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J Biol Chem. 2014 Dec 12;289(50):35015-28. doi: 10.1074/jbc.M114.591826. Epub 2014 Oct 23.

Dramatic potentiation of the antiviral activity of HIV antibodies by cholesterol conjugation.

Author information

1
From Ceinge Biotecnologie Avanzate S.C.R.L., Via Gaetano Salvatore 486, 80145 Napoli (NA), Italy, the Laboratory of Virus Molecular Biology, University of Gdansk, 80-822 Gdansk, Poland.
2
the School of Life Sciences and Nottingham Digestive Diseases Centre Biomedical Research Unit, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom.
3
From Ceinge Biotecnologie Avanzate S.C.R.L., Via Gaetano Salvatore 486, 80145 Napoli (NA), Italy.
4
From Ceinge Biotecnologie Avanzate S.C.R.L., Via Gaetano Salvatore 486, 80145 Napoli (NA), Italy, the Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via Pansini 5, 80131 Napoli (NA), Italy.
5
the Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, 81100 Caserta (CE), Italy.
6
the Viral Pseudotype Unit, Infectious Diseases and Allergy group, School of Pharmacy, University of Kent, Kent ME4 4TB, United Kingdom.
7
the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, and.
8
From Ceinge Biotecnologie Avanzate S.C.R.L., Via Gaetano Salvatore 486, 80145 Napoli (NA), Italy, JV Bio, Via Gaetano Salvatore 486, 80145 Napoli (NA), Italy a.pessi@jvbio.com.

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.

KEYWORDS:

Antibody Engineering; Cholesterol; Human Immunodeficiency Virus (HIV); Lipid Raft; Membrane Fusion; Molecular Evolution; Viral Immunology; Virus Entry

PMID:
25342747
PMCID:
PMC4263897
DOI:
10.1074/jbc.M114.591826
[Indexed for MEDLINE]
Free PMC Article

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