Exposure to the Epstein-Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo.

Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20-50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity.

Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.

Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity.

Recent findings indicate that the ubiquitin-proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain-derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin-independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE-SJL mice is caused by a dramatic shift in the balance between constitutive and ß1i(high) immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, ß1i is increased in resident CNS cells, whereas ß5i is imported by infiltrating lymphocytes through the blood-brain barrier. Peptidyl epoxyketone specifically inhibits brain-derived ß1i(high) immunoproteasomes in vitro (kobs/[I] = 240 M(-1)s(-1)), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the ß1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.

Clinical and experimental studies of multiple sclerosis in Russia: experience of the leading national research centers.

Mechanisms of axonal damage and adaptive capacity in multiple sclerosis (MS), including cortical reorganization, have been actively studied in recent years. The lack of regenerative capabilities and the irreversibility of neurodegeneration in MS are critical factors for the optimization of MS treatment. In this study, we present the results of clinical and basic studies in the field of MS by two leading Russian centers. Clinical and neuroimaging correlations show that spinal damage in MS is accompanied by functional reorganization of the cerebral cortex, which is determined not only by the efferent component but also by the afferent component. Comparative analysis of MS treatment with both interferon ß1b (IFN-ß1b) and IFN-ß1a at a dosage of 22 µg for 3 years through subcutaneous administration and glatiramer acetate showed equally high efficiency in reducing the number of exacerbations in relapsing-remitting MS and secondary-progressive MS. We demonstrate a reduced risk of disability in relapsing-remitting MS and secondary-progressive MS patients in all groups treated with IFN-ß1 and glatiramer acetate. MS appears to be a disease that would greatly benefit from the development of personalized therapy; thus, adequate molecular predictors of myelin degradation are greatly needed. Therefore, novel ideas related to the viral hypothesis of the etiology of MS and new targets for therapeutic intervention are currently being developed. In this manuscript, we discuss findings of both clinical practice and fundamental research reflecting challenges and future directions of MS treatment in the Russian Federation.

Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.

Role of κ→λ light-chain constant-domain switch in the structure and functionality of A17 reactibody.

The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Šresolution. It was found that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the VL and VH domains. These VL/VH domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.

Heavy-light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation.

The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.

Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase.

Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-ß-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 µM and 20 µM and kcat of 115 and 92 s(-1). Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications.

Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo.

The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD(50) of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.

Liposome-encapsulated peptides protect against experimental allergic encephalitis.

Multiple sclerosis (MS) is a severe inflammatory and neurodegenerative disease with an autoimmune background. Despite the variety of therapeutics available against MS, the development of novel approaches to its treatment is of high importance in modern pharmaceutics. In this study, experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats has been treated with immunodominant peptides of the myelin basic protein (MBP) encapsulated in mannosylated small unilamellar vesicles. The results show that liposome-encapsulated MBP(46-62) is the most effective in reducing maximal disease score during the first attack, while MBP(124-139) and MBP(147-170) can completely prevent the development of the exacerbation stage. Both mannosylation of liposomes and encapsulation of peptides are critical for the therapeutic effect, since neither naked peptides nor nonmannosylated liposomes, loaded or empty, have proved effective. The liposome-mediated synergistic effect of the mixture of 3 MBP peptides significantly suppresses the progression of protracted EAE, with the median cumulative disease score being reduced from 22 to 14 points, compared to the placebo group; prevents the production of circulating autoantibodies; down-regulates the synthesis of Th1 cytokines; and induces the production of brain-derived neurotrophic factor in the central nervous system. Thus, the proposed formulation ameliorates EAE, providing for a less severe first attack and rapid recovery from exacerbation, and offers a promising therapeutic modality in MS treatment.

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics.

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (V(L)) and variable heavy (V(H)) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.

Combinatorial antibody library from multiple sclerosis patients reveals antibodies that cross-react with myelin basic protein and EBV antigen.

Multiple sclerosis (MS) is a widespread neurodegenerative autoimmune disease with unknown etiology. It is increasingly evident that, together with pathogenic T cells, autoreactive B cells are among the major players in MS development. The analysis of myelin neuroantigen-specific antibody repertoires and their possible cross-reactivity against environmental antigens, including viral proteins, could shed light on the mechanism of MS induction and progression. A phage display library of single-chain variable fragments (scFvs) was constructed from blood lymphocytes of patients with MS as a potential source of representative MS autoantibodies. Structural alignment of 13 clones selected toward myelin basic protein (MBP), one of the major myelin antigens, showed high homology within variable regions with cerebrospinal fluid MS-associated antibodies as well as with antibodies toward Epstein-Barr latent membrane protein 1 (LMP1). Three scFv clones showed pronounced specificity to MBP fragments 65-92 and 130-156, similar to the serum MS antibodies. One of these clones, designated E2, in both scFv and full-size human antibody constructs, was shown to react with both MBP and LMP1 proteins in vitro, suggesting natural cross-reactivity. Thus, antibodies induced against LMP1 during Epstein-Barr virus infection might act as inflammatory trigger by reacting with MBP, suggesting molecular mimicry in the mechanism of MS pathogenesis.

Design of targeted B cell killing agents.

B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents.

Catalytic antibodies: balancing between Dr. Jekyll and Mr. Hyde.

The immunoglobulin molecule is a perfect template for the de novo generation of biocatalytic functions. Catalytic antibodies, or abzymes, obtained by the structural mimicking of enzyme active sites have been shown to catalyze numerous chemical reactions. Natural enzyme analogs for some of these reactions have not yet been found or possibly do not exist at all. Nowadays, the dramatic breakthrough in antibody engineering and expression technologies has promoted a considerable expansion of immunoglobulin's medical applications and is offering abzymes a unique chance to become a promising source of high-precision "catalytic vaccines." At the same time, the discovery of natural abzymes on the background of autoimmune disease revealed their beneficial and pathogenic roles in the disease progression. Thus, the conflicting Dr. Jekyll and Mr. Hyde protective and destructive essences of catalytic antibodies should be carefully considered in the development of therapeutic abzyme applications.

Strategies for induction of catalytic antibodies toward HIV-1 glycoprotein gp120 in autoimmune prone mice.

Tremendous efforts to produce an efficient vaccine for HIV infection have been unsuccessful. The ability of HIV to utilize sophisticated mechanisms to escape killing by host immune system rises dramatic problems in the development of antiviral therapeutics. The HIV infection proceeds by interaction of coat viral glycoprotein gp120 trimer with CD4(+) receptor of the lymphocyte. Thus this surface antigen may be regarded as a favorable target for immunotherapy. In the present study, we have developed three different strategies to produce gp120-specific response in autoimmune prone mice (SJL strain) as potential tools for production "catalytic vaccine". Therefore (i) reactive immunization by peptidylphosphonate, structural part of the coat glycoprotein, (ii) immunization by engineered fused epitopes of gp120 and encephalogenic peptide, a part of myelin basic protein, and (iii) combined vaccination by DNA and corresponding gp120 fragments incorporated into liposomes were investigated. In the first two cases monoclonal antibodies and their recombinant fragments with amidolytic and gp120-specific proteolytic activities were characterized. In the last case, catalytic antibodies with virus neutralizing activity proved in cell line models were harvested.

Recognition and degradation of myelin basic protein peptides by serum autoantibodies: novel biomarker for multiple sclerosis.

The pathologic role of autoantibodies in autoimmune disease is widely accepted. Recently, we reported that anti-myelin basic protein (MBP) serum Abs from multiple sclerosis (MS) patients exhibit proteolytic activity toward the autoantigen. The aim of this study is to determine MBP epitopes specific for the autoantibodies in MS and compare these data with those from other neuronal disorders (OND), leading to the generation of new diagnostic and prognostic criteria. We constructed a MBP-derived recombinant "epitope library" covering the entire molecule. We used ELISA and PAGE/surface-enhanced laser desorption/ionization mass spectroscopy assays to define the epitope binding/cleaving activities of autoantibodies isolated from the sera of 26 MS patients, 22 OND patients, and 11 healthy individuals. The levels of autoantibodies to MBP fragments 48-70 and 85-170 as well as to whole MBP and myelin oligodendrocyte glycoprotein molecules were significantly higher in the sera of MS patients than in those of healthy donors. In contrast, selective reactivity to the two MBP fragments 43-68 and 146-170 distinguished the OND and MS patients. Patients with MS (77% of progressive and 85% of relapsing-remitting) but only 9% of patients with OND and no healthy donors were positive for catalysis, showing pronounced epitope specificity to the encephalitogenic MBP peptide 81-103. This peptide retained its substrate properties when flanked with two fluorescent proteins, providing a novel fluorescent resonance energy transfer approach for MS studies. Thus, anti-MBP autoantibody-mediated, epitope-specific binding and cleavage may be regarded as a specific characteristic of MS compared with OND and healthy donors and may serve as an additional biomarker of disease progression.

Anti-idiotypic antibody mimics proteolytic function of parent antigen.

Functional imaging of subtilisin Carlsberg active center by the idiotypic network yielded a catalytic anti-idiotypic antibody with endopeptidase, amidase, and esterase activities. A monoclonal antibody inhibitory to subtilisin (Ab1 5-H4) was employed as the template for guiding the idiotypic network to produce the catalytic anti-idiotypic Ab2 6B8-E12. Proteolytic activity of 6B8-E12 was demonstrated by zymography using self-quenched fluorescein-BSA conjugate and in a coupled assay detecting Ab2-dependent RNase A inactivation. Cleavage of peptide substrates by 6B8-E12 revealed distinct patterns of hydrolysis with high preference for aromatic residues before or after the scissile bond. Catalytic activity of Ab2 was inhibited by phenylmethylsulfonyl fluoride, a mechanism-based inhibitor of serine hydrolases. 5-H4 and 6B8-E12 were cloned, produced in Escherichia coli as single-chain variable fragments (scFvs), and purified. Kinetic parameters for amidolytic and esterolytic activities were similar in Ab2 and its scFv derivative. Although the antigen-specific portion of 6B8-E12 possesses no primary structure similarity to subtilisin, it mimics proteolytic and amidolytic functions of the parental antigen, albeit with 4 orders of magnitude slower acceleration rates. The lack of detectable endopeptidase activity of 6B8-E12 scFv raises interesting issues concerning general evolution of catalytic activity. The in silico 3D models of Ab1 and Ab2 revealed strong structural similarity to known anti-protease antibodies and to abzymes, respectively. These results indicate that the idiotypic network is capable, to a significant extent, of reproducing catalytic apparatus of serine proteases and further validate the use of imaging of enzyme active centers by the immune system for induction of abzymes accelerating energy-demanding amide bond hydrolysis.

Routes to covalent catalysis by reactive selection for nascent protein nucleophiles.

Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.

Induction of a protein-targeted catalytic response in autoimmune prone mice: antibody-mediated cleavage of HIV-1 glycoprotein GP120.

We have induced a polyclonal IgG that degrades the HIV-1 surface antigen, glycoprotein gp120, by taking advantage of the susceptibility of SJL mice to a peptide-induced autoimmune disorder, experimental autoimmune encephalomyelitis (EAE). Specific pathogen-free SJL mice were immunized with structural fragments of gp120, fused in-frame with encephalitogenic peptide MBP(85-101). It has resulted in a pronounced disease-associated immune response against antigens. A dramatic increase of gp120 degradation level by purified polyclonal IgG from immunized versus nonimmunized mice has been demonstrated by a newly developed fluorescence-based assay. This activity was inhibited by anti-mouse immunoglobulin antibodies as well as by Ser- and His-reactive covalent inhibitors. A dominant proteolysis site in recombinant gp120 incubated with purified polyclonal IgG from immunized mice was shown by SDS-PAGE. The SELDI-based mass spectrometry revealed that these antibodies exhibited significant specificity toward the Pro484-Leu485 peptide bond. The sequence surrounding this site is present in nearly half of the HIV-I variants. This novel strategy can be generalized for creating a catalytic vaccine against viral pathogens.

Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen.

Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.