Epitope insertion into variable loops of HIV-1 gp120 as a potential means to improve immunogenicity of viral envelope protein

We report on the properties of a set of HIV-1 IIIB Env mutants carrying a l inear gp41 epitope insertion (LLELDKWASL) in the V1, V2, V3 or V4 variable loop. Insertion of the epitope, which is defined by the HIV-I neutralizing MAb 2F5, was well tolerated in the V1, V2 and V4 loops, as these mutants we re properly expressed, retained reactivity to conformation-dependent monocl onal antibodies and exhibited patterns similar to the parental Env molecule . However, insertion of this epitope in the V3 loop was associated with dra stically reduced protein expression. Relative to parental Env molecule, the V1, V2 and V4 insertion mutants demonstrated significantly increased bindi ng to mAb 2F5 in vitro. To evaluate immunogenicity, mice and guinea pigs we re immunized with plasmid expression vectors for the mutant proteins. For b oth mice and guinea pigs, all four mutants elicited anti-gp120 antibody res ponses. In mice the V1 and V3 insertion mutants, but neither the V3 or V4 i nsertion mutant nor the parental env, elicited significant titers against t he epitope peptide, whereas in guinea pigs, V2 insertion mutant was most ef fective in eliciting anti-2F5 peptide antibody responses. While original V2 2F5 insertion mutant failed to elicit anti-2F5 peptide responses in mice, studies with 14 additional V2 insertion mutants revealed several insertion sites at which the epitope was able to induce epitope-specific antibody res ponses. This indicates that the precise position at which the epitope inser tion takes place dictates the ability of the mutant to induce the epitope-s pecific antibody responses. When tested for virus neutralization activity, the guinea pig sera that contain high titers of anti-2F5 peptide antibody f ailed to enhance the virus neutralizing activity, suggesting that the confi guration of 2F5 epitope plays a critical role in inducing neutralizing anti body responses. The results from this study may have potential implications with respect to modification of the HIV-1 Env molecule for the purpose of improving HIV-I Env immunogenicity. (C) 1999 Elsevier Science Ltd. All righ ts reserved.