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Immunogenic structure of the influenza virus hemagglutinin
Cell, Vol. 28, 477-487,
March
1982,
Copyright
0 1982
by MIT
Immunogenic Structure of the Influenza Virus Hemagglutinin Nicola Green, Hannah Alexander, Arthur Olson, Stephen Alexander, Thomas M. Shinnick, J. Gregor Sutcliffe and Richard A. Lerner Committee for the Study of Molecular Genetics and the Department of lmmunopathology Research Institute of Scripps Clinic La Jolla, California 92037
Summary We chemically synthesized 20 peptides corresponding to 75% of the HA1 molecule of the influenza virus. Antibodies to the majority (18) of these peptides were capable of reacting with the hemagglutinin molecule. These 18 peptides are not confined to the known antigenlc determinants of the hemagglutinin molecule, but rather are scattered throughout its three-dimensional structure. In contrast, antibody raised to intact hemagglutinin did not react with any of the 20 peptides. Taken together these results suggest that the immunogenicity of an intact protein molecule is not the sum of the immunogenicity of Its pieces. Introduction From the considerable amount of work carried out on the immunogenicity of proteins, the general conclusion has been that an immune response is directed against a small number of antigenic determinants confined to a few loci in the molecule. These are predominantly made up of higher order structures composed of amino acid sequences brought into proximity by folding or looping of the protein chain (“conformational determinants”) (Benjamini et al., 1972; Crumpton, 1974; Atassi, 1975). Arnon and her colleagues were able to synthesize a conformation-dependent determinant of lysozyme that elicited antibodies reactive with the native molecule (“antiloop antibodies”) (Arnon and Sela, 1969; Arnon et al., 1971). In contrast with the point of view that most antigenic regions of a protein are dependent on complex conformations, recent studies have demonstrated the feasibility of the use of antisera prepared against chemically synthesized short linear peptides to study structural and biological aspects of native proteins, indicating that at least some antigenic determinants arise from conformations adoptable by these peptides (Arnon, 1980; Sutcliffe et al., 1980; Walter et al., 1980; Green et al., 1981; Lerner et al., 1981 a, 1981 b). Such antipeptide antisera have distinct advantages. Since they are raised by immunization with defined linear polypeptides, we know the exact region within the native molecule with which the antisera react. Thus any biological or biochemical perturbation achieved by such antisera can be understood with a precision not
heretofore possible in studies of structure-function relations. In addition, these sera contain a heterogeneous population of antibodies with respect to antigen affinity and immunoglobulin class or subclass, and are therefore not likely to be restricted in their ability to manifest a biological effect. We have used antisera to 20 peptides encompassing 75% of the HA1 molecule to study the immunogenic structure of the influenza virus hemagglutinin. The influenza hemagglutinin is the major viral surface antigen. It is a strain-specific glycoprotein. and can be seen by electron microscopy to form spikes that radiate from the usually spherical lipid envelope. Each spike is a trimer in which the monomers consist of two polypeptide chains, HA1 (46,000-65,000 daltons, depending on the virus strain) and HA2 (21 ,OOO30,000 daltons) (reviewed by Laver, 1973; Schulz, 1973). HA1 and HA2 are synthesized as a single polypeptide precursor (HA) that is subsequently cleaved at the plasma membrane to form the disulfidelinked two-chain monomer (Lazarowitz et al., 1971; Skehel, 1972; Hay, 1974). Both HA1 and HA2 contribute to the fibrous, stemlike conformation that forms the proximal membrane attachment end of the hemagglutinin molecule, while HA1 alone forms the distal globular region (Wilson et al., 1981). In its trimeric form the hemagglutinin is responsible for the attachment of virus to the host cell, and a number of studies have shown that it is antibody to the hemagglutinin that blocks infection, both in vivo and in vitro (Laver and Kilbourne, 1966; Laver, 1973; Schulz, 1973; Dowdle et al., 1974; Schulman, 1975; Virelizier, 1975; Breschkin et al., 1981). The influenza hemagglutinin is well known for its ability to undergo antigenie variation. Although minor variation can occur throughout the length of its primary sequence, recent evidence suggests that certain sites of the hemagglutinin molecule may be particularly important in the elicitation of major antigenic alterations that permit escape from existing immune defenses (antigenic drift) (Laver et al., 1980; Webster and Laver, 1980; Wiley et al., 1981). We wished to carry out an in-depth analysis of the immunogenicity of a single molecule of known structure when presented to the immune system as short linear sequences. We chose to study the immunogenicity of peptide domains of the influenza virus X47 (H3N2) hemagglutinin for two reasons. Not only was the DNA sequence of the X47 hemagglutinin gene known (Min Jou et al., 1980), but also the recent solution of the crystallographic structure to 3 A of the closely related A/Hong Kong hemagglutinin (X31, H3N2) (Wilson et al., 1981) allowed us to identify the position of our synthetic peptides within the molecular structure, and permitted us to correlate the precise location of specific antigenic determinant8 with the reactivity of antibodies prepared against them. When
Cdl 478
presented in pieces, most regions of the protein molecule were immunogenic and elicited antibodies reactive with the hemagglutinin. Thus the conclusion from classical studies, that the immunogenicity of a protein is confined to a few loci, does not hold when a molecule is presented as short sequences. Results Characteristics of Peptldes Selected for Synthesis Since most studies indicate that under natural conditions HA1 is the major target of the immune System (reviewed by Laver, 1973; Schulman, 19751, we have focused our attention on this portion of the chain. The HA1 of influenza strain X47 contains 329 amino acid residues (Min Jou et al., 1980). We have selected a series of 20 partially overlapping peptides that cover 75% of the HA1 chain. Figure 1 shows the position of these peptides on the linear sequence of X47 HA1 . In Figure 2 the position of synthetic peptides in the prototype hemagglutinin monomer structure of A/ Hong Kong/88 (X31 1 (Wilson et al., 1981) is shown. Sequence data have revealed 21 changes between X31 and X47 in HA1 , and 4 in HA2 (Verhoeyen et al., 1980); both are H3N2. The 95% sequence homology of X47 to X31, including all cysteine disulfide bridges, indicates that the structures will be very similar (Min Jou et al., 1980; Verhoeyen et al., 1980; Wilson et al., 1981). Thus we have analyzed our peptides with reference to the X31 structure. Figures 2A and 2B show two views of the HA monomer in which all synthesized peptides are indicated. In Figures 2C-2L selected individual peptides are positioned on the monomer to illustrate structural 1
-16 (~~KTIIALSYIFCLVFA)
20
40
Y
80
2
-o’-
3
100
DGINCTLID;LLLGDPHCDGFQNEKWDLFVERSKAFSNCYPYDVPDYANGGSSAC
-g1 Y-
60
bDLPGNDNNSTATLCLGHHANGTLVKTiTN~IE~N~TELVQSSSTGKICNNPIIRIL Y-lY-d-
Y
- l, -6 -Y
regions that were synthesized. In individual views the monomer is differentially rotated around its long axis to allow the best assessment of the position of the synthetic peptide. Starting at the amino terminus of HA1 , peptide 2 (Figure 2C; residues l-36) begins at the membrane-attachment end of the hemagglutinin and continues outward in an extended conformation along the long axis of the molecule. The carboxyterminal region of this peptide forms a laterally oriented looplike structure that overlaps both the ascending and descending limbs of the HA2 chain. In the trimeric structure, this loop would be on the surface with some residues inaccessible. Peptide 3 (Figure 2D; residues 15-53) contains a segment of the extended conformation of peptide 2, the entire laterally oriented loop, and additional residues in an extended conformation that lead up to a site of intrachain disulfide bonding between residues 53 and 278. The next two peptides, 4 (Figure 2E; residues 39-65) and 7 (Figure 2F; residues 53-87) contain the sequences surrounding residue 53. This region, and its paired segment surrounding residue 278, form a bulge that is external in the trimer and that has been proposed as a possible antigenic site in the native molecule (Laver et al., 1980; Wiley et al., 1981). Beginning at residue 65, the carboxy-terminal region of peptide 7 forms a short disulfide loop (residues 65-77) and enters a three-turn helical structure, which is continued in the overlapping peptide 10 (Figure 2G; residues 76-l 11). The four subsequent peptides lie within an eight-stranded antiparallel P-sheet structure that extends from residues 117 to 282. Peptide 11 (Figure 2H; residues 105-140) overlaps peptide 10 in the helical structure, then forms a short, external loop
,g-
Y
-7-3-Y 120
140
11
12
Y-12y-13-
-1‘l -15 --16
160 180 200 KRGPDSGFF~RLMILYKSG~TYPVQ~~NNDNSDKLYi~VKKPSTD~EQ~LYVQ~GK~STK~~TIIPNVG~ -17-c Y-l-12-13-13-c 16 -IS-14-Y
220
240 260 280 KP~KGLSSicISI~IVK~GDILVINSN~NLI~RGYF~~KSSIM~D~IG~S~ECITPNGSI~NDKPFQN~~ -19-c
300
320 ITYGACPKY;KQNTLKLAT&RNVPEKOTR
Figure 1. Amino Acid Sequence As Predicted from the Nucleotide
of X47 HA1 Sequence
Nucleotide sequence data are from hlin Jou et al. (1980). Regions of the protein selected for synthesis are underlined and numbered. C or Y at one end: addition of cysteine or tyrosine not found in the primary sequence.
z;gthetic
Influenza
Virus
Peptides
parallel to the long axis of the hemagglutinin molecule. This area is possibly involved in two antibody-binding sites (Laver et al., 1980; Wiley et al., 1981). Peptide 12 (Figure 21; residues 130-l 51) encompasses a region that forms another loop (residues 135-149) protruding horizontally from the structure. This region is thought to be of antigenic significance (Laver et al., 1980; Webster and Laver, 1980; Wiley et al., 1981). Peptide 17 (Figure 2J; residues 174-l 96) forms an a! helix at the distal end of the molecule. This helix has been proposed as part of an antigenic site in the native structure (Wiley et al., 1981). Peptide 19 (Figure 2K; residues 201-227) lies on an external face of the monomer and crosses laterally from the protruding loop structure of peptide 12 to the opposite side of the globular region. However, in the trimer, some of residues 202-217 of peptide 19 are buried at the interface formed by juxtaposition of the globular regions of the three monomers. Finally, peptide 20 (Figure 2L; residues 306-329) contains the carboxyl terminus of HA1 , and is located in the stem region of the molecule. It forms interchain interactions with HA2. The Majority of Antipeptide Antibodies Are Capable of Reacting with the Intact Hemagglutinin An enzyme-linked immunosorbent assay (ELISA) was used to test rabbit antisera for their ability to react with the peptide used for immunization, with peptides that contain sequences overlapping the peptide used for immunization, with hemagglutinin isolated from virus by bromelain cleavage and with intact X47 virus. Antibody titers are expressed as the reciprocal of the antiserum dilution that binds 50% of 5 pmole of antigen. In Figure 3 the results of such assays for 20 antiHA1 -peptide antisera are shown. All the peptides tested were immunogenic; titers against the peptides used for immunization ranged from 5 to 1280 (Figure 3). At the highest concentration, antisera to unrelated peptides did not score in the ELISA, and thus correction for background was not necessary. Most often these antibodies were also capable of reacting with overlapping peptides, although the titer against an overlapping peptide was usually significantly lower than the titer against the peptide used for immunization. In general, however, peptides in the area of the protruding loop formed by residues 135-l 49 did not exhibit reactivity with over-
Figure 2. (Over/ear) Stereoscopic Hemagglutinin Monomers
Display
of the Positions
of Sequences
lapping peptides. Of the 20 anti-HA1 -peptide antisera, 15 recognized HA1 determinants as they are present in hemagglutinin isolated from X47 virus by bromelain cleavage (Figure 3, X47 HA column). The five peptides (8, 9, 13, 14, 16) that induced antibodies unreactive with purified hemagglutinin cluster into two regions of the linear sequence (see Figure 1). Peptides 8 and 9 span residues 76-90, while peptides 13, 14 and 16 overlap in the region of the loop formed by residues 135-l 49. Most antisera showed the same reactivity with intact virus as they did with purified hemagglutinin. The four exceptions were: antisera to peptides 9, 13 and 16, which were nonreactive when assayed against purified hemagglutinin but which were positive (albeit low-titered) when assayed against intact X47 virus; and antiserum to peptide 12, which had a titer of 10 against purified hemagglutinin but which was negative against X47 virus. None of the antipeptide antibodies reacted with unrelated synthetic peptides or with the noncrossreacting hemagglutinin A/ PR/8/34 (Hl Nl) (data not shown), again indicating the specificity of the reaction. A structural representation of the ELISA data on the hemagglutinin trimer is shown in Figure 4. Figure 4A depicts the positions of all peptides synthesized, while Figure 46 shows peptides that were capable of eliciting antibodies reactive with the hemagglutinin and Figure 4C shows peptides that elicited antibodies unreactive with the hemagglutinin. Because of overlapping peptides that contain reactive as well as unreactive determinants, the loop region formed by residues 135-l 49 (Figure 4C, arrow) is shown in both Figures 48 and 4C. Peptides 13 (residues 132-l 48) and 14 (residues 140-l 471, which lie almost entirely within this loop region, elicited antibodies unreactive with the hemagglutinin molecule; thus they are shown in Figure 4C. However, peptides containing this region plus additional sequences on either side of the loop (11 [residues 105-l 401. 12 [residues 130-l 511 and 15 [residues 140-l 561) did elicit antibodies that reacted with the hemagglutinin molecule, and are therefore shown in Figure 48. The positive reactions observed with these sera may be caused by antigenic determinants lying outside the loop region. Alternatively, the presence of additional residues could facilitate folding of the loop sequence into a more favorable conformation. The ELISA results demonstrating the ability of anti-
Corresponding
to Synthetic
HA1 Peptides
in the Tertiary
Structure
of the
(A and 6) Pink: all HA1 residues synthesized. Yellow: HA1 residues not selected for synthesis. Blue: HA2 chain. (C-L) Representation of selected individual peptides. Yellow: indicated HA1 peptide. Red: side chains of indicated peptide. Light blue: residues of HA1 chain not contained in the indicated peptide. Dark blue: HA2 chain. (C) Peptide 2. residues l-36. (D) Peptide 3, residues 15-53. (E) Peptide 4. residues 39-65. (F) Peptide 7. residues 53-67. (G) Peptide 10, residues 76-l 11. (H) Peptide 11, residues 105-140. 6) Peptide 12. residues 130-l 51. (J) Peptide 17, residues 174-l 96. (K) Peptide 19. residues 201-227. (L) Peptide 20. residues 306-329. Residue numbers correspond to the sequence of Min Jou et al. (1960). The 1966 Hong Kong hemagglutinin structure was determined by Wilson et al. (1961).
Cell 480
Synthetic 481
Influenza
Virus Peptides
Cell 482
Figure
3. Reactivity
of Antipeptide
Sera with HA1 Peptides
and Intact
Hemagglutinin
Larger peptides consisting of 34 or more amino acids were Injected in two forms, uncoupled (free) and coupled to keyhole (KLH) through cysteine residues GKLH). Peptides 16 and 17 were insoluble in aqueous buffers and were injected uncoupled.
limpet hemocyanin All other peptides
Titers in ELISA are expressed as the reciprocal of the serum dilution that binds 50% of 5 pmole of antigen (stippled boxes). A noncrossreacting rabbit antiserum (prepared against a hepatitis B envelope peptide) was used in each assay to assess background activity. Diagonal lines: no reactivity. Open boxes: the particular antiserum-antigen combination has not been tested. X47 HA: purified hemagglutinin isolated by bromelain cleavage. X47 virus: intact virus.
HA1 -peptide antibodies to recognize intact hemagglutinin were confirmed by immunoprecipitation studies of ‘251-labeled X47 hemagglutinin isolated from virus by bromelain cleavage. Figure 5 shows SDS-polyacrylamide gel electrophoresis of immunoprecipitates in which representative anti-HA1 -peptide antibodies were reacted with iodinated hemagglutinin. Control serum prepared against a peptide unrelated to hemagglutinin showed no reactivity above nonspecific trapping of radioactivity (Figure 5, lane a), while an antiserum that contained specificities for a number of viral proteins (raised by immunization with X47 virus; Figure 5, lane b) and two anti-HA1 -peptide sera (Figure 5. lanes c and d) precipitated proteins with apparent molecular weights of 57.000 and 22,000 daltons, corresponding to HA1 and HA2, respectively. Similar results have been obtained in immunoprecipitation studies of the total protein from disrupted virus, in that all antisera reactive with virus in the ELBA specifically precipitated HA1 and HA2 (data not shown). HA2 is precipitated because it is disulfide-linked to HAl;
when the HA1 and HA2 chains are separated by elution from polyacrylamide gels, only HA1 remains precipitable with anti-HA1 -peptide sera (data not shown). Antibodies against X47 Virus Do Not React with the Synthetic Peptides The data show that the majority of antipeptide antibodies are capable of recognizing the HA1 molecule. We also performed the converse experiment. Rabbit antiserum against the intact X47 virus was tested for reactivity with individual synthetic peptides (Figure 3). The antiviral antibody did not react with any of the synthetic peptides. Discussion A reasonable prediction based on the literature would be that antipeptide antibodies would react with the native molecule only if the peptide represented a domain seen during an immune response directed
Synthetic 483
Figure
4.
The view (A) Pink: (B) Pink: (0 Pink: protruding
Influenza
Virus Peptides
Dual Stereoscopic
Displays
of the Positions
of Synthetic
Peptides
in the Hemagglutinin
Trimer
on the right looks down from the top of the molecule through the long axis. all HA1 peptides synthesized. Yellow: HA1 residues not contained in synthesized peptides. Blue: HA2 chain. HA1 peptides eliciting antibodies capable of reacting with the hemagglutinin. Yellow: all other HA1 residues. Blue: HA2 chain. HA1 peptides eliciting antibodies that were unreactive with the hemagglutinin. Yellow: all other HA1 residues. Blue: HA2 chain. loop formed by residues 135-l 49.
An
Cell 484
a
b
c
d
45K+ 30K+ 14.3K-*
Figure 5. Immunoprecipitation Antibodies
of X47 Hemagglutinin
by Antipeptide
‘251-labeled hemagglutinin isolated by bromelain cleavage (1 X 10’ cpm) was reacted with 10 PI of one of the following: control serum specific for a peptide unrelated to influenza (lane a); serum raised by immunization with intact X47 virus (lane b); anti-peptideantiserum (lane c): or anti-peptideantiserum (lane d). Precipitates were collected with Staphylococcus aureus and subjected to electrophoresis on a 5%-l 7% SDS-polyacrylamide gel prior to autoradiography.
against native protein. Consequently, the usefulness of antipeptide antibodies would be predicated on locating those domains seen during an immune response directed against native proteins. In the case of the influenza HA1 , these areas have been mapped by analysis of variants (Laver et al., 1980; Webster and Laver, 1980; Willey et al., 1981). We thus expected that peptides that were capable of eliciting antibodies reactive with HA1 would cluster to those mapped regions. This was not the case. Our basic finding is that synthetic peptides encompassing about 75% of the HA1 sequence, and corresponding on the intact HA1 molecule to a diverse array of secondary structures within the native target, generate antibodies reactive with the hemagglutinin. Probably for most proteins, the sole requirement for generation of antipeptide antibodies reactive with native molecules is that the peptide be represented on the surface of the structure. All the peptides in this study that elicited antibodies reactive with hemagglutinin have at least a portion that is on the surface of the molecule, as indicated by crystallographic analysis. As we have previously noted (Lerner et al., 1981 b), the fact that most of the peptides contain proline may be significant. In contrast with peptides, immunization with the intact hemagglutinin may result in the preferential recognition of complex determinants by the immune system. The fact that antibodies to intact virions do not recognize any of our peptides, which represent 75% of the primary HA1 sequence, supports this assertion. This may be a consequence of constraints
in the way larger protein molecules are presented. Perhaps the potential of the relatively unrestricted immunological repertoire is narrowed by a limited presentation system, which we can somewhat circumvent by presenting the molecule in pieces. If this is true, then the issue is not whether native antigens and peptides give comparable titers to a given determinant, but rather whether the elicited immunological specificities are to different determinants. It already seems likely that short peptides may induce antibodies that could not be raised by the native structure. One can think in similar terms about the evolution of changes in the HA1 molecule. This molecule, of course, evolves in the face of the entire immunological potential of the host, but the changes we see may only reflect nonlethal mutations sufficient to evade antibodies generated during ordinary presentation of antigens during infection. Presumably, the chemically synthesized peptides are able to elicit antibodies reactive with the native molecule because they exist for part of the time in a conformation that is the same as or similar to that which occurs in the intact protein (Sachs et al., 1972; Lerner et al., 1981 b). The immunological repertoire for a given peptide must be much larger than the few conformations that approximate the native form, but the magnitude of that repertoire is unknown and will probably differ from peptide to peptide. At any rate, considerations of immunological diversity must take into account not only all possible immunogens, but also all possible conformations. Along these lines, it is interesting to note that our antibodies to the C terminus of HA1 have the highest titer. This may simply reflect the observation that the C-terminal amino acids of HA1 appear flexible on the x-ray structure. The last few amino acids of HA1 may adopt a number of conformations and may therefore be recognized by antipeptide antisera to several different peptide conformations. One way of looking at the problem is that the bulk of the hemagglutinin serves as a “carrier” for the C-terminal peptide, a situation not too remote from that which one obtains when the peptide is coupled to keyhole limpet hemocyanin. Antisera to five of the HA1 peptides (8, 9, 13, 14, 16) showed no (or very little) reactivity for the hemagglutinin molecule. Since the titers of these five antisera fell within the normal range when tested against the immunizing peptide, their lack of reactivity with the hemagglutinin must be explained on the basis of the hemagglutinin structure. The five peptides define two regions of the hemagglutinin molecule, and, as is shown in Figure 4C, a considerable proportion of each region is highly accessible in the trimeric structure. Peptides 8 and 9 overlap and span the segment from residues 76 to 90. We note that the oligosaccharideattachment site Asns4 may affect the ability of antipeptide sera to recognize this region. Also, neither 8 nor 9 contains a proline. The second group of peptides that elicited anti-
z;;thetic
Influenza
Virus
Peptides
bodies unreactive with hemagglutinin was 13, 14 and 16. Their shared sequence forms a protruding loop (residues 135-l 49; Figure 4C, arrow) that has been shown to be an important region involved in antigenic variation (Laver et al., 1980; Webster and Laver, 1980; Wiley et al., 1981). One possible explanation for the failure of antisera to these three peptides to react with hemagglutinin is that coupling to the carrier through the cysteine at position 140 prevented the peptide from taking the native loop conformation. Such an effect was not observed, however, with other peptides in this study having internal cysteines. Antibodies to peptides within this segment of polypeptide chain were not capable of reacting with overlapping peptides. This observation indicates that in this region, neighboring residues may greatly alter a peptide’s antigenic conformation. Surprisingly, peptide 15, which is entirely contained within peptide 16, was successful in eliciting antibodies reactive with the hemagglutinin. Perhaps peptide 16 (36 residues) is large enough that it assumes a stable conformation unlike its structure within HA1 . Alternatively, the low solubility of peptide 16 may prevent its assuming the conformation found in the intact molecule. One additional explanation that could account for the inability of certain antipeptide antisera to recognize the intact hemagglutinin lies in the high degree of genetic variability characteristic of this molecule. The virus preparations used to obtain purified hemagglutinin and the intact virus used for ELBA were different, the latter having been adapted to growth in mice. Since neither represented a stock identical to the one used to obtain the DNA sequence data, it is possible that both had undergone some degree of genetic variation. Such variation seems more likely to have occurred in the loop region, since changes in the loop are more frequently selected with antibodies (Laver et al., 1980; Webster and Laver, 1980). Recent studies by others have used proteolytic fragments and synthetic peptides to study the immunogenicity of HA1 (Jackson et al., 1982). Jackson and his colleagues synthesized a decapeptide corresponding to the loop formed by residues 140-l 50 of the A/Hong Kong/68 (X31) strain of influenza. Although, as in our study, they were unable to demonstrate that antibody to this region significantly bound to hemagglutinin, they did show that antibody to the intact hemagglutinin bound to the synthetic peptide. In results similar to ours, Arnon was able to elicit antibodies to a synthetic HA1 peptide that were capable of binding to and neutralizing virus (R. Arnon, personal communication). Experimental
Procedures
Syntheels of Peptides For this study, peptides were synthesized by J. K. Chang, who used the solid-phase methods developed by Merrifield and his colleagues (Marglin and Merrifield. 1970). Each synthetic peptide was subjected to acid hydrolysis in vacua (6 N HCI. at 11O’C for 72 hr) and the
amino acid composition was determined. No attempt was made to remove multimeric forms, since the sole usa of the peptides was as immunogens. Peptides were synthesized according to the amino acid sequence predicted from the nucleotide sequence &tin Jou et al., 1980). When necessary. an additional cysteine (for the purpose of coupling to the protein carrier) or an additional tyrosine (for labeling purposes). or both, were added to the sequence. Coupling of Synthetic Peptides to Carrier Protein Peptides were coupled to the carrier protein keyhole limpet hemocyanin (KLH) through the cysteine of the peptide. with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as the coupling reagent (Liu et al., 1979). The peptides were dissolved in phosphate-buffered saline (pH 7.51, 0.1 M sodium borate buffer (pH 9.0) or 1 .O hi sodium acetate buffer (pH 4.0). The pH for the dissolution of the peptide was chosen to optimize peptide solubility. The content of free cysteine for soluble peptides was determined by Ellman’s method (Ellman, 1959) and ranged from 30% to 90%. For each peptide. 4 mg KLH in 0.25 ml of 10 mM sodium phosphate buffer (pH 7.2) was reacted with 0.7 mg MBS (dissolved in dimethyl formamide) and stirred for 30 min at room temperature. The MBS was added dropwise to ensure that the local concentration of formamide was not too high, as KLH is insoluble in ~30% formamide. The reaction product, KLH-MB. was then passed through Sephadex G25 equilibrated with 50 mM sodium phosphate buffer (pH 6.0) to remove free MBS. KLH recovery from peak fractions of the column eluate (monitored by OD& was estimated to be approximately 80%. KLH-MB was then reacted with 5 mg peptide dissolved in 1 ml of the chosen buffer. The pH was adjusted to 7-7.5 and the reaction was stirred for 3 hr at room temperature. Coupling efficiency was monitored with radioactive peptide by dialysis of a sample of the conjugate against phosphate-buffered saline, and ranged from 8% to 60%. Preparation of Antlpeptkfe and Antiviral Antibodies Rabbits were immunized according to the following schedule: first, 200 pg peptide-coupled KLH in complete Freund’s adjuvant (1:1) subcutaneously on day 0: second, 200 pg in incomplete Freund’s adjuvant (1 :l) subcutaneously on day 14; and finally, 200 pg with 4 mg alum intraperitoneally on day 21. Animals were bled 4 and 5 weeks after the first injection. After the initial course of injections, the rabbits were boosted with 200 pg peptide-coupled KLH with 4 mg alum at 5 week intervals, and bled 1 and 2 weeks following the injection. Large peptides were sometimes injected uncoupled (1 mg per injection). Antiviral antisera were prepared according to the same protocol, with each rabbit receiving 700 pl X47 allantoic fluid per injection. Enzyme-Linked lmmunosorbent Assay Reactivity of the various antipeptide antisera was determined in a two step ELISA. Antibody was bound to antigen immobilized on polystyrene plates, and the antigen-antibody complex was detected with an enzyme-labeled goat antirabbit IgG. This “sandwich” complex was measured enzymatically by the addition of enzyme substrate. Five picamoles of antigen in 0.1% bovine serum albumin in phosphatebuffered saline were added per well of a 96 well microtiter plate and dried overnight at 37°C. (For the the purpose of calculating 5 pmole of antigen. the molecular weight of pure hemagglutinin. in its glycosylated trimeric form, was taken as 224,640 daltons. In assays with Intact X47 virus as a target, HA1 was assumed to be 20% of the viral protein content [Schulz, 19731.) The dried antigen was then fixed to the plates with 50 pl per well of methanol for 5 min at room temperature. Before adding the antibodies. we coated the plates for 4 hr with 3% bovine serum albumin in phosphate-buffered saline to block nonspecific absorption of antibodies to the plate. Serial dilutions (1:2) of each antiserum were prepared in minimal essential medium containing 10% fetal calf serum, 25 pl of serum dilution were added per well and the plates were incubated overnight at room temperature. The unbound antibody was then washed off with water. The antigen-antibody complex was reacted with 25 pl per
Cell 466
well of 1 :lOO dilution of goat antirabbit IgG coupled to glucose oxidase CTernynck and Avranmeas, 1977). Excess goat antirabbit antibody was washed off with water, and the reaction was developed by adding 50 f.&Iper well of developing solution (25 ml of 0.1 M sodium phosphate buffer [pH 6.01; 3 ml of 20% glucose; 200 cl of 0.1% horseradish peroxidase: 200 ~1 of chromogen [2,2’-azino-di<3-athylbenzthiazolin-sulfonat)] at 20 mg/ml in sodium phosphate buffer [pH 6.01). The plates were read in Titertek Multiskan at A,.. and the values presented are the reciprocal of the serum dilution at the 50% point of the dilution curve. lmmunoprecipitation lmmunoprecipitation with formalin-fixed S. aureus. and polyacrylamide gel electrophoresis of immunoprecipitates, were carried out according to procedures we have used previously (Lerner et al., 1981 a). ‘251-IabeIed X47 hemagglutinin was suspended in RIPA (0.15 M NaCI, 10 mM sodium phosphate [pH 7.51. 1% Nonidet P-40,0.05% sodium deoxycholate, 0.1% SDS) and reacted (5 x 10’ cpm per reaction) at 0°C with 10 pl of test serum or control rabbit serum for 1 hr. Precipitates were collected with S. aureus. and pellets were washed with RIPA. then twice with 500 mM LiCI. 100 mM Tris (pH 8.5). For gel electrophoresis. washed pellets were suspended in gel loading buffer, boiled, centrifuged to remove S. aureus and subjected to electrophoresis on a 5%-l 7% acrylamide-SDS gel, and the gel was autoradiographed. Acknowledgments We thank Drs. I. A. Wilson, D. C. Wiley and J. J. Skehel for providing the x-ray coordinates for the HA1 molecule and for introducing us to Dr. Arthur Olson. We thank Brian Browne and Suzie Seaver for excellent technical assistance. This is publication no. 2598 from the Research Institute of Scripps Clinic. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
October
28. 1981;
revised
December
2. 1981
defined
antiviral
vaccines.
Arnon, R. and Sela, M. (1969). Antibodies lysozyme provoked by a synthetic antigen Acad. Sci. USA 62, 163-l 70.
Tissue
sulfhydryl
groups.
Arch.
Biochem.
Ann. Rev.
to a unique region in conjugate. Proc. Nat.
Arnon, R.. Maron. E., Sela. M. and Anfinsen. C. B. (1971). Antibodies reactive with native lysozyme elicited by a completely synthetic antigen. Proc. Nat. Acad. Sci. USA 68, 1450-l 455. Atassi, M. Z. (1975). Antigenic structure of myoglobin: the complete immunochemical anatomy of a protein and conclusions relating to antigenic structures of proteins. Immunochemistry 72. 423-438. Benjamini, E., Michaeli. D. and Young, J. D. (1972). Antigenic determinants of proteins of defined sequences. Curr. Topics Microbial. Immunol. 58, 85-134. Breschkin. A. M.. Ahern. J. and White, D. 0. (1981). Antigenic determinants of influenza virus hemagglutinin VIII. Topography of the antigenic regions of influenza virus hemagglutinin determined by competitive radioimmunoassay with monoclonal antibodies. Virology 7 13, 130-l 40. Crumpton, M. J. (1974). Protein antigens: the molecular bases of antigenicity and immunogenicity. In The Antigens, 2, M. Sela, ed. (London: Academic Press). pp. l-78. Dowdle, W. R.. Downie. J. C. and Laver. W. G. (1974). Inhibition of virus release by antibodies to surface antigens of influenza viruses. J. Virol. 13, 269-275.
Bio-
Green, N., Shinnick, T. M., Witte. 0.. Ponticelli, A., Sutcliffe, J. G. and Lerner. R. A. (1981). Sequence-specific antibodies show that maturation of Moloney leukemia virus envelope polyprotein involves removal of a COOH-terminal peptide. Proc. Nat. Acad. Sci. USA 78, 6023-6027. Hay, A. J. (1974). envelope. Virology
Studies on the formation 60, 398-418.
of the influenza
virus
Jackson, D. C.. Murray, J. M., Brown, L. E. and White D. 0. (1982). lmmunochemical properties of influenza virus hemagglutinin and its fragments. In ICN-UCLA Symposia, D. Nayak. ed. (New York: Alan R. Liss). in press. Laver. W. G. (1973). Res. 18, 57-103
The polypeptides
of influenza
viruses.
Adv. Virus
Laver, W. G., and Kilbourne, E. D. (1966). Identification in a recombinant influenza virus of structural proteins derived from both parents. Virology 30, 493-501. Laver. W. G.. Air, G. M., Dopheide, T. A. and Ward, C. W. (1980). Amino acid sequence changes in the haemagglutinin of A/Hong Kong (H3N2) influenza virus during the period 1968-77. Nature 283, 454457. Lazarowitz, S. G.. Cornpans. R. W., and Choppin, P. W. (1971). Influenza virus structural and nonstructural proteins in infected cells and their plasma membranes. Virology 46, 830-843. Lerner, R. A., Green, N., Alexander, H., Liu, F.-T., Sutcliffe. J. G. and Shinnick. T. M. (1981 a). Chemically synthesized peptides predicted from the nucleotide sequence of the hepatitis B virus genome elicit antibodies reactive with the native envelope protein of Dane particles. Proc. Natl. Acad. Sci. USA 78, 3403-3407. Lerner. R. A., Sutcliffe. J. G.. and Shinnick. T. M. (1981 b). Antibodies to chemically synthesized peptides predicted from DNA sequences as probes of gene expression. Cell 23, 309-310. Liu, F.-T., Zinnecker. M., Hamaska, T. and Katz, D. H. (1979) New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of o-amino acids and immunochemical characterization of such conjugates. Biochemistry 18. 690-697. Marglin. A. and Merrifield. R. B. (1970). Chemical synthesis tides and proteins. Ann. Rev. Biochem. 39, 841-866.
References Arnon, R. (1980). Chemically Microbial. 34, 593-618.
Ellman, G. L. (1959). phys. 82, 7077.
of pep-
Min Jou. W., Verhoeyen. M.. Devos, R.. Saman, E.. Fang, R., Huylebroeck, D.. Fiers, W., Threlfall, G.. Barber, C.. Carey, N. and Emtage, S. (1980). Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA. Cell 19, 683-696. Sachs, D. H.. Scheehter. A. M., Eastlake, A. and Anfinsen. C. B. (1972). An immunologic approach to the confirmational equilibrium of polypeptides. Proc. Nat. Acad. Sci. USA 89, 3790-3794. Schulman. J. L. (1975). Immunology of influenza. Viruses and Influenza. E. D. Kolbourne, ed. (New Press), pp. 373-393. Schulz, I. T. (1973). 18, l-55.
Structure
Skehel, J. J. (1972). Polypeptide cells. Virology 49, 23-30.
of the influenza synthesis
virion.
in influenza
In The Influenza York: Academic Adv. Virus
Res.
virus-infected
Sutcliffe. J. G.. Shinnick, T. M.. Green, N.. Liu. F.-T., Niman, H. L. and Lerner, R. A. (1980) Chemical synthesis of a polypeptide predicted from nucleotide sequence allows detection of a new retroviral gene product. Nature 287, 801-805. Ternynck, T. and Avranmeas. S. (1977). Conjugation of pbenzoquinone treated enzymes with antibodies and Fab fragments. Immuncchemistry 14, 767-774. Verhoeyen. M., Fang, R.. Min Jou, W.. Devos, R., Huylebroeck. D., Saman. E. and Fiers. W. (1980). Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/ Victoria/3/75. Nature 286, 771-776.
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Virus Peptides
Virelizier, J. L. (1975). Host defenses against influenza virus: the role of antihaemagglutinin antibody. J. Immunol. 7 15. 434-439. Waiter, G.. Scheidtmann. K.-H., Carbone. A., Laudano, Doolittle. Ft. F. (1980). Antibodies specific for the carboxy terminal regions of simian virus 40 large tumor antigen. Acad. Sci. USA 77, 5197-5200.
A. P. and and aminoProc. Nat.
Webster, Ft. G. and Laver. W. G. (1980). Determination of the number of nonoverlapping antigenic areas in Hong Kong (H,Nz) influenza virus hemagglutinin with monoclonal antibodies and the selection of variants with potential epidemiological significance. Virology 104, 139-l 48. Wiley, D. C., Wilson, I. A. and Skehel, J. J. (1981). Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289, 373-378. Wilson, I. A., Skehel. J. J. and Wiley, haemagglutinin membrane glycoprotein olution. Nature 289, 388-373.
D. C. (1981). of influenza
Structure virus at
of the res-
3A
March
1982,
Copyright
0 1982
by MIT
Immunogenic Structure of the Influenza Virus Hemagglutinin Nicola Green, Hannah Alexander, Arthur Olson, Stephen Alexander, Thomas M. Shinnick, J. Gregor Sutcliffe and Richard A. Lerner Committee for the Study of Molecular Genetics and the Department of lmmunopathology Research Institute of Scripps Clinic La Jolla, California 92037
Summary We chemically synthesized 20 peptides corresponding to 75% of the HA1 molecule of the influenza virus. Antibodies to the majority (18) of these peptides were capable of reacting with the hemagglutinin molecule. These 18 peptides are not confined to the known antigenlc determinants of the hemagglutinin molecule, but rather are scattered throughout its three-dimensional structure. In contrast, antibody raised to intact hemagglutinin did not react with any of the 20 peptides. Taken together these results suggest that the immunogenicity of an intact protein molecule is not the sum of the immunogenicity of Its pieces. Introduction From the considerable amount of work carried out on the immunogenicity of proteins, the general conclusion has been that an immune response is directed against a small number of antigenic determinants confined to a few loci in the molecule. These are predominantly made up of higher order structures composed of amino acid sequences brought into proximity by folding or looping of the protein chain (“conformational determinants”) (Benjamini et al., 1972; Crumpton, 1974; Atassi, 1975). Arnon and her colleagues were able to synthesize a conformation-dependent determinant of lysozyme that elicited antibodies reactive with the native molecule (“antiloop antibodies”) (Arnon and Sela, 1969; Arnon et al., 1971). In contrast with the point of view that most antigenic regions of a protein are dependent on complex conformations, recent studies have demonstrated the feasibility of the use of antisera prepared against chemically synthesized short linear peptides to study structural and biological aspects of native proteins, indicating that at least some antigenic determinants arise from conformations adoptable by these peptides (Arnon, 1980; Sutcliffe et al., 1980; Walter et al., 1980; Green et al., 1981; Lerner et al., 1981 a, 1981 b). Such antipeptide antisera have distinct advantages. Since they are raised by immunization with defined linear polypeptides, we know the exact region within the native molecule with which the antisera react. Thus any biological or biochemical perturbation achieved by such antisera can be understood with a precision not
heretofore possible in studies of structure-function relations. In addition, these sera contain a heterogeneous population of antibodies with respect to antigen affinity and immunoglobulin class or subclass, and are therefore not likely to be restricted in their ability to manifest a biological effect. We have used antisera to 20 peptides encompassing 75% of the HA1 molecule to study the immunogenic structure of the influenza virus hemagglutinin. The influenza hemagglutinin is the major viral surface antigen. It is a strain-specific glycoprotein. and can be seen by electron microscopy to form spikes that radiate from the usually spherical lipid envelope. Each spike is a trimer in which the monomers consist of two polypeptide chains, HA1 (46,000-65,000 daltons, depending on the virus strain) and HA2 (21 ,OOO30,000 daltons) (reviewed by Laver, 1973; Schulz, 1973). HA1 and HA2 are synthesized as a single polypeptide precursor (HA) that is subsequently cleaved at the plasma membrane to form the disulfidelinked two-chain monomer (Lazarowitz et al., 1971; Skehel, 1972; Hay, 1974). Both HA1 and HA2 contribute to the fibrous, stemlike conformation that forms the proximal membrane attachment end of the hemagglutinin molecule, while HA1 alone forms the distal globular region (Wilson et al., 1981). In its trimeric form the hemagglutinin is responsible for the attachment of virus to the host cell, and a number of studies have shown that it is antibody to the hemagglutinin that blocks infection, both in vivo and in vitro (Laver and Kilbourne, 1966; Laver, 1973; Schulz, 1973; Dowdle et al., 1974; Schulman, 1975; Virelizier, 1975; Breschkin et al., 1981). The influenza hemagglutinin is well known for its ability to undergo antigenie variation. Although minor variation can occur throughout the length of its primary sequence, recent evidence suggests that certain sites of the hemagglutinin molecule may be particularly important in the elicitation of major antigenic alterations that permit escape from existing immune defenses (antigenic drift) (Laver et al., 1980; Webster and Laver, 1980; Wiley et al., 1981). We wished to carry out an in-depth analysis of the immunogenicity of a single molecule of known structure when presented to the immune system as short linear sequences. We chose to study the immunogenicity of peptide domains of the influenza virus X47 (H3N2) hemagglutinin for two reasons. Not only was the DNA sequence of the X47 hemagglutinin gene known (Min Jou et al., 1980), but also the recent solution of the crystallographic structure to 3 A of the closely related A/Hong Kong hemagglutinin (X31, H3N2) (Wilson et al., 1981) allowed us to identify the position of our synthetic peptides within the molecular structure, and permitted us to correlate the precise location of specific antigenic determinant8 with the reactivity of antibodies prepared against them. When
Cdl 478
presented in pieces, most regions of the protein molecule were immunogenic and elicited antibodies reactive with the hemagglutinin. Thus the conclusion from classical studies, that the immunogenicity of a protein is confined to a few loci, does not hold when a molecule is presented as short sequences. Results Characteristics of Peptldes Selected for Synthesis Since most studies indicate that under natural conditions HA1 is the major target of the immune System (reviewed by Laver, 1973; Schulman, 19751, we have focused our attention on this portion of the chain. The HA1 of influenza strain X47 contains 329 amino acid residues (Min Jou et al., 1980). We have selected a series of 20 partially overlapping peptides that cover 75% of the HA1 chain. Figure 1 shows the position of these peptides on the linear sequence of X47 HA1 . In Figure 2 the position of synthetic peptides in the prototype hemagglutinin monomer structure of A/ Hong Kong/88 (X31 1 (Wilson et al., 1981) is shown. Sequence data have revealed 21 changes between X31 and X47 in HA1 , and 4 in HA2 (Verhoeyen et al., 1980); both are H3N2. The 95% sequence homology of X47 to X31, including all cysteine disulfide bridges, indicates that the structures will be very similar (Min Jou et al., 1980; Verhoeyen et al., 1980; Wilson et al., 1981). Thus we have analyzed our peptides with reference to the X31 structure. Figures 2A and 2B show two views of the HA monomer in which all synthesized peptides are indicated. In Figures 2C-2L selected individual peptides are positioned on the monomer to illustrate structural 1
-16 (~~KTIIALSYIFCLVFA)
20
40
Y
80
2
-o’-
3
100
DGINCTLID;LLLGDPHCDGFQNEKWDLFVERSKAFSNCYPYDVPDYANGGSSAC
-g1 Y-
60
bDLPGNDNNSTATLCLGHHANGTLVKTiTN~IE~N~TELVQSSSTGKICNNPIIRIL Y-lY-d-
Y
- l, -6 -Y
regions that were synthesized. In individual views the monomer is differentially rotated around its long axis to allow the best assessment of the position of the synthetic peptide. Starting at the amino terminus of HA1 , peptide 2 (Figure 2C; residues l-36) begins at the membrane-attachment end of the hemagglutinin and continues outward in an extended conformation along the long axis of the molecule. The carboxyterminal region of this peptide forms a laterally oriented looplike structure that overlaps both the ascending and descending limbs of the HA2 chain. In the trimeric structure, this loop would be on the surface with some residues inaccessible. Peptide 3 (Figure 2D; residues 15-53) contains a segment of the extended conformation of peptide 2, the entire laterally oriented loop, and additional residues in an extended conformation that lead up to a site of intrachain disulfide bonding between residues 53 and 278. The next two peptides, 4 (Figure 2E; residues 39-65) and 7 (Figure 2F; residues 53-87) contain the sequences surrounding residue 53. This region, and its paired segment surrounding residue 278, form a bulge that is external in the trimer and that has been proposed as a possible antigenic site in the native molecule (Laver et al., 1980; Wiley et al., 1981). Beginning at residue 65, the carboxy-terminal region of peptide 7 forms a short disulfide loop (residues 65-77) and enters a three-turn helical structure, which is continued in the overlapping peptide 10 (Figure 2G; residues 76-l 11). The four subsequent peptides lie within an eight-stranded antiparallel P-sheet structure that extends from residues 117 to 282. Peptide 11 (Figure 2H; residues 105-140) overlaps peptide 10 in the helical structure, then forms a short, external loop
,g-
Y
-7-3-Y 120
140
11
12
Y-12y-13-
-1‘l -15 --16
160 180 200 KRGPDSGFF~RLMILYKSG~TYPVQ~~NNDNSDKLYi~VKKPSTD~EQ~LYVQ~GK~STK~~TIIPNVG~ -17-c Y-l-12-13-13-c 16 -IS-14-Y
220
240 260 280 KP~KGLSSicISI~IVK~GDILVINSN~NLI~RGYF~~KSSIM~D~IG~S~ECITPNGSI~NDKPFQN~~ -19-c
300
320 ITYGACPKY;KQNTLKLAT&RNVPEKOTR
Figure 1. Amino Acid Sequence As Predicted from the Nucleotide
of X47 HA1 Sequence
Nucleotide sequence data are from hlin Jou et al. (1980). Regions of the protein selected for synthesis are underlined and numbered. C or Y at one end: addition of cysteine or tyrosine not found in the primary sequence.
z;gthetic
Influenza
Virus
Peptides
parallel to the long axis of the hemagglutinin molecule. This area is possibly involved in two antibody-binding sites (Laver et al., 1980; Wiley et al., 1981). Peptide 12 (Figure 21; residues 130-l 51) encompasses a region that forms another loop (residues 135-149) protruding horizontally from the structure. This region is thought to be of antigenic significance (Laver et al., 1980; Webster and Laver, 1980; Wiley et al., 1981). Peptide 17 (Figure 2J; residues 174-l 96) forms an a! helix at the distal end of the molecule. This helix has been proposed as part of an antigenic site in the native structure (Wiley et al., 1981). Peptide 19 (Figure 2K; residues 201-227) lies on an external face of the monomer and crosses laterally from the protruding loop structure of peptide 12 to the opposite side of the globular region. However, in the trimer, some of residues 202-217 of peptide 19 are buried at the interface formed by juxtaposition of the globular regions of the three monomers. Finally, peptide 20 (Figure 2L; residues 306-329) contains the carboxyl terminus of HA1 , and is located in the stem region of the molecule. It forms interchain interactions with HA2. The Majority of Antipeptide Antibodies Are Capable of Reacting with the Intact Hemagglutinin An enzyme-linked immunosorbent assay (ELISA) was used to test rabbit antisera for their ability to react with the peptide used for immunization, with peptides that contain sequences overlapping the peptide used for immunization, with hemagglutinin isolated from virus by bromelain cleavage and with intact X47 virus. Antibody titers are expressed as the reciprocal of the antiserum dilution that binds 50% of 5 pmole of antigen. In Figure 3 the results of such assays for 20 antiHA1 -peptide antisera are shown. All the peptides tested were immunogenic; titers against the peptides used for immunization ranged from 5 to 1280 (Figure 3). At the highest concentration, antisera to unrelated peptides did not score in the ELISA, and thus correction for background was not necessary. Most often these antibodies were also capable of reacting with overlapping peptides, although the titer against an overlapping peptide was usually significantly lower than the titer against the peptide used for immunization. In general, however, peptides in the area of the protruding loop formed by residues 135-l 49 did not exhibit reactivity with over-
Figure 2. (Over/ear) Stereoscopic Hemagglutinin Monomers
Display
of the Positions
of Sequences
lapping peptides. Of the 20 anti-HA1 -peptide antisera, 15 recognized HA1 determinants as they are present in hemagglutinin isolated from X47 virus by bromelain cleavage (Figure 3, X47 HA column). The five peptides (8, 9, 13, 14, 16) that induced antibodies unreactive with purified hemagglutinin cluster into two regions of the linear sequence (see Figure 1). Peptides 8 and 9 span residues 76-90, while peptides 13, 14 and 16 overlap in the region of the loop formed by residues 135-l 49. Most antisera showed the same reactivity with intact virus as they did with purified hemagglutinin. The four exceptions were: antisera to peptides 9, 13 and 16, which were nonreactive when assayed against purified hemagglutinin but which were positive (albeit low-titered) when assayed against intact X47 virus; and antiserum to peptide 12, which had a titer of 10 against purified hemagglutinin but which was negative against X47 virus. None of the antipeptide antibodies reacted with unrelated synthetic peptides or with the noncrossreacting hemagglutinin A/ PR/8/34 (Hl Nl) (data not shown), again indicating the specificity of the reaction. A structural representation of the ELISA data on the hemagglutinin trimer is shown in Figure 4. Figure 4A depicts the positions of all peptides synthesized, while Figure 46 shows peptides that were capable of eliciting antibodies reactive with the hemagglutinin and Figure 4C shows peptides that elicited antibodies unreactive with the hemagglutinin. Because of overlapping peptides that contain reactive as well as unreactive determinants, the loop region formed by residues 135-l 49 (Figure 4C, arrow) is shown in both Figures 48 and 4C. Peptides 13 (residues 132-l 48) and 14 (residues 140-l 471, which lie almost entirely within this loop region, elicited antibodies unreactive with the hemagglutinin molecule; thus they are shown in Figure 4C. However, peptides containing this region plus additional sequences on either side of the loop (11 [residues 105-l 401. 12 [residues 130-l 511 and 15 [residues 140-l 561) did elicit antibodies that reacted with the hemagglutinin molecule, and are therefore shown in Figure 48. The positive reactions observed with these sera may be caused by antigenic determinants lying outside the loop region. Alternatively, the presence of additional residues could facilitate folding of the loop sequence into a more favorable conformation. The ELISA results demonstrating the ability of anti-
Corresponding
to Synthetic
HA1 Peptides
in the Tertiary
Structure
of the
(A and 6) Pink: all HA1 residues synthesized. Yellow: HA1 residues not selected for synthesis. Blue: HA2 chain. (C-L) Representation of selected individual peptides. Yellow: indicated HA1 peptide. Red: side chains of indicated peptide. Light blue: residues of HA1 chain not contained in the indicated peptide. Dark blue: HA2 chain. (C) Peptide 2. residues l-36. (D) Peptide 3, residues 15-53. (E) Peptide 4. residues 39-65. (F) Peptide 7. residues 53-67. (G) Peptide 10, residues 76-l 11. (H) Peptide 11, residues 105-140. 6) Peptide 12. residues 130-l 51. (J) Peptide 17, residues 174-l 96. (K) Peptide 19. residues 201-227. (L) Peptide 20. residues 306-329. Residue numbers correspond to the sequence of Min Jou et al. (1960). The 1966 Hong Kong hemagglutinin structure was determined by Wilson et al. (1961).
Cell 480
Synthetic 481
Influenza
Virus Peptides
Cell 482
Figure
3. Reactivity
of Antipeptide
Sera with HA1 Peptides
and Intact
Hemagglutinin
Larger peptides consisting of 34 or more amino acids were Injected in two forms, uncoupled (free) and coupled to keyhole (KLH) through cysteine residues GKLH). Peptides 16 and 17 were insoluble in aqueous buffers and were injected uncoupled.
limpet hemocyanin All other peptides
Titers in ELISA are expressed as the reciprocal of the serum dilution that binds 50% of 5 pmole of antigen (stippled boxes). A noncrossreacting rabbit antiserum (prepared against a hepatitis B envelope peptide) was used in each assay to assess background activity. Diagonal lines: no reactivity. Open boxes: the particular antiserum-antigen combination has not been tested. X47 HA: purified hemagglutinin isolated by bromelain cleavage. X47 virus: intact virus.
HA1 -peptide antibodies to recognize intact hemagglutinin were confirmed by immunoprecipitation studies of ‘251-labeled X47 hemagglutinin isolated from virus by bromelain cleavage. Figure 5 shows SDS-polyacrylamide gel electrophoresis of immunoprecipitates in which representative anti-HA1 -peptide antibodies were reacted with iodinated hemagglutinin. Control serum prepared against a peptide unrelated to hemagglutinin showed no reactivity above nonspecific trapping of radioactivity (Figure 5, lane a), while an antiserum that contained specificities for a number of viral proteins (raised by immunization with X47 virus; Figure 5, lane b) and two anti-HA1 -peptide sera (Figure 5. lanes c and d) precipitated proteins with apparent molecular weights of 57.000 and 22,000 daltons, corresponding to HA1 and HA2, respectively. Similar results have been obtained in immunoprecipitation studies of the total protein from disrupted virus, in that all antisera reactive with virus in the ELBA specifically precipitated HA1 and HA2 (data not shown). HA2 is precipitated because it is disulfide-linked to HAl;
when the HA1 and HA2 chains are separated by elution from polyacrylamide gels, only HA1 remains precipitable with anti-HA1 -peptide sera (data not shown). Antibodies against X47 Virus Do Not React with the Synthetic Peptides The data show that the majority of antipeptide antibodies are capable of recognizing the HA1 molecule. We also performed the converse experiment. Rabbit antiserum against the intact X47 virus was tested for reactivity with individual synthetic peptides (Figure 3). The antiviral antibody did not react with any of the synthetic peptides. Discussion A reasonable prediction based on the literature would be that antipeptide antibodies would react with the native molecule only if the peptide represented a domain seen during an immune response directed
Synthetic 483
Figure
4.
The view (A) Pink: (B) Pink: (0 Pink: protruding
Influenza
Virus Peptides
Dual Stereoscopic
Displays
of the Positions
of Synthetic
Peptides
in the Hemagglutinin
Trimer
on the right looks down from the top of the molecule through the long axis. all HA1 peptides synthesized. Yellow: HA1 residues not contained in synthesized peptides. Blue: HA2 chain. HA1 peptides eliciting antibodies capable of reacting with the hemagglutinin. Yellow: all other HA1 residues. Blue: HA2 chain. HA1 peptides eliciting antibodies that were unreactive with the hemagglutinin. Yellow: all other HA1 residues. Blue: HA2 chain. loop formed by residues 135-l 49.
An
Cell 484
a
b
c
d
45K+ 30K+ 14.3K-*
Figure 5. Immunoprecipitation Antibodies
of X47 Hemagglutinin
by Antipeptide
‘251-labeled hemagglutinin isolated by bromelain cleavage (1 X 10’ cpm) was reacted with 10 PI of one of the following: control serum specific for a peptide unrelated to influenza (lane a); serum raised by immunization with intact X47 virus (lane b); anti-peptideantiserum (lane c): or anti-peptideantiserum (lane d). Precipitates were collected with Staphylococcus aureus and subjected to electrophoresis on a 5%-l 7% SDS-polyacrylamide gel prior to autoradiography.
against native protein. Consequently, the usefulness of antipeptide antibodies would be predicated on locating those domains seen during an immune response directed against native proteins. In the case of the influenza HA1 , these areas have been mapped by analysis of variants (Laver et al., 1980; Webster and Laver, 1980; Willey et al., 1981). We thus expected that peptides that were capable of eliciting antibodies reactive with HA1 would cluster to those mapped regions. This was not the case. Our basic finding is that synthetic peptides encompassing about 75% of the HA1 sequence, and corresponding on the intact HA1 molecule to a diverse array of secondary structures within the native target, generate antibodies reactive with the hemagglutinin. Probably for most proteins, the sole requirement for generation of antipeptide antibodies reactive with native molecules is that the peptide be represented on the surface of the structure. All the peptides in this study that elicited antibodies reactive with hemagglutinin have at least a portion that is on the surface of the molecule, as indicated by crystallographic analysis. As we have previously noted (Lerner et al., 1981 b), the fact that most of the peptides contain proline may be significant. In contrast with peptides, immunization with the intact hemagglutinin may result in the preferential recognition of complex determinants by the immune system. The fact that antibodies to intact virions do not recognize any of our peptides, which represent 75% of the primary HA1 sequence, supports this assertion. This may be a consequence of constraints
in the way larger protein molecules are presented. Perhaps the potential of the relatively unrestricted immunological repertoire is narrowed by a limited presentation system, which we can somewhat circumvent by presenting the molecule in pieces. If this is true, then the issue is not whether native antigens and peptides give comparable titers to a given determinant, but rather whether the elicited immunological specificities are to different determinants. It already seems likely that short peptides may induce antibodies that could not be raised by the native structure. One can think in similar terms about the evolution of changes in the HA1 molecule. This molecule, of course, evolves in the face of the entire immunological potential of the host, but the changes we see may only reflect nonlethal mutations sufficient to evade antibodies generated during ordinary presentation of antigens during infection. Presumably, the chemically synthesized peptides are able to elicit antibodies reactive with the native molecule because they exist for part of the time in a conformation that is the same as or similar to that which occurs in the intact protein (Sachs et al., 1972; Lerner et al., 1981 b). The immunological repertoire for a given peptide must be much larger than the few conformations that approximate the native form, but the magnitude of that repertoire is unknown and will probably differ from peptide to peptide. At any rate, considerations of immunological diversity must take into account not only all possible immunogens, but also all possible conformations. Along these lines, it is interesting to note that our antibodies to the C terminus of HA1 have the highest titer. This may simply reflect the observation that the C-terminal amino acids of HA1 appear flexible on the x-ray structure. The last few amino acids of HA1 may adopt a number of conformations and may therefore be recognized by antipeptide antisera to several different peptide conformations. One way of looking at the problem is that the bulk of the hemagglutinin serves as a “carrier” for the C-terminal peptide, a situation not too remote from that which one obtains when the peptide is coupled to keyhole limpet hemocyanin. Antisera to five of the HA1 peptides (8, 9, 13, 14, 16) showed no (or very little) reactivity for the hemagglutinin molecule. Since the titers of these five antisera fell within the normal range when tested against the immunizing peptide, their lack of reactivity with the hemagglutinin must be explained on the basis of the hemagglutinin structure. The five peptides define two regions of the hemagglutinin molecule, and, as is shown in Figure 4C, a considerable proportion of each region is highly accessible in the trimeric structure. Peptides 8 and 9 overlap and span the segment from residues 76 to 90. We note that the oligosaccharideattachment site Asns4 may affect the ability of antipeptide sera to recognize this region. Also, neither 8 nor 9 contains a proline. The second group of peptides that elicited anti-
z;;thetic
Influenza
Virus
Peptides
bodies unreactive with hemagglutinin was 13, 14 and 16. Their shared sequence forms a protruding loop (residues 135-l 49; Figure 4C, arrow) that has been shown to be an important region involved in antigenic variation (Laver et al., 1980; Webster and Laver, 1980; Wiley et al., 1981). One possible explanation for the failure of antisera to these three peptides to react with hemagglutinin is that coupling to the carrier through the cysteine at position 140 prevented the peptide from taking the native loop conformation. Such an effect was not observed, however, with other peptides in this study having internal cysteines. Antibodies to peptides within this segment of polypeptide chain were not capable of reacting with overlapping peptides. This observation indicates that in this region, neighboring residues may greatly alter a peptide’s antigenic conformation. Surprisingly, peptide 15, which is entirely contained within peptide 16, was successful in eliciting antibodies reactive with the hemagglutinin. Perhaps peptide 16 (36 residues) is large enough that it assumes a stable conformation unlike its structure within HA1 . Alternatively, the low solubility of peptide 16 may prevent its assuming the conformation found in the intact molecule. One additional explanation that could account for the inability of certain antipeptide antisera to recognize the intact hemagglutinin lies in the high degree of genetic variability characteristic of this molecule. The virus preparations used to obtain purified hemagglutinin and the intact virus used for ELBA were different, the latter having been adapted to growth in mice. Since neither represented a stock identical to the one used to obtain the DNA sequence data, it is possible that both had undergone some degree of genetic variation. Such variation seems more likely to have occurred in the loop region, since changes in the loop are more frequently selected with antibodies (Laver et al., 1980; Webster and Laver, 1980). Recent studies by others have used proteolytic fragments and synthetic peptides to study the immunogenicity of HA1 (Jackson et al., 1982). Jackson and his colleagues synthesized a decapeptide corresponding to the loop formed by residues 140-l 50 of the A/Hong Kong/68 (X31) strain of influenza. Although, as in our study, they were unable to demonstrate that antibody to this region significantly bound to hemagglutinin, they did show that antibody to the intact hemagglutinin bound to the synthetic peptide. In results similar to ours, Arnon was able to elicit antibodies to a synthetic HA1 peptide that were capable of binding to and neutralizing virus (R. Arnon, personal communication). Experimental
Procedures
Syntheels of Peptides For this study, peptides were synthesized by J. K. Chang, who used the solid-phase methods developed by Merrifield and his colleagues (Marglin and Merrifield. 1970). Each synthetic peptide was subjected to acid hydrolysis in vacua (6 N HCI. at 11O’C for 72 hr) and the
amino acid composition was determined. No attempt was made to remove multimeric forms, since the sole usa of the peptides was as immunogens. Peptides were synthesized according to the amino acid sequence predicted from the nucleotide sequence &tin Jou et al., 1980). When necessary. an additional cysteine (for the purpose of coupling to the protein carrier) or an additional tyrosine (for labeling purposes). or both, were added to the sequence. Coupling of Synthetic Peptides to Carrier Protein Peptides were coupled to the carrier protein keyhole limpet hemocyanin (KLH) through the cysteine of the peptide. with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as the coupling reagent (Liu et al., 1979). The peptides were dissolved in phosphate-buffered saline (pH 7.51, 0.1 M sodium borate buffer (pH 9.0) or 1 .O hi sodium acetate buffer (pH 4.0). The pH for the dissolution of the peptide was chosen to optimize peptide solubility. The content of free cysteine for soluble peptides was determined by Ellman’s method (Ellman, 1959) and ranged from 30% to 90%. For each peptide. 4 mg KLH in 0.25 ml of 10 mM sodium phosphate buffer (pH 7.2) was reacted with 0.7 mg MBS (dissolved in dimethyl formamide) and stirred for 30 min at room temperature. The MBS was added dropwise to ensure that the local concentration of formamide was not too high, as KLH is insoluble in ~30% formamide. The reaction product, KLH-MB. was then passed through Sephadex G25 equilibrated with 50 mM sodium phosphate buffer (pH 6.0) to remove free MBS. KLH recovery from peak fractions of the column eluate (monitored by OD& was estimated to be approximately 80%. KLH-MB was then reacted with 5 mg peptide dissolved in 1 ml of the chosen buffer. The pH was adjusted to 7-7.5 and the reaction was stirred for 3 hr at room temperature. Coupling efficiency was monitored with radioactive peptide by dialysis of a sample of the conjugate against phosphate-buffered saline, and ranged from 8% to 60%. Preparation of Antlpeptkfe and Antiviral Antibodies Rabbits were immunized according to the following schedule: first, 200 pg peptide-coupled KLH in complete Freund’s adjuvant (1:1) subcutaneously on day 0: second, 200 pg in incomplete Freund’s adjuvant (1 :l) subcutaneously on day 14; and finally, 200 pg with 4 mg alum intraperitoneally on day 21. Animals were bled 4 and 5 weeks after the first injection. After the initial course of injections, the rabbits were boosted with 200 pg peptide-coupled KLH with 4 mg alum at 5 week intervals, and bled 1 and 2 weeks following the injection. Large peptides were sometimes injected uncoupled (1 mg per injection). Antiviral antisera were prepared according to the same protocol, with each rabbit receiving 700 pl X47 allantoic fluid per injection. Enzyme-Linked lmmunosorbent Assay Reactivity of the various antipeptide antisera was determined in a two step ELISA. Antibody was bound to antigen immobilized on polystyrene plates, and the antigen-antibody complex was detected with an enzyme-labeled goat antirabbit IgG. This “sandwich” complex was measured enzymatically by the addition of enzyme substrate. Five picamoles of antigen in 0.1% bovine serum albumin in phosphatebuffered saline were added per well of a 96 well microtiter plate and dried overnight at 37°C. (For the the purpose of calculating 5 pmole of antigen. the molecular weight of pure hemagglutinin. in its glycosylated trimeric form, was taken as 224,640 daltons. In assays with Intact X47 virus as a target, HA1 was assumed to be 20% of the viral protein content [Schulz, 19731.) The dried antigen was then fixed to the plates with 50 pl per well of methanol for 5 min at room temperature. Before adding the antibodies. we coated the plates for 4 hr with 3% bovine serum albumin in phosphate-buffered saline to block nonspecific absorption of antibodies to the plate. Serial dilutions (1:2) of each antiserum were prepared in minimal essential medium containing 10% fetal calf serum, 25 pl of serum dilution were added per well and the plates were incubated overnight at room temperature. The unbound antibody was then washed off with water. The antigen-antibody complex was reacted with 25 pl per
Cell 466
well of 1 :lOO dilution of goat antirabbit IgG coupled to glucose oxidase CTernynck and Avranmeas, 1977). Excess goat antirabbit antibody was washed off with water, and the reaction was developed by adding 50 f.&Iper well of developing solution (25 ml of 0.1 M sodium phosphate buffer [pH 6.01; 3 ml of 20% glucose; 200 cl of 0.1% horseradish peroxidase: 200 ~1 of chromogen [2,2’-azino-di<3-athylbenzthiazolin-sulfonat)] at 20 mg/ml in sodium phosphate buffer [pH 6.01). The plates were read in Titertek Multiskan at A,.. and the values presented are the reciprocal of the serum dilution at the 50% point of the dilution curve. lmmunoprecipitation lmmunoprecipitation with formalin-fixed S. aureus. and polyacrylamide gel electrophoresis of immunoprecipitates, were carried out according to procedures we have used previously (Lerner et al., 1981 a). ‘251-IabeIed X47 hemagglutinin was suspended in RIPA (0.15 M NaCI, 10 mM sodium phosphate [pH 7.51. 1% Nonidet P-40,0.05% sodium deoxycholate, 0.1% SDS) and reacted (5 x 10’ cpm per reaction) at 0°C with 10 pl of test serum or control rabbit serum for 1 hr. Precipitates were collected with S. aureus. and pellets were washed with RIPA. then twice with 500 mM LiCI. 100 mM Tris (pH 8.5). For gel electrophoresis. washed pellets were suspended in gel loading buffer, boiled, centrifuged to remove S. aureus and subjected to electrophoresis on a 5%-l 7% acrylamide-SDS gel, and the gel was autoradiographed. Acknowledgments We thank Drs. I. A. Wilson, D. C. Wiley and J. J. Skehel for providing the x-ray coordinates for the HA1 molecule and for introducing us to Dr. Arthur Olson. We thank Brian Browne and Suzie Seaver for excellent technical assistance. This is publication no. 2598 from the Research Institute of Scripps Clinic. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
October
28. 1981;
revised
December
2. 1981
defined
antiviral
vaccines.
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3A