Proc. Natl. Acad. Sci. USAVol. 77, No. 3, pp. 1622-1626, March 1980Medical Sciences

Specificity of human antibodies to oncovirus glycoproteins:Recognition of antigen by natural antibodies directed againstcarbohydrate structures

(humoral immunity/heterophil antibody/radioimmunoprecipitation/retroviruses)

HARRY W. SNYDER, JR., AND ERWIN FLEISSNER

Laboratory of Viral Oncology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021

Communicated by Robert A. Good, December 26, 1979

ABSTRACT Antibodies in human sera from healthy indi-viduals were shown to be reactive with highly purified70,00OA-totn envelope glycoprotein (gp70) of the simian sar-coma virus-simian sarcoma-associated virus (SSV-SSAV)complex in radioimmunoprecipitation assays under certainconditions. The specificity of the reaction was analyzed in ab-sorption tests with normal human serum proteins, assays of viralgp7O antigenicity after exposure to exo- and endoglycosidasesor trypsin, and carbohydrate hapten inhibition studies. On thebasis of the results obtained in these experiments we haveconcluded that immune recognition of SSV-SSAV gp7O can bemediated by naturally occurring heterophil antibodies in humansera that are reactive by virtue of binding to the carbohydratemoiety of the viral gp7O molecules. The results are consistentwith the idea that the antibodies in. question are elicited as aresult of exposure to many natural substances possessing widelycrossreacting antigens and are not a result of widespread in-fection of man with replication-competent oncoviruses.

The potential role of oncoviruses in the development of somehuman malignancies, particularly leukemia, remains an in-triguing and unresolved issue. There are reports in the literaturethat some human tissues contain proviral sequences and canexpress, depending on the assays employed, reverse transcrip-tase activity, virus-like RNA, viral proteins, and even mor-phological structures resembling virions (with or without as-sociated infectivity). However, these findings must be consid-ered together with negative data from other investigatorsseeking similar evidence (see reviews, refs. 1 and 2). In theabsence of a clearly defined oncovirus of human origin, anumber of investigators have approached this problem by in-vestigating human sera for naturally occurring antibodies tooncoviruses of other species. The results obtained have variedfrom positive to negative or equivocal (3-19). Attempts to re-solve discrepancies in the published data have centered arounddiscussions of the lability of certain viral protein probes (14) andthe use of special reaction conditions influencing human anti-body binding to viral antigens (13, 14). By extending our earlierstudies on the reactivity of human antibodies with intact on-covirus virions (9, 10) we hoped to reconcile the disparate resultsand to provide further insight into the nature of the antibodieswe were detecting. In the studies presented here we show thathuman antibodies are capable of immunoprecipitating thepurified envelope glycoprotein (gp70) from the primate on-covirus simian sarcoma virus-simian sarcoma-associated virus(SSV-SSAV). We also present evidence that the reaction in-volves combinations of naturally occurring heterophil anti-

bodies, which bind by virtue of their crossreaction with thecarbohydrate moiety of the viral glycoprotein. Studies byBarbacid et al. (20) have led to similar conclusions.

MATERIALS AND METHODSCells, Viruses, and Viral Proteins. The source of normal rat

kidney cells producing SSV and SSAV and the methods usedfor maintaining cell cultures and for preparation of purifiedradiolabeled virions have been described (9). For experimentsdescribed here, [3H]glucosamine-labeled virions were obtainedfrom cultures incubated for 18 hr in the presence of mediumcontaining 50 ,iCi of D-[6-3H]glucosamine (10-30 Ci/mmol,New England Nuclear; 1 Ci = 3.7 X 1010 becquerels) perml.

For preparation of soluble radiolabeled viral 70,000-daltonglycoprotein (gp7O), purified virions were subjected to deter-gent lysis in a buffer containing 0.05 M NaCI, 0.02 M Tris-HCl(pH 7.4), 0.5% Nonidet P40, 0.5% sodium deoxycholate, and10% Traysylol protease inhibitor (10,000 kallikrein inactivatorunits per ml) for 30 min at 370C. After the viral lysates weresubjected to centrifugation at 100,000 X g for 60 min in a type50 rotor, the supernatant fractions containing soluble viralproteins, including radiolabeled gp7O, were collected and storedat -70'C until used.

Purification of gp7O after dialyzing the above proteins againstthe appropriate column buffer was accomplished (i) by phos-phocellulose chromatography of the above lysates followed byeither hydroxylapatite (21) or DEAE-cellulose chromatography(22) or (ii) by concanavalin A affinity chromatography followedby sizing on Sephadex G-150 (23). In each case gp7O was pu-rified to apparent homogeneity as determined by Na-DodSO4/polyacrylamide gel electrophoresis in one of two gelsystems based on the procedure of Laemmli (24): cylindricalgels of 15% acrylamide (25) or step-gradient slab gels of 7.5-12.5% acrylamide (26).

Serological Assays. Human sera were obtained from normalblood donors and laboratory personnel at Memorial Sloan-Kettering Cancer Center and from donors in Tubingen, WestGermany (provided by R. Kurth).

Details of our radioimmunoprecipitation (RIP) assay againstintact virus have been published (9). The conditions used forRIP of soluble purified gp7O were those of Kurth and Mikschy(14). For competition radioimmunoassays (RIAs) aliquots oftest serum diluted to the last point at which 100% of the gp7Oprobe was precipitable were incubated with serial dilutions of

Abbreviations: SSV-SSAV, simian sarcoma virus-simian sarcoma as-

sociated virus; gp7O, 70,000-dalton glycoprotein; RIP, radioimmu-noprecipitation; RIA, radioimmunoassay.

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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competing antigen (1 hr, 370C) prior to addition of labeledgp70. This level of antibody was chosen to enhance the abilityof the assay to detect the greatest variety of antigenic deter-minants.Enzyme Treatments of SSV-SSAV gp7O. Approximately

250 ng of [3H]glucosamine-labeled gp7O purified by phos-phocellulose and hydroxylapatite chromatography was incu-bated for 16 hr at 370C in phosphate-buffered saline containingCa2+ and Mg2+ with 10 units of Vibrio cholerae neuraminidase(Behring, Somerville, NJ), 0.06 units of Diplococcus pneumo-niae endoglycosidase D, and 0.1 unit of Streptomyces griseusendoglycosidase H (Miles). An equal amount of gp7O was in-cubated with trypsin-TPCK (Worthington) at 20 ,gg of enzymeper ml for 2 hr at 37"C followed by addition of an excess ofsoybean trypsin inhibitor (Miles) for 16 hr at 370C. An equalamount of untreated control gp7O was incubated in the abovebuffer under the same conditions.

A B

gp370O- *W

p30 -_I-4-

FIG. 2. NaDodSO4 step-gradient gel electrophoresis ofSSV-SSAV gp7O preparations.(A) gp7O-containing fraction ofvirus bound to phosphocelluloseand eluted with 0.1-0.2 M NaCl.(B) gp7O from the phosphocel-lulose eluate bound to hydroxyl-apatite and eluted with 0-0.1 Mpotassium P04. Total protein ineach preparation was radioiodi-nated in vitro prior to electro-phoresis. Individual proteinbands were visualized by auto-radiography.

RESULTSPurification of SSV-SSAV gp7O and Demonstration of Its

Reactivity with Natural Human Antibodies. In an attemptto radiolabel and purify gp7O in such a way that its reactivitywith human antibodies was retained, we began by precursor-labeling SSV-SSAV in tissue culture to high specific activitywith [3H]glucosamine (105 trichloroacetic acid-precipitablecpm per ,g of total viral protein were obtained). Purified vir-ions were lysed with Nonidet P40 and the lysate was centri-fuged at 100,000 X g for 60 min. Soluble gp7O in the high-speedsupernatant served as a radiochemically (though not bio-chemically) pure probe that required no further manipulation(e.g., in vitro radioiodination) before testing its reactivity withnatural human antibodies. By direct precipitation more than75% of normal human sera was found to be reactive with thesolubilized gp7O probe, and the antibody titers fell over a widerange, as demonstrated by the titration curves of selected serashown in Fig. 1.

Purification of the [3H]glucosamine-labeled gp7O from themixture of viral proteins was performed by several differentprocedures: phosphocellulose chromatography followed byeither hydroxylapatite or DEAE-cellulose chromatography,

100

-om 60-._._QLO 50-

ECL 40-

or concanavalin A affinity chromatography followed bySephadex G-150 filtration. At each stage of the purificationprocedures, both the chemical purity and antigenicity weremonitored, the former by in vitro radioiodination and Na-DodSO4 slab gel electrophoresis (e.g., Fig. 2) and the latter byprecipitation with reactive human sera (Fig. 3). Our resultsshow that, although the absolute amounts of gp7O precipitatedby the test sera are somewhat lower with the purified probe,there is no question that the purified gp70s are reactive withthe antibodies in these sera. We have obtained results similarto those in Fig. 3 by using purified human immunoglobulinsand F(ab')2 fragments. These data confirm the finding of Kurthand Mikschy (14) that, under their reaction conditions, anti-bodies in normal human sera can be detected that are capableof precipitating purified SSV-SSAV gp7O.

Because each of our purified gp70s was reactive with thehuman antibodies, we could not ascribe discrepant results be-tween laboratories to antigen lability during purification.Therefore, further studies of the nature of the antibodies re-active in this assay were performed in order to resolve thequestion of specificity.

'D

(U)G)

a.E-1a.E

5 10 20 40 80 160Dilution-'

FIG. 1. RIP titrations of five normal human sera and goat anti-SSV-SSAV antiserum (X- X) with 5 ng of solubilized [3H]glu-cosamine-labeled gp7O.

5 10 20 40 80 160 5 10 20 40 80 160Dilution-'

FIG. 3. RIA titrations of two human sera with 5 ng of solubilizedunpurified [3H]glucosamine-labeled SSV-SSAV gp7O (0, 0), labeledgp7O bound to phosphocellulose and eluted with 0.1-0.2 M NaCl (A,A), gp7O purified on phosphocellulose followed by DEAE-cellulosechromatography (-, o), gp7O purified on phosphocellulose followedby hydroxylapatite chromatography (-, 0), and gp7O purified byconcanavalin A affinity chromatography followed by Sephadex G-150filtration (V, v).

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Apparent Contribution of Natural Heterophil Antibodiesto the RIP Reaction. Although the human antibody reactioncould be shown with both purified immunoglobulin and puri-fied gp70 and some evidence for viral specificity of these an-tibodies has been published (14), the widespread occurrenceof the antibodies in the human population suggested that theymight still be heterophil antibodies that are capable of cross-reacting with many antigens in nature. In a seroepidemiologicalstudy we observed that the incidence of human antibodies toSSV-SSAV gp7O varied significantly as a function of age: cordsera were largely unreactive, maximum incidence of reactivitywas observed between 5 and 20 yr of age, and there was a slowdrop in incidence thereafter (Fig. 4). It has been observed thatmany "natural" heterophil antibodies conform to this agepattern (27-29). Barbacid et al. (20) have demonstrated thatmany natural substances such as components of animal sera,extracts of bacteria, and even glycogen share some gp70 de-terminants recognized by human sera. We have confirmed thataddition of fetal calf serum proteins to the RIP reaction inter-feres with precipitation of the gp70 probe. Interference couldnot be attributed to proteolysis of our probe in the presence ofthe serum proteins because (i) both the treated and untreatedprobes exhibited identical electrophoretic profiles after analysisin NaDodSO4 gels and (ii) the titers of hyperimmune goatanti-SSV-SSAV antiserum with both probes were identical. Thefetal calf serum protein interference could be mimicked bysubstituting horse, rabbit, swine, or chicken serum proteins, butnot proteins from anti-gp70 antibody-negative human sera. Weinferred from our observation that the interfering capacity offetal calf serum was eliminated after passage over a column ofconcanavalin A-Sepharose that a serum glycoprotein or gly-colipid was the source of the crossreacting antigen. The mostprobable explanation for these results seemed to be that we weredetecting combinations of naturally occurring heterophil an-tibodies by virtue of their crossreaction with the viral gp7Omolecules.

Relative Contributions of Carbohydrate and Protein to theRIA Reactions. The fact that most chemically defined heter-ophil antigens have been shown to be associated with glycolipid(30, 31) suggested that the human antibodies might be bindingto the carbohydrate moiety of the gp7O. Our preparations ofpurified [3H]glucosamine-labeled gp70 were particularly wellsuited for analyzing the contribution of carbohydrate compo-

100-

80A

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C.,

40-

20-

020 40 60 80

Age, yrFIG. 4. Relative distribution of SSV-SSAV gp7O-precipitating

antibodies according to age of normal individuals. Each point repre-sents a minimum of 15 sera at the indicated average age.

0

6 2- B

xElg 1 -

O

020 40 60 80

FractionFIG. 5. NaDodSO4 gel electrophoresis of phosphocellulose-

hydroxylapatite purified [3H]glucosamine-labeled SSV-SSAV gp7O(A), an identical amount of gp7O after exo- and endoglycosidasetreatment (B), and an identical amount of gp7O after trypsin treat-ment (C).

nents to the overall reactivity. Equal quantities of purified gp70were treated with (i) a mixture of exo- and endoglycosidases,(ii) trypsin, or (iii) buffer only. NaDodSO4 gel electrophoreticanalysis of the three preparations is shown in Fig. 5. Over 90%of the [3H]glucosamine counts was removed from the gp7Omolecule by the glycosidase treatment. The sizes of the releasedoligosaccharides, expected to be approximately Mr 500041500(32), were too small to allow retention in these gels. There wasno apparent proteolytic activity in the glycosidase preparations,as determined by lack of any effect on electrophoretic mobilityand serological properties of SSV-SSAV p30 (not shown).Trypsinization of the probe resulted in loss of more than 60%of the gp7O-associated [3H]glucosamine cpm. Some cpm re-mained associated with the protein fragments distributedthroughout the length of the gel, whereas other cpm were as-sociated with much smaller fragments and free glycopeptidesthat migrated out of the gel.When enzyme-treated and untreated preparations of gp7O

were analyzed in parallel, the carbohydrate-associated radio-activity in all three probes was found to be still precipitable byhuman antibodies (Fig. 6). Reactive sera could be divided intotwo general classes. One class recognized the intact probe, thetrypsin-treated probe, and the glycosidase-treated probe withequal efficiency (Fig. 6A). The target antigen of this class ofantibody clearly seemed to be carbohydrate. The second classof sera showed some diminution in reactivity with the en-zyme-treated probes compared with the intact glycoprotein(Fig. 6B). Although this result could be taken as evidence thatboth carbohydrate and protein determinants are involved withthis class of antibodies, it is more likely that this class of anti-bodies is reactive with carbohydrate determinants that are (i)at or near glycosidase cleavage sites or (ii) contributed to in partby tertiary structures of the oligosaccharides that are alteredafter glycosidase or trypsin treatment of the intact glycoprotein.

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60-

50

40

30-

20

X 10

0)

a 70-Eg 60-' 50-

40-

30-

20-

10-

A

10 40 160Dilution-'

FIG. 6. RIA titrations of two human sera (A and B) with purified[:1Hlglucosamine-labeled gp7O (0), glycosidase-treated gp7O (-), andtrypsin-treated gp70 (-).

Given the known heterogeneity of carbohydrate antibodies inthe human population (ref. 33; also see below), the distinctionsobserved in Fig. 6 were not unexpected.

Further evidence that carbohydrate antibodies are involvedin these reactions was obtained in hapten inhibition studies.Various saccharides were tested for their ability to compete withlabeled viral glycoprotein for binding antibodies in limitingdilutions of test human sera. Representative data for two seraand three sugars are shown in Fig. 7 and data for six sera and10 sugars are summarized in Table 1. Partial but significantinhibition with different saccharides was observed among thesera tested. Slopes of the inhibition curves were, in general,rather shallow, implying a low affinity for the antibodies. It wastherefore not always possible to achieve a clear plateau levelof inhibition within the limitations of the assay, nor was itpossible to determine whether a given antibody was specificfor a particular saccharide, one with a similar structure, or an

antigen defined by the specific linkage between two sugars (33).

100- e

80-

60

C

0

40-

20-

0 ,0.01 0.1 1.0

Carbohydrate, MFIG. 7. Competition RIA of antibodies in limiting dilutions of

two human sera (open and solid symbols) for binding 5 ng of purified[3H]gp7O while in the presence of the indicated concentrations ofN-acetyl-galactosamine (triangles), melibiose (circles), and maltose(squares).

Table 1. Carbohydrate hapten inhibition studiesMaximum competition, %

Competitor A B C D E F

Mannose 34 0 0 50 2 41N-Acetyl-

galactosamine 20 50 0 45 40 66N-Acetyl-

glucosamine 10 50 6 32 25 20Lactose 16 20 29 96 100 18Glucose 20 17 5 45 21 25Maltose 30 30 30 59 25 41Raffinose 20 42 26Melibiose 20 41 12 50 44 34Galactose 40 42 60 36 29 25Fucose 46 55 29 32

An example of representative data is in Fig. 7. A, B, C, etc., referto human sera.

Nonetheless it was clear in these experiments that there werequalitatively different patterns of inhibition exhibited amongthe sera tested. This is supportive of other evidence for in-volvement of heterogeneous anticarbohydrate antibodies in thissystem.

DISCUSSIONNatural immunity encompasses antibodies reactive with variousdeterminants, either supplied by diet and intestinal tract bac-teria or due to subclinical infections (28, 38, 34). Autologousantigens may also contribute in some instances (33). Such nat-ural antibodies often crossreact with broadly reactive heterophilantigens, of which Forssman antigen, present in tissues of manyspecies as well as on both Gram-positive and Gram-negativebacteria, is the classic example (28, 30). These antigens are oftenassociated with glycolipids (30, 31). Human heterophil anti-bodies reactive with blood group substances can be traced toexposure to carbohydrate determinants present in the cell wallsof intestinal flora (27, 28, 31, 34). Humans usually develop suchblood group isoagglutinins within the first year of life; titerspeak at 5-20 yr and decline slowly thereafter (27, 28). Thispattern of age-related titer is found for other classes of naturalantibodies as well (27-29). The natural human antibodies re-active with SSV-SSAV gp70 display many of the propertiesassociated with heterophil antibodies: they are present in themajority of individuals, display a typical pattern of age-relatedreactivity, crossreact with other glycoproteins, and appear tobe directed against certain carbohydrate structures of the gp7Omolecule.

Addition of oligosaccharides to viral glycoproteins is a host-dependent process (35-40). Thus, glycosylation can varyqualitatively among cells used to produce virus and may confercell-coded antigenicity to a virus-coded protein backbone.Barbacid et al. (20) have shown that glycoproteins purifiedfrom several type-C viruses grown in human cells were notrecognized by human natural antibodies; in contrast, these gp70molecules when purified from the same virus grown in canine,feline, or rodent cells were reactive. These findings have beeninterpreted as evidence for a cellular rather than viral originof the antigenic determinants recognized by human serum inthe type-C viral envelope glycoproteins.

Glycosylation of viral proteins has also been shown to dependin part on the viral genetic origin of these proteins: in infectedmouse cells the Rauscher murine leukemia virus gp7O receivesa large complex oligosaccharide that the Sindbis virus envelopeprotein apparently cannot accept (32). This is consistent withour finding that gp7O of SSV-SSAV grown in rat cells is reactivewith natural human antibodies, whereas glycoproteins of

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Sindbis and vesicular stomatitis virus, produced in the samecells, are not (9, 41). Virus-specific differences in glycosylationcan occur among different oncoviruses as well. Thus, it hasrecently been found that the envelope glycoproteins of eco-tropic and dualtropic (MCF) murine leukemia viruses exhibitqualitative differences in their carbohydrate constituents whengrown in the same cell type (M. Kemp and N. Famulari, per-sonal communication). Similar considerations may explain ourobservation that particular oncovirus isolates are unreactivewith natural human antibodies, even when grown in nonhumancells. Examples are baboon-endogenous virus (BEV) from minkand bat cells and the related feline endogenous virus (RD1 14)produced in cat cells (ref. 41; unpublished data).Our results emphasize the need for extensive controls in de-

termining the specificity of human antibody recognition of viralprotein determinants, especially when sensitive RIP assays areused. In this respect, the recognized influence of neoplasia andchemotherapy on levels of natural heterophil antibodies (29,42) may necessitate some re-evaluation of data suggesting el-evated titers of oncoviral antibodies in patients with Hodgkindisease (18). The present data do not exclude the possibility thatantibodies in some individuals may result from direct exposureto oncoviruses. However, our results argue that the antibodiespresent in a considerable proportion of the human populationthat are capable of precipitating oncoviral gp7O are probablynot in fact due to such exposure.

Excellent technical assistance was provided by K. Phillips and S.Rosen. H.W.S. is a Special Fellow of the Leukemia Society of America.This research was conducted under support from Grant IM-91 fromthe American Cancer Society, National Cancer Institute Core GrantCA-08748, and National Institutes of Health Biomedical ResearchSupport Grant RR-05534.

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