Characterization of gp120 Hydrolysis by IgA Antibodies from Humans without HIV Infection

Planque, Stephanie; Mitsuda, Yukie; Taguchi, Hiroaki; Salas, María; Morris, Mary Kate; Nishiyama, Yasuhiro; Kyle, Robert H.; Okhuysen, Pablo C.; Escobar, Miguel A.; Hunter, Robert L.; Sheppard, Haynes W.; Hanson, Carl V.; Paul, Sudhir

doi:10.1089/aid.2007.0081

Cited by 36 publications

(69 citation statements)

References 48 publications

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“…The 421-433 CD4BS region, however, is a B cell SAg epitope for which preimmune human BCRs and secreted Abs produced without exposure to gp120 already express a binding site encoded by V H germ line genes without a requirement for adaptive sequence diversification (7,10,11,13,71). Preimmune Abs from mice possess a similar capability (71).…”

Section: Discussionmentioning

confidence: 99%

“…Noncovalent SAg binding by preimmune Abs, however, is characterized by low-to-moderate binding strength (12). Most gp120-binding preimmune Abs from humans without infection display poor or no HIV neutralizing activity (13). Patients with the autoimmune disease lupus and no HIV infection produce increased amounts of Abs to the 421-433 CD4BS region (14).…”

mentioning

confidence: 99%

“…The proteolytic reaction imparts improved antigen inactivation potency to Abs (22). We reported the neutralization of HIV by secretory IgA from humans without infection, an Ab class distinguished by the ability to catalyze the hydrolysis of gp120 selectively because of initial noncovalent recognition of the 421-433 CD4BS region (13).…”

mentioning

confidence: 99%

“…From mutagenesis and sequence identity studies, the gp120-binding site of preimmune Abs, in contrast, is composed mainly of the V H domain FR1 and FR3 (10,11,37). As certain preimmune Abs express HIV neutralizing activity attributable to recognition of the 421-433 region (13), the FR-dominated site must recognize the native state of this CD4BS epitope expressed on the viral surface.…”

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confidence: 99%

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Toward Effective HIV Vaccination

Nishiyama

Planque

Mitsuda

et al. 2009

Journal of Biological Chemistry

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We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288 -306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (V H ) domain framework (FR) residues. Substitution of the FR cavity V H Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and V H FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from V H 1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Toward Effective HIV Vaccination

Nishiyama

Planque

Mitsuda

et al. 2009

Journal of Biological Chemistry

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“…Following SDS electrophoresis under reducing conditions, the gels were electroblotted and FLAG-E2 was stained with: (a) goat anti-E2 polyclonal IgG (1:2000; BioDesign) followed by peroxidase-conjugated rabbit anti-goat IgG (1:1000; Pierce), (b) mouse anti-E2 monoclonal IgG (1:2000; Sigma) followed by peroxidase-conjugated goat anti-mouse IgG (1:2000; Sigma), or (c) peroxidase-conjugated mouse anti-FLAG monoclonal IgG (1:2000; Sigma). The intensity of the intact FLAG-E2 band (68 kDa) was quantified in arbitrary volume units (AVU, pixel intensity ϫ band area) (36). Hydrolysis was computed as ((AVU Diluent Ϫ AVU IgG ) ϫ 100/ (AVU Diluent )).…”

Section: Methodsmentioning

confidence: 99%

Antigen-specific Proteolysis by Hybrid Antibodies Containing Promiscuous Proteolytic Light Chains Paired with an Antigen-binding Heavy Chain

Sapparapu

Planque

Nishiyama

et al. 2009

Journal of Biological Chemistry

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Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located ϳ11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid IgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit. Antibodies (Abs)2 are composed of light and heavy chain subunits linked by intra-and inter-chain disulfide bonds. The noncovalent antigen binding site of Abs is formed mainly by amino acids located in the complementarity determining regions of the light and heavy chain variable domains (V L and V H domains). Physiological Ab-antigen binding reactions require both Ab subunits. The individual light and heavy chains can bind antigens independent of each other, but the binding affinity of the isolated subunits is often lower than the intact Abs from which they are derived (1-4). From crystallography analyses of Ab-antigen complexes, it appears that antigen contact areas with the V H domain are somewhat greater than the V L domain (5, 6). Recombinant IgG Abs composed of the heavy chain drawn from antigen-specific IgGs paired with irrelevant light chains retain antigen binding activity, albeit at reduced levels (1, 3).Following the initial noncovalent antigen binding step, some Abs proceed to catalyze hydrolysis of peptide bonds (7-12). The chemical catalysis step entails nucleophilic attack on the electrophilic carbonyl of peptide bonds by serine protease-like sites present in Ab V domains followed by hydrolysis of the covalent reaction intermediate if a water molecule is available (13-15). Unlike reversible binding, the catalytic function offers a means to permanently inactivate the antigen by its hydrolysis into smaller fragments. Reversibly binding Abs bind the antigen stoichiometrically (e.g. 2 antigen molecules/IgG molecule). As catalysts are reusable, a single catalytic Ab molecule can hydrolyze multiple antigen molecules. This offers the possibility of increased antigen neutralizing potency. Therefore, there is considerable interest in developing catalytic Abs directed to individual polypeptide antigens. The serine protease-like activity is a heritable trait encoded by germline Ab V genes, and Abs in the preimmune repertoire can hydrolyze peptides with diverse sequence promiscuously (13,14,16,17). However, the adaptive immune system has evolved to maximize noncovalent binding affinity of Abs over the course of B cell differentiation. Physiological immune mechanisms do not favor retention and improvement of the catalytic function. B cell clonal proliferation is driven by antigen binding to B cell receptors (Abs associated with signal transducing proteins). A...

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Antibody Engineering

Paul¹,

Planque²

2011

Encyclopedia of Life Sciences

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The antigen‐binding site of antibodies is composed of the heavy and light chain variable domains (V domains). Therapeutic human antibodies specific for individual antigen can be obtained by inducing adaptive B cell development in transgenic animal or by test‐tube affinity maturation of cloned antibody V domain repertoires. The V domains are recloned as full‐length antibodies to improve their pharmacokinetic behaviour and incorporate effector functions residing in the constant domains. Assembly of the V domains into multivalent constructs improves the binding avidity. Linkage to enzymes, toxins or delivery proteins imparts novel functions to the constructs. Emerging engineering strategies include the development of antibodies with catalytic activity and antibodies that can target intracellular antigens. Key Concepts: Antibodies are highly adaptive structures. They are encoded by germline genes that have diversified over evolutionary time and then undergo further antigen‐driven adaptive development over the life time of an individual organism. The antigen combining site is composed of the antibody light and heavy chain subunit variable domains. The constant domain contributes effector functions such complement activation and Fc receptor binding. Monoclonal antibodies have emerged as a major class of biological drugs for various diseases. The efficacy and safety of monoclonal antibody drugs depend on their antigen recognition affinity, specificity and in vivo pharmacokinetics. Understanding the natural process underlying adaptive sequence diversification has permitted isolation of monoclonal antibodies meeting the criteria for therapeutic applications. Antibody display and cloning methods have enabled identification of individual antibodies with defined antigenic specificities from vast repertoires. Improvement of antibody functions can be attained in the test tube by rational or random mutagenesis coupled with directed selection of the mutants. Catalytic antibodies combining the specificity of the antigen‐binding site with the turnover capability of enzymatic sites have emerged as a viable second generation technology for novel antibody applications.

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Characterization of gp120 Hydrolysis by IgA Antibodies from Humans without HIV Infection

Cited by 36 publications

References 48 publications

Toward Effective HIV Vaccination

Toward Effective HIV Vaccination

Antigen-specific Proteolysis by Hybrid Antibodies Containing Promiscuous Proteolytic Light Chains Paired with an Antigen-binding Heavy Chain

Antibody Engineering

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