Biochem. J. (2006) 399
(483–491) (Printed in Great Britain)
Design of immunogens that present the crown of the HIV-1 V3 loop in a conformation competent to generate 447-52D-like antibodies
Kausik CHAKRABORTY*, Venuka DURANI*, Edward Roshan MIRANDA*, Michael CITRON†, Xiaoping LIANG†, William SCHLEIF†, Joseph G. JOYCE*†1 and Raghavan VARADARAJAN*†‡1
*Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India, †Merck Research Laboratories, West Point, PA 19486, U.S.A., and ‡Chemical Biology Unit, Jawaharlal Center for Advanced Scientific Research, Jakkur, P.O., Bangalore 560 004, India
Key words: HIV-1, immunogen design, neutralizing antibody, stability, thioredoxin, V3 loop.
gp120 is a subunit of the envelope glycoprotein of HIV-1. The third variable loop region of gp120 (V3 loop) contains multiple immunodominant epitopes and is also functionally important for deciding cell-tropism of the virus. 447-52D is a monoclonal antibody that recognizes the conserved tip of the V3 loop in a b-turn conformation. This antibody has previously been shown to neutralize diverse strains of the virus. In an attempt to generate an immunogen competent to generate 447-52D-like antibodies, the known epitope of 447-52D was inserted at three different surface loop locations in the small, stable protein Escherichia coli Trx (thioredoxin). At one of the three locations (between residues 74 and 75), the insertion was tolerated, the resulting protein was stable and soluble, and bound 447-52D with an affinity similar to that of intact gp120. Upon immunization, the V3 peptide-inserted Trx scaffold was able to generate anti-V3 antibodies that could compete out 447-52D binding to gp120. Epitope mapping studies demonstrated that these anti-V3 antibodies recognized the same epitope as 447-52D. Although the 447-52D-type antibodies were estimated to be present at concentrations of 50–400 µg/ml of serum, these were not able to effect neutralization of strains like JRFL and BAL but could neutralize the sensitive MN strain. The data suggest that because of the low accessibility of the V3 loop on primary isolates such as JRFL, it will be difficult to elicit a V3-specific, 447-52D-like antibody response to effectively neutralize such isolates.
Abbreviations used: HRV, human rhino virus; mAb, monoclonal antibody; NHisTrx, Escherichia coli thioredoxin with an N-terminal hexahistidine tag; 33NHisTrxV3, NHisTrx with residues 305–320 of JRFL HIV-1 gp120 inserted between residues 33 and 34; 74NHisTrxV3(307), same as 74NHisTrxV3 but with additional mutations I307C/Y318C; 74NHisTrxV3(308), same as 74NHisTrxV3 but with additional mutations H308C/F317C; 74NHisTrxV3, same as 33NHisTrxV3 but with insertion between residues 74 and 75; 83NHisTrxV3, same as 33NHisTrxV3 but with insertion between residues 83 and 84; Ni-NTA, Ni2+-nitrilotriacetate; RU, response units; SPR, surface plasmon resonance; TCLA, T-cell line adapted; Trx, thioredoxin.
1Correspondence may be addressed to either of the authors (email varadar@mbu.iisc.ernet.in or joseph_joyce@merck.com).
Received 20 April 2006/4 July 2006; accepted 7 July 2006
Published as BJ Immediate Publication 7 July 2006, doi:10.1042/BJ20060588
The Biochemical Society, London ©2006
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