Background:  Immunogens capable of inducing antibodies that neutralize HIV-1 are an essential component of a preventative vaccine. The HIV-1 envelope (Env) contains conserved neutralizing epitopes, defined by monoclonal antibodies isolated from infected individuals. We hypothesize that directed molecular evolution can create novel Env variants that render these neutralizing epitopes more immunogenic.

Methods:  In vitro homologous DNA recombination is used to create novel chimeric variants of the Env protein from wild type env sequences encoding clade B gp120 and gp120 core, as well as consensus clade A, B, C and Group M gp140. Variants are characterized by the binding profile of human monoclonal antibodies. Rabbits are immunized using electroporation of plasmid DNA followed by boosting with a heterologous adjuvanted gp120 protein. Sera and purified IgG are used to measure neutralization activity against panels of clade B and nonclade B viruses using a pseudovirus entry assay.

Results:  Chimeric Env variants created by directed molecular evolution exhibited novel antigenic properties based on antibody binding with respect to the parental genes from which they were derived. Analyses of the antibody responses in rabbits showed that anti-Env titers were quite reproducible within groups of animals (coefficient of variation of ~50%), and that neutralization titers to SF162 were well-correlated to the overall anti-Env titers. Neutralizing antibodies to JRCSF did not correlate as well with the antibody titers, and appeared to require greater maturation for effective neutralization activity. Neutralization was investigated using a panel of pseudoviruses derived from 6 primary clade B isolates and 6 nonclade B viruses. Five novel chimeric gp120 variants showed improved neutralization activity on both sets of viruses compared to the 5 parental sequences tested in parallel. Neutralization activity given by novel chimeric gp120 core sequences was also greater that that given by a parental gp120 core. A second round of directed molecular evolution has been applied to the initial set of improved chimeric variants. Initial results indicate that properties that characterized the improved immunogenicity were enhanced.

Conclusions:  Directed molecular evolution can be used to improve the immunogenicity of the gp120 form of HIV-1 Env. Additional rounds of directed molecular evolution provide a reiterative and logical way to enhance these incremental improvements.

Keywords: Envelope; Antibody; Neutralization