13th International HIV Drug Resistance Workshop 8–12 June 2004, Tenerife Sur-Costa Adeje, Canary Islands, Spain

As a validated target for anti-HIV drugs, the highly conserved Heptad Repeat 1 (HR1) of gp41 is also a potential target for neutralizing antibodies in either a therapeutic or prophylactic setting. Though no HR1- directed neutralizing monoclonal antibodies have been described to date, we have now identified such an antibody using phage display. Bacteriophage bearing human single-chain antibodies (scFvs) were selected for binding to the gp41 HR1 region, which is the target of T20. scFvs encoded by bound phage were expressed, purified and screened using a high-throughput HIV entry assay. One scFv, D5, inhibited entry in this assay and also blocked HIV replication in singleand multiple-cycle assays. Upon conversion into a human IgG1, D5 retained antiviral potency equivalent to that of the scFv on a molar basis.

Biochemical studies demonstrated that the D5 epitope maps to the C-peptide binding groove on HR1. Further, D5 prevented binding of an epitope-tagged C peptide to HR1 in vitro, suggesting that D5 blocks HIV entry via a T20-like mechanism.

D5 IgG1 neutralized a variety of HIV isolates, but potency varied considerably and some isolates were almost completely resistant. Resistant isolates contained amino acid polymorphisms in the HR1 region. However, these polymorphisms did not confer resistance when transferred to a sensitive isolate, and in biochemical studies D5 bound equally well to wildtype and polymorphic gp41 mimetics. Thus, D5 resistance was caused by regions of the envelope glycoprotein outside HR1.

This study marks the first time that a synthetic antigen has been used to select a broadly-neutralizing monoclonal antibody. Discovery of D5 provides proof-ofconcept that the gp41 HR1 region is accessible to human IgG, that an IgG directed at HR1 can block HIV entry, and that it is possible to design synthetic antigens bearing an HR1-derived neutralizing epitope. Collectively, these observations lay the foundation for identification of therapeutic mAbs directed at HR1 and for design of immunogens capable of eliciting antibodies in vaccinees.

PRESENTING AUTHOR: MD Miller

ACKNOWLEDGEMENTS: The HIV Antibody Discovery Team includes Z An¹, E Bianchi², D Bramhill¹, G Ciliberto², J Cook¹, R Cortese², D Eckert¹, E Emini¹, R Hampton¹, D Hazuda¹, R Hrin¹, S Jarantow¹, J Joyce¹, P Kim¹, S Lane³, S Lennard³, C Lloyd³, D Lowe³, M Lu¹, P Lu¹, M McElhaugh¹, J Osbourn³, M Patel¹, A Pessi², W Schleif¹, J Shiver¹, W Strohl¹, T Vaughan³ and H Zhang¹.

¹Merck Research Laboratories, West Point, Pa. ; ²IRBM P. Angeletti, Pomezia, RM, Italy; ³Cambridge Antibody Technology, Cambridge, UK.

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2004-06-08
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