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Peptide Engineering and AIDS Vaccine Design

A.C. Satterthwait, R.H. Zhang, E. Cabezas, Y. Xu, E.D. Santos, G. Liu, J.C. Calvo, S.M. Chen, C. Weiss*

* Food and Drug Administration, Bethesda, MD

This past year, we concentrated our efforts on research on a vaccine for AIDS. As is well known, development of an AIDS vaccine has eluded investigators for more than a decade. One problem is the high mutation rate of HIV type 1 (HIV-1). Another problem is that HIV-1 protects vulnerable sites on its surface from antibodies. Our goals have been to detect sites on the virus that are required for infection and to mimic these sites with synthetic peptides for use as a vaccine. The synthetic approach may be the only way to induce neutralizing responses to vulnerable sites that might be only transiently exposed during infection.

Our working hypothesis has been that a synthetic vaccine should mimic not only fine structure but also the conformation of neutralizing sites. To fold peptides, we developed semiautomated synthetic methods for replacing structure-defining hydrogen bonds with covalent mimics. Covalent hydrogen-bond mimics have demonstrably stabilized peptides as reverse turns, -helices, and probably medium-sized loops. We then showed that a malaria peptide could be converted from an immunologically inactive form to an active form by constraining the conformation of the peptide.

The determination of neutralizing sites on HIV-1 suitable for vaccine development, however, has confounded researchers. We are approaching this problem in 2 ways. First, we stabilized short, overlapping peptides from the HIV-1 envelope protein as -helices and had the peptides screened for HIV-1 inhibitory activity. This step revealed an -helical peptide that had more activity (>10-fold) than did the linear peptide. This finding further validates our method for stabilizing -helices. The peptide comes from a conserved region of HIV-1, a situation that makes the peptide particularly interesting. The peptide is being modified for further drug and vaccine tests.

The second approach makes use of a strategy we developed with the malaria peptide. This approach is based on determining and mimicking the epitopes recognized by the most potent HIV-1 neutralizing monoclonal antibodies available (Fig. 1). One of these, MAb 58.2, discovered by Repligen Corp., binds the V3 loop on gp120, cross-reacts with North American/European Clade B isolates, and reportedly neutralizes macrophage-tropic strains that survive initial infection. Conformational screening of MAb 58.2 with several rounds of peptides constrained with a hydrogen-bond mimic revealed a loop peptide that binds about 1600 times better than does the corresponding linear peptide. The higher affinity of the loop indicates that its solution structure mimics the conformation bound by the antibody. This finding has been confirmed in part by nuclear magnetic resonance spectroscopy in our laboratory and by x-ray crystallography by R. Stanfield and I. Wilson of the Department of Molecular Biology.

We linked the loop peptide and the corresponding linear peptide to a "universal" T-cell epitope to form multiple antigen-presentation systems (Fig. 1). Rabbit hyperimmune antisera to the loop multiple antigen-presentation systems but not to the linear multiple antigen-presentation systems bound recombinant gp120, the HIV-1 coat protein, indicating that the high-affinity loop mimics the HIV epitope far better than the linear peptide does. An important question, however, was whether the rabbits had been able to mimic the conformational specificity and/or activity of the potent MAb 58.2 in their polyclonal responses. To assess this, we first developed a conformational scan with 10 linear and constrained peptides with a wide range of affinities for MAb 58.2.

We detected 1 loop antiserum with a MAb 58.2--like profile and a second loop antiserum with a completely different profile. This finding was a surprise, because we had expected similar profiles for each rabbit, reflective of a diverse polyclonal response. Instead, we observed sharply differentiated profiles indicative of a narrow and highly developed clonal responses. M. Wang and P. Parren, Department of Immunology, then showed that the MAb 58.2--like antiserum neutralized HIV-1_MN, whereas the other antiserum did not.

These results have several implications. First, they establish a role for conformational scans in evaluating immune responses to synthetic vaccines. Second, they establish the importance of the loop conformation for any AIDS vaccine based on the V3 epitope. More generally, they verify a method and routes to achieving polyclonal responses that closely mimic the conformational preferences and neutralizing activities of specifically selected monoclonal antibodies, including the most potent neutralizing antibodies.

PUBLICATIONS

Cabezas, E., Satterthwait, A.C. The NMR structure of a V3 loop peptide that binds tightly to a monoclonal antibody that potently neutralizes HIV-1. In: Peptides: Chemistry, Structure and Biology. Proceedings of the 15th American Peptide Symposium. Tam, J.P., Kaumaya, P.T.P. (Eds.). ESCOM, Leiden, the Netherlands, in press.

Dovalsantos, E.Z., Cabezas, E., Satterthwait, A.C. Alpha-helix nucleation between peptides with a covalent hydrogen bond mimic. In: Peptides: Chemistry, Structure and Biology. Proceedings of the 15th American Peptide Symposium. Tam, J.P., Kaumaya, P.T.P. (Eds.). ESCOM, Leiden, the Netherlands, in press.

Zhang, R., Xu, Y., Nakamura, G.R., Berman, P.W., Satterthwait, A.C. Conformational mapping of a neutralizing epitope on the C4 region of HIV-1 gp120 with cyclic and bicyclic peptides. In: Peptides: Chemistry, Structure and Biology. Proceedings of the 15th American Peptide Symposium. Tam, J.P., Kaumaya, P.T.P. (Eds.). ESCOM, Leiden, the Netherlands, in press.