Specific HIV gp120-cleaving Antibodies Induced by Covalently Reactive Analog of gp120

Chemical Immunology Research Center, Department of Pathology, University of Texas, Houston Medical School, 77030, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2003; 278(22):20429-35. DOI: 10.1074/jbc.M300870200
Source: PubMed


We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314-28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and Km for this reaction was approximately 200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization.

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Available from: Sudhir Paul, Oct 08, 2015
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    • "Binding of the anti-V3 monoclonal antibodies 447-52D and 268-DIV by E-gp120 was reduced compared to non-electrophilic gp120, suggesting selectively reduced accessibility or partial destruction of the gp120 V3 epitopes deriving from structural perturbations attendant to electrophile insertion (Nishiyama et al., 2009). Interestingly, E-gp120 spontaneously formed covalently bonded oligomers by virtue of an intermolecular reaction between the phosphonate electrophile and a naturally occurring gp120 nucleophilic site (Paul et al., 2003; Nishiyama et al., 2005, 2009). Similar nucleophilic sites serve as the catalytic sites of enzymes, and such sites were originally assumed to exist only in catalytic biomolecules. "
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    ABSTRACT: The immunodominant epitopes expressed by the HIV-1 envelope protein gp120 are hypermutable, defeating attempts to develop an effective HIV vaccine. Targeting the structurally conserved gp120 determinant that binds host CD4 receptors (CD4BD) and initiates infection is a more promising route to vaccination, but this has proved difficult because of the conformational flexibility of gp120 and immune evasion mechanisms used by the virus. Mimicking the outer CD4BD conformational epitopes is difficult because of their discontinuous nature. The CD4BD region composed of residues 421-433 (CD4BD(core)) is a linear epitope, but this region possesses B cell superantigenic character. While superantigen epitopes are vulnerable to a small subset of spontaneously produced neutralizing antibodies present in humans without infection (innate antibodies), their non-covalent binding to B cell receptors (BCRs) does not stimulate an effective adaptive response from B cells. Covalent binding at naturally occurring nucleophilic sites of the BCRs by an electrophilic gp120 (E-gp120) analog is a promising solution. E-gp120 induces the synthesis of neutralizing antibodies the CD4BD(core). The highly energetic covalent reaction is hypothesized to convert the abortive superantigens-BCR interaction into a stimulatory signal, and the binding of a spatially distinct epitope at the traditional combining site of the BCRs may furnish a second stimulatory signal. Flexible synthetic peptides can detect pre-existing CD4BD(core)-specific neutralizing antibodies. However, induced-fit conformational transitions of the peptides dictated by the antibody combining site structure may induce the synthesis of non-neutralizing antibodies. Successful vaccine targeting of the CD4BD will require a sufficiently rigid immunogen that mimics the native epitope conformation and bypasses B cell checkpoints restricting synthesis of the neutralizing antibodies.
    Frontiers in Immunology 12/2012; 3:383. DOI:10.3389/fimmu.2012.00383
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    • "The following equations were applied to compute initial velocity (V obs,I ), steady-state velocity (V obs,S ) and the burst amplitude (A; corresponding to the minimum active site concentration) for the reaction: [AMC] = V obs,S t + A(1 − e −k obs t ), and, V obs,I = k obs A, where k obs and t represent, respectively, the observed first-order rate constant and time. Cleavage of biotinylated proteins was analyzed by SDS-gel electrophoresis followed by densitometry of electroblots stained with streptavidin-peroxidase (Paul et al., 2003, 2004). Immunostaining of gp120 fragments was done using anti-gp120 Abs conjugated to horseradish peroxidase (1:1000; Fitzgerald, Concord, MA). "
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    ABSTRACT: Antibodies (Abs) to the superantigenic determinant of HIV gp120 (gp120(SAg)) are potential protective agents against HIV infection. We report that the light chain subunits of Abs cloned from lupus patients using phage library methods bind and hydrolyze gp120(SAg) independent of the heavy chain. Unlike frequent gp120(SAg) recognition by intact Abs attributable to V(H) domain structural elements, the isolated light chains expressed this activity rarely. Four light chains capable of gp120(SAg) recognition were identified by fractionating phage displayed light chains using peptide probes containing gp120 residues 421-433, a gp120(SAg) component. Three light chains expressed non-covalent gp120(SAg) binding and one expressed gp120(SAg) hydrolyzing activity. The hydrolytic light chain was isolated by covalent phage fractionation using an electrophilic analog of residues 421-433. This light chain hydrolyzed a reporter gp120(SAg) substrate and full-length gp120. Other peptide substrates and proteins were hydrolyzed at lower rates or not at all. Consistent with the expected nucleophilic mechanism of hydrolysis, the light chain reacted selectively and covalently with the electrophilic gp120(SAg) peptide analog. The hydrolytic reaction entailed a fast initial step followed by a slower rate limiting step, suggesting rapid substrate acylation and slow deacylation. All four gp120(SAg)-recognizing light chains contained sequence diversifications relative to their germline gene counterparts. These observations indicate that in rare instances, the light chain subunit can bind and hydrolyze gp120(SAg) without the participation of the heavy chain. The pairing of such light chains with heavy chains capable of gp120(SAg) recognition represents a potential mechanism for generating protective Abs with enhanced HIV binding strength and anti-viral proteolytic activity.
    Molecular Immunology 05/2007; 44(10):2707-18. DOI:10.1016/j.molimm.2006.12.005 · 2.97 Impact Factor
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    • ". The immunogen employed to generate the MAbs, E-gp120, differs from conventional gp120 preparations in that it contains self-assembled oligomers (Paul et al., 2003). The oligomers may expose epitopes not customarily encountered in monomer gp120 preparations. "
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    ABSTRACT: Conventional antibodies react with antigens reversibly. We report the formation of unusually stable complexes of HIV gp120 and nucleophilic antibodies raised by immunization with an electrophilic HIV gp120 analog (E-gp120). The stability of the complexes was evident from their very slow dissociation in a nondenaturing solvent (approximate t(1/2) 18.5 days) and their resistance to dissociation by a denaturant commonly employed to disrupt noncovalent protein-protein binding (sodium dodecyl sulfate). Kinetic studies indicated time-dependent and virtually complete progression of the antibody-gp120 complexes from the initial noncovalent state to a poorly dissociable state. The antibodies to E-gp120 displayed improved covalent reactivity with an electrophilic phosphonate probe compared to control antibodies, suggesting their enhanced nucleophilicity. One of the stably binding antibodies neutralized the infectivity of CCR5-dependent primary HIV strains belonging to clades B and C. These findings suggest the feasibility of raising antibodies capable of long-lasting inactivation of antigens by electrophilic immunization.
    Journal of Molecular Recognition 09/2006; 19(5):423-31. DOI:10.1002/jmr.795 · 2.15 Impact Factor
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