite.

TheHAstemisamoreconservedregionwhichhasbeen

shownto berecognizedbydifferentfamiliesof broadly

neutralizingantibodiesthatrecognizemultiplesubtypes

ingroup1[31–34],group2[35]and,insomecases,both

group 1 and group 2 influenza A viruses [36,37,38].

Recently, Pappas et al. showed that most individuals

makeantibodiesthatbroadlyrecognizegroup1subtypes.

ThispublicantibodyresponsereliesexclusivelyontheH

chain,whichisencodedbyVH1-69alleleswith

phenyl-alanineatposition54andashortHCDR3withtyrosineat

position98.Interestingly,throughthe reconstructionof

found that high affinity binding was achieved in most

casesbyasinglemutation[39].Thesefindingsexplain

thehighfrequencyofantibodiesspecificforthestemof

group1HAs.Incontrast,antibodiesthatrecognizeboth

group 1 andgroup 2 HAs are muchless frequentsince

they use differentVH/VL genes. Furthermore they are

generated through a complex developmental pathway,

beingfirstselected for theirgerm-linereactivity against

group 1 and subsequently acquiring group 2 reactivity

throughsomaticmutations[36].Thelatterfinding

sug-geststhattherarepan-influenzaAneutralizingantibodies

might be elicited using an appropriate heterologous

prime-boost strategy.

TheidentificationoftheHAstemasaconservedsiteof

influenza HAhas raisedthepossibilityofdevelopinga

universalinfluenza vaccine [40].Two approacheshave

been developed towards thisambitious goal. Thefirst

involvesaprime-booststrategyusingchimericHA

mole-cules carryingthe samestem combined with

heterolo-gousheads[41,42]andisbasedontheclassicalprinciple

oftheoriginalantigenicsinthatrepresentsinthiscasea

natural mechanism of immunofocusing. The second

approach of epitope-focused vaccinology involves the

productionofheadlessHAs [43](Figure1).Inarecent

report, Yassine et al.used iterative cycles of

structure-baseddesign toproduceastabilizedstemimmunogen,

HA-SS,geneticallyfusedtoferritinnanoparticles,which

is recognized by stem-specific monoclonal antibodies

and elicits, in mice and ferrets, broadly cross-reactive

antibodies[44].Ingeneral,theassembliesofmultiple

copiesofsubunitantigensinwell-orderedarrays,suchas

in the case of ferritin orVLPs, offer the advantage of

mimickingtherepetitivenessofmostnaturalpathogens

surfaceproteinspotentiallyprovidingimprovedantigen

stability and immunogenicity [45,46]. In an another

study,Impagliazzoetal.usedarationaldesigncombined

withalibraryapproach togenerateaHAstemantigen

called mini-HAthat protectsmiceandnon-human

pri-matesfromlethalityandsymptoms[47].Itshouldbe

noted that in both studies the neutralizing antibody

levels induce by the vaccine were modest, suggesting

thatinvivoprotectionmaybeachievedthrougha

com-bination of neutralization and Fc-dependent effector

function, which is known to be elicited by anti-stem

antibodies [36,48,49]. The headless HA vaccine has

been shown to workfor group 1 influenza viruses and

should beextendedtogroup2 viruses,andpossiblyto

influenza B, in order to fully realize the dream of a

universalinfluenzavaccine.

Factors

that

limit

the

antibody

response:

the

challenge

of

HIV-1

Aneffectiveantibodyresponseislimitedbythe

frequen-cy ofantigen-specificBcells in thenaı¨ve repertoire,by

thenumberofmutationsrequiredtoreachhighaffinityor

breadth,andbytheavailabilityofTcellhelp.Thus,an

idealvaccineshouldcontainasufficientlyhighnumberof

T and B cell epitopes and should be formulated to

effectively prime the germinal center reaction [50]. In

addition,thefindingthatpreexistingimmunitycanshape

theantibodyresponsetonewstructurallyrelatedantigens

[51–53]suggestsmodalitiesofprime-boostimmunization

to optimizetheantibodyresponse[54].

Recent technological advances are increasinglyused to

investigate thedevelopmental pathways leading to

po-tentandbroadly neutralizingantibodies.Theseinclude

theindepthanalysisoftheantibodyresponsethroughthe

isolationofmultiplecellswithinthesamelineage,

possi-bly implemented by next generation sequencing and

deconvolutionoftheserumantibodyrepertoirethrough

LC–MS/MSanalysis[39,55,56].Thesystematic

recon-structionofthegenealogytreesofantibodylineageshas

shownthatneutralizingantibodiestoinfluenzaorHRSV

mature rapidly [39] or may not even need affinity

maturation, while somatic mutations are critical to

achieve breadth[23,36].Thesituationisstrikingly

dif-ferentinthecaseofHIV-1,wherevirusesandBcells

co-evolve over a period of years, leading to a slow and

infrequent development of broadly neutralizing

antibo-dies[57].Inthelastdecademanynewbroadly

neutraliz-ing antibodies of high potency have been isolated and

newsitesofvulnerabilityontheEnvproteinhavebeen

defined.Importantly,inthelastfewyearscryo-EMand

crystallographywereusedtofinallysolvethestructureof

a stabilized soluble Env trimer molecule (i.e. BG505

SOSIP), alone or in combination with several broadly

neutralizingantibodies[58–60].Theseconstructsarenow

considered a relevant mimic of the native functional

trimer being recognized preferentially by neutralizing

antibodies. The HIV-1 broadly neutralizing antibodies

are found only in a fraction of infected individual and

have a slow and complex developmental pathway or

derive from very rare precursors characterized by

ex-tremely long HCDR3. These observations have led to

the proposal of a prime-boost strategy with different

antigens that target the naı¨ve precursors and different

stagesoftheantibodydevelopmentalpathwaydefinedas

‘Bcell-lineage vaccinedesign’[61].

These hurdles represent a significant challenge to the

development of an effective HIV-1 vaccine. However,

these efforts contributed to the development of novel

vaccine design strategiesthatrepresentafertile ground

for significant advances in the generation of new and

bettervaccines againstotherpathogens.

Acknowledgements

WethankSiroBianchiforthehelpinthepreparationofthefigure.

TheworkinLanzavecchia’slaboratoryissupportedbytheEuropean ResearchCouncil(grantno.250348IMMUNExploreand670955 BROADimmune),theSwissNationalScienceFoundation(grantno. 160279),theSwissVaccineResearchInstitute.

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