title: Dramatic potentiation of the antiviral activity of HIV antibodies by cholesterol conjugation.
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.
authors.affiliation: 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.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Lacek
authors.affiliation: 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.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Urbanowicz
authors.affiliation: From Ceinge Biotecnologie Avanzate S.C.R.L., Via Gaetano Salvatore 486, 80145 Napoli (NA), Italy.
authors.forename: Fulvia
authors.initials: F
authors.lastname: Troise
authors.affiliation: 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.
authors.forename: Claudia
authors.initials: C
authors.lastname: De Lorenzo
authors.affiliation: the Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, 81100 Caserta (CE), Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Severino
authors.affiliation: the Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Via Vivaldi 43, 81100 Caserta (CE), Italy.
authors.forename: Antimo
authors.initials: A
authors.lastname: Di Maro
authors.affiliation: 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.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tarr
authors.affiliation: the Viral Pseudotype Unit, Infectious Diseases and Allergy group, School of Pharmacy, University of Kent, Kent ME4 4TB, United Kingdom.
authors.forename: Francesca
authors.initials: F
authors.lastname: Ferrara
authors.affiliation: the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, and.
authors.forename: Alexander
authors.initials: A
authors.lastname: Ploss
authors.affiliation: the Viral Pseudotype Unit, Infectious Diseases and Allergy group, School of Pharmacy, University of Kent, Kent ME4 4TB, United Kingdom.
authors.forename: Nigel
authors.initials: N
authors.lastname: Temperton
authors.affiliation: 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.
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Ball
authors.affiliation: 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.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Nicosia
authors.affiliation: From Ceinge Biotecnologie Avanzate S.C.R.L., Via Gaetano Salvatore 486, 80145 Napoli (NA), Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Cortese
authors.affiliation: 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.
authors.forename: Antonello
authors.initials: A
authors.lastname: Pessi
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism*; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D057809:HEK293 Cells; D015483:HIV Antibodies / Q000276:immunology* / Q000378:metabolism*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D009500:Neutralization Tests
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D015483:HIV Antibodies; D002784:Cholesterol
keywords: Antibody Engineering; Cholesterol; Human Immunodeficiency Virus (HIV); Lipid Raft; Membrane Fusion; Molecular Evolution; Viral Immunology; Virus Entry