A second neutralizing epitope of B19 parvovirus implicates the spike region in the immune response.

Vol.65,No. 12

A

Second Neutralizing Epitope of

B19

Parvovirus Implicates the

Spike Region

in the Immune

Response

KOHJIYOSHIMOTO,1 STEPHEN ROSENFELD,1 NORBERTFRICKHOFEN,' DOUGLAS KENNEDY,2 ROBERTHILLS,3 SACHIKO KAJIGAYA,1AND NEAL S.

YOUNG'*

CellBiologySection, ClinicalHematology Branch, NationalHeart, Lung, and BloodInstitute, Bethesda, Maryland 20892,1 and Bureau ofBiologics, Drugs Directorate,2 and BureauofChemicalSafety,

Food Directorate,3 Ottawa, Ontario KJA OL2, Canada

Received12 July 1991/Accepted 16 September 1991

We used 18 monoclonal antibodies against B19 parvovirus to identify neutralizing epitopeson the viral

capsid. Of the 18 antibodies, 9 had in vitro neutralizing activityin abonemarrowcolony culture assay.The overlapping polypeptide fragments spanning the B19 structural proteins were produced in a pMAL-c

Escherichia coliexpressionsystemandusedtoinvestigatethebinding sitesof the neutralizing antibodies. One of thenine neutralizing antibodies reacted withboth VP1 andVP2capsid proteins and asingle polypeptide

fragmenton animmunoblot,identifyingalinearneutralizing epitope between amino acids 57 and 77 of the VP2

capsid protein. Eight of nineneutralizing antibodies failedtoreactwith eitherofthe capsidproteinsor any

polypeptide fragments, despite reactivities with intact virions in a radioimmunoassay, suggesting that

additionalconformationally dependent neutralizing epitopes exist.

Parvoviruses, the smallest DNA-containing animal

vi-ruses, are responsible for a wide variety of diseases in

vertebrates (18). In humans, diseases caused by B19

parvo-virusinclude erythema infectiosum (fifth disease) in children andapolyarthralgia syndrome in adults (20), transient

aplas-tic crisis inpersonswithunderlyinghemolysis (15),

sponta-neousabortion and hydrops fetalis inuteroinfection (1, 2, 9), andpersistent infection in immunocompromised hosts like patients with AIDS and those receiving chemotherapy for leukemia (11).

Theperiod of viremia is short in individuals with normal immune function and ends with formation of specific anti-bodies to the virus (16). In persistent infection, pooled humanimmunoglobulin is effective therapy (6). Despite the evident importance of antibody for viral clearance and resolutionofinfection, the humoralresponsetoB19 hasnot been well characterized and only a single neutralizing

epitope has been reported (17). We studied the reactivities of

a panel of anti-B19 neutralizing monoclonal antibodies

(MAbs) by using bacterium-expressed polypeptide frag-ments of the B19 capsid proteins. We identified a second

neutralizing epitope on the VP2 capsid protein. However, most of the MAbs appeared to recognize conformational ratherthanlinearepitopes.

MAbsweremadebyamodifiedcell fusionmethod(7, 10).

BALB/c mice wereimmunized intraperitoneally with 50 pLg

ofpurified B19 parvovirus in Freund's complete adjuvant and thenreceivedtwoboosterimmunizations of the antigen in Freund's incomplete adjuvant at 14-day intervals with a

trial bleeding atday 40. The mice subsequently received a

boosterintravenously 4 days before cell fusion. Spleen cells from the mice were fused with murine myeloma cell line Sp2/0-AG14 (AmericanType Culture Collection, Rockville,

Md.) bypolyethylene glycol 1500 (Sigma ChemicalCo., St.

Louis, Mo.). The hybridomas were cultured in Dulbecco's

modified Eagle medium containing 20% fetal bovine serum,

0.1 mM hypoxanthine, 0.4 ,uM aminopterin, 16 puM

thymi-*Corresponding author.

dine(HAT medium supplement; Sigma), and feeder layers of MEA(continuous BALB/ctissuegammairradiatedat12,000 rads), and 21 days after cell fusion, the supernatants of hybridomas were tested by radioimmunoassay against B19

parvovirus (3). For theradioimmunoassay, 6.4-mm-diameter polystyrene beads were coated by immersion ingoat anti-humanmuchain (Tago, Inc., Burlingame, Calif.) diluted in

0.05 M carbonate-bicarbonatebuffer, pH 9.6, atroom tem-perature for 3 h, and then washed withphosphate-buffered salinecontaining 0.05% Tween 20(PBST). The beads were

incubated for3 hat37°Cwith200 pul of humanserumknown tobeanti-B19parvovirus immunoglobulin M(IgM)antibody positive. Afterwashing with PBST, 200 p1ofpurifiedvirus,

diluted in PBST containing 10% fetal bovine serum to its working concentration, was added to the beads for 3 h at 37°Candtheywerewashedagain. The beadswereincubated with 200 ,ul of hybridoma supernatant for 18 h at room temperature and then incubated with

1251I-labeled

sheep anti-mouse immunoglobulin (Amersham Corp., Arlington Heights, Ill.)asthe secondantibodyatroomtemperaturefor 1 h. After washing of the beads, the radioactivity was

measured with a Gamma 4000 gamma radiation counter (BeckmanInstruments, Inc., Fullerton, Calif.).

Asummaryof the MAbs derivedfrom cell fusion is shown

in Table 1. Eighteenclones, named MAbs A, B, C, D, E, F, G, H, I, K, L, M, N,0,P, Q, R,S,reactive with B19 inthe

radioimmunoassay were isolated and cloned twice by the

limiting-dilution method. Established hybridoma clones

wereinjected into the peritoneal cavities ofPristane-treated

BALB/c mice, and ascites fluids were collected. Antibody

subclasses were determinedby enzyme-linked

immunosor-bentassayusingaHybridomaSubisotyping kit (Calbiochem

Co., SanDiego, Calif.).

In vitro neutralizing activity was assessed by measuring

the ability of the antibodytopreventvirus-induced toxicity

inaCFU-Eassay(2, 12). Serum obtained fromapatient with

sickle cell diseaseand transientaplastic crisisduringarecent B19 parvovirus epidemic was used as a virus source, and bone marrow mononuclear cells derived from normal

indi-vidualswereusedastargetcells. Asanegativecontrol, cells 7056

JOURNALOF VIROLOGY, Dec. 1991,p. 7056-7060 0022-538X/91/127056-05$02.00/0

CopyrightC 1991, AmericanSocietyforMicrobiology

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TABLE 1. Summary of MAbs

MAb Subclass CFU-Ea

(%)Reactivity

in immunoblot

A IgG2a 0 NDb

B IgG2a 92 None

C IgGl 0 ND

D IgGl 100 None

E IgGl 65 VPF6, SF3

F IgGl 82 None

G IgGl 0 ND

H IgGl 0 ND

I IgGl 0 ND

K IgGl 108 None

L IgG2a 0 ND

M IgGl 0 ND

N IgG2b 5 ND

0 IgGl 0 ND

P IgGl 81 None

Q IgGl 95 ND

R IgG2a 90 None

S IgGl 88 ND

aTheCFU-E assay results are means of each three experiments at an

antibody dilution of 1:75. % CFU-E was calculated with following formula % CFU-E = [colony number/(colony number of negative control - colony number ofpositivecontrol)] x 100.

bND, not done.

wereculturedwithout virus, andas apositivecontrol, cells werecultured with virus alone. With this assay, 9ofthe 18 clones (B, D, E, F, K, P, Q,R, and S)were neutralizingat titers between 1:150 and 1:3,750. Theneutralizing activities were between 65 and 108% at an antibody dilution of 1:75 (Table 1).

To determine the epitopes recognized by neutralizing antibodies, the recombinant VP1 and VP2 capsid proteins and the 11fusion proteins containing B19 parvovirus struc-tural protein polypeptide fragments were prepared and

immunoblotting

wasperformed. Preparation of the recombi-nantVP1 andVP2capsidproteinswasdescribedpreviously (8). The expression vectors were constructed as follows. DNA.segments encoding 11 recombinant proteins were amplified from pYT103c (a nearly full-length B19 cloned DNA) withadequateprimers by polymerase chain reaction. Polymerase chain reactions were performed by

using

a

standard protocol in a total volume of 100 ,ul

containing

polymerase chain reaction buffer (10mMTris-HCl[pH 8.3], 50 mM KCI, 1.5 mM

MgCl2,

0.01%

gelatin),

the four deoxynucleoside

triphosphates

at200,uMeach,2.5UofTaq

polymerase (Perkin

Elmer

Cetus, Norwalk,

Conn.),

ade-quate primers (21-mer), and pYT103c (a template). After purification of the

amplified

DNAsfromanagarose

gel, they

were inserted into a StuI site of maltose-binding protein vectorpMAL-c (Fig. 1A), which carriedatermination codon created

by

insertion ofanXbaI linker (New

England

Bio-labs, Inc., Beverly, Mass.) into a blunt-ended EcoRI site.

TABLE 2. Amino acidsequences of shortfragmentsof VPF6

Fragment _aminoNo. of_acids Sequence_Sqec

SF1 18 TFSRQFLIPYDPEHHYKV

SF2 19 YDPEHHYKVFSPAASSCHN

SF3 21 FSPAASSCHNASGKEAKVCTI

SF4 20 ASGKEAKVCTISPIMGYSTP

The sequencesof inserted portionswereconfirmedbyusing

a Sequenase kit (United States Biochemical). The fusion proteins ofB19 capsid sequences and maltose-binding pro-tein prepared for immunoblotting were designated VPF1, VPF2, VPF3, VPF4, VPF5, VPF6, VPF7, VPF8, VPF9, VPF10, and VPF11. Figure 1B shows the relationship of thesepolypeptides tothe amino acid sequences of the B19 capsid proteins. The B19-specific portion of each fusion protein ranged from 56 to 111 amino acids, and all 11 together spantheentire capsid protein sequence.

Thereactivity patternsofMAbs and VP1 and VP2capsid proteins and fusion proteins on immunoblot were analyzed byusing the materials andmethodsofthe Protein ImageKit (United States Biochemical). The isolated empty capsids wereboiled in sample loading buffer for 3 min. Escherichia colitransfected withpMAL-cfusion plasmidswascollected afterinduction with 0.3 mM

isopropyl-p-D-thiogalactopyran-oside. Bacteria were lysed in sample loading buffer and boiledfor 3min. Proteins were separated byelectrophoresis through asodium dodecyl sulfate

(SDS)-8%

polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride membranes in transfer buffer containing 25 mM Tris and 192 mM glycine at 100 mA for 16 h. Membranes were blocked with 1% blocking solution, incubated with MAbs diluted 1:1,000, and incubated with alkaline phos-phatase-conjugated goat anti-mouse IgG (BRL). Antibody incubations were performed for 1 h at room temperature, andthe membranes were washedthree times between incu-bations with a salt buffer containing 0.1% Tween 20. Anti-body binding was detected with Lumiphos (United States Biochemical) chemiluminescent alkaline phosphate sub-strate.

OnlyasingleneutralizingMAb, E, showed reactivity with bothVP1 and VP2 capsid proteins (Fig. 2). Since the entire sequenceof VP2is contained within VP1, MAb E recognizes a linear epitope of the VP2 capsid protein. MAb E reacted with fusion proteins of the expected sizes (53 to 54 kDa) on immunoblot, and thisreactivity was restricted to the VPF6 fusion protein (Fig. 3A and B). Todetermine in further detail the epitope recognized by MAb E, four short fragments of VPF6 were prepared by the same method and designated SF1, SF2, SF3, and SF4 (Table 2). When the reactivitiesof thesefour short fragments of VPF6 were tested by immuno-blot, only SF3 reacted withMAb E (Fig. 4), indicatingthat theepitope recognizedby MAb E waslocalizedto 21 amino acids lying between residues 57 and 77 ofVP2.

The genome of the B19 parvovirus isapproximately5.4kb long and contains two major open reading frames (5). The left open reading frame encodes the 71-kDa nonstructural protein (NS), and the right open readingframe encodes the two capsid proteins VP1 and VP2. The entire sequence of VP2 iscontained within VP1; the twoamino acid sequences are identical except for the amino-terminal 226 residues of VP1. Invirions, VP1representsonly about5% of the capsid proteins and theremainderis composed ofVP2 (3,4). While no detailed structural dataare available for B19 parvovirus, the structure of the related canine parvovirus (CPV) has been solved to the atomic level by X-ray crystallography (19).While thestructuralrelationshipof this virustoB19 can be extrapolated only tentatively, the amino acid sequences ofthecapsidproteins share41% homologyand 19%identity and mostmajor structural features appeartobe conserved in B19 parvovirus (Fig. 5). A single neutralizing epitope has been reported for B19 parvovirus and mapped to residues 328 to 344 of VP2 (17). This region

corresponds

to amino acids355 to373of CPV and contributes tothe spike onthe

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7058 NOTES

polyl inker

A

&aIE

a 2.26

p tac

rrnD

ter inator r

Amp

lact

ori

Sac I kpn I Eag I haF I

I

11

1

I

I

I

Sft I

I

i

Polyt Inker: aUE...TCGAGC TCG GTA CCC GGCCGGGGA TCC ATC GAGCCT ACC CCT

hfa# I Xba I Sal I Pst I

I

I

1

II

1I

find Ill

I

I

GAATTC AGTAAA ACC CTCGATGGA TCC TCT AGA GTCGAC CTG CAGGCA AGC TTG...IacZa

B

781 a.s.

I

I

VP2

F

VPFI I- | VPF6

I-VPF2 I VPF7 |- I

[image:3.612.130.477.74.481.2]

VPF3

|--VPF4

VPF5

VPF8

I-VPF9 I

VPFIO

I-VPFII I

FIG. 1. (A) B19-maltose-binding proteinfusionproteinswereprepared byinsertion of B19capsidgenefragmentsintoplasmid pMAL-c attheStuI restriction site.(B) Overlapping fragments ofthe B19 structuralproteingenesareshown with amino acid(a.a.)numbers and their positions within thesequencesofVP1 and VP2. From the aminoterminus,thepositionsof thepolypeptides in the amino acid sequence of the VP1 structuralproteinare asfollows: VPF1, 0to94; VPF2,52to142; VPF3,122to177; VPF4,158to227; VPF5, 187to265;VPF6,228 to334; VPF7, 314to423; VPF8, 403 to513; VPF9,493to603;VPF10,583to693;andVPF11,673to781.

threefoldaxis of thevirion. None ofourneutralizing MAbs

recognized this epitope, but MAb E recognized a region

from residues 57to77 of VP2 correspondingto71 to91 on CPV. Both neutralizing epitopes are also present on VP1.

These amino acids of CPV also fold to the spike. Two neutralizing epitopes mapping to residues Ala-300 and Asn-93 have been identified on CPV (14). Despite their

separationin the linearsequenceof thecapsid proteins, both of theseresidues alsomaptothespikeonthe threefold axis

and they may correspond structurally to the neutralizing regions identified on B19 parvovirus.

Of the nine neutralizing MAbs that we studied, only one

reacted with the capsid proteins and the peptide fragments

onanimmunoblot. These MAbswereoriginally screenedby

theradioimmunoassay, and under these conditions the

con-formationalstructureof the virion should bepreserved. It is

likely that the eight unreactive MAbs recognize

conforma-tionalepitopesthatarenolongerpresentonthe

membrane-transferred proteinin an immunoblot.

We havereportedthegenerationofneutralizingantiserum in animals immunized with B19 empty capsids containing

both VP1 and VP2 butnotVP2 alone(8). Theseresultsare

surprising given the identification of two neutralizing epitopes on VP2. It is suspected that the presence ofVP1 modulates theantigenicity of the viral particle and that the

antigenicityof the thoseepitopesistoolowtoberecognized

Eco RI

I

I

VPI

J. VIROL.

a

I

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1 2

200kd

-97

-68- -

83 kd

mp5-58

43

-1 2 3 4

200 kd

-97

-

68-43

-26

-FIG. 2. Thereactivitiesof MAb E and B19 capsid proteins were tested by immunoblotting. Ten microliters of purified empty capsids containingVP1and VP2capsid proteins were loaded on an SDS-polyacrylamide gel and blotted onto a polyvinylidene difluoride membrane. Lane 1 was probedwithMAb K, and lane 2 was probed with MAb E. kd, kilodaltons.

in theabsenceof VP1. The clinicalobservation that patients with persistent infection generate a specific antibody re-sponse that fails to neutralize the virus supports the idea that the neutralizing epitopes are of inherently low antigenicity. VP1may also directly affect theconformational structureof theneutralizing epitopes by its role in the formation of the structuralfeatures of B19, such as the spike on the threefold axis.

Determination of neutralizing epitopes is important for vaccinedevelopment. In some cases, linear epitopes recog-nized by neutralizing antibodies may be able to induce a

A.

200 kd

-97

-68

-

26-FIG. 4. The reactivity ofMAb E with shortfragmentsofVPF6 was analyzed by immunoblotting. Five microliters of bacterial lysates containing short fragments of VPF6 was loaded onto an

SDS-polyacrylamide gelandblottedonto apolyvinylidene difluoride membrane. Lanes: 1, SF1;2, SF2;3, SF3;4, SF4. Themembrane wastreated with MAb E.kd, kilodaltons.

1.MTSVNSAEASTGAGGGGSNSVKSMWSEGATFSA 33 1 MSDGAVQPDGGQPAVRNERATGSGNGSGGGGGGGSGGVG... ISTGTFNN 47

34 48

NSVTCTFSRQFL.IPYDPEHHPVSPIMG

QTEFKFLENGWVEITANSSRLVHLNHPESENYRRVVVNNMDKTAV|GNMA 8297

83 YS .. TPWRYLDFNALNLFFSPLEFQHLIENYGSIAPDALTVTISE 125

98 LDDIHAQIVTPWSLVDANAWGVWFNPGDWQLIVNTMSELHLVSFEQEIFN 147 126 IAVKDVTDK. TGGGVQV.TDSTTGRLCMLVDHEYKYPYVLGQGQDTLAPE 173 148 VVLKTVSESATQPPTKVYNNDLTASLMVALDSNNTMPFTPAAMRSETLGF 197

1 2 3 4 5 6 7 8 9 10 11

174 LPIWVYFPPQYAYL...TVGDVNTQGISGDSKKL.... ASEESAFYVL 214

198 YPWKPTIPTPWRYYFQWDRTLIPSHT.GTSGTPTNIYHGTDPDDVQFYTI 246

215 EHS.SFQLLGTGGTASMSYKFPPVPPENLEGCSQHFYEMYNPLYGSRLGV 263 247 ENSVPVHLLRTGDEFATGTFFFDCKPCRLTHTWQTNRALGLPPFLNSLPQ 296

0

43 -26

-264 PDTLGG...DPKFRSLTHEDHA...IQPQNFMPGPLVNSVSTKEGD 303

297 SEG4TNFGDIGVQQDKRRGVTQMGNTNYITEATIMRPAEVGYSAPYYSFE 346

304 SSNTGAGKALTGLSTGTSQNTRI6 EE YVTGINAIS 353 347 ASTQGPFKTPIAAGRGGAQTDENQAADGNPRYAFGRQHGQK..TTTTTGET 394

B. 1 2 3 4 5 6 7 8 9 10 11

200 kd-

_W

9

97-s

451 QPPPQIFLKILPQ.SGPIGGIKSMGITTLVQYAVGIMTVTMTFKLGPRKA 499

493 NCPGQLFVKVAPNLTNEYDPDASANMSRIVTYSDFWWKGKLVFK.AKLRA 541

68

-26

-FIG. 3. Antigen epitope recognized by MAb E detected by immunoblotting. Five microliters of bacterial lysates containing

each B19capsidprotein fragmentwasloadedonto

SDS-polyacryl-amide gels and blotted onto polyvinylidene difluoride membranes Lanes: 1, VPF1; 2, VPF2; 3, VPF3; 4, VPF4; 5, VPF5; 6, VPF6; 7, VPF7; 8, VPF8; 9, VPF9; 10, VPF10; 11,VPF11. Themembranes

weretreated with MAbsE(A)and K(B). kd, kilodaltons.

500 TGRWNPQPGVYPPHAAGHLPYVLYDPTATDAKQHHRHGYEKPEELWTAKS 549

[image:4.612.395.493.81.194.2]

542 SHTWNPIQQM. SINVDNQFNYV..PSNIGGMKIVYEKSQLAPRKLY.... 584

FIG. 5. Comparisonof amino acidsequencesofthe VP2capsid proteinsof B19 and CPValignedformaximumhomology. Thelast

five amino acids of the B19 sequence do not contribute to the alignmentandarenotshown.Shadingin the upper line indicatesthe

binding sites of MAbs E and BEll. Shading in the lower line indicates neutralizing epitopesofCPV (residuesAla-300and

Asn-93). Theamino acids marked with stars arelocatedoutside ofthe CPV virion.

354 HGQTTYGNAEDKEYQQGVGRFPNEKEQLKQLQGLNMHTY..FPNKGTQQY 401

395 PERFTYIAHQDTGRYPEGDWIQNINFNLPVTNDNVLLPTDPIGGKTGINY 444

402 TDQIERPLMVGSVWNRRALHYESQLWSKIPNLDDSFKTQFAALGGW.GLH 450

445 TNIFNTYGPLTALNNVPPVYPNGQIWDK..EFDTDLKPRLHVNAPFVCQN 492

---a-mmmumm-mmb---Mm---maim

-_Mwmlq.__ lomppqmmw

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7060 NOTES

protective immune response in vivo. Unfortunately, the

neutralizing epitopes of many viruses are conformational,

and in thesecasespeptide vaccinesmaybe of limiteduse.In

addition, peptides are often poorly immunogenic. Despite

these drawbacks, the safety and simplicity of synthetic peptides make them attractive materials for vaccines. Fur-ther characterization of the neutralizing region we have

identified may allowits use as apeptide vaccine.

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