The concept of molecular mimicry holds that an antigen mimic reproduces some or all of the important molecular contacts that an antigen makes in binding to its “cognate” antibody. In such instances, a common assumption is made that there is a connection between molecular mimicry and immunogenic mimicry, which supposes that the closer an immunogen comes to emulating the three-dimensional structure of the original epitope, the greater the likelihood that it will elicit antibodies that cross-react with that epitope. However, this assumption has largely been untested by structural comparisons of immunogenic mimics and their cognate epitopes in complex with their corresponding antibodies.
With the advent of peptide library technology, the concept of immunogenic mimicry has been applied to vaccine development with limited success. For example, immunogenic mimicry has been reported for cross-reactive peptides that bind to antibodies against carbohydrate20
and protein linear epitopes.17,18,19
However, the structural basis of such mimicry has not been analyzed. Importantly, most anti-protein antibodies recognize discontinuous epitopes,29,30,31,32
but as yet, unrelated, cross-reactive peptides for such antibodies have not been clearly shown to act as immunogenic mimics of their cognate antigens.
The structure presented here is the first example of a peptide bound to an antibody elicited against a discontinuous protein epitope. We have characterized the mode of peptide binding to b12, and have compared it to models proposed for b12-gp120 interaction based on extensive mutagenesis of b1211
Our previous hypothesis was that B2.1 might resemble the D-loop of gp120, based on sequence homology,21
and the requirement of Asp 279 of the D-loop for binding to b12.9,11
However, our analysis of the structure of B2.1 synthetic peptide bound to Fab b12, together with the Ala substitution data on phage-displayed B2.1 indicate that, besides Arg 13, only a single cluster of residues, whose core comprises three contiguous CBRs (Phe 7, Ser 8, Asp 9), is in direct contact with b12 Fab. Thus, B2.1 “mimic” at most one to two sub-sites on gp120, and not the entire b12 epitope. Our immunization studies with the B2.1 peptide also support this conclusion in showing that B2.1 does not elicit detectable cross-reactivity with gp120. The recently-determined co-crystal structure of Fab b12 bound to a gp120 core has revealed that gp120 binds a site removed from the B2.1 binding site, and provides proof that the B2.1 peptide and gp120 interact with b12 by very different mechanisms (pers. comm., P. Kwong and T. Zhou, Vaccine Research Center, NIH, Bethesda, MD).
Ala substitution studies on Fab b12, particularly for light-chain CDR L1 residues Arg L29, Arg L31, and Arg L32, also suggested a shared mechanism of binding between B2.1 and gp12011
(see and Supplementary Table 2
). These residues are required for b12 binding to B2.1 via
critical contacts with Phe P7 and Asp P9. The involvement of these residues in binding to gp120 is also now unclear, as CDR-L1 does not appear to contact gp120 (pers. comm., P. Kwong and T. Zhou). Our study has also revealed that Ala substitution of residues in b12 that do not contact B2.1, such as Asp100f, Asn100g, Tyr100h and Tyr100i of CDR-H3, can also affect binding of b12 to both B2.1 and gp12011
(Supplementary Table 2
). These residues are most likely required to maintain the b12 structure, at least, for B2.1 peptide binding. We previously supposed that there must be some overlap in the binding sites for B2.1 and gp120, since there is reciprocal cross-reactivity between gp120 and the B2.1; each antigen can block 100% of b12 binding to the other.21
However, shows that the structure of CDR-H3 changes somewhat on Fab binding to B2.1; perhaps this or some other structural change induced by peptide binding affects the ability of b12 to bind gp120. Alternatively, the two antigen binding sites on b12 may be close enough or overlap sufficiently to sterically hinder the approach of the alternate antigen when the other ligand is already bound. Our present conclusion, that B2.1 does not mimic the b12-epitope on gp120 and binds to b12 by a different mechanism, is consistent with our recent results with three other MAbs, for which crystal structures of the original Fab-cognate antigen complexes showed that they also bind to discontinuous epitopes33,34,35
(unpublished data M. I., L. Craig, A. M., M. M. and J.K.S.). Nevertheless, in all four cases, the peptide ligands selected from phage display peptide libraries competed with the original cognate antigen for antibody binding. However, none of the peptide mimics elicited antibodies that were cross-reactive with their cognate antigens. The crystal structure of the Fab-peptide complex described here has allowed us to differentiate contact residues from those required to maintain the structure of the peptide. The structure has also elucidated that only a highly restricted portion of the B2.1 sequence directly contacts b12; therefore, B2.1 cannot reproduce the many critical contacts that b12 very likely makes with the gp120 epitope.
Although other immunogenic-mimic peptides have been described,13,14,15,16,17,18,19,20
nostructures of these peptides in complex with their antibodies have been determined. Thus, the degree to which they faithfully mimic the contacts that the corresponding cognate epitopes make with the same antibodies is unknown. Only one published study has tested the hypothesis that a peptide can mimic the gross structure and antibody contacts made by a cognate epitope.36
In this study, which involves a peptide that cross-reacts with a carbohydrate epitope, the corresponding crystal structures of the peptide and oligosaccharide individually complexed with the Fab of an anti-carbohydrate antibody showed that both ligands bind in the same overall region of the Fab, but by different structural mechanisms, but the Fab uses different residues to contact each antigen. Interestingly, while the intrinsic affinities of the Fab for each antigen differed by only two-fold, entropic vs.
enthalpic contributions of the carbohydrate and peptide to binding were very different. Furthermore, another study compared complexes of the HIV-1 neutralizing antibody 2G12 with a phage-display-selected, gp120-cross-reactive peptide and with oligosaccharide fragments corresponding to the 2G12 epitope on gp120; these studies revealed only slight overlap in the binding footprints on 2G12 for the two antigens (A.M., D.A. Calarese, C.N. Scanlan, K.C. Chow, R. Kunert, R.L. Stanfield, H. Katinger, D.R.B., I.AW. and J.K.S., manuscript submitted). Thus, in those cases, as in the results presented here, it appears that, in screening peptide libraries, antibodies select peptides to fit their cognate paratope, or part of it, but may accomplish binding through very different contacts for peptide and cognate epitope. While these two studies did not test the peptide ligands for immunogenic mimicry, our immunization studies here with the B2.1 peptide clearly showed its failure to elicit gp120-binding antibodies.
In contrast, a recent study by Dorgham et al.37
describe b12-binding peptides, similar to B2.1, that behave as immunogenic mimics of gp120. Using the b12 antibody to screen a phage-displayed peptide library, the authors identified peptides bearing a binding motif, V(W/F)SD, which is similar to the one we previously described,21
and is shared by B2.1. Although b12 binding to free, or KLH-conjugated peptide was not observed, immunization of mice with whole phage bearing the peptide elicited weak gp120 cross-reactivity, but no HIV-1-neutralizing activity; moreover, the anti-peptide antibodies in the anti-phage sera were neither measured, nor tested for competition with the b12 antibody. These results37
contrast with the immunization results reported here, for which strong anti-peptide titers were, in fact, obtained, yet no appreciable gp120 cross-reactivity was observed. Moreover, this study showed that the critical binding residues in B2.1 that were recognized by the highest titer, anti-peptide sera were quite different from those recognized by the b12 antibody (). It is possible that the difference in gp120 reactivity of anti-peptide antibodies between the two studies is due to sequence difference that flank the shared, ‘(Aromatic) SD’ motif in these immunogens . Another possibility is that the contribution of the protein scaffold to the peptide structure is different for the two coat proteins to which the peptides are fused (pIII vs
. pVIII). Nevertheless, the work of Dorgham et al.37
is of interest as it suggests that b12-binding peptides may be able to elicit some, albeit weak, gp120 cross-reactivity. However, in order to conclude that the serum antibodies, or cloned scFvs, produced by phage-immunized mice specifically bind the b12 epitope on gp120, it should be demonstrated that b12 blocks their cross-reactivity with gp120.
Our experience with b12 and several other antibodies that are known to bind discontinuous protein epitopes highlights the difficulties in using peptides to mimic this type of epitope. While cross-reactive, neutralizing and even protective antibody responses have been elicited by peptide “mimics” of linear and carbohydrate epitopes, the work presented here illustrates the necessity and value of direct structural evidence to elucidate the basis of antigenic and/or immunogenic mimicry of discontinuous protein epitopes.