The immune response to Epstein-Barr nuclear antigen: Conformational and structural features of antibody binding to synthetic peptides

MolecularImmunology, Vol. 21, No. 11,

pp.1047-1054.1984

Printed in Great Britain

0

0161-5890184 $3.00 + 0.00 1984 Pergamon PressLtd

THE IMMUNE RESPONSE TO EPSTEIN-BARR NUCLEAR ANTIGEN: CONFORMATIONAL AND STRUCTURAL FEATURES OF ANTIBODY BINDING TO SYNTHETIC PEPTIDES” GARY RHODES, RICHARD HOUGHTEN,-~ JOSEPH P. TAULANE,$. DENNIS CARSON

Departments

and JOHN VAUGHAN§ of Basic and Clinical Research and tMolecular

Biology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.; and SDepartment of Chemistry, University of California, San Diego, La Jolla, CA 92093, U.S.A. (Accepted

10 Jury 1984)

Abstract-Naturally developing human antibodies to the Epstein-Barr nuclear antigen recognize synthetic peptides containing sequences from the unusual glycine-alanine region of this protein. We tested antibody binding to a series of peptides of from five to 20 amino acids in length. Peptides as small as seven amino acids could bind but optimal results required chain lengths of 15. Binding was extremely sensitive to small changes in the length and sequence of the peptide, and also to the temp of the reaction. The changes can be ascribed to two factors: (1) deletion of the site of antigen binding and (2) loss of peptide secondary structure.

INTRODUCTION

The Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and also has been associated with two human malignancies. Essentially all humans are infected by EBV during childhood or adolescence. After the acute phase of infection, which may or may not be symptomatic, the virus persists in a small percentage of the Blymphocytes (Kleff et al., 1982). In this state, the viral DNA replicates along with the cellular DNA (Nonoyama and Pagano, 1972; Henderson et al., 1983) and expresses only a small number of the viral proteins (probably less than a half dozen). The best characterized of these is the Epstein-Barr nuclear antigen (EBNA). * This is publication number 3582BCR from the Research Institute of Scripps Clinic, La Jolla, CA. This research was supported in part by NIH grants AM21175 and AM07144. § Dedication by John Vaughan: When I was in Elvin’s laboratory, 1951-1953, he was just beginning his seminal work with the inhibition of anti-dextran antibodies by oligosaccharides of varying sizes and linkages, which provided the first estimate of the size of the combining site on antibody molecules. This work has stood the test of time and has been developed further by other laboratories. Our contribution here refocuses on this subject through studies of antibodies to oligopeptides in a virus-encoded protein, emphasizing the complexities presented by the secondary structures in the peptides. Understanding these complexities may be important both for understanding differences in the ways in which certain patient groups make immune responses and for developing synthetic peptides for effective immunization. O& dedication of ihis paper to Elvin on his 70th birthdav includes mv oersonal thanks to him for the beginning’he gave me’in’ my career and our appreciation together for the beginning he gave us to understanding the antigen-binding site.

EBNA can be visualized by immunofluorescence in the nucleus of all Epstein-Barr transformed cell lines (Reedman et al., 1973). The DNA which codes for this protein has been partially sequenced (Heller et al., 1982). The EBNA protein contains a remarkable stretch of 239 amino acids that are either glycine or alanine (Hennessy and Kieff, 1983; Hennessy et al., 1983). This glycine-alanine region comprises about 30% of the total protein. The persistence of the virus in its host must continuously stimulate the immune response because antibody titers to the viral capsid antigen (VCA) and the EBNA protein remain at high levels throughout life. Thus, most of the adult population is a source of anti-EBNA antisera and the antibodies are a marker of prior infection by the virus. We have shown (manuscript in preparation) that the anti-EBNA antibodies can be assayed in a standard enzyme-linked immunosorbent assay (ELISA) in which the wells are coated with synthetic peptides of 16-21 amino acids comprising sequences from the glycine-alanine region of EBNA. We have synthesized peptides from areas throughout the repeating region. One, designated peptide P62, has the highest activity when tested on over 100 human sera. We are interested in the nature of the EBNA/antiEBNA reaction because of the potential clinical significance and because patients with certain autoimmune diseases seem to mount a quantitatively or qualitatively different response to the EBNA protein (Catalan0 et al., 1979, 1980; Billings et al., 1983). The system also presents a unique opportunity to study the specificity of human antibodies to a protein of an infectious virus. To this end, we synthesized a series of peptides of

1047

GARY

1048

et al.

RHODES

increasing chain length and have measured the reactivity of each synthetic antigen with human antiEBNA antibodies. As peptide length is shortened from 20 to nine amino acids, its ability to be recognized by antibody also diminishes. The decrease appears to be due to two effects: (1) deletion of specific amino acid sequences to which the antibody binds and. (2) a change in the conformation of the peptide antigen. By manipulating the secondary structure of the peptide with temp changes one can partially uncouple these two effects. Thus, as previously emphasized by Goodman (1969), the minimum size of the peptides recognized is not solely defined by the dimensions of the antibody combining site or the amino acid sequence but also depends on the minimum size of the peptide necessary for it to assume secondary configurations necessary for antibody binding.

plates coated we have not bition concns constant but indication of reaction. Inhibition

with solid-phase peptide. Therefore attempted to interpret the 50% inhias measuring an average equilibrium merely used this value as a qualitative the strength of the antibody-inhibitor

at various temps

Plates were coated with peptide at room temp for 30 min. The BSA quenching solution was added and the plate incubated at the temperature specified for 90 min. At the same time, sera at a ?&u dilution was incubated at the appropriate temperature with peptide as described above. The BSA solution was discarded and the antibody-inhibitor solution was added to the plate and incubated for 90 min at the specified temp. After that time, the plates were returned to room temp, washed and subsequent treatment was identical to that described earlier.

MATERIALS AND METHODS

Circular dichroism

ELISA assay of antibody binding Standard 96-well microtiter plates (Costar) were coated for 15-30 min with a concn of 10 kg/ml peptide in BBS (0.05 M sodium borate, 0.14 M NaCl, pH 8.3) unless otherwise noted. The peptide solutions were discarded and excess binding sites were quenched for 30 min using a 1% bovine serum albumin (BSA) solution in BBS. This solution was discarded and 100 ~1 of human or rabbit antiserum diluted in BBS was added for 1 hr at room temp. The wells were washed with BBS containing 0.5% Tween 20 and then a solution of alkaline phosphatase coupled goat anti-human (or anti-rabbit when appropriate) antibody (Tago) was added at a dilution of 1/400. After 1 hr at room temp the wells were washed and incubated with 100 ~1 of a pnitrophenyl phosphate solution (1 mgiml in 0.05 M NaHCOa, pH 9.8, 1.0 mM MgCQ. The O.D. of the solution at 40.5 nm was determined after 1 hr [also see Chen et al. (1984)]. Inhibition peptide

of

antibody

binding

by

(CD) measurements

Spectra were taken using a Cary 61 spectropolarimeter interfaced and automated with a DEC 11102 Computer. The data in Fig. 1 are the average of 10 successive scans. The temp during these measurements was not controled and was approximately 22°C. The sequence of the unordered peptide, F12, is ile-met-ser-asp-glu-gly-pro-gly-thr-gly-asn-gly-leugly-glu-cys.

I

a competing

8 Y

This assay is identical with the standard ELISA described earlier, except that the antibody and competing peptide were incubated at room temp for 30 min with 5 times the final concn of peptide desired and then were diluted with BBS and incubated for another hour. This solution was then added to the P62 peptide coated plate. This procedure was used to assure that antibody-peptide binding would reach an equilibrium value even at the lowest peptide concns. However the same results were obtained for several sera if antibody and competing peptide were simply added at the final concn desired and incubated for an hour. Although the antibody-inhibitor preincubation solutions were at equilibrium, this was not necessarily still true when this solution was added to the

J

PEPTIOE

F12

I

200

I

220

WAVELENGTH

240

260

(nm)

Fig. 1. Circular dichroism spectra of peptides P62 and F12. Both peptides were at a concn of 1 mgiml in a solution of 10 mM sodium phosphate, 0.14 M NaCl, pH 6.8. The mean residue ellipticity, [0], is expressed in degreescm*idmole.

The immune

response

to Epstein-Barr

Materials Rabbit antibodies to the peptides were prepared by coupling the peptide to keyhole limpet hemocyanin (Green et al., 1982) and immunizing the rabbit with 1 mg coupled peptide in Freund’s complete adjuvant followed by a boost with the same amount of peptide 1 month later in Freund’s incomplete adjuvant. The animals were bled after a further month. Serum S62 was made against peptide P62 while serum S60 was prepared against another sequence from within the glycine-alanine region of EBNA and cross-reacted with peptide P62. All human sera are from rheumatoid arthritis patients with the exception of serum N6 which was from a normal control group. Data analysis Values of 50% inhibition were estimated by drawing a smoothed curve through the data points and interpolating. The results are repeatable to within a factor of 2 or 3. The major source of variation is probably due to temp variations from day to day. RESULTS

We have synthesized peptides, each 16-22 amino acids in length, from multiple areas throughout the 239 amino acid glycine-alanine region of the EBNA protein. The abilities of these peptides to bind to anti-EBNA antibodies in over 100 human sera were tested (manuscript in preparation). We can summarize these findings as: (1) all VCA+ sera have antibodies that will recognize at least one of the glycine-alanine peptides, (2) no VCA- sera recognize these peptides, and (3) individual sera exhibit preference for different peptides. One of these glycine-alanine peptides, P62, generally displayed the highest activity with most sera and was chosen as the prototype peptide for these studies. The sequence of P62 is shown in Table 1. The CD spectrum of the parent peptide and this is shown in Fig. 1. The main features of this spectrum are a minimum at 222 nm and a peak at 213 nm.

Table P62

1. Sequences

nuclear

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antigen

These attributes indicate a substantial amount of secondary structure in solution. For comparison, the spectrum of another peptide, F12, of a completely different sequence is also shown. The featureless curve above 210 nm is indicative of a random conformation and is the more usual result obtained with peptides of this size. The surprising finding is that the characteristic pattern of peptide P62 was observed with every peptide we tested which contains the glycine-alanine sequence from the EBNA region but not with any peptides from outside this region. The actual secondary structure of the peptide cannot be decided entirely from the CD spectrum. The minimum at 222 nm probably indicates some beta structure although some of the unique structures first discovered in poly(gly) are also possible. An alpha-helix is less likely. Studies with model peptides [Brack and Spach (1972) and see Discussion] also support this view. The presence of a negative absorption band at 200 nm may indicate either that there is also some disordered conformation present or that the conformation at this temp is highly ordered and the negative band is due to the formation of some left-handed conformations. Thus, the model of peptide structure which emerges from the CD spectrum is that of a relatively rigid structure which may or may not be in equilibrium with a less ordered state. The highly ordered structure of all the glycine-alanine peptides in solution at 20°C may explain the efficient binding of the peptides to naturally developing human antiEBNA antibodies. Peptide P62 consists of 20 amino acids of which the first nine are directly repeated (Table 1). The synthetic peptides Dl, D2 and D3 delete three amino acids from the N-terminus of the preceding peptide, thus removing increasing portions of the sequence symmetry of P62. We tested the D-series, in the solid phase of the ELISA, against human and rabbit sera (Fig. 2). Antibody binding to peptide Dl was nearly the same as that to the parent peptide P62 for all sera tested, although two-four-fold higher concns of the peptide were necessary to

of the synthetic

peptides

AGAGGGAGG*AGAGGGAGGAG

Dl D2 D3

GGGAGGAGAGGGAGGAG AGGAGAGGGAGGAG AGAGGGAGGAG GGGAG AGGGAG GAGGGAG AGAGGGAG GAGAGGGAG

A5 A6 A7 AS A9 The * indicates glycine.

the junction

of the direct

repeat

in P62. Abbreviations:

A, alanine;

G,

GARY RHODES et al

1050 (a)

(b)

Table

2. Concn of competing peptide which gives 50% inhibition of antibody binding to peptide P62 Concn

producing

50% inhibition

Competing

Peptlde

concn

(pg/mL)

peptide

Sera

P62

Dl

D2

D3

TJ VM cv N6 JC

0.05 0.3 0.1 0.6 1

0.05 0.3 0.1 0.6 1

0.1 0.4 0.1 0.6 1

200 3 10 3 >500

(cl

60.

40 20

./’

IL, i/

o-

_-!. IO

Peptide

Fig. 2. Binding

of selected

All data are expressed

20

concn

40

(@g/ml)

sera to peptides

Dl, D2 and D3.

as the percentage

of binding

observed when the plates are coated with peptide P62. The plates were coated with: (a) peptide Dl, (b) peptide D2, and (c) peptide D3. The sera in each panel are: (U) S62, (A-A) TJ, (0-O) VM and (B-W) CV.

saturate the plate (see Fig. 2). In contrast, there was no binding to solid-phase D3 for any serum except S62, a rabbit antiserum raised against peptide P62. The results for peptide D2 were intermediate and seemed to depend on the serum tested. Thus, antibody recognition of this series of peptide is severely depressed as its length was shortened from 20 to 11 amino acids. The sequence symmetry of the peptide assures that all sequences of four-eight amino acids which are present in peptide P62 are also in D3, with the exception of those across the junction of the repeat. This suggests that it is not simply the loss of a particular amino acid sequence that is causing the decrease of peptide binding and that another factor is contributing to the decline in antibody recognition. One possibility is that the conformation of the peptide is changed when it binds to the solid phase. We tested this possibility by inhibiting the binding of antibody to solid-phase P62 by increasing concns of competing peptides in solution. In these experiments the antibody was first incubated with the peptide in solution for an hour before adding it to a microtiter plate coated with P62. The results of two representative experiments are shown in Fig. 3 and the results with several sera are summarized in Table 2. The inhibitory action of peptide Dl (17 amino acids long) upon antibody binding to P62 is

indistinguishable from the inhibitory effect of P62 itself. This is true for all sera tested. More interestingly, peptide D3, which is only 11 amino acids in length, inhibited two of the sera, VM and N6, about as well as the larger peptides did. This strong inhibitory effect occurred despite the fact that neither of the sera bound efficiently solid-phase D3. We interpret these data as indicating that the peptides must be of a certain minimum length of about 15 amino acids to maintain their secondary structure when binding to the plastic surface of the microtiter plates. We next turned our attention to determining the minimal antigen size necessary for recognition. By comparing the activity of various glycine-alanine peptides with a number of sera we determined that the sequence gly-ala-gly-gly-gly must be important in the recognition of many sera. Thus we constructed the group of peptides A5-A9 in Table 1. Peptide A5 has five amino acids and the series increases by one amino acid up to the nine amino acids of peptide A9. From the data with the Dpeptides we expected that no antibody would bind to solid-phase peptides of this length and, indeed, this was the case. More importantly, the data in Table 3 show that most all sera tested were inhibited by peptide A9 although very high concns were required (>lOO-fold higher than the concns of P62 or Dl needed for equivalent inhibition). In addition, three sera were inhibited by A8 and one by A7. None were inhibited by the shorter peptides. If the decrease in binding activity of the peptides is partly due to loss of secondary structure, one Table

3. Inhibition of antibody binding to peptide 100 wgiml competing peptide % of uninhibited

P62 by

activity

Sera

A5

A6

A7

A8

A9

TJ VM cv JC S62 S60

96 93 92 94 X6 106

80 81 93 92 96 109

91 51 93 60 89 93

74 17 72 88 95 84

31 9 20 74 78 53

The immune response to Epstein-Barr

-4

-3

-2

-I

0

I

-4

2

log [inhibitor

concn

1051

nuclear antigen

-3

-2

-I

0

I

2

tpg/ml)l

Fig. 3. Inhibition of binding of sera TJ and N6 to solid-phase peptide P62. Panel (a) serum TJ and (b) serum N6. The inhibiting peptides are (U) P62, (A-A) Dl, (0-D) D2, and (m-m) D3. would expect to see those same results if the peptide structure is perturbed by other means. This can be done by changing the temp of the peptide solution. In general, an ordered peptide structure would be disrupted or melted as the temp rises. We first tested several sera for the ability to bind to solid-phase peptide P62 at four temps. One of the sera, TJ, produced the same level of antibody binding (within 7%) at all four temps tested. Other sera reacted differently to temp. Serum VM bound best at 8°C and binding fell off monotonically with temp, while serum N6 had its maximum binding at 18°C and was lower at both higher and lower temps. We assume the complex behaviour of the latter two sera reflect changes in the antibody molecule because solid-phase peptide is expected to be immobile. Since these changes were not present in serum TJ, it was chosen for peptide inhibition analysis. Table 4 displays the inhibitory activity of a single concn of all peptides at four different temps. In each case, the inhibitory power of the peptide decreased with increasing temp. Note that while peptides A8 and A9 inhibit at the lowest temp (8°C) there was no evidence of any inhibition at this temp with peptide A7. Also note that peptide A9 (nine amino acids) was a slightly better inhibitor than peptide D3 (11 amino acids) at each temp. Peptide A9 covers the same region as D3 except that it has one more amino acid at the amino terminal end and three amino acids less at the carboxy terminal end. We feel that serum TJ probably binds to the amino terminal portion of both peptides and removing amino acids from this region shortens the primary antibody-

binding site. It would be interesting to delete peptide A9 from the other (carboxy terminal) end in order to define more exactly the minimal antigen site. Based on the current data, we can place an upper limit of eight amino acids as the size of the antigenic determinant. A complimentary series of experiments determined the concn of competing peptide needed to produce 50% inhibition in antibody binding to solidphase P62. These data are shown in Table 5. All the peptides required higher concns for equivalent inhibition as the temp increased. The smaller peptides were more affected by temp changes. For example, the amount of P62 required for equivalent inhibition increased four-fold as the temp was increased from 8 to 18°C. In contrast, the same temp increase required a change of greater than 600-fold for equivalent inhibition by peptide D3. Thus, temp can have a dramatic effect on the efficiency with which a peptide is bound to an antibody. DISCUSSION

The classical studies of Kabat and coworkers (Kabat, 1966) defined the size of the antigenbinding site as equivalent to five-six glucose residues for anti-dextran antibodies, or of the order of 3 nm, assuming the most extended conformation of the antigen. An estimate of the combining site of induced rabbit antibodies to synthetic homopolypeptides indicated a size of four-eight amino acids, depending on the system studied (Goodman, 1969; Schechter et al., 1966). Kabat has also suggested that antibody combining sites can have different

Table 4. Inhibition of serum TJ binding to peptide P62 by 100 kg/ml competing peptide at several temps Temp (“C) 8 18 30 37

% of uninhibited activity A5

A6

A7

A8

A9

D3

D2

Dl

87 90 84 90

98 95 94 101

95 94 89 87

60 77 84 86

18 29 52 79

21 44 61 79

2 5 14 39

0 0 2 3

GARY RHODESet al.

1052

Table 5. Concentration of competing peptide which gives 50% inhibition of serum TJ binding to peptide P62 Concn producing 50% inhibition Temp (“C) Peptide P62 Dl D2 D3

8

18

30

31

0.005 0.005 1.0 0.3

0.02 0.02 3.0 190

0.05 0.2 20.0 >400

0.3 1.0 300 > 1000

geometries, depending on the structure of the antigen. Antibodies to internal portions of a polymer antigen might be expected to have an extended groove-like structure of perhaps 3 nm whereas those directed at the ends would have a pocket-like structure of half this size (Kabat, 1976; Kabat, 1978). The results presented here probe the binder site of naturally developing human antibodies directed at the interior portion of a viral protein. At lower temps and in solutions, where conformational fluctuations of the peptides are minimized, there is no difference between peptides of 20 and 17 amino acids in length and little change in reducing this to 14 amino acids. Thus, depending on peptide conformation, one can estimate that the total functional combining site is approximately 2-3 nm. Since we still see some inhibition with peptides containing only eight amino acids the primary site extends for 1 nm. These estimates are consistent with X-ray structural studies of antibodies. Although there are no data on antigen-antibody complexes, binding of haptens to crystalline antibodies has partially defined the binding sites of two antibodies (Davies and Metzger, 1983; Poljak, 1978; Davies et al., 1975; Kabat, 1976). One finds extended grooves and smaller pockets of the expected dimensions. The size of the peptides and the temp and solvent conditions are known to influence the structure of peptides in solution. A systematic study of the chain length dependence of gamma-methyl glutamic acid in dimethylformamide solvent (Goodman et al., 1960) showed that a helical structure formed during the increase from heptapeptide to nonapeptide. This same transition occurs in oligo(ala) around 10 residues in dichloroacetic acid (Goodman et al., 1966) but is not complete even with peptides as long as 22 amino acids with lysine oligomers (Yaron et al., 1971). Generally, structures are also less stable in water than in the less polar solvents. Thus the structures of peptides of lo-20 amino acids are expected to be quite sensitive to changes in size and solvent conditions. Several groups have reported structural studies of polymers of repeating sequence of glycine and alanine which have been synthesized as models of silk fibroin (Anderson et al., 1972) and collagen

(Doyle et al., 1970; Anderson et al., 1970). The most systematic study has been that of Brack and Spach (1972), who synthesized a series of polymers of the form poly(ala,-gly,). In the solid state, the polymers composed of alanine were alpha-helical and those containing mostly glycine were disordered. In solution poly(ala) was alpha-helical but poly(alazgly) was in a beta antiparallel form. The more glycine-rich polymers had another fixed structure which is neither an alpha-helix nor a beta structure. Even though the solvents used here were not physiological, these results illustrate two points: (1) structural changes can occur as the sequence of a peptide change, and (2) structural changes also occur during the transition from solution to solid state. Much of the work on the binding of peptides to higher-titer anti-protein antibodies has been reviewed by Benjamini et al. (1972). Most of the studies to determine the effects of changes in the sequence of peptides binding antibody have been interpreted as indicating the fine specificity of the antibody-binding site. Some of these results can equally well be explained by unexpected alterations in antigen structure that alters binding (Goodman, 1969). The best example of this is the studies of antibody directed against the tobacco mosaic virus coat protein (Benjamini et al., 1968; Young et al., 1967). Part of the antibody response to this protein is against the C-terminal decapeptide. Shortening this to the C-terminal pentapeptide drastically decreases antibody binding, but adding five alanine residues to the N-terminus of the pentapeptide restores binding to decapeptide levels, even though the alanine is unrelated to the original sequence. Poly(ala) is known to favor the alpha-helix (Brack and Spach, 1972) and this may stabilize a peptide conformation necessary for antibody binding. Up to this point we have been discussing peptide conformations involving only one peptide chain. Another possibility is that dimer or even multimer formation can occur between peptide molecules. This is especially likely if the conformations involved are beta structures which require a tight chain turn if there is no association between different peptide chains. There are hints that intermolecular interactions may occur. The far-u.v. spectra of some of our peptides show a red shift as the concn of the peptides is increased from 10 to 50 M (unpublished). In addition, higher solution concns are required to saturate a microtiter plate as the peptide length is shortened (see Fig. 2), even though the amount of peptide in solution is vastly greater than the amount bound to the plate. Both of these are perhaps clues that intermolecular peptide interactions may provide a structure necessary for antibody binding. Resolution of these points will require further experiments. Immune interactions at the molecular level involve binding of an antigen in a defined sequence

The immune response to Epstein-Barr (primary structure) and in a defined conformation (secondarv or tertiarv \ i i structure). / The immune response to protein antigens has traditionally been interpreted as being directed against the primary, secondary or tertiary structure of the protein. This classification scheme mavi be valid for uroteins which have a well-defined overall structure at physiological temps and solutions. Clearly, it breaks down for peptide antigens which have a more dynamic structure. For examule. one would describe antibody to peptide D3 as directed at the primary structure if assayed at 8°C but at the secondary structure if assayed at 30°C. In this paper we present direct evidence that small changes in chain length, sequence and temp can drastically alter the lability _of synthetic eBNA peptides to bind to human antibody. The simplest interpretation of these data is that there are peptide conformations which the anti-EBNA antibodies do not recognize. Careful comparison of antibody binding with changes in antigen conformation induced by temp can be used to distinguish between the simultaneous changes in sequence and conformation which can occur when amino acid substitutions and deletions are placed in oligopeptides. Precisely defining these parameters is a necessary prerequisite for the development of immunogenic synthetic peptides for vaccination. Acknowledgements-We

thank Drs Pojen Chen, Richard Sportsman and Sherman Fong for helpful discussions and Jean Valbracht for excellent technical assistance. We are indebted to Drs Arlinghaus and R. Smith of the Johnson and Johnson Biotechnology Center for providing peptides Dl, D2 and D3. The comments of Dr S. J. Singer were valuable and are greatly appreciated. REFERENCES

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Kabat E. A. (1966) The nature of an antigenic determinant. J. Zmmun. 97, l-11. Kabat E. A. (1976) Structural Concepts in Immunology and Immunochemistry, pp. 119-166. Holt, Rinehart & Winston, New York. Kabat E. A. (1978) The structural basis of antibody complementarity. Adv. Protein Chem. 32, l-75. Kieff E., Dambauah T., Heller M., King W.. Cheung A.. van Santen V., hummel M., Beisel e., FennewalUd S.: Hennessy K. and Heineman T. (1982) The biology and chemistry of Epstein-Barr virus. J. infect. Dis. 146. 506-517: . Nonoyama M. and Pagan0 J. (1972) Separation of Epstein-Barr virus DNA from large chromosomal DNA in non-virus-producing cells. Nature New Biol. 238, 169-171. Poljak R. J. (1978) Correlations between three-dimensional structure and function of immunoglobulins. CRC Crit. Rev. Biochem.

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Reedman B. M. and Klein G. (1973) Cellular localization of an Epstein-Barr virus associated complement-fixing

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