Anti-human immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize HIV-1

Abstract

The conserved membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is an important lead for vaccine design. However, immunogens that bear MPER epitopes so far have not elicited neutralizing antibodies in laboratory animals. One explanation is that the immunogens fail to recreate the proper molecular environment in which the epitopes of 2F5 and 4E10 are presented on the virus. To explore this molecular environment, we used alanine-scanning mutagenesis across residues 660 to 680 in the MPER of a pseudotyped variant of HIV-1(JR-FL), designated HIV-1(JR2), and examined the ability of 2F5 and 4E10 to neutralize the Ala mutant viruses. The results show that the only changes to produce neutralization resistance to 2F5 occurred in residue D, K, or W of the core epitope (LELDKWANL). Likewise, 4E10 resistance arose by replacing one of three residues; two (W and F) were in the core epitope, and one (W) was seven residues C-terminal to these two (NWFDISNWLW). Importantly, no single substitution resulted in resistance of virus to both 2F5 and 4E10. Surprisingly, 8 out of 21 MPER Ala mutants were more sensitive than the parental pseudovirus to 2F5 and/or 4E10. At most, only small differences in neutralization sensitivity to anti-gp120 monoclonal antibody b12 and peptide T20 were observed with the MPER Ala mutant pseudoviruses. These data suggest that MPER substitutions can act locally and enhance the neutralizing activity of antibodies to this region and imply a distinct role of the MPER of gp41 during HIV-1 envelope-mediated fusion. Neutralization experiments showing synergy between and T20 and 4E10 against HIV-1 are also presented. The data presented may aid in the design of antigens that better present the MPER of gp41 to the immune system.

FIG. 1.

Alanine scan of L660 to W680 of gp160_JR2 (only gp41 is shown). The three amino acid differences between JR2 and JR-FL are shown underlined. Each A represents one Ala mutant. W672 was replaced with Phe (F) and Tyr (Y) in addition to Ala. The numbering scheme follows that of the HxB2 strain.

FIG. 2.

Neutralization of the 2F5-resistant HIV-1_JR2 mutant pseudovirus D664A by T20 in the presence and absence of 2F5 or D50. Virus was preincubated with different concentrations of T20 in the presence or absence of a molar excess of either 2F5 or D50 (1 μM constant throughout) and then added to U87.CD4.CCR5 cells. Luciferase activity was measured after 72 h. The sequence of T20 is shown below the graph with the 2F5 epitope indicated, as well as the approximate region to which D50 binds, according to a previous study (21). Control experiments showed no effect of 1 μM D50 or 2F5 on the infectivity of the JR2 mutant D664A.