Focused dampening of antibody response to the immunodominant variable loops by engineered soluble gp140

Immunization studies with modified gp120 monomers using a hyperglycosylation strategy, in which undesired epitopes are masked by the selective incorporation of N-linked glycans, were described in a previous paper (Selvarajah S, et al., J Virol 2000; 79: 12148-12163). In this report, we applied the hyperglycosylation strategy to soluble uncleaved gp140 trimers to improve the antigenic and immunogenic profile in the context of a trimeric conformation of the immunogen. The JR-FL gp140 gene was added upstream of a soluble trimerization domain of chicken cartilage matrix (CART) protein and expressed predominantly as a trimer and called gp140-CART wild-type. In the hyperglycosylated gp140-CART mCHO(V) mutant, four extra sugar attachment motifs on the variable loops helped mask epitope recognition by monoclonal antibodies specific to the variable loops. The gp140-CART mCHO(V) mutant and gp140-CART wild-type soluble trimer protein were used to immunize rabbits. The gp140-CART mCHO(V) immune sera had reduced antibody response to the variable loops compared to gp140-CART wild-type immune sera as shown by peptide reactivity, competition assays, and the reduced ability of sera to neutralize SF162 virus (a variable loop neutralization-sensitive virus). The antibody response to the CD4 binding site was retained in the gp140-CART mCHO(V) mutant immune sera similar to gp140-CART wild-type immune sera. The results demonstrate that the strategy of hyperglycosylation is clearly useful in the context of a compact form of Env immunogen such as the soluble gp140 trimer in dampening responses to variable loops while maintaining responses to an important epitope, the CD4 binding site. However, the results also show that in order to elicit broadly neutralizing antibodies that target conserved epitopes, the soluble gp140 trimer immunogen template will require further modifications.