Vol. 31, No. 1INFECTION AND IMMUNITY, Jan. 1981, p. 436-4440019-9567/81/010436-09$02.00/0

Immune Response After Exposure to Varicella Zoster Virus:Characterization of Virus-Specific Antibodies and Their

Corresponding AntigensHANS J. ZWEERINKt* AND BEVERLY J. NEFF

Division of Virus and Cell Biology Research, Merck Institute for Therapeutic Research, West Point,Pennsylvania 19486

Fourteen varicella zoster virus antigens were identified that induce antibodiesduring primary and recurrent infections. These antigens, which included themajor nucleocapsid polypeptide (molecular weight, 155,000) and three glycopro-teins (molecular weights, 130,000, 88,000, and 60,000, respectively) plus a numberof minor antigens, were identified in radioimmunoprecipitation assays, using[35S]methionine-labeled extracts of cells infected with varicella zoster virus andsera from patients with primary and recurrent viral infections. No significant andreproducible differences were observed between early convalescent sera fromcases with natural chicken pox and sera from cases with zoster in their ability toreact with viral antigens. Sera that were taken many years after episodes ofchicken pox still retained their ability to react with the major viral antigens.

Varicella-zoster virus (VZV), in common withother herpesviruses, causes primary infections(varicella or chicken pox) and occasionally arecurrent infection, herpes zoster, usually seenin aging adults (13, 21). The immune mecha-nisms responsible for the prevention of and therecovery from primary and recurrent infectionsare poorly understood. Such understanding re-quires the characterization of viral antigens, es-pecially those at the surface of infected cells thatinteract with the immune system.Growth of VZV in tissue culture is limited,

and the virus remains cell associated. Thismakes it very difficult to obtain sufficient quan-tities of purified virions for analyses and to assesstheir degree of purity. A recent report (22) de-scribed the purification of VZV and the analysisof its constituent polypeptides by sodium dode-cylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Although at least 31 polypeptideswere identified, only 16 polypeptides were foundin the precipitate obtained by reacting purifiedvirions with immune serum.The purpose of this work was to characterize

those viral antigens that induce significant levelsof antibodies in individuals with primary andrecurrent VZV infections. Antigens and antibod-ies were characterized by immunoprecipitationassays as described previously (14). Extracts of[35S]methionine-labeled WI-38 cells infectedwith VZV were reacted with a number of sera,and precipitated viral antigens were separatedby SDS-PAGE and visualized by fluorography.

t Present address: Department of Immunology, Merck In-stitute for Therapeutic Research, Rahway, NJ 07065.

MATERIALS AND METHODSMedium, buffers, and solutions. EBME-FC10

medium is Eagle basal metabolic medium with Earlesalts plus 10% fetal calf serum. NET buffer consists of0.05 M tris(hydroxymethyl)aminomethane-0.005 Methylenediaminetetraacetate-0.15 M NaCl-0.1 mMphenylmethylsulfonyl fluoride (pH 7.2), NET-T bufferis NET buffer containing 0.05% Triton X-100, and TEbuffer consists of 0.01 M tris(hydroxymethyl)amino-methane-0.0025 M ethylenediaminetetraacetate (pH7.5). Phosphate-buffered saline (PBS) consists of 0.04M sodium phosphate-0.15 M NaCl (pH 7.2), and PBS-KCl is PBS with 0.005 M KCl. SP buffer consists of0.01 M Tris (pH 6.8)-2% SDS-2% /-mercaptoethanol-20% glycerol-0.02% bromophenol blue. Five or 20%(wt/vol) sucrose was prepared in 0.05 M tris(hy-droxymethyl)aminomethane-1 M NaCl-0.01 M ethyl-enediamine-tetraacetate-0.15% Sarcosyl (pH 7.5).

Cells. Human diploid fibroblasts (WI-38) at 26 to29 population doublings were grown in EBME-FC10medium.

Virus strains and sera. The strains of VZV wereisolated from vesicle fluids obtained from clinical casesof varicella and grown in WI-38 cells (Table 1). Thestrains were identified at the third or fourth passagelevel, using cell-free virus obtained by disruption ofthe cells (2, 4), in plaque reduction neutralization tests.Paired acute and convalescent human sera obtainedfrom clinical cases of varicella were used as well ashorse hyperimmune serum made with a heterologousstrain of VZV. All of the isolates were negative whentested for pleuropneumonia-like organisms and otherextraneous agents.

Sera were obtained from cases with clinical varicellaor zoster and from normal seropositive and -negativeadults (Table 2).

Virus infection. Seed stocks of virus-infected cellswere added to confluent monolayers of WI-38 cells (48h after planting) at a ratio of 1:10 or 1:20 (infected to

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VZV-SPECIFIC ANTIGENS AND ANTIBODIES 437

TABLE 1. VZV strains usedVZV strain Date of vesicle fluid Passage levela

KMcC 4/11/66 4KMcC 4/11/66 46AW 4/09/66 5AW 4/09/66 26LB 1/11/79 28RH 4/01/68 28

a All strains were passaged in WI-38 cells.

TABLE 2. Sera used for immunoprecipitation ofVZVpolypeptides

Serum des- IAHA' Descriptionignation titer

V1-A <2 Acute serum from child with vari-cella

V1-C 2512 Early convalescent serum fromchild with varicella (6 wk afterrash)

V2-C >512 Early convalescent serum fromchild with varicella (5 wk afterrash)

79187 512 Early convalescent serum fromchild with varicella (2 wk afterrash)

79058 1,024 Early convalescent serum fromadult with varicella

V3 21,024 Early convalescent serum fromadult with zoster

79100 512 Early convalescent serum fromadult with zoster (14 days aftershingles)

MSD-2 256 Serum from adult with long pastchicken pox

MSD-4 <2 Serum from adult with long pastchicken pox

MSD-14 <2 Serum from adult with long pastchicken pox

HSV-AC 256 Acute serum from child with pri-mary oral herpetic infection(HSV-specific neutralizing titer,<16)

HSV-CONV 512 Convalescent serum from abovechild (HSV-specific neutralizingtiter, 64)

a Immune adherence hemagglutination assay (23).

uninfected cells) and incubated at 36°C. Cytopatho-genicity, typical of varicella, affecting 30 to 60% of thecell sheet was observed in these cultures after approx-imately 72 h.Labeling of infected cells. WI-38 cells were in-

fected with VZV as described above, and after 72 hthey were trypsinized. These cells were combined withfreshly trypsinized uninfected WI-38 cells at a ratio of1:4, replanted in 10-cm petri dishes (5 x 106 cells perdish) in EBME-FC10, and incubated at 36°C in a CO2incubator (17). When cytopathology involving approx-imately 50% of the culture was observed (generally 24h later), the medium was removed and the cells werewashed twice with PBS followed by the addition of 5ml of medium containing 0.45 mg of methionine perliter (3% of the normal methionine concentration),nonessential amino acids, and 2% dialyzed fetal calfserum. One hour later [35S]methionine (400 Ci/mmol;Amersham Corp.) was added to 30 ftCi/ml, and thecells were incubated at 37°C for 16 h. After the addi-

tion of 5 ml of complete medium for 1 h, cells werewashed with PBS and removed from the surface of thepetri dish with 0.02% ethylenediaminetetraacetate and0.05% NaHCO3 in PBS-KCl containing 0.1 mM N-a-p-tosyl-L-lysine-chloromethyl ketone to inhibit pro-teases. Cells were pelleted, washed once in PBS, di-vided into six samples per petri dish (approximately106 cells per sample), pelleted, and stored at -70°C.Unlabeled uninfected cells were also harvested withethylenediaminetetraacetate and stored as two pelletsper 10-cm petri dish (ca. 3 x 106 cells per sample).

Cells were labeled with D-[U-"C]glucosamine (10,uCi/ml; >200 mCi/mmol; Amersham Corp.) by incu-bating them for 16 h in Eagle basal medium containing20% of the normal glucose concentration and 2% di-alyzed fetal calf serum. They were harvested andstored as described above.

Immunoprecipitation. Protein A-bearing staphy-lococcus immunoadsorbant (IgGsorb) was boughtfrom the Enzyme Center, Inc. (Boston, Mass.). It wasreconstituted to yield a 10% (vol/vol) suspension andkept at -70°C in 2-ml aliquots. Before use, the bacteriawere washed as described (12, 14) and resuspended inNET-T. Frozen cell pellets (106 35S-labeled infectedcells or 3 x 106 unlabeled uninfected cells) were sus-pended in 200 pl of NET buffer. Triton X-100 anddeoxycholate were each added to 1%, and the suspen-sion was kept at 4°C for 15 min. Nuclei were removedby centrifugation at 700 x g for 6 min, and the super-natant was preadsorbed for 60 min at 22°C with 20 p1of normal rabbit serum, followed by the addition of100 ,ul of protein A-bearing staphylococcus (20%, vol/vol) for 15 min at 22°C. This mixture was centrifugedat 6,500 x g for 2 min, and the pellet was discarded.Samples, 20 p1, of diluted serum (generally 1:25) werepreadsorbed for 15 min at 22°C with 20 ,ul of extractprepared from uninfected, unlabeled WI-38 cells. Tothis was added 20 ,ul of labeled extract (preadsorbedwith normal rabbit serum) and this mixture was in-cubated for 60 min at 22°C, followed by the additionof 50 ,A of protein A-bearing staphylococcus suspen-sion (10% vol/vol). After incubation for 30 min at22°C, complexes were pelleted at 6,500 x g for 2 minand washed three times with 1.0 ml of NET-T buffer.The final pellet was resuspended in 70 ,ul of SP bufferand placed in boiling water for 3 min to dissociate theantigen-antibody complexes, the protein A-bearingstaphylococcus was removed by centrifugation, andsupematants were applied to SDS-polyacrylamidegels.

Preparation of nuclear extracts. The nuclearpellet (see above) was washed with NET-T, resus-pended in 20 pl of NET buffer containing 0.1% SDS,and sonicated twice for 20 s with a Kontes sonicator.The sonicated material was diluted to 100/l with NETbuffer and preadsorbed with normal rabbit serum asdescribed above.SDS-PAGE. Samples were electrophoresed in 10%

discontinuous slab gels for 5 h at 110 V (15). Gels werefixed for 30 min in a mixture of methanol, acetic acid,and water (5:1:5) and soaked overnight in 7% aceticacid. Next, the gels were impregnated with 2,5-diphen-yloxazole (22.4 g per 100 ml of dimethyl sulfoxiderinsed with water, dried, and exposed to Kodak RoyalX-Omat film (for details, see reference 1). Molecularweight markers (Pharmacia Fine Chemical, Inc.) were

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438 ZWEERINK AND NEFF

phosphorylase b (94,000), albumin (67,000), ovalbumin(43,000), carbonic anydrase (30,000), and trypsin inhib-itor (20,100). They were labeled with 14C-acetic anhy-dride by resuspending the contents of a Pharmaciavial in 1 ml of phosphate buffer (0.3 M; pH 7.2)followed by the addition of 500,Ci of "4C-labeledacetic anhydride (60 to 120 mCi/mmol; AmershamCorp). The mixture was incubated for 30 min at roomtemperature, and labeled proteins were lyophilizedafter their purification by chromatography on a P-10column (Pharmacia Fine Chemicals). Molecularweights of 100,000 and higher were estimated relativeto the herpes simplex virus type 1 (HSV-1) nucleocap-sid polypeptide and the A and B glycopolypeptides(19, 20; Zweerink and Stanton, submitted for publica-tion).

Lectin-affinity chromatography. Cell extractswere fractionated on columns with the lectin fromLens culinaris coupled to Sepharose 4B. Lectin(Boehringer Mannheim Corp.), 30 mg, was reactedwith 4 g of cyanogen bromide-activated Sepharose 4B(Pharmacia Fine Chemicals), and columns (made in 3-ml syringes) with a volume (wet material) of approxi-mately 0.5 ml were run at room temperature with aflow rate of 12 ml/h. [35S]methionine-labeled infectedcells (approximately 2 x 106) were suspended in 0.5 mlof NET buffer plus 1% Triton X-100. Nuclei wereremoved, and the cytoplasmic extract was applied tothe column. First the column was washed with NET-T buffer adsorbed material was eluted with the samebuffer containing 10 mg each of glucose and a-methyl-D-mannoside per ml (both carbohydrates were usedfor more efficient elution) or 3 M NH4SCN. Fractionsof 1.2 ml were collected and 20-[d aliquots werecounted in Aquasol (New England Nuclear Corp.).

Isolation of VZV nucleocapsids. Infected cellswere labeled overnight with 32P (carrier-free; Amer-sham Corp.) at 50 uCi/ml in phosphate-free Eaglebasal medium or with [3S]methionine as describedabove. The supernatant was removed, and the cellswere washed once with PBS and scraped with a rubberpoliceman into PBS. They were pelleted and washedonce with PBS. The pellet (approximately 10' cells)was suspended in 1 ml ofTE buffer before the additionof Triton X-100 to 1% and ribonuclease T, (50 U;Calbiochem). After 10 min in ice, nuclei were removed(1,500 rpm for 5 min), the cytoplasmic extract waskept at room temperature for 10 min, and Sarcosylwas added to 0.15%. The sample was placed on a 10-ml 5 to 20% sucrose gradient that had been layeredover a 1-ml cushion of CsCl (p = 1.5 g/cm3) in anSW41 tube. The gradient was centrifuged for 3 h at35,000 rpm and 20°C, fractions of 0.5 ml each werecollected from the bottom of the tube, and the radio-activity in a 20-,ul aliquot of each sample was deter-mined by Cerenkov radiation (for 32P) or in Aquasol(for [3S]methionine). Fractions containing viral nu-cleocapsids were combined and dialyzed against NETbuffer.

Electron microscopy. A drop of purified VZVnucleocapsids in NET buffer (see above) was placedfor 1 h on carbon-coated 200-mesh copper grids. Thebuffer was removed, and the grids were negativelystained with 2% phosphotungstic acid (pH 6.2) andexamined in a Philips EM300 electron microscope atmagnifications of 20,000 and 50,000.

RESULTSVZV polypeptides in infected cells. Often,

intracellular virus-specific polypeptides can beidentified by comparing electropherograms ofradiolabeled polypeptides in infected and unin-fected cells (9). This approach could not be usedfor VZV, since virus-specific polypeptide synthe-sis is not extensive and host-specific proteinsynthesis continues.

Therefore, VZV-specific polypeptides were

analyzed by immunoprecipitation. Cytoplasmicextracts from [35S]methionine-labeled unin-fected WI-38 cells and WI-38 cells infected withVZV strain KMcC (passage 46) were incubatedwith acute and early convalescent sera from a

child with clinical varicella, and precipitated an-

tigens were analyzed by SDS-PAGE and fluo-rography.

Figure 1 shows that at least 14 polypeptidesranging in molecular weight from less than20,000 to approximately 200,000 were precipi-tated from infected cell extracts with immuneserum, but not with acute serum, and were notprecipitated from uninfected cells. Polypeptide

I-)

...o

r,-> >~>o-I T)1 r)

94 > ~|X;-¶ § -5 ;!

b7K

~7A43K s. ~_

30K >

20.1 K---O-

FIG. 1. Fluorograms of polypeptides precipitatedby acute (V1-A) and convalescent (Vl-C) sera fromcytoplasmic extracts of uninfected WI-38 cells (UN)or WI-38 cells infected with VZVKMcC (passage 46).(A) Film exposure, 4 days; (B) film exposure, 1 day.

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VZV-SPECIFIC ANTIGENS AND ANTIBODIES 439

3 occasionally migrated as three separate bands(see Fig. 1B), and the material designated aspolypeptide 4 always migrated as a broad band.Polypeptide 4 could represent either severalpolypeptide species or a single polypeptide spe-cies with variable carbohydrate side chains (seebelow). The polypeptide labeled "A" is an arti-fact caused by the accumulation of the immu-noglobulin heavy chain in that region of the gel.Some precipitation of polypeptides 2 and 7 andthe smallest of the three polypeptide 3 speciesby acute serum was often observed. This couldbe due to either nonspecific precipitation or thefact that acute serum contained low levels ofantibodies against these polypeptides. Poly-peptide 7A was precipitated from uninfectedcells with both sera and from infected cells withcontrol serum. However, precipitation was heav-iest when infected cell extracts were reacted withimmune serum; no further attempts were madeto determine the origin of this polypeptide.Characterization of viral polypeptides.

Glycopolypeptides were identified by exposinginfected cells to [14C]glucosamine during viralgrowth. Figure 2, gel B, shows that this carbon_hydrate is incorporated into polypeptides 3, 4,and 7. The glycoprotein nature of these polypep-tides was confirmed by fractionating cytoplas-mic extracts of [35S]methionine-labeled infectedcells on a column with the lectin from L. culi-naris. Three fractions were obtained: the unad-sorbed material, adsorbed material that elutedwith a-methyl-mannoside and glucose, and ad-sorbed material that eluted with 3 M NH4SCN(fractions A, B, and C, respectively, in Fig. 3).Each fraction was reacted with immune serum,and the precipitated polypeptides were analyzedby SDS-PAGE. Figure 2, gel D, shows that thematerial in fraction B contained viral polypep-tides 3 and 4 and a lesser amount of 7 and thatfraction C was enriched for polypeptide 7. Theunadsorbed material (Fig. 2, gel C) containedmainly polypeptides 2 and 4 and small amountsof polypeptides 3 and 7.As will be shown below, polypeptide 2 is the

major nucleocapsid component. Its presence inthe lectin-adsorbed material is probably due toincomplete removal of glycoproteins from thenucleocapsids and binding of these complexes tothe lectin. Complete dissociation of the glyco-proteins (especially polypeptide 7) from the lec-tin requires strong denaturing conditions (3 MNH4SCN). Failure of cell glycoprotein materialto bind to the column (see Fig. 2, gel C) probablyreflects heterogeneity in glycosylation.The nucleocapsid polypeptides were identified

after isolating nucleocapsids from the cytoplasmof infected cells by centrifugation in sucrosedensity gradients. For identification purposes

_0 -- I'

FIG. 2. Fluorograms of VZVglycoproteins. (A) Cy-toplasmic extract of WI-38 cells infected with VZVKAMcC (passage 46) and labeled with [14C]glucosa-mine was reacted with acute serum (Vl-A); (B) sameextract reacted with immune serum (VI-C); (C, D,and E) fractions A, B, and C from Fig. 3 (labeled with[35S]methionine) reacted with immune serum (Vl-C).

viral deoxyribonucleic acid was labeled with 32p.Most of the 32P-labeled material that was asso-ciated with structures large enough to enter thegradient sedimented on top of the CsCl cushion(Fig. 4). By electron microscopy it was shownthat this material consisted of nucleocapsids(Fig. 5). A small amount of radiolabeled materialsedimented more slowly (see arrow in Fig. 4).This also is the position to which HSV nucleo-capsids sedimented.

Nucleocapsid polypeptides were identified bylabeling infected or uninfected WI-38 cells with[35S]methionine, and the cytoplasmic extractswere fractionated by sedimentation in sucrosedensity gradients. The material at the interfacebetween CsCl- and sucrose-containing viral nu-cleocapsids (see Fig. 5) was dialyzed againstNET buffer and pelleted at 35,000 rpm for 3 h,and the polypeptide composition was deter-mined by SDS-PAGE. Figure 6 (gel B) showsthat polypeptide 2 was the major constituent ofnucleocapsids; smaller amounts of the glycopro-teins 3, 4, and 7 and the low-molecular-weightpolypeptides 10, 12, and 14 were also found incores. None of these polypeptides was present infractionated cytoplasmic extract from unin-fected cells (Fig. 6, gel A). Possibly glycoproteinswere incompletely removed from the nucleocap-sids by detergent treatment, whereas polypep-tides 10, 12, and 14 could be minor-constituentnucleocapsid polypeptides. Nucleocapsid poly-peptide 2 was the major viral antigen in isolatednuclei of infected cells (data not shown).

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0

06

5-

4

B

NH4SCN

10 20 30 40 50

FRACTION NUMBERFIG. 3. L. culinaris affinity chromatography of the cytoplasmic extract of WI-38 cells infected with VZV

KMcC (passage 46) and labeled with (35S]methionine. (Peak A) Unadsorbed material; (peak B) materialeluted with the carbohydrates (CHO) a-methyl-mannoside and glucose; (peak C) material eluted with 3 MNH4SCN.

5-

PoX 3_-

2

cscIIi

5 10 15 20FRACTION NUMBER (0.5 ML)

FIG. 4. Isolation of VZVnucleocapsid. WI-38 cellsinfected with VZV KMcC (passage 46) were labeledwith 32P, and the cytoplasmic extract was fraction-ated on a sucrose gradient that was layered over a

cushion of CsCl, as described in the text.

Coelectrophoresis of polypeptides precipi-tated from VZV-infected cells with those precip-itated from cells infected with HSV-1 showedthat the major nucleocapsid polypeptides ofeach virus migrated at the same rate, suggestingidentical molecular weights. Similarly, VZV gly-coprotein 3 coelectrophoresed with the major Aand B HSV-1 glycoprotein complex (19, data notshown).The properties of the VZV polypeptides and

their molecular weights are summarized in Ta-ble 3.Polypeptides in various VZV isolates. It

has been reported for HSV that there are differ-ences in the polypeptides (relative amounts andmolecular weights) of the two serotypes (HSV-1 and HSV-2) and the various isolates withineach serotype (5, 8). We investigated whetherthis was also the case for VZV. WI-38 cells wereinfected with four different VZV isolates, strainsKMcC (passage 46), AW (passage 26), LB (pas-sage 28), and RH (passage 28), and exposed to[35S]methionine. Viral polypeptides were precip-itated with immune serum and analyzed bySDS-PAGE. Figure 7 shows no differences in

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VZV-SPECIFIC ANTIGENS AND ANTIBODIES 441

A1

$

*.*s.B

_L I

FIG. 5. Electron micrograph of nucleocapsids isolated from the interface between CsCl and sucrose (seeFig. 4). (A) and (B) are two different magnifications. Bar, 100 nm.

TABLE 3. Summary of VZVpolypeptidesNo. Mol wt Property

94K -*

- 2-3""4

1 -200,0002 155,0003 130,0004 88,0005 73,0006 67,0007 60,0007a 57,0008 39,0009 37,00010 34,500

11 33,00012 31,50043K- ->

30K--

10----i2

20.1 K -*-

FIG. 6. Fluorograms of polypeptides incleocapsids that were isolated by sucrose-lmentation (see text) from the cytoplasmic[35S]methionine-labeled WI-38 cells infe

13 22,50014 17,500

Major nucleocapsid polypeptideGlycoproteinGlycoprotein

GlycoproteinPossibly host cell specific

Possibly minor nucleocapsidpolypeptide

Possibly minor nucleocapsidpolypeptide

Possibly minor nucleocapsidpolypeptide

the migration rate or the relative amounts of themajor polypeptides. Differences in the relativeamounts of the minor polypeptides were ob-served, but these were not reproducible, andthey were also observed with different prepara-

- 14 tions of the same isolate (cf. Fig. 1 and 7).It was also shown that passaging of VZV in

WI-38 cells did not alter the polypeptide pattern.

VZVnu-CsCl sedi-extract ofcted with

VZVKMcC (passage 46). For comparison, uninfectedcells were fractionated and analyzed in a similarfashion. (A) Uninfected cells; (B) infected cells.

A B

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442 ZWEERINK AND NEFF

(DI

y

NC N

3-<

2-

7\: 531I',-7. IN

fe1

raf-

IS

FIG. 7. Fluorograms of polypejby immune serum (V1-C) from extrinfected with VZV KMcC (passage26), LB (passage 28), and RH (pas

No differences were observesKMcC at passage levels 6 and 4passage levels 6 and 26 (data nReactivity ofvarious sera

peptides. Immunoprecipitatiused in this paper can be usedantigens in infected cells, or coitated antigens can serve as incific antibodies in sera. This reqof macromolecular aggregatesmore than one polypeptide srtracts from infected cells. We Isuch aggregates, if present, doproblem in extracts prepared frwith HSV (Zweerink and Sta]mun., in press). The possible pgates in extracts of VZV-infecvestigated as follows. Extractsscribed in Materials and Methwith control serum and immuand after centrifugation at 10min, and the precipitated polyjalyzed by SDS-PAGE and fluimmunoprecipitation pattern opeptides and the major polypel7 was not affected by high-spe4except that there was a small,decrease in the amount of precapsid polypeptide 2 (data ndemonstrates that large strucicompletely solubilized virions a

sufficient quantities to influen(

Extracts from WI-38 cells infected with VZVKMcC (passage 46) were reacted with sera fromindividuals with clinical varicella or zoster (seeTable 2). These were early convalescent sera

x: with high IAHA titers (-512) from three chil-dren (V1-C V2-C, and 79157) and one adult(79058) with varicella and from two adults withzoster (V3 and 79100). In addition, three sera

3 - - 94 K from adults with long past chicken pox (MSD-2,Z. -4 67 K -4, and -14) were used. IAHA titers of two of

these sera were low, but one (MSD-2) had a titerof 256. All sera precipitated the major nucleo-

_-- 43 K capsid polypeptide, the three glycopolypeptides,and a number of minor polypeptides (Fig. 8).Qualitative differences were observed in the pre-

-- 30K cipitation of some of the minor polypeptides bythe various sera. However, these minor differ-enes were not reproducible with differentbatches of infected cells. Serum MSD-2 (longpast chicken pox; IAHA, 256) reacted stronglywith all viral antigens, whereas the two other

,tides precipitated similar sera (MSD-10 and -14; IAHA, <2) were,acts of WI-38 cells only reactive with the major antigens (the nu-e 46), AW (passage cleocapsid polypeptide and the three glycopro--sage 28). teins). Figure 8 also shows that acute and early

convalescent sera from a child with a primaryI between VZV herpetic infection (increase in HSV-specific neu-W6 or VZV AW at tralizing antibodies, <1:16 to 1:64) and naturallyot shown). immune to varicella (VZV-specific IAHA titerswith VZV poly- in these sera, 256 and 512, respectively) had theon analyses as same pattern of reactivity in both sera and withto identify viral VZV polypeptides. If herpesvirus-specific anti-onversely, precip- bodies could react with VZV antigens, one woulddicators for spe- expect increased precipitation with the earlyuires a minimum convalescent serum compared with acute serum.that consist of

)ecies in the ex-have shown thatnot constitute a^om cells infectednton, Infect. Im-resence of aggre-ted cells was in-prepared as de-ods were reactedine serum before0,000 x g for 60peptides were an-uorography. Theof the minor poly-ptides 2, 3, 4, anded centrifugationbut reproducible,,cipitated nucleo-ot shown). Thistures such as in-are not present ince these assays.

DISCUSSIONAt least 14 polypeptides were precipitated

from VZV-infected cells with immune sera. Withthe possible exception of one (polypeptide 7A),they were virus coded because they were precip-itated preferentially by immune sera from ex-tracts of infected cells. Sera in the acute phaseof varicella were used for controls in these stud-ies. It has been our experience (14; unpublisheddata) that these sera contain few if any antibod-ies that can be measured in the assays used.They offer the advantage (unless one has earliersera from the same patients) of being matchedwith early convalescent sera. Results with theacute serum of V2-A (see Table 2) were identicalto those with serum V1-A.

It is unlikely that any host cell-specific poly-peptides were trapped by the protein A-bear-ing staphylococcus-antibody-antigen complexes.This was shown by reacting immune serum witha mixture of extracts from unlabeled infectedcells and [35S]methionine-labeled uninfected

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VZV-SPECIFIC ANTIGENS AND ANTIBODIES

<~ C) ~

I I ' lx u-)-C.-

Y*- C> >

T-r

c< m r\J 3D rl

C*, - 0 GcT (1U) (:) C:n>- rl-. 2

63K-7

FIG. 8. Fluorograms ofpolypeptides precipitated from WI-38 cells infected with VZV KMcC (passage 46)

by sera from patients with chicken pox or zoster and individuals with long past chicken pox (see Table 2).HSV-AC and HSV-CONV are acute and convalescent sera, respectively, from a child with primary oral

herpes lesions. 79100A and 79100B are the same gel but exposed for 3 and 1 days, respectively.

cells. The precipitated radioactive polypeptidesdid not contain any of the virus-specific polypep-tides that are indicated in Fig. 1 (data notshown). Four of the major precipitated polypep-tides were characterized further: one was thenucleocapsid polypeptide; the other three wereglycoproteins, presumably representing enve-

lope antigens. They corresponded to glycopro-teins described by Grose (7). A fourth glycopro-tein (molecular weight, 45,000) described byGrose was not observed by us.

The molecular weight of VZV deoxyribonu-cleic acid has been reported to be 100 x 106 (11),with a coding capacity of 5 x 10' daltons ofpolypeptides. The total molecular weight of theVZV polypeptides in Table 2 is approximately106, which is 20% of the maximum coding capac-ity. Several of the radioactive bands in the gelsmay have contained more than one polypeptidespecies (see, for example, polypeptide 3 in Fig.1), and minor polypeptide species may have beenoverlooked. Furthermore, polypeptides that didnot induce specific antibodies during naturalinfections will not be detected by the methodsused in this paper. It is likely that VZV poly-peptide synthesis is regulated as is the case forother herpes-viruses (10). [35S]methionine wasadded to cultures late during infection at a timeof moderate cytopathic effect, when only part ofthe viral genome was expressed. One might alsoexpect differences in the extent to which viralreplication had proceeded in the various cultures

used for labeling, which would explain why dif-ferent batches of infected cells varied somewhatin their content of precipitated viral polypep-tides.No differences in the relative amounts and the

molecular weights of the major polypeptides infour different isolates of VZV were observed.Similar results were obtained for two viralstrains that had been passaged 6 and 46 times,respectively, in WI-38 cells. This is in contrastto HSV, for which differences between viralisolates ofthe same serotype have been observed(5, 8). Stability between VZV isolates has alsobeen observed at the level of its deoxyribonucleicacid: variations of the restriction endonucleaseprofiles of a number of isolates of both HSVserotypes have been observed repeatedly (3, 18),whereas such profiles are much more stable forisolates of VZV (16; our unpublished data).

Sera from patients with chicken pox (a pri-mary infection) and herpes zoster (a recurrentinfection) contained the same antibodies asjudged by the immunoprecipitation assays, andsera from normal adults who presumably hadhad varicella during childhood still containedantibodies against the major nucleocapsid poly-peptide and the three glycoproteins.The identification of several viral glycopro-

teins that induce antibodies during primary in-fection and recurrences provides a rationale forselecting antigens to be included in a potentialVZV subunit vaccine. Furthermore, it provides

U.) (I

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444 ZWEERINK AND NEFF

an immunochemical approach for the evaluationof antibody responses to immunization with can-didate vaccine strains of VZV.

ACKNOWLEDGMENTS

We thank B. Wolanski for preparing the electron micro-graph, R. E. Weibel and A. A. Gershon for sera and clinicalobservations, and L. Stanton, H. Winterbottom, D. Morton,and K. Guckert for technical assistance.

LITERATURE CITED

1. Bonner, W. M., and R. A. Laskey. 1974. A film detectionmethod for tritium-labelled proteins and nucleic acidsin polyacrylamide gels. Eur. J. Biochem. 46:83-88.

2. Brunell, P. A. 1967. Separation of infectious varicella-zoster virus from human embryonic lung fibroblasts.Virology 31:723-734.

3. Buchman, T. G., B. Roizman, G. Adams, and B. H.Stover. 1978. Restriction endonuclease fingerprintingof herpes simplex virus DNA: a novel epidemiologicaltool applied to a nosocomial outbreak. J. Infect. Dis.138:488-498.

4. Calnek, B. W., S. B. Hitchner, and H. K. Adldinger.1970. Lyophilization of cell-free Marek's disease herpes-virus and a herpesvirus from turkeys. Appl. Microbiol.20:723-726.

5. Cassai, E. N., M. Sarmiento, and P. G. Spear. 1975.Comparison of the virion proteins specified by herpessimplex virus types 1 and 2. J. Virol. 16:1327-1331.

6. Gibson, W., and B. Roizman. 1972. Proteins specifiedby herpes simplex virus. VIII. Characterization andcomposition of multiple capsid forms of subtypes 1 and2. J. Virol. 10:1044-1052.

7. Grose, C. 1980. The synthesis of glycoproteins in humanmelanoma cells infected with varicella-zoster virus. Vi-rology 101:1-9.

8. Heine, J. W., R. W. Honess, E. Cassai, and B. Roiz-man. 1974. Proteins specified by herpes simplex virus.XII. The virion polypeptides of type 1 strains. J. Virol.14:640-651.

9. Honess, R. W., and B. Roizman. 1973. Proteins specifiedby herpes simplex virus. XI. Identification and relativemolar rates of synthesis of structural and nonstucturalherpes virus polypeptides in the infected cell. J. Virol.12:1347-1365.

10. Honess, R. W., and B. Roizman. 1974. Regulation ofherpesvirus macromolecular synthesis. I. Cascade reg-

INFECT. IMMUN.

ulation of the synthesis of three groups of viral proteins.J. Virol. 14:8-19.

11. Iltis, J. P., J. E. Oakes, R. W. Hyman, and F. Rapp.1977. Comparison of the DNAs of varicella-zoster vi-ruses isolated from clinical cases of varicella and herpeszoster. Virology 82:345-352.

12. Kessler, S. W. 1975. Rapid isolation of antigens from cellswith a staphylococcal protein A-antibody adsorbent:parameters of the interaction of antibody-antigen com-plexes with protein A. J. Immunol. 115:1617-1624.

13. Luby, J. P. 1973. Varicella-zoster virus. J. Invest. Der-matol. 61:212-222.

14. Machamer, C. E., E. C. Hayes, S. D. Gollobin, L. K.Westfall, and H. J. Zweerink. 1980. Antibodiesagainst the measles matrix polypeptide after clinicalinfection and vaccination. Infect. Immun. 27:817-825.

15. Maizel, J. V., Jr. 1971. Polyacrylamide gel electropho-resis of viral proteins, p. 179-246. In C. K. Maramoroschand H. Koprowsky (ed.), Methods in virology, vol. 5.Academic Press, Inc., New York.

16. Oakes, J. E., J. P. Iltis, R. W. Hyman, and F. Rapp.1977. Analysis by restriction enzyme cleavage of humanvaricella-zoster virus DNAs. Virology 82:353-361.

17. Schmidt, N. J., and E. H. Lennette. 1976. Improvedyields of cell-free varicella-zoster virus. Infect. Immun.14:709-715.

18. Skare, J., W. P. Summers, and W. C. Summers. 1975.Structure and function of herpesvirus genomes. I. Com-parison of five HSV-1 and two HSV-2 strains by cleav-age of their DNA with Eco R I restriction endonuclease.J. Virol. 15:726-732.

19. Spear, P. G., 1976. Membrane proteins specified byherpes simplex viruses. I. Identification of four glyco-protein precursors and their products in type 1-infectedcells. J. Virol. 17:991-1008.

20. Spear, P. G., and B. Roizman. 1972. Proteins specifiedby herpes simplex virus. V. Purification and structuralproteins of the herpes virion. J. Virol. 9:143-159.

21. Taylor-Robinson, D., and A. E. Caunt. 1972. Varicellavirus. Virol. Monogr. 12:27.

22. Wolff, M. H. 1978. The proteins of varicella-zoster virus.Med. Microbiol. Immunol. 166:21-28.

23. Yamada, A., S. Ogino, Y. Asano, T. Otsuka, M. Tak-ahashi, K. Baba, and H. Yabuuchi. 1979. Compari-son of 4 serological tests-complement fixation, neu-tralization, fluorescent antibody to membrane antigenand immune adherence hemagglutination-for assay ofantibody to varicella-zoster (V-Z) virus. Biken J. 22:55-60.

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