Comparison of Four Distinct Idiotypes of Monoclonal Antibodies against Hen’s Egg Ovomucoid

Two monoclonal antibodies (mAb 41B3 and mAb 51B3), directed against hen’s egg ovomucoid (OM) and different from those we previously reported (mAb 23E5 and mAb 32A8), were prepared and purified. A competitive radioimmunoassay using ¹²⁵I-labeled OM showed that both mAb 41B3 and mAb 51B3 could bind all three homologous domains (domains I (DI), II (DII) and III (Dili)) of OM and that they reacted most efficiently with Dill. To analyze the paratope specificities of these four mAbs, a sandwich assay and a competitive radioimmunoassay were done. Only the pair mAb 41B3 and mAb 51B3 could not simultaneously bind the OM or domains in the sandwich assay. Only mAb 41B3 inhibited the binding of mAb 51B3 to antigens and vice versa in the competitive radioimmunoassay. These results suggest that mAb 41B3 and mAb 51B3 recognized a closely related site distinct from the epitopes for mAb 23E5 or mAb 32A8. These mAbs may be useful for general studies on epitopes of protein antigens as well as for analyses of the antigenic determinants of OM.     

Optimum Length and Flexibility of Reovirus Attachment Protein σ1 Are Required for Efficient Viral Infection

Reovirus attachment protein σ1 is an elongated trimer with head-and-tail morphology that engages cell-surface carbohydrate and junctional adhesion molecule A (JAM-A). The σ1 protein is comprised of three domains partitioned by two flexible linkers termed interdomain regions (IDRs). To determine the importance of σ1 length and flexibility at different stages of reovirus infection, we generated viruses with mutant σ1 molecules of altered length and flexibility and tested these viruses for the capacity to bind the cell surface, internalize, uncoat, induce protein synthesis, assemble, and replicate. We reduced the length of the α-helical σ1 tail to engineer mutants L1 and L2 and deleted midpoint and head-proximal σ1 IDRs to generate ΔIDR1 and ΔIDR2 mutant viruses, respectively. Decreasing length or flexibility of σ1 resulted in delayed reovirus infection and reduced viral titers. L1, L2, and ΔIDR1 viruses but not ΔIDR2 virus displayed reduced cell attachment, but altering σ1 length or flexibility did not diminish the efficiency of virion internalization. Replication of ΔIDR2 virus was hindered at a postdisassembly step. Differences between wild-type and σ1 mutant viruses were not attributable to alterations in σ1 folding, as determined by experiments assessing engagement of cell-surface carbohydrate and JAM-A by the length and IDR mutant viruses. However, ΔIDR1 virus harbored substantially less σ1 on the outer capsid. Taken together, these data suggest that σ1 length is required for reovirus binding to cells. In contrast, IDR1 is required for stable σ1 encapsidation, and IDR2 is required for a postuncoating replication step. Thus, the structural architecture of σ1 is required for efficient reovirus infection of host cells.     

Sub-Domains of Ricin��s B Subunit as Targets of Toxin Neutralizing and Non-Neutralizing Monoclonal Antibodies

The B subunit (RTB) of ricin toxin is a galactose (Gal)−/N-acetylgalactosamine (GalNac)-specific lectin that mediates attachment, entry, and intracellular trafficking of ricin in host cells. Structurally, RTB consists of two globular domains with identical folding topologies. Domains 1 and 2 are each comprised of three homologous sub-domains (α, β, γ) that likely arose by gene duplication from a primordial carbohydrate recognition domain (CRD), although only sub-domains 1α and 2γ retain functional lectin activity. As part of our ongoing effort to generate a comprehensive B cell epitope map of ricin, we report the characterization of three new RTB-specific monoclonal antibodies (mAbs). All three mAbs, JB4, B/J F9 and C/M A2, were initially identified based on their abilities to neutralize ricin in a Vero cell cytotoxicty assay and to partially (or completely) block ricin attachment to cell surfaces. However, only JB4 proved capable of neutralizing ricin in a macrophage apoptosis assay and in imparting passive immunity to mice in a model of systemic intoxication. Using a combination of techniques, including competitive ELISAs, pepscan analysis, differential reactivity by Western blot, as well as affinity enrichment of phage displayed peptides, we tentatively localized the epitopes recognized by the non-neutralizing mAbs B/J F9 and C/M A2 to sub-domains 2α and 2β, respectively. Furthermore, we propose that the epitope recognized by JB4 is within sub-domain 2γ, adjacent to RTB’s high affinity Gal/GalNAc CRD. These data suggest that recognition of RTB’s sub-domains 1α and 2γ are critical determinants of antibody neutralizing activity and protective immunity to ricin.     

Characterization of New Alternaria alternata–Specific Rat Monoclonal Antibodies

In this study, three different rat hybridoma cell lines secreting monoclonal antibodies (mAbs) recognizing the spores from Alternaria alternata, a plant pathogenic fungus, contaminant of food products and important cause of both allergic rhinitis and asthma, have been characterized. These three mAbs are all of IgM isotype. Two antibodies, A1 and F10, were cross-reactive antibodies recognizing spores from Alternaria, Cladosporium, Penicillium, Aspergillus and Stachybotrys genera, but not the yeasts Saccharomyces cerevisiae or Candida albicans. Competitive and sandwich assays demonstrated that these two mAbs were directed against the same or very close repetitive(s) epitope(s). A1-based sandwich ELISA efficiently detected this epitope in various mould (but not yeast)-soluble extracts prepared from strains grown in the laboratory. Moreover, this A1-based sandwich ELISA detected its cognate epitope in air and dust samples obtained from dwellings. The third antibody, E5, recognized only the spores of Alternaria and the phylogenetically very close Ulocladium botrytis. This E5 antibody is directed against a repetitive epitope found in Alternaria and Ulocladium laboratory extracts and can be used in a sandwich assay for the quantification of these moulds. Therefore, E5 antibody is a promising tool for the development of Alternaria-Ulocladium-specific immunoassays, while A1 and F10 could be interesting tools for the quantification of the total mould biomass.     

Preparation of Monoclonal Antibodies against Human Ventricular Myosin Light Chain 1 （HVMLC1） for Functional Studies

Using purified recombinant human ventricular myosin light chain 1 (HVMLC 1) as the antigen,three monoclonal antibodies,designated C8,C9 and B 12,were prepared.Immunoblot experiments demonstratedthat all monoclonal antibodies could react with the ventricular myosin light chain 1 isolated from differentsources,such as human,rat or pig.It was also demonstrated that C8 was directed against the NN part of theN-fragment (amino acid 1-40) of HVMLC1,and both C9 and B12 against the C-fragment (amino acid 99-195).The affinity constants of C8,C9 and B12 were 3.20×10~8,8.60×10~7 and 1.77×10~8 M~(-1),respectively,determined by non-competitive ELISA.The isotype of B12 was determined as lgG2a,whereas the isotype ofboth C8 and C9 were IgG1.In the presence of C9 or B12,the actin-activated Mg~(2 )ATPase activity of myosinwas greatly inhibited,but there was almost no effect on the Mg~(2 )ATPase activity for C8.B12 and C9 alsoinhibited the superprecipitation of porcine cardiac native actomyosin (myosin B) and reconstituted actomyosin,but C8 did not.The results indicate that all three monoclonal antibodies could bind the intact myosin molecule,but B12 and C9 might more easily react with epitopes located in the C-fragment of HVMLC1.The inhibitoryeffects of B 12 and C9 on ATPase activity and superprecipitation assays show that light chain 1,particularlythe C-fragment domain,is involved in the modulation of the actin-activated Mg~(2 )ATPase activity of myosinand,as a consequence,plays an essential role in the interaction of actin and myosin.     

Structure of Reovirus σ1 in Complex with Its Receptor Junctional Adhesion Molecule-A

Viral attachment to specific host receptors is the first step in viral infection and serves an essential function in the selection of target cells. Mammalian reoviruses are highly useful experimental models for studies of viral pathogenesis and show promise as vectors for oncolytics and vaccines. Reoviruses engage cells by binding to carbohydrates and the immunoglobulin superfamily member, junctional adhesion molecule-A (JAM-A). JAM-A exists at the cell surface as a homodimer formed by extensive contacts between its N-terminal immunoglobulin-like domains. We report the crystal structure of reovirus attachment protein σ1 in complex with a soluble form of JAM-A. The σ1 protein disrupts the JAM-A dimer, engaging a single JAM-A molecule via virtually the same interface that is used for JAM-A homodimerization. Thus, reovirus takes advantage of the adhesive nature of an immunoglobulin-superfamily receptor by usurping the ligand-binding site of this molecule to attach to the cell surface. The dissociation constant (K_D) of the interaction between σ1 and JAM-A is 1,000-fold lower than that of the homophilic interaction between JAM-A molecules, indicating that JAM-A strongly prefers σ1 as a ligand. Analysis of reovirus mutants engineered by plasmid-based reverse genetics revealed residues in σ1 required for binding to JAM-A and infectivity of cultured cells. These studies define biophysical mechanisms of reovirus cell attachment and provide a platform for manipulating reovirus tropism to enhance vector targeting.     

Targeted In Vivo Inhibition of Specific Protein–Protein Interactions Using Recombinant Antibodies

With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated “silencing” represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein–protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.     

Structure of Serotype 1 Reovirus Attachment Protein σ1 in Complex with Junctional Adhesion Molecule A Reveals a Conserved Serotype-Independent Binding Epitope

Mammalian orthoreoviruses use glycans and junctional adhesion molecule A (JAM-A) as attachment receptors. We determined the structure of serotype 1 reovirus attachment protein σ1 alone and in complex with JAM-A. Comparison with the structure of serotype 3 reovirus σ1 bound to JAM-A reveals that both σ1 proteins engage JAM-A with similar affinities and via conserved binding epitopes. Thus, σ1–JAM-A interactions are unlikely to explain the differences in pathogenesis displayed by these reovirus serotypes.     

Development of Lateral-flow Immunoassay for WSSV with Polyclonal Antibodies Raised against Recombinant VP （19＋28） Fusion Protein

We have developed a sensitive and rapid lateral-flow immunoassay (LFIA) for WSSV, using colloidal gold as an indicator. The fusion protein, VP (19 28), was expressed in E. coli, purified and used to prepare polyclonal antibodies. The purified anti-VP (19 28) IgG were conjugated with colloidal gold. Unconjugated anti-VP (19 28) IgG and goat anti-rabbit IgG were immobilized on nitrocellulose membranes. After assembly, three groups (5 individual animals in each group) of shrimp samples were tested which included healthy, moribund and dead shrimps. For each group, three different tissues (body juices, gills and hepatopancreas) were tested at the same time. In parallel, all the samples were also analyzed using PCR for comparison. Out of 45 samples tested, 30 were detected as positive while 15 were classified as negative. The results of LFIA correlate with those obtained by the PCR analysis, indicating that these two detection methods have the same efficacy in the limited number of samples tested in this preliminary study.     

Monoclonal Antibodies against Aβ42 Fibrils Distinguish Multiple Aggregation State Polymorphisms in Vitro and in Alzheimer Disease Brain

Amyloidogenic proteins generally form intermolecularly hydrogen-bonded β-sheet aggregates, including parallel, in-register β-sheets (recognized by antiserum OC) or antiparallel β-sheets, β-solenoids, β-barrels, and β-cylindrins (recognized by antiserum A11). Although these groups share many common properties, some amyloid sequences have been reported to form polymorphic structural variants or strains. We investigated the humoral immune response to Aβ42 fibrils and produced 23 OC-type monoclonal antibodies recognizing distinct epitopes differentially associated with polymorphic structural variants. These mOC antibodies define at least 18 different immunological profiles represented in aggregates of amyloid-β (Aβ). All of the antibodies strongly prefer amyloid aggregates over monomer, indicating that they recognize conformational epitopes. Most of the antibodies react with N-terminal linear segments of Aβ, although many recognize a discontinuous epitope consisting of an N-terminal domain and a central domain. Several of the antibodies that recognize linear Aβ segments also react with fibrils formed from unrelated amyloid sequences, indicating that reactivity with linear segments of Aβ does not mean the antibody is sequence-specific. The antibodies display strikingly different patterns of immunoreactivity in Alzheimer disease and transgenic mouse brain and identify spatially and temporally unique amyloid deposits. Our results indicate that the immune response to Aβ42 fibrils is diverse and reflects the structural polymorphisms in fibrillar amyloid structures. These polymorphisms may contribute to differences in toxicity and consequent effects on pathological processes. Thus, a single therapeutic monoclonal antibody may not be able to target all of the pathological aggregates necessary to make an impact on the overall disease process.     

Development of a Cell-Based Assay Measuring the Activation of FcγRIIa for the Characterization of Therapeutic Monoclonal Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.     

A Novel Universal Neutralizing Monoclonal Antibody against Enterovirus 71 That Targets the Highly Conserved “Knob” Region of VP3 Protein

Hand, foot and mouth disease caused by enterovirus 71(EV71) leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4) were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the “knob” region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.     

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Background

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique.

Methodology/Principal Findings

After transplantation with CD34⁺CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2^−/−IL-2Rγc^−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19⁺CD27⁺ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively.

Conclusion/Significance

This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.     

Modern Approaches to Analysis of Protein–Ligand Interactions

Biophysics - Abstract—The capabilities of computer analysis of the conformational properties of proteins and the nature of their interactions with ligands are rapidly growing due to the...