Immunity

Article

Selective CD4

T Cell Help for Antibody Responses to a Large Viral Pathogen:Deterministic Linkage of Speciﬁcities

Alessandro Sette,

Magdalini Moutaftsi,

Juan Moyron-Quiroz,

Megan M. McCausland,

D. Huw Davies,

Robert J. Johnston,

Bjoern Peters,

Mohammed Raﬁi-El-Idrissi Benhnia,

Julia Hoffmann,

Hua-Poo Su,

Kavita Singh,

David N. Garboczi,

Steven Head,

Howard Grey,

Philip L. Felgner,

and Shane Crotty

Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology (LIAI), La Jolla, CA 92037, USA

Division of Infectious Diseases, Department of Medicine, University of California, Irvine, CA 92697, USA

DNA Array Core Facility and Consortium for Functional Glycomics, the Scripps Research Institute, 10550 N Torrey Pines Road, La Jolla,CA 92037, USA

Structural Biology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Twinbrook 2, 12441 Parklawn Drive, Rockville, MD 20852, USA *Correspondence:shane@liai.orgDOI 10.1016/j.immuni.2008.04.018

SUMMARY

Antibody responses are critical components of pro-tective immune responses to many pathogens, butparameters determining which proteins are targetedremain unclear. Vaccination with individual MHC-II-restricted vaccinia virus (VACV, smallpox vaccine)epitopes revealed that CD4

T cell help to B cellswas surprisingly nontransferable to other virion pro-tein speciﬁcities. Many VACV CD4

T cell responsesidentified in an unbiased screen targeted antibodyvirion protein targets, consistent with deterministiclinkage between specificities. We tested the deter-ministic linkage model by efficiently predicting newvacciniaMHCIIepitopes(830%improvedefficiency).Finally, we showed CD4

T cell help was limiting for neutralizing antibody development and protectiveimmunity in vivo. In contrast to the standard model,these data indicate individual proteins are the unitofBcell-Tcellrecognitionforalargevirus.Therefore,MHCrestrictionisakeyselectiveeventfortheantivi-ral antibody response and is probably important for vaccine development to large pathogens.

INTRODUCTION

Vaccines are one of the most cost-effective medical treatmentsin modern civilization ( Rappuoli et al., 2002 ). Vaccinia virus(VACV) is the viral species used as the human smallpox vaccine.The smallpox vaccine has been extraordinarily effective, havingbrought about the worldwide eradication of smallpox disease( Fenner et al., 1988 ). The smallpox vaccine is generally consid-ered the gold standard of vaccines, and elucidating the immu-nobiology underlying the protection provided by the smallpoxvaccine will continue to reveal vaccinology principles that canbe applied to future vaccine development against other infec-tious scourges. However, identifying and analyzing the finespecificities of the adaptive immune response to a large patho-gen—suchasapoxvirus—isconfoundedbyanumberoffactors,notleastofwhichisthestarkmagnitudesofthepotentialpeptidetargetsoftheTcellresponsesandthepotentialproteintargetsof theantibody responses.Asaresultofthesechallenges,wepos-sess only a piecemeal understanding of the fine specificities of Tcellandantibodyresponsestoanylargepathogenandthereforehave a thin understanding of the roles of each fine specificity inprotective immunity, thereby limiting our ability to rationallydesign new vaccines against large and complex pathogens. Although neutralizing antibodies are of primary importance inthe protection from smallpox provided by the smallpox vaccinein animal models ( Belyakov et al., 2003; Edghill-Smith et al.,2005; Galmiche et al., 1999; Lustig et al., 2005 ) and humans( Amanna et al., 2006; Demkowicz et al., 1992 ), CD4

T cellsand CD8

T cells are also of great value ( Amanna et al., 2006;Fang and Sigal, 2005; Tscharke et al., 2005; Xu et al., 2004 ).Here, we have focused on understanding the relationship be-tween antibody and CD4

T cell responses to vaccinia virus inmice, as part of a strategy to elucidate the value of individualfine specificities, potential interrelationships between thosespecificities, and the underlying immunobiological and virologi-cal parameters that determine the emergence of protectiveimmune responses to a small subset of all possible specificities.

RESULTSExquisitely Selective Antigen-Speciﬁc T Cell Help

Infection of mice with VACV results is an acute infection charac-terized by several days of high viral replication and viral loadsof>10

infectiousvirions;thisisfollowedbyastrongadaptiveim-mune response and viral clearance in 1–2 weeks ( Amanna et al.,2006; Harrington et al., 2002; Xu et al., 2004 ). IgG responses toVACV are fully dependent on CD4

T cell help, as shown by theabsence of VACV IgG in MHC-II-deﬁcient mice ( Figure 1 A andXuetal.[2004] ).Werecentlyidentiﬁed 14 VACVMHCII epitopesafter VACV infection of B6 mice ( Moutaftsi et al., 2007 ). CD4

Tcells of each speciﬁcity expressed CD40L after stimulation withcognatepeptide ( Figure1B),indicating theircompetencetopro-vide B cell help. In an effort to boost the antiviral antibody

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responses to VACV infection, we increased the available CD4

TcellhelpbyimmunizingmicewiththeVACVI1

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MHCIIepitope(oneofthefirstepitopesidentifiedfromaVACVvirionprotein),in-fecting the mice with VACV, and finally monitoring the subse-quent antiviral antibody responses. Vaccinating with I1

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MHC II epitope resulted in a strong 10-fold increase in the totalVACV antibody response, as measured by a standard VACVELISA ( Figure 2 A). Unexpectedly, virus neutralizing antibodytiters were unimproved in VACV-infected mice preimmunizedwith I1

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when compared to unprimed mice ( Figure 2B). Al-thoughI1isaviralvirioncoreproteinandthereforenotitselfaneu-tralizingantibodytarget,Il-speciﬁcCD4

Tcellswereexpectedtoprovide intermolecular help to all B cells specific for VACV viralparticle proteins and thereby boost neutralizing antibody titers( Janeway et al., 2005 ). Surprisingly, when serum samples wereprobed for the detailed antigen specificities of the antibody re-sponsewithvacciniaproteinmicroarrays,wefoundtheincreasedantibodyresponsewas exclusively against I1and not other VACVproteins( Figures2C–2H).IgGspecificforvirioncoreproteinI1wasincreased 1930% (p < 0.0004) ( Figure 2E). A10 is a second majorcoreproteinpresentalongwithI1inVACVvirions,andA10isatar-get of the antiviral IgG response in infected B6 mice (see below).Neverthelessthe strength of the anti-A10 IgG responsewas unal-teredinI1MHCIIepitopevaccinatedmice( Figure2F).TheIgGre-sponsestotwovacciniasurfacemembraneproteinvirioncompo-nents,D8andH3,alsoexhibitednoimprovement( Figures2Gand2H).Selectiveincreaseinanti-I1IgGwasstillobservedatamemorytimepoint( Figure2I).Thus,antigen-specificCD4

Tcellsappeared

Figure1. VacciniaVirusCD4

HelperTCellsand Helper T Cell Dependent Antibodies

(A) Quantitative ELISA of anti-VACV IgG (

g/ml),day 30 after infection in wild-type (WT) and MHCII-deﬁcient (

H2-Ab

) mice. The graph showsmean ± SEM.(B) Splenocytes from day 10 VACV

-infectedmice were incubated with CD11c

dendritic cells(DCs) pulsed with VACV peptides (L4, J4, etc.,names corresponding to the VACV protein fromwhich each peptide epitope was derived). Cellswere incubated for 6 hr and then stained for intra-cellularIFN

andCD40L.GatedCD4

CD62L

lym-phocytes are shown, and percentages quantiﬁedare IFN

CD40L

of CD4

CD62L

. DCs infectedwith VACV

(MOI = 5) for 2 hr prior to addition of splenocyteswereusedforquantiﬁcationofthetotalanti-VACV CD4

T cell response (bottom right,‘‘VACV

DCs’’). Background levels were deter-minedwithuninfectedDCs(‘‘neg’’).Low-frequency A28-speciﬁc response was only detectable byELISPOT (not shown). Results are representativeof three (A) and four (B) independent experiments.

to provide help only to B cells of pairedprotein speciﬁcity, not virion speciﬁcity.

Antibody Response SelectivityDriven by Antigen-Speciﬁc CD4

T Cells

To directly establish the role of antigen-speciﬁc CD4

T cells in the selective tar-geting of individual viral virion proteins for antibody responses,we performed CD4

T cell adoptive-transfer experiments.CD4

T cells were puriﬁed from donor mice immunized withI1

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MHC II epitope, and cells were transferred into unimmu-nized recipient mice. Recipient mice were infected with VACVand examined for VACV antibody responses. Mice receivingprimed CD4

T cells responded with a dramatic increase inanti-I1 IgG (1210% enhancement, p < 0.0001) ( Figures 3 A–3C),whereas the antibody responses to other viral virion proteinspeciﬁcities remained unchanged ( Figures 3 A, 3B, 3D, and 3E),starkly ignorant of the CD4

T cells transferred from the VACVI1

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-vaccinated mice.This observation of paired targeting by the CD4 T cell and an-tibody response indicates that CD4

T cell help is preferentiallyprovided to B cells with the identical protein speciﬁcity, eventhough the viral particle exists as a solid physical structure as-sembled from greater than 75 distinct viral protein components( Chung et al., 2006; Condit et al., 2006; Moss, 2001; Reschetal.,2006 )andthisstructureisexpectedtofunctionasauniﬁedimmunologicaltargetforBcellsandBcell-Tcell(B-T)interaction( Janewayetal.,2005;Milichetal.,1987;RussellandLiew,1979;Scherle and Gerhard, 1986 ). Naked unwrapped viral nucleopro-tein core particles are released from dying infected cells andwould be expected to function in the same manner.To determine whether these I1 core protein results were gen-eralizable, we tested the properties of several additional VACVCD4

T cell responses shown inFigure 1. We chose MHC II epi-topes from H3 and D8 because these major virion surface

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transmembrane proteins are key IgG targets ( Amanna et al.,2006; Davies et al., 2005b ) and are ostensibly recognized by Bcells as prominent components of the surface of whole virions.Inaddition, wetestedthevacciniaL4

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MHCIIepitope,rep-resenting a second major viral particle core component, tocom-

Figure 2. Selective Protein-Speciﬁc CD4

T Cell Help to B Cells after VACV Infection

Mice peptide vaccinated with VACV I1

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MHC IIepitope were then infected with vaccinia virus. (A)shows Vaccinia-speciﬁc IgG responses in VACV-infected mice primed with adjuvant alone (‘‘mockprime,’’ open circles), I1-primed mice subse-quently infected with VACV (squares), I1-primedonly mice (‘‘X’’ symbol), and uninfected controlmice (‘‘I’’ symbol) were measured by ELISA. n =4/group. Error bars represent ± SEM. As shownin (B), virus neutralizing antibody titers (PRNT

)were measured in I1-primed and unprimed mice(p > > 0.05). I1

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MHC II peptide-primed mice(‘‘+,’’ n = 4) and mock-primed mice (‘‘

,’’ adju-vant-only prime; n = 4) were tested after VACV in-fection.Shownismean±SEM.(C)–(I)showresultswith sets of VACV proteins were synthesized andprinted in microarray format for generation of VACV proteome arrays that could be probedwith serum samples (seeExperimental Proce-dures ). VACV proteome microarrays were probedwith serum day 7 after VACV infection from miceprimed with I1

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MHC II epitope or mockprimed.(C)and(D)showIgG responses ofindivid-ual representative mice (C) primed with I1

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MHC II epitope or (D) mock primed. Stars indicatethe anti-I1 signal. Error bars indicate range of rep-licates. (E)–(H) show antibody responses to indi-vidual vaccinia-virus protein determinants afterVACV

infection in groups of I1

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MHC II pep-tide-primed mice (‘‘+,’’ n = 4) and mock-primedmice (‘‘

,’’ adjuvant-only prime. n = 4). Quantita-tion of anti-I1 IgG ([E], p < 0.0004), anti-A10 IgG([F], p >> 0.05), anti-D8 IgG ([G], p >> 0.05), andanti-H3 IgG ([H], p >> 0.05) concentrations weredetermined. Graphs show mean ± SEM (I) IgG re-sponses in I1-primed and not primed mice at day30 after VACV infection. Anti-I1 IgG (p < 0.02),anti-A10 (p >> 0.05), and anti-D8 (p >> 0.05) re-sponses were measured. *p < 0.05, **p < 0.01,and ***p < 0.001. Data are representative of ﬁveindependent experiments.

pare with the responses to the I1 coreprotein. Antiviral IgG responses were en-hanced after VACV infection of miceprimed with any one of the three newVACV MHC II epitopes ( Figure 4 A).VACV proteome microarray serologicalanalysis again revealed a remarkable se-lectivity of the antibody response in pep-tide-vaccinated animals. In H3

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MHC-II-epitope-primed animals, anti-H3IgG was increased dramatically (48-foldincrease, p < 0.0001) ( Figure 4B). H3 isa known target of neutralizing antibodies( Davies et al., 2005b; Lin et al., 2000 ),and virus neutralizing antibody titers were selectively increasedin mice with H3-speciﬁc CD4

T cells ( Figure 4C, p < 0.02). Anti-A10 IgG concentrations were unaltered ( Figure 4B), con-ﬁrming and expanding the results ﬁrst obtained for I1 ( Figure 2 ). Anti-H3 IgM concentrations were also selectively increased

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