Directed Evolution of Peptide Inhibitors of HIV-1 Entry.

摘要

The conflict between HIV-1 and the host immune system plays out over a time-scale of months and years, and on a grander scale in the co-evolution of lentiviruses and the immune systems of their host species. Directed evolution of HIV-1 entry inhibitors using controlled randomization together with a display system offers a means of recapitulating one side of this conflict in vitro on an accelerated time-scale. To address limitations in existing display systems, we constructed a vector (pDQ1) integrating phage-display and mammalian-expression systems. This vector displays on phage when expressed in bacteria, and as an Fc-fusion when expressed in tissue culture, thus accelerating the iterative process of randomization, display, and characterization. We demonstrated the utility of this vector in the evolution of a CD4-mimetic peptide. The HIV-1 envelope glycoprotein (Env) is the sole viral protein expressed on the surface of the virion, and it is subject to neutralization by the host antibody response. In adapting to this pressure Env has acquired inherent resistance to neutralization: Env has evolved such that its accessible epitopes are poorly conserved and its conserved epitopes are poorly accessible. Two epitopes are constrained by the need to interact with host proteins: the receptor- and coreceptor-binding sites. Access to these conserved sites is limited for molecules the size of antibodies. Targeting the binding sites with receptor-mimetic peptides sidesteps accessibility issues in a way not possible for antibodies, while retaining access to the molecular toolbox of display technologies. Using the pDQ1 vector, we adapted a natively-expressed version of a CD4-mimetic previously developed using non-natural amino acids. Development of a high-affinity natively-expressible peptide allowed for the fusion of this element to a tyrosine-sulfated CCR5 coreceptor-mimetic peptide to generate a double-mimetic peptide. Optimization of linker length and direction allowed for simultaneous binding of the double-mimetic to receptor- and coreceptor-binding sites. We characterized the CD4-mimetic and the double-mimetic peptides with binding, receptor-complementation and neutralization assays. Inclusion of a coreceptor-mimetic peptide improved avidity and eliminated CD4-like enhancement of viral infection. Our studies indicate that there is significant advantage to simultaneously targeting both conserved Env epitopes.