Protease Cleavage Sites in HIV-1 gp120 Recognized by Antigen Processing Enzymes Are Conserved and Located at Receptor Binding Sites

Abstract

The identification of vaccine immunogens able to elicit broadly neutralizing antibodies (bNAbs) is a major goal in HIV vaccine research. Although it has been possible to produce recombinant envelope glycoproteins able to adsorb bNAbs from HIV-positive sera, immunization with these proteins has failed to elicit antibody responses effective against clinical isolates of HIV-1. Thus, the epitopes recognized by bNAbs are present on recombinant proteins, but they are not immunogenic. These results led us to consider the possibility that changes in the pattern of antigen processing might alter the immune response to the envelope glycoprotein to better elicit protective immunity. In these studies, we have defined protease cleavage sites on HIV gp120 recognized by three major human proteases (cathepsins L, S, and D) important for antigen processing and presentation. Remarkably, six of the eight sites identified in gp120 were highly conserved and clustered in regions of the molecule associated with receptor binding and/or the binding of neutralizing antibodies. These results suggested that HIV may have evolved to take advantage of major histocompatibility complex (MHC) class II antigen processing enzymes in order to evade or direct the antiviral immune response.

HIV-1 Envelope Proteins and V1/V2 Domain Scaffolds with Mannose-5 to Improve the Magnitude and Quality of Protective Antibody Responses to HIV-1

“…Immunization or boosting with V1/V2 scaffolds may be the most effective way to improve the relative magnitude of antibody responses of the V1/V2 domain. In previous studies, we demonstrated that the V1/V2 domain possesses a highly conserved cathepsin D cleavage site that may diminish the immunogenicity of key epitopes in the V1/V2 domain by proteolytic degradation in vivo (93). Deletion of this site might also enhance the immunogenicity of this region.…”

Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

“…Since binding of gp120 to CD4 requires elements of gp120's inner domain, outer domain and the bridging sheet, the presence of the disulfide bridge in the inner domain likely restricted the inherent flexibility of the molecule thereby affecting the accessibility needed for initial “docking” of CD4 to gp120, and hence the lower affinity of the stabilized gp120 for CD4 [17]. The affinity between gp120 (wild-type or other forms) and CD4 (4-domain, 2-domain or CD4-IgG2), measured previously particularly via non-SPR based methods [22], [23], showed sub-nanomolar affinity. The observed relative difference in affinity via non-SPR methods could be due to differences in the strains of the gp120 used, the expression system and level of purity of the gp120 protein, the buffer systems used and/or their linear range of quantitation.…”

Increased Functional Stability and Homogeneity of Viral Envelope Spikes through Directed Evolution

“…Most likely then the mutations in Env cause it to assume a more closed conformation in which protease cleavage sites are less accessible. It is possible that digestion of Env immunogens in vivo restricts elicitation of certain Abs [72], [73], so incorporation of comb-mut mutations into Env trimer-based immunogens might offer a level of protection against such degradation. We note that the specific enzymes used here would not be encountered at the site of vaccination, but comb-mut Env trimers are resistant to a cocktail of multiple proteases with different specificities so the effect may be more general.…”