Abstract
The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane-proximal external region of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization; first, they bind and preconcentrate at the viral membrane through their long, hydrophobic CDRH3 loops, and second, they form a high affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol-conjugated antibodies bind to lipid raft domains on the membrane, and because of this enrichment, they show increased antiviral potency. In particular, we find that cholesterol conjugation (i) rescues the antiviral activity of CDRH3-mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane-binding HIV antibody D5 10-100-fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears to be a general strategy to boost the potency of antiviral antibodies, and, because membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies.
Highlights
Some HIV-neutralizing antibodies have an antigen-binding site with dual specificity, for the plasma membrane and a viral epitope
We show here that this is the case and that cholesterol conjugation can increase the antiviral activity of another HIV antibody, D5, which is devoid of membrane affinity
We have shown here that the site-specific addition of cholesterol to an HIV antibody endows it with affinity for the lipid raft domain of the cell membrane and, because of this enrichment, potentiates the antibody-neutralizing activity
Summary
Some HIV-neutralizing antibodies have an antigen-binding site with dual specificity, for the plasma membrane and a viral epitope. Two independent studies have shown that mutagenesis of the hydrophobic residues at the tip of the CDRH3 or ablation of the apex of the same loop can completely abolish the neutralizing activity of 2F5 and 4E10 while inducing no change in the affinity for the peptide epitope [5,6,7,8]. The same studies have shown that the CDRH3 residues promote binding of the antibodies to synthetic lipid bilayers [6, 7], including liposomes that mimic the lipid composition of the HIV-1 membrane [9] These results have led to the proposal of a two-step mechanism for HIV neutralization by 2F5 [5, 6] and 4E10 [6]. We show here that this is the case and that cholesterol conjugation can increase the antiviral activity of another HIV antibody, D5, which is devoid of membrane affinity
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