Helper T-cell Epitope Immunodominance Associated with Structurally Stable Segments of Hen Egg Lysozyme and HIV gp120

J. theor. Biol. (2000) 203, 189}201 doi:10.1006/jtbi.1999.1056, available online at http://www.idealibrary.com on

Helper T-cell Epitope Immunodominance Associated with Structurally Stable Segments of Hen Egg Lysozyme and HIV gp120 SAMUEL J. LANDRY* Department of Biochemistry, ¹ulane ;niversity School of Medicine, 1430 ¹ulane Avenue, New Orleans, ¸A 70112, ;.S.A. (Received on 8 March 1999, Accepted in revised form on 12 November 1999)

Although many antigen sequences potentially can bind to the MHCII proteins, only a small number of epitopes dominate the immune response. Additional mechanisms of processing, presentation or recognition must restrict the immune response against a large portion of the antigen. A highly signi"cant correlation is found between epitope immunodominance and local structural stability in hen egg lysozyme. Since antigen proteins are likely to retain substantial native-like structure in the processing compartment, protease action may be focused on regions that are most readily accommodated in the protease active sites, and thus, the intervening sequence are preferentially presented. Immunodominance also correlates with sequence conservation as expected from the constraints imposed by structure. These results suggest that the three-dimensional structure of the antigen limits the immune response against some antigen segments. For HIV gp120, a substantial improvement in the accuracy of epitope prediction is obtained by combining rules for MHCII binding with a restriction of the predicted epitopes to well-conserved sequences. ( 2000 Academic Press

Introduction Helper T-cell epitope immunodominance within a protein antigen potentially arises from biases in any of several steps in cellular immune recognition: antigen processing into peptides, presentation of peptides in MHCII, and recognition of MHCII}peptide complexes by T-cell receptors. T-cell receptor recognition of an MHCIIrestricted peptide epitope has long been appreciated as a requirement for immune surveillance, and this is the basis for elimination or inactivation of T cells speci"c for self-epitopes. T-cell restimulation assays suggested that immune surveillance is constrained by peptide sequence recognition by the MHCII protein * E-mail: [email protected] 0022}5193/00/070189#13 $35.00/0

(DeLisi & Berzofsky, 1985; Rothbard & Taylor, 1988; Rothbard et al., 1988; Hill et al., 1991; De Magistris et al., 1992), and sequencing of MHCIIbound peptides found the same patterns in the naturally processed peptides (Rudensky et al., 1991; Chicz et al., 1992; Hunt et al., 1992; Falk et al., 1994). A direct test of binding ability with peptides presented by M13 phage con"rmed that MHCII recognize a sequence motif characterized by the presence and proper spacing of hydrophobic &&anchor'' residues in the peptides that "t into pockets of the MHCII groove (Hammer et al., 1994). Flanking sequences also can in#uence loading (Moudgil et al., 1996) and stability (Nelson et al., 1994) of MHCII complexes. The context of processing and presentation can a!ect the nature of the MHCII}peptide complex, resulting in the stimulation of di!erent T-cell ( 2000 Academic Press

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subsets by the same antigen sequence (Bhayani & Paterson, 1989; Viner et al., 1995). The dominance or crypticity of many epitopes is imposed at the level of proteolytic processing of the antigen. Cryptic epitopes may be de"ned as epitopes which, in the context of a peptide, can restimulate T-cells after immunization with the peptide but cannot restimulate T cells after immunization by the authentic antigen. A cryptic epitope evidently satis"es the requirements of binding to the MHCII protein but normally does not become available during processing of the native antigen. Circumstances that promote Tcell responses to cryptic epitopes may alter the proteolytic processing pattern of the antigen. The presence or absence of a key proteolytic activity could a!ect the generation of a given epitope (Takahashi et al., 1989; Vidard et al., 1991; Fineschi et al., 1995; Bevec et al., 1996). Alternatively, an epitope could be exposed or protected from proteolysis by bound MHCII protein (Donermeyer & Allen, 1989; Mouritsen et al., 1992), immunoglobulin (Manca et al., 1985, 1988; Simitsek et al., 1995) or other protein (Dong et al., 1994). In a similar manner, the structural context of a T-cell epitope within the antigen itself could control exposure to proteases (Landry, 1997; Vanegas et al., 1997). Evidence presented here indicates that the three-dimensional structure of the antigen limits access by processing proteases. This aspect of antigen processing provides a framework for understanding the dependence of immunodominance on the structural context of the antigen as well as the epitope. Local antigen instability is likely to be an important determinant of endoproteolytic cleavage of antigens. Lysosomal enzymes available to carry out processing, including cathepsins B, C, E, L, and S, exhibit only modest amino acid sequence speci"city (McKay et al., 1983; Towatari & Katunuma, 1983; Bromme et al., 1989; Kageyama et al., 1995). The tendency for proteases to nick between well-folded domains has been employed by biochemists for many years (HoK gberg-Raibaud & Goldberg, 1977; Vas et al., 1990; Witkowski et al., 1991). In particular, papain, a member of the family of proteases that contains several cathepsins, selectively cleaves between domains of proteins, such as immunoglobulins and myosin. Polyprotein processing

probably also depends on the resistance of well-folded domains to the action of redundant processing enzymes (Friedman et al., 1994; Kageyama et al., 1995). Even in single-domain proteins, proteolytic nick sites tend to occur in regions exhibiting high crystallographic Bfactors (Novotny & Bruccoleri, 1987; Hubbard et al., 1991). Thornton and co-workers have proposed that unstructured or unstable segments are preferred proteolytic nick sites because they can be molded to "t protease active sites (Hubbard et al., 1991, 1994). A relationship between the locations of T-cell epitopes and structurally unstable sites in proteins was identi"ed recently (Landry, 1997). Local structural instability can be gauged by inspection of crystallographic B-factors (Ringe & Petsko, 1986), NMR relaxation parameters (Kay et al., 1989), and amide hydrogen/deuterium exchange (HX) rates (Bai et al., 1995). The number and locations of well-ordered, stable segments is similar to the number and locations of T-cell epitopes in lysozyme, staphylococcal nuclease, and cytochrome c (Landry, 1997). The epitopes tend to partially overlap the unstable regions and lie on their C-terminal side. This pattern was suggested to arise from preferential proteolytic cleavage at unstable segments followed by presentation of the N-terminal peptide of each C-terminal cleavage product. Of the various biophysical indices for local instability, HX appeared to be most useful for the prediction of T-cell epitopes. HX indicates conformational #uctuations that expose amide protons to exchange with solvent deuterons (Bai et al., 1995). Since global unfolding of these model proteins occurs on the time scale of months, sites in the native proteins for which HX exchange occurs in less than a few hours undergo local conformational #uctuations. These sites exhibiting fast to moderate HX might more easily conform to the substrate-binding site of a protease, and thus they are associated with adjacent T-cell epitopes. In this paper, a statistical analysis reveals a highly signi"cant correlation between T-cell epitopes and local structural stability in hen egg lysozyme, although the strength of the correlation is clearly tempered by other factors, such as MHCII protein binding and T-cell receptor recognition. Since detailed structural information

IMMUNODOMINANCE OF STABLE SEGMENTS

often is lacking for medically important antigen proteins, hydropathy and sequence conservation were tested as correlated to T-cell immunogenicity for lysozyme and human immunode"ciency virus (HIV) envelope glycoprotein gp120. Based on these results, sequence conservation appears to be particularly useful as a predictor of immunodominance. Methods Amino acid sequence conservation was estimated by identity to a consensus sequence. Sequences for lysozyme from 70 organisms and gp120s from 23 HIV strains were aligned with Clustal W (Thompson et al., 1994) version 1.6 implemented on an IBM-compatible personal computer. The consensus sequence was determined by assigning the most popular residue for each position, and then conservation at each position was estimated as the number of sequences identical to the consensus residue divided by the total number of sequences in the set. A sample alignment and analysis of conservation is presented in Table 1. Conservation was assigned a value of 0 at positions where the consensus sequence contained a gap introduced by the alignment. Data for positions lacking a counterpart in the sequence of gp120 from HIV strain HXBc2 were discarded. Correlations of helper T-cell epitopes with local antigen instability were evaluated using Pearson correlation coe$cients (Daniel, 1991) with published T-cell epitope maps and structural data on the antigens using the feature provided in Excel (Microsoft). Each residue was assigned an &&epitope score'' (1 or 0) based on whether or not it is within a helper T-cell epitope or determinant core (in the case lysozyme). Epitope scores for residues appearing in more than one epitope or determinant core were incremented by one for each additional epitope that included it. Thus, a single amino acid position may accumulate a score greater than one, for example, if it appears in overlapping epitopes described for two strains of mice having di!erent haplotypes or for a single strain of mice and an infected human. For the analysis of hen egg lysozyme, epitope data were from the study identifying epitope determinant cores by Gammon

191

et al. (1991) in three strains of mice. For the analysis of HIV gp120, epitope data were from mapping studies in mice, humans, and monkeys that employed peptides corresponding to at least 40% of the gp120 sequence (Hale et al., 1989; Schrier et al., 1989; Wahren et al., 1989; Sjolander et al., 1996; Lekutis et al., 1997). This strategy entails several assumptions: (i) all peptides compete equally well for binding to the MHCII, (ii) the more readily processed peptides are more likely to be presented, and (iii) the MHCII haplotype does not a!ect the pool of available peptides. This model is taken as a "rst approximation, with the appreciation that none of these features can be fully correct. The "rst two assumptions are favored by the promiscuity of MHCII proteins and the low sequence speci"city of processing proteases. However, catalysis of peptide exchange by HLA-DM biases loading toward peptides having the highest a$nity for the MHCII (Kropshofer et al., 1996; Weber et al., 1996). Thus, for a given haplotype, the single best peptide may be preferentially presented, and biased antigen processing could be hidden from the analysis. By using data from several haplotypes, the spectrum of epitopes might more faithfully re#ect the pool of available peptides. The HX protection factor indicates the resistance of a given amide proton to exchange with solvent deuterons due to the presence of secondary and tertiary structure. Typically, HX protection is measured by dissolving the protein in D O 2 and monitoring the loss of the 1H-NMR signal. Unprotected amide protons exchange in approximately 0.1 s, whereas amide protons involved in stable hydrogen-bonded secondary structure may survive for months. The protection factor is de"ned as the rate of exchange of the corresponding amino acid amide in an unfolded model peptide divided by the rate of exchange in the protein. Due to the cooperativity of protein structure stabilization, the rate of exchange probably re#ects local transitions between fully folded and fully unfolded conformations, with exchange occurring essentially only in the unfolded conformation (Bai et al., 1995). Thus, the amide proton exchange rate e!ectively samples the equilibrium of local unfolding. As suggested by Thornton and co-workers, proteolytic nick sites may tend to occur in regions that can be distorted into

192

TABLE 1 Sequence conservation in the gp120 segment 447}468 (HXBc2) 450

460

S S S S S S S S S S S S S S S S S S S S S S S

N N N N N N N N N N N N N N N N N N N N N N N

I I I I I I I I I I I I I I I I I I I I I I I

T T T T T T T T T T T T T T T T T T T T T T T

G G G G G G G G G G G G G G G G G G G G G G G

L L L I L L L L L L L L L L L L L L L L L L L

L L L L L L I I L I L L I I L L L L L L L L L

L L L L L L L L L L L L L L L L L L L L L L L

T T T T T T T T T T T T T T T T T T T T T T T

R R R R R R R R R R R R R R R R R R R R R R R

D D D D D D D D D D D D D D D D D D D D D D D

G G G G G G G G G G G G G G G G G G G G G G G

G G G V G G G G G V G G G G G G G G G G G G G

T D } G } G G G } G S S S D T I N N N D N K }

} } } } } } } } } } } } } } } } N S N K N } }

} } } } } } } } } } } } } } } } N N N E N } }

N N N N N D N N N N N N N N N N N N N N N N N

N N N N N A I N G N N N N N V G G E G S G E S

T S S T S N N S S S S S S S T T S S S T S S T

K A T S T S E E N T T T T E } } } } } } } E E

} } } } } } } } T } } } } } } } } } } } } } }

} } } } } T } } } } } } } } } } } } } } } } }

} } } } } N } } } } } } } } } } } } } } } } }

} } } } } } } } } } } } } } N N } } } } } } T

} } } } } G } } } } } } } } D D } } } } } } E

N N N N N N S N N N N N N S T T } } } T } I T

E E E E E E Q E E E E E E E E E E E E E E E E

T T T T I T I T T T T T I T V V I I I I I I I

F F F F F F F F F F F F F F F F F F F F F F F

Amino acid } A C D E F G H I K L M N P Q

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 23 0 0

0 0 0 0 0 0 0 0 23 0 0 0 0 0 0

0 0 0 0 0 0 0 0 1 0 0 0 0 0 0

0 0 0 0 0 0 23 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 1 0 22 0 0 0 0

0 0 0 0 0 0 0 0 5 0 18 0 0 0 0

0 0 0 0 0 0 0 0 0 0 23 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 23 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 23 0 0 0 0 0 0 0 0

0 0 0 0 0 0 21 0 0 0 0 0 0 0 0

4 0 0 3 0 0 5 0 1 1 0 0 4 0 0

18 0 0 0 0 0 0 0 0 1 0 0 3 0 0

18 0 0 0 1 0 0 0 0 0 0 0 4 0 0

0 0 0 1 0 0 0 0 0 0 0 0 22 0 0

0 1 0 0 2 0 5 0 1 0 0 0 11 0 0

0 0 0 0 0 0 0 0 0 0 0 0 2 0 0

7 1 0 0 5 0 0 0 0 1 0 0 1 0 0

22 0 0 0 0 0 0 0 0 0 0 0 0 0 0

22 0 0 0 0 0 0 0 0 0 0 0 0 0 0

22 0 0 0 0 0 0 0 0 0 0 0 1 0 0

20 0 0 0 0 0 0 0 0 0 0 0 2 0 0

19 0 0 2 1 0 1 0 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0 1 0 0 0 12 0 0

0 0 0 0 22 0 0 0 0 0 0 0 0 0 1

0 0 0 0 0 0 0 0 10 0 0 0 0 0 0

0 0 0 0 0 23 0 0 0 0 0 0 0 0 0

S. J. LANDRY

Isolate U455 SF1703 IBNG Z321 92RW020.5 92UG031.7 92UG037.8 UG275A UG273A V1191A DJ264A DJ263A DJ258A KENYA SF2 SF2B13 LAI HXB2R GP160EN NY5CG NL43 JRCSF P896

0 23 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 22 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 23 0 0 0

23 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 2 0 0

0 3 2 0 0 0

0 1 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 2 0 1 0 0

0 15 6 0 0 0

0 2 6 0 0 0

0 0 1 0 0 0

0 0 1 0 0 0

0 0 0 0 0 0

0 0 1 0 0 0

0 0 0 0 0 0

0 2 4 0 0 0

0 0 0 0 0 0

0 0 11 2 0 0

0 0 0 0 0 0

Concensus

S

N

I

T

G

L

L

L

T

R

D

G

G

G

}

}

N

N

S

}

}

}

}

}

}

N

E

T

F

Isolate U455 SF1703 IBNG Z321 92RW020.5 92UG031.7 92UG037.8 UG275A UG273A V1191A DG264A DG263A DG258A KENYA SF2 SF2B13 LAI HXB2R GP160EN NY5CG NL43 JRCSF P986

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 0 0 1 0 1 1 0 0 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 0 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1

0 0 0 1 0 1 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 0 0 1 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0

0 1 1 0 1 0 0 1 1 1 1 1 1 1 0 0 1 1 1 0 1 1 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 1 1 1 1 1 0 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0

1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 0 1 0 1 1 1 1 1 0 1 0 0 0 0 0 0 0 0 0

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0

0

0

0

0

0

0

0

Conservation

1.00 1.00 1.00 0.96 1.00 0.96 0.78 1.00 1.00 1.00 1.00 1.00 0.91 0.22

0.98 0.48 0.65

0.52 0.96 0.48 1.00

IMMUNODOMINANCE OF STABLE SEGMENTS

R S T V W Y

193

194

S. J. LANDRY

a conformation compatible with the recognition site of the protease (Hubbard et al., 1991). A locally unstable segment of approximately 12 residues was deemed suitable (Hubbard et al., 1994). Given that both proteases and MHCII proteins recognize short segments of a polypeptide, both data sets were smoothed using moving-window averages of comparably sized segments. This correlation strategy evaluates the covariance of data for protein segments corresponding to the size of the respective functional units. Results Inspection of the smoothed HX protection factors and epitope scores plotted as a function of the lysozyme amino acid sequence reveals a similar number of peaks (Fig. 1, panels d and e). The Pearson correlation coe$cient (r) gives an indication of how the two properties are related. Values range from 0, no correlation, to 1, perfect correlation, or !1, perfect anticorrelation. For example, a positive r is expected if epitopes tend to occur at the point of maximum structural stability. The value of r for epitope scores and HX protection factors was !0.21. A better correlation was sought by o!setting one data set relative to the other by an integer number of residues. This manipulation has the e!ect of correlating epitopes with HX protection in the adjacent segment toward the N- or C-terminus. The value of r grew to 0.22 when the epitope scores were o!set #8 residues (toward the C-terminus) and to an even more signi"cant negative value of !0.53 (window-size 17) when epitope scores were o!set !6 residues (toward the N-terminus) (Fig. 1, panel i). The negative value of r simply re#ects the opposing directions of variance in the respective data sets, i.e. epitopes correlate with low HX protection in segments located six residues toward the N-terminus. Such a correlation is expected if proteolytic cleavage of the antigen at an unstable site coincides with loading of the adjacent C-terminal peptide into the MHCII protein. This pattern of side-by-side processing and presentation may be superimposed on a more basic mechanism of antigen processing in which local instability promotes the proteolytic destruction of epitopes, and local stability protects epitopes against destruction.

Although the correlation between epitope scores and HX protection factors is highly signi"cant, HX protection factors explain only a modest fraction of the variance in epitope scores. The probability that epitope scores and HX protection factors are unrelated is extremely low, P(10~30. However, local instability explains only part of the variance in epitope scores. The strength of the correlation between unstable segments and epitopes can be assessed by the coe$cient of determination (r2) (Daniel, 1991). The correlation coe$cient of !0.53 obtained for lysozyme (o!set"!6) translates to an r2 of 0.28, which means that 28% of the variance in T-cell epitope scores is explained by the correlation with amide protection factors. The remaining 72% of the variance must be explained by other factors, including selectivity at the level of peptide binding to MHC proteins. The X-ray crystallographic B-factor (or temperature factor) provides another measure of local instability that potentially marks sites of facile proteolytic cleavage and adjacent T-cell epitopes. B-factors weight the contribution of the respective atoms during quantitative re"nement of the crystal structure. Since disordered atoms are assigned high B-factors, we expect an inverse relationship between B-factors and HX exchange protection. In the plots of smoothed B-factors and epitope scores vs. sequence, a similar number of peaks are observed, but the relationship between the two properties is not readily apparent (Fig. 1, panels c and e). However, analysis of the correlation coe$cients with various o!sets reveals that epitopes tend to occur eight residues C-terminal from disordered sites, as indicated by an r of 0.51 with an epitope score o!set of !8 (Fig. 1, panel h). This correlation is essentially the same as that between epitope scores and HX protection factors except that the behavior of r vs. o!set is inverted by the relationship between B-factors and HX protection factors. Whether analysed by HX protection factors or crystallographic B-factors, local instability appears to direct presentation of adjacent peptides as helper T-cell epitopes. Since neither HX protection factors nor crystallographic B-factors are available for many important antigens, indirect measures of structural stability/instability were investigated for

IMMUNODOMINANCE OF STABLE SEGMENTS

195

FIG. 1. Pro"les of structural parameters and epitope scores for hen egg lysozyme (left panels), and plots of correlation coe$cient vs. epitope score o!set (right panels). T-cell epitopes tend to occur in stable segments and approximately six residues C-terminal from unstable segments. For the pro"les, structural parameters were smoothed with a 7-residue averaging window, and epitope score was smoothed with a 3-residue window. T-cell epitope scores were generated using the determinant cores identi"ed by Gammon et al. (1991). HX data were from Radford et al. (1992). Crystallographic B-factors for backbone amide nitrogens were from PDB "le 2LYM (Kundrot & Richards, 1987). The hydropathy plot uses the Kyte and Doolittle scale and was prepared using the ProtScale module of the Expasy Tools (http://expasy.ch) with entry LYC CHICK. ~ The identity pro"le was prepared as described in the text. Plots of correlation coe$cient vs. o!set were calculated using epitope scores smoothed with an 11-residue window. Similar plots were obtained with epitope scores smoothed with windows ranging from three to 11 residues. Structural data were smoothed with window sizes indicated by symbol according to the key. 17 (}m}); 13 (}] #}); 9 (}# }); 5 (*j*).

a correlation with T-cell epitope scores. A positive correlation of epitope scores and hydrophobicity could arise from the tendency for hydrophobic amino acids to pack into the hydrophobic core of the structure where they are protected from proteolysis. A positive correlation

might also result from a requirement for hydrophobic residues as anchors for binding to MHCII proteins. Data for lysozyme reveal a similar number of peaks in the pro"les of hydropathy and epitope score (Fig. 1, panels b and e). The correlation yields an r of 0.62 for an o!set of #2,

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suggesting that epitopes tend to occur near the middle of hydrophobic sites (Fig. 1, panel g). Well-ordered protein segments are likely to be evolutionarily conserved due to structural constraints. Conservation at each residue position of lysozyme was evaluated as the fraction of identity with the most popular amino acid in a collection of 70 lysozyme sequences. To establish the relationship of sequence identity and order, the identity pro"le was correlated with HX protection and B-factors. Sequence identity correlates reasonably well with HX protection (r"0.52) and with B-factors (r"!0.55). These correlations are maximal for o!sets equal to zero (data not shown). T-cell epitopes tend to occur in regions of elevated sequence identity (Fig. 1, panel a). The correlation yields an r of 0.44 for zero o!set and !0.50 for an o!set of !8 (Fig. 1, panel f ). Thus, sequence conservation serves as an indirect measure of local instability and reveals the same relationship to peptide presentation, in which epitopes tend to occur within well-conserved sequences that immediately follow poorly conserved sequences. The lysozyme example suggests that hydropathy and sequence conservation may be used to predict epitopes in medically important antigens. When the analysis was applied to the HIV envelope glycoprotein, gp120, neither hydropathy nor sequence conservation were found to correlate strongly with epitope scores for gp120 (r&0.1). Nevertheless, helper T-cell epitopes tend to cluster around regions of low sequence conservation (Figs 2 and 3). Epitopes are conspicuously absent from the center of V1/V2 and a poorly conserved segment of C3. Discussion The importance of helper T-cell epitope immunodominance for vaccine design and treatment of autoimmune disease warrants investigation of its mechanism. Most studies that identi"ed immunodominant sequences employed synthetic peptides to restimulate T-cells that were primed against the full-length antigen by vaccination or other exposure (StevanovmH c & Rammensee, 1996). Immunodominant epitopes may be de"ned as immune determinants expressed by

naturally processed and presented peptides that elicit T cell proliferation. Abundant T cell clones are identi"ed by the proliferative response induced by restimulation with a synthetic peptide mimic. Thus, the experimentally observed T-cell response to a given peptide could be modulated at any of several steps: antigen processing, natural or synthetic peptide presentation, or T-cell receptor recognition. Antigen sequences are discriminated at the level of binding to the MHCII molecule. Early work suggested that sequences able to form amphipathic helices were preferentially presented, presumably because this structure is more favorably accommodated in the MHCII proteins (DeLisi & Berzofsky, 1985; Rothbard & Taylor, 1988; Rothbard et al., 1988; Hill et al., 1991; De Magistris et al., 1992). However, crystal structures of HLA-DR1 revealed that peptides bind to MHC proteins in a more extended polyproline type II (PPII) conformation (Brown et al., 1993; Stern et al., 1994; Jardetzky et al., 1996). Perhaps the most important feature of the binding interaction is the array of hydrogen bonds between the MHC peptide-binding groove and the backbone amide and carbonyl groups of the peptides (Stern et al., 1994; Jardetzky et al., 1996). As this is a constant feature of all peptides, it does not provide a basis to distinguish one sequence from another. The MHC peptide-binding grooves have &&speci"city pockets'' which are able to differentiate between various sequences (Brown et al., 1993; Stern et al., 1994). Some of the early T-cell restimulation studies suggested peptide binding to HLA-DR1 is governed by the position and spacing of hydrophobic residues (DeLisi & Berzofsky, 1985; Rothbard & Taylor, 1988; Rothbard et al., 1988; Hill et al., 1991; Kropshofer et al., 1991). In recent years, peptides presented on HLA-DR1 have been sampled by direct sequencing (Rudensky et al., 1991; Chicz et al., 1992; Hunt et al., 1992; Falk et al., 1994; Hammer et al., 1994). The upshot of all of these studies is that peptides bearing two or three properly spaced hydrophobic residues are compatible with binding to the MHCII protein. This constraint cannot account for the small number of immunodominant epitopes observed for naturally processed antigens (StevanovmH c & Rammensee, 1996).

IMMUNODOMINANCE OF STABLE SEGMENTS

197

FIG. 2. Pro"les of structural features, proteolytic nick sites, and predicted and observed helper T-cell epitopes for HIV gp120. Proteolytic cleavage sites for staphylococcal V8 protease and trypsin were determined by Nygren et al. (1988). The enzyme responsible for cleavage in the V3 loop (indicated by the asterisk) was not identi"ed in the study by Stephens et al. (1990). The hydropathy plot was prepared with the HIV gp160 sequence ENV HV1H2 as described in the legend for Fig. 1. ~ The pro"le of sequence conservation was prepared with a collection of 23 HIV gp120 sequences as described in the Methods section. Pro"les of hydropathy and conservation were smoothed with a 17-residue averaging window. B-factors for the backbone amide nitrogen atoms were from the coordinate "le for the gp120}CD4-antibody complex provided by Wayne Hendrickson (Kwong et al., 1998). B-factors for deleted or disordered residues were assigned a value of 100 As . The EpiMatrix T-cell epitope prediction (http://www.EpiMatrix.com/HIV) utilized the sequence designated HIV-1 env B HXB2R, Epi~ ~ ~ Matrix matrix-based clustering, class II motifs for an unweighted population, a 20-amino-acid clustering frame with 10-residue overlaps, and a 20% binding probability cuto!. The EpiMatrix prediction was &&"ltered'' by eliminating epitopes for sequences with below-average sequence conservation (indicated by the horizontal line in panel C). Application of this constraint improves epitope prediction on the basis of MHCII binding motifs. Experimental T-cell epitopes tend to be excluded from variable regions and cluster in conserved regions #anking the variable loops.

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Although antigen sequences are bound to MHC proteins in an extended PPII conformation (Jardetzky et al., 1996), immunodominant sequences are limited neither to sequences that adopt an extended conformation in the native antigen, nor to sequences that are most conformationally labile. The present analysis shows that T-cell epitopes tend to occur in stable wellfolded parts of the protein, and where the threedimensional structure of the antigen is known, the T-cell epitopes are found in a variety of structural contexts (data not shown). The importance of hydrophobic anchor residues for MHCII binding could bias T-cell epitopes to sequences disposed on the protein interior. This notion is consistent with the tendency observed here for epitopes to occur in structurally stable, hydrophobic segments. The antigen may have to be at least partially unfolded for the epitope to become available for binding to the MHC protein. Conformationally labile antigen segments may be excluded from MHC binding by proteolytic degradation. Most or all proteolytic degradation of antigens is thought to be carried out by cathepsins in an endosomal-lysosomal compartment. Several of the cathepsins are homologous to papain, which binds an 4-residue stretch of the substrate in an extended conformation (Turk et al., 1997). Antigen segments will have varying abilities to conform to protease active sites and thus exhibit a range of susceptibility to proteolytic attack, with the most conformationally labile segments being preferentially degraded. This pattern of degradation would promote presentation of antigen segments that are most resistant to proteolytic attack. The combined action of proteolysis, MHCII selectivity, and catalysis of peptide exchange by HLA-DM contribute to presentation of polypeptide segments derived from the structural core of the antigen molecule. Although many proteolytic fragments may transiently bind to the MHCII, the HLA-DM molecule catalyses discharge from the MHCII of antigen segments that are not bound tightly (Urban et al., 1994; Kropshofer et al., 1996; Weber et al., 1996). Weakly bound segments are constantly returned to the milieu of proteolytic enzymes where they can be degraded. The antigen is progressively unfolded as it is degraded, exposing hydrophobic segments

which bind more e!ectively to the MHCII protein. These segments are resistant to discharge catalyzed by HLA-DM, and they are protected against degradation by the MHC protein. Thus, the most well-structured and evolutionarily conserved antigen segments are preferentially presented. The correlation of helper T-cell epitopes and structural stability is less clear cut for HIV gp120 than for hen egg lysozyme. Nevertheless, epitopes tend to be excluded from disordered regions such as V1/V2, and epitopes tend to cluster on the #anks of disordered regions. Low-resolution and unsystematic sampling in the epitope-mapping studies may partially explain the poor statistical correlation of epitopes and structural data for HIV gp120. T-cell epitope data for gp120 is of much lower resolution than that for lysozyme due to the use of fewer and larger peptides in the proliferation assays. In addition, the epitope data have biases. Many of the epitopes in the HIV Immunology Database were identi"ed in nonsystematic mapping studies in which epitopes were predicted, for example on the basis of amphipathicity, and then con"rmed by restimulation assays. The present analysis excludes extreme cases, but the two epitope-mapping studies in mice sampled only 48% (Hale et al., 1989) and 51% (Sjolander et al., 1996) of the gp120 sequence. Many HIV gp120 epitopes in humans were de"ned with T-cells from patients or volunteers who had been exposed to unde"ned HIV strains. Therefore, peptides used for restimulation may not correspond to the immunizing epitopes and could be recorded as false negatives. In the present analysis, the epitope data were not corrected for over-representation of particular haplotypes in the human and non-human primate studies; thus, large epitope scores could re#ect strong biases in peptide loading rather than processing. Despite the poor correlation of epitopes and sequence conservation, an improved prediction of helper T-cell epitopes in HIV gp120 is provided by the combined application of sequence conservation and MHCII protein-binding motifs. The Epimatrix algorithm is a World Wide Web-based algorithm that identi"es clustered protein sequences that are compatible with binding to MHC proteins (De Groot et al., 1997). De Groot et al. evaluated the success of the

FIG. 3. Ribbon diagrams of core gp120 (Kwong et al., 1998). In (a), positions are colored red if found in at least two helper T-cell epitopes. In (b), positions are colored by B-factor, ranging from blue (low B-factor, stable) to orange (high B-factor, unstable). T-cell epitopes cluster in well-ordered regions #anking the variable loops.

IMMUNODOMINANCE OF STABLE SEGMENTS

EpiMatrix approach in terms of the number of predicted peptides required to identify known epitopes. The sample contained 158 known epitopes in 133 proteins, giving a frequency of approximately 1 epitope per protein. The success rate was 44% in a high-stringency analysis (predicting the single highest scoring 10-mer peptide), and it was 85% using a pool of the ten highestscoring peptides in a given protein. A low-stringency EpiMatrix analysis of MHCII binding motifs for gp120 (HXB2r strain) reveals a broad distribution of potential epitopes that poorly matches the experimental epitope score (Fig. 2, cf. panels g and e). However, when potential epitopes in regions of below-average conservation are excluded, the pro"le of predicted epitopes more closely resembles the pro"le of experimentally observed epitopes (Fig. 2, cf. panels f and e). This study reveals one predictable feature, in contrast to the numerous aleatory features (Moudgil et al., 1996), of helper T-cell epitope immunodominance. The relationship between antigen structure and helper T-cell epitope immunodominance should be considered in the design and improvement of subunit vaccines. I thank Peter Kwong and Wayne Hendrickson for access to the coordinate "le of the gp120-CD4-antibody complex prior to PDB release. This work was supported by NIH grants R01-AI42350 and R21AI42702.

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immune response and may alter the way in which the total antigen is processed.