Interactions between lipids and human anti-HIV antibody 4E10 can be reduced without ablating neutralizing activity.
Human 4E10 is one of the broadest-specificity, HIV-1-neutralizing monoclonal antibodies known, recognizing a membrane-proximal linear epitope on gp41. The lipid cross-reactivity of 4E10 has been alternately suggested either to contribute to the apparent rarity of 4E10-like antibody responses in HIV infections, through elimination by B-cell tolerance mechanisms to self-antigens, or to contribute to neutralization potency by virus-specific membrane binding outside of the membrane-proximal external region (MPER). To investigate how 4E10 interacts with membrane and protein components, and whether such interactions contribute to neutralization mechanisms, we introduced two mutations into 4E10 Fv constructs, Trp to Ala at position 100 in the heavy chain [W(H100)A] and Gly to Glu at position 50 in the light chain [G(L50)E], selected to disrupt potential lipid interactions via different mechanisms. Wild-type and mutant Fvs all bound with the same affinity to peptides and monomeric and trimeric gp140s, but the affinities for gp140s were uniformly 10-fold weaker than to peptides. 4E10 Fv binding responses to liposomes in the presence or absence of MPER peptides were weak in absolute terms, consistent with prior observations, and both mutations attenuated interactions even further, as predicted. The W(H100)A mutation reduced neutralization efficiency against four HIV-1 isolates, but the G(L50)E mutation increased potency across the same panel. Electron paramagnetic resonance experiments showed that the W(H100)A mutation, but not the G(L50)E mutation, reduced the ability of 4E10 to extract MPER peptides from membranes. These results show that 4E10 nonspecific membrane binding is separable from neutralization, which is achieved through specific peptide/lipid orientation changes.
Xu
Hengyu
H
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109, USA.
Song
Likai
L
Kim
Mikyung
M
Holmes
Margaret A
MA
Kraft
Zane
Z
Sellhorn
George
G
Reinherz
Ellis L
EL
Stamatatos
Leonidas
L
Strong
Roland K
RK
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003460:Crystallization; D003461:Crystallography; D000939:Epitopes / Q000737:chemistry; D015483:HIV Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009500:Neutralization Tests; D010455:Peptides / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000939:Epitopes; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; D007141:Immunoglobulin Fc Fragments; D008563:Membrane Lipids; D010455:Peptides