Yukie Mitsuda

Neutralization of genetically diverse Hiv-1 strains by Iga antibodies to the gp120–cd4-binding site from long-term survivors of Hiv infection

Objective:To identify an HIV epitope suitable for vaccine development. Design:Diverse HIV-1 strains express few structurally constant regions on their surface vulnerable to neutralizing antibodies. The mostly conserved CD4-binding site (CD4BS) of gp120 is essential for host cell binding and infection by the virus. Antibodies that recognize the CD4BS are rare, and one component of the CD4BS, the 421–433 peptide region, expresses B-cell superantigenic character, a property predicted to impair the anti-CD4BS adaptive immune response. Methods:IgA samples purified from the plasma of patients with HIV infection were analyzed for the ability to bind synthetic mimetics containing the 416–433 gp120 region and full-length gp120. Infection of peripheral blood mononuclear cells by clinical HIV isolates was measured by p24 ELISA. Results:IgA preparations from three patients with subtype B infection for 19–21 years neutralized heterologous, coreceptor CCR5-dependent subtype A, B, C, D, and AE strains with exceptional potency. The IgAs displayed specific binding of a synthetic 416–433 peptide mimetic dependent on recognition of the CD4-binding residues located in this region. Immunoadsorption, affinity chromatography, and mutation procedures indicated that HIV neutralization occurred by IgA recognition of the CD4BS. Conclusion:These observations identify the 421–433 peptide region as a vulnerable HIV site to which survivors of infection can produce powerful neutralizing antibodies. This indicates that the human immune system can bypass restrictions on the adaptive B cell response to the CD4BS, opening the route to targeting the 421–433 region for attaining control of HIV infection.

Physiological IgM Class Catalytic Antibodies Selective for Transthyretin Amyloid

Background: Some antibodies express serine protease activity. Transthyretin misfolding causes accumulation of pathogenic amyloid. Results: Constitutively produced IgM but not IgG class antibodies selectively hydrolyzed and dissolved misfolded transthyretin without hydrolyzing physiologically folded transthyretin. Some IgMs were oligoreactive with amyloids and superantigens. Conclusion: Catalytic IgMs may clear misfolded TTR and delay amyloidosis. Significance: The innate antibody repertoire is a source of selective catabodies to toxic proteins. Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded β-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetrameric TTR (phyTTR). IgM class B cell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid β peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function.

Toward Selective Covalent Inactivation of Pathogenic Antibodies A PHOSPHONATE DIESTER ANALOG OF VASOACTIVE INTESTINAL PEPTIDE THAT INACTIVATES CATALYTIC AUTOANTIBODIES

We report the selective inactivation of proteolytic antibodies (Abs) to an autoantigen, the neuropeptide vasoactive intestinal peptide (VIP), by a covalently reactive analog (CRA) of VIP containing an electrophilic phosphonate diester at the Lys20 residue. The VIP-CRA was bound irreversibly by a monoclonal Ab that catalyzes the hydrolysis of VIP. The reaction with the VIP-CRA proceeded more rapidly than with a hapten CRA devoid of the VIP sequence. The covalent binding occurred preferentially at the light chain subunit of the Ab. Covalent VIP-CRA binding was inhibited by VIP devoid of the phosphonate diester group. These results indicate the importance of noncovalent VIP recognition in guiding Ab nucleophilic attack on the phosphonate group. Consistent with the covalent binding data, the VIP-CRA inhibited catalysis by the recombinant light chain of this Ab with potency greater than the hapten-CRA. Catalytic hydrolysis of VIP by a polyclonal VIPase autoantibody preparation that cleaves multiple peptide bonds located between residues 7 and 22 essentially was inhibited completely by the VIP-CRA, suggesting that the electrophilic phosphonate at Lys20 enjoys sufficient conformational freedom to react covalently with Abs that cleave different peptide bonds in VIP. These results suggest a novel route to antigen-specific covalent targeting of pathogenic Abs.

Metal-dependent amyloid β-degrading catalytic antibody construct

Catalytic antibodies (catabodies) that degrade target antigens rapidly are rare. We describe the metal-dependence of catabody construct 2E6, an engineered heterodimer of immunoglobulin light chain variable domains that hydrolyzes amyloid β peptides (Aβ) specifically. In addition to the electrophilic phosphonate inhibitor of serine proteases, the metal chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline completely inhibited the hydrolysis of Aβ by catabody 2E6. Formation of catabody-electrophilic phosphonate inhibitor adducts was unaffected by EDTA, suggesting that the metal exerts a favorable effect on a catalytic step after the initial catabody nucleophilic attack on Aβ. The EDTA inactivated catabody failed to disaggregate fibrillar Aβ, indicating the functional importance of the Aβ hydrolytic activity. Treating the EDTA-inactivated catabody with Zn(2+) or Co(2+) restored the Aβ hydrolytic activity, and Zn(2+)-induced catabody conformational transitions were evident by fluorescence emission spectroscopy. The studies reveal the absolute catabody dependence on a metal cofactor.

Toward Effective HIV Vaccination: INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY*

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 (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421–433 region peptide probe. The CDRs and VH 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 VH1 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.

Antibodies to a Superantigenic Glycoprotein 120 Epitope as the Basis for Developing an HIV Vaccine

Failure to induce synthesis of neutralizing Abs to the CD4 binding determinant (CD4BD) of gp120, a central objective in HIV vaccine research, has been alternately ascribed to insufficient immunogen binding to Abs in their germline V region configuration expressed as BCRs, insufficient adaptive mutations in Ab V regions, and conformational instability of gp120. We employed peptide analogs of gp120 residues 421–433 within the CD4BD (CD4BDcore) to identify Abs produced without prior exposure to HIV (constitutive Abs). The CD4BDcore peptide was recognized by single-chain Fv fragments from noninfected humans with lupus that neutralized genetically diverse strains belonging to various HIV subtypes. Replacing the framework region (FR) of a VH4-family single-chain Fv with the corresponding VH3-family FRs from single-chain Fv JL427 improved the CD4BDcore peptide-binding activity, suggesting a CD4BDcore binding site outside the pocket formed by the CDRs. Replacement mutations in the FR site vicinity suggested the potential for adaptive improvement. A very small subset of serum CD4BDcore-specific serum IgAs from noninfected humans without autoimmune disease isolated by epitope-specific chromatography neutralized the virus potently. A CD4BDcore-specific, HIV neutralizing murine IgM with H and L chain V regions (VH and VL regions) free of immunogen-driven somatic mutations was induced by immunization with a CD4BDcore peptide analog containing an electrophilic group that binds B cells covalently. The studies indicate broad and potent HIV neutralization by constitutive Abs as an innate, germline-encoded activity directed to the superantigenic CD4BDcore epitope that is available for amplification for vaccination against HIV.

Towards Covalent Vaccination IMPROVED POLYCLONAL HIV NEUTRALIZING ANTIBODY RESPONSE INDUCED BY AN ELECTROPHILIC gp120 V3 PEPTIDE ANALOG

Rare monoclonal antibodies (Abs) can form irreversible complexes with antigens by enzyme-like covalent nucleophile-electrophile pairing. To determine the feasibility of applying irreversible antigen inactivation by Abs as the basis of vaccination against microbes, we studied the polyclonal nucleophilic Ab response induced by the electrophilic analog of a synthetic peptide corresponding to the principal neutralizing determinant (PND) of human immunodeficiency virus type-1 (HIV) gp120 located in the V3 domain. Abs from mice immunized with the PND analog containing electrophilic phosphonates (E-PND) neutralized a homologous HIV strain (MN) ∼50-fold more potently than control Abs from mice immunized with PND. The IgG fractions displayed binding to intact HIV particles. HIV complexes formed by anti-E-PND IgG dissociated noticeably more slowly than the complexes formed by anti-PND IgG. The slower dissociation kinetics are predicted to maintain long-lasting blockade of host cell receptor recognition by gp120. Pretreatment of the anti-PND IgG with a haptenic electrophilic phosphonate compound resulted in more rapid dissociation of the HIV-IgG complexes, consistent with the hypothesis that enhanced Ab nucleophilic reactivity induced by electrophilic immunization imparts irreversible character to the complexes. These results suggest that electrophilic immunization induces a sufficiently robust nucleophilic Ab response to enhance the anti-microbial efficacy of candidate polypeptide vaccines.

Specific Amyloid β Clearance by a Catalytic Antibody Construct

Background: Naturally occurring catalytic antibodies (catabodies) can hydrolyze peptide bonds. Results: A catabody engineered from innate immunity principles hydrolyzed amyloid β (Aβ) specifically, dissolved Aβ aggregates, and cleared brain Aβ deposits without evident toxicity. Conclusion: The catabody could potentially be developed as a therapy for Alzheimer disease. Significance: The innate catabody repertoire may be a source of useful catabodies to toxic amyloids. Classical immunization methods do not generate catalytic antibodies (catabodies), but recent findings suggest that the innate antibody repertoire is a rich catabody source. We describe the specificity and amyloid β (Aβ)-clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Aβ C terminus noncovalently and hydrolyzed Aβ rapidly, with no reactivity to the Aβ precursor protein, transthyretin amyloid aggregates, or irrelevant proteins containing the catabody-sensitive Aβ dipeptide unit. The catabody dissolved preformed Aβ aggregates and inhibited Aβ aggregation more potently than an Aβ-binding IgG. Intravenous catabody treatment reduced brain Aβ deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aβ hydrolysis appears to be an innate immune function that could be applied for therapeutic Aβ removal.

Constant Domain-regulated Antibody Catalysis

Background: Some antibody variable domains express nucleophilic catalytic sites. Results: The same variable domains displayed superior catalysis when expressed in the IgM compared with the IgG constant domain scaffold; all monoclonal IgMs were catalytic, and polyclonal IgM was more catalytic than IgG. Conclusion: The constant domains regulate variable domain catalysis. Significance: Catalysis may be a first line immune function expressed prior to adaptive antibody induction. Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies.

Deficient synthesis of class-switched, Hiv-neutralizing antibodies to the Cd4 binding site and correction by electrophilic gp120 immunogen

Objective:HIV is vulnerable to antibodies that recognize a linear CD4 binding site epitope of gp120 (CLIN), but inducing CLIN-directed antibody synthesis by traditional vaccine principles is difficult. We wished to understand the basis for deficient CLIN-directed antibody synthesis and validate correction of the deficiency by an electrophilic gp120 analog (E-gp120) immunogen that binds B-cell receptors covalently. Methods:Serum antibody responses to a CLIN peptide and full-length gp120 epitopes induced by HIV infection in humans and immunization of mice with gp120 or E-gp120 were monitored. HIV neutralization by monoclonal and variable domain-swapped antibodies was determined from tissue culture and humanized mouse infection assays. Results:We describe deficient CLIN-directed IgG but not IgM antibodies in HIV-infected patients and mice immunized with gp120 accompanied by robust synthesis of IgGs to the immunodominant gp120 epitopes. Immunization with the E-gp120 corrected the deficient CLIN-directed IgG synthesis without overall increased immunogenicity of the CLIN or other gp120 epitopes. E-gp120-induced monoclonal IgGs neutralized diverse HIV strains heterologous to the immunogen. A CLIN-directed IgG neutralized HIV more potently compared to its larger IgM counterpart containing the same variable domains, suggesting obstructed access to HIV surface-expressed CLIN. An E-gp120-induced IgG suppressed HIV infection in humanized mice, validating the tissue culture neutralizing activity. Conclusion:A CLIN-selective physiological defect of IgM→IgG class-switch recombination (CSR) or restricted post-CSR B-cell development limits the functional utility of the humoral immune response to gp120. The E-gp120 immunogen is useful to bypass the restriction and induce broadly neutralizing CLIN-directed IgGs (see Supplemental Video Abstract, http://links.lww.com/QAD/A551).