Production and characterization of the monoclonal antibodies to <Emphasis Type="Italic">Bacillus anthracis</Emphasis> protective antigen

Anthrax, a cosmopolitan acute infectious disease affecting animals and humans, is a category A bioterrorism threat agent because of the high resistance of Bacillus anthracis spores to adverse environmental factors and their easy production. In this work, we have obtained a representative panel of 20 monoclonal antibodies to the anthrax protective antigen, a key component of the pathogenic exotoxin. A sandwich ELISA for quantification of the protective antigen using these antibodies was developed. Six pairs of monoclonal antibodies are able to detect the protective antigen in blood serum with a detection limit of 1 ng/ml.     

Incorporation of the protective antigen of Brucella abortus into liposomes and the immunogenic properties of the antigen-containing liposomes

The incorporation of B. abortus protective antigen into liposomes and its localization in liposomes have been found to depend on the lipid composition of liposomes. After the injection of the protective antigen conjugated with negatively charged liposomes humoral response is more pronounced than after the injection of the protective antigen incorporated into neutral liposomes. The immunization of guinea pigs with antigen-containing liposomes ensures the production of "incomplete antibodies" in the animals in high titers.     

Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin

A less than adequate therapeutic plan for the treatment of anthrax in the 2001 bioterrorism attacks has highlighted the importance of developing alternative or complementary therapeutic approaches for biothreat agents. In these regards passive immunization possesses several important advantages over active vaccination and the use of antibiotics, as it can provide immediate protection against Bacillus anthracis. Herein, we report the selection and characterization of several human monoclonal neutralizing antibodies against the toxin of B. anthracis from a phage displayed human scFv library. In total 15 clones were selected with distinct sequences and high specificity to protective antigen and thus were the subject of a series of both biophysical and cell-based cytotoxicity assays. From this panel of antibodies a set of neutralizing antibodies were identified, of which clone A8 recognizes the lethal (and/or edema) factor binding domain, and clones F1, G11, and G12 recognize the cellular receptor binding domain found within the protective antigen. It was noted that all clones distinguish a conformational epitope existing on the protective antigen; this steric relationship was uncovered using a sequential epitope mapping approach. For each neutralizing antibody, the kinetic constants were determined by surface plasmon resonance, while the potency of protection was established using a two-tier macrophage cytotoxicity assay. Among the neutralizing antibodies identified, clone F1 possessed the highest affinity to protective antigen, and provided superior protection from lethal toxin in the cell cytotoxicity assay. The data presented provide the ever-growing arsenal of immunological and functional analysis of monoclonal antibodies to the exotoxins of anthrax. In addition it grants new candidates for the prophylaxis and therapeutic treatment against this toxin.     

Schistosoma mansoni: antigenic community between schistosomula surface and adult worm incubation products as a support for concomitant immunity

The use of protective monoclonal antibodies has enabled us to demonstrate antigenic community between a 38-kDa schistosomula surface molecule and a 115-kDa component derived from adult worms. Injection of adult worms in rats also led to the production of antibodies specific for the 38-kDa antigen, suggesting that the 115-kDa adult worm molecule could act as an inducer of the protective immune response raised against young invading parasites.     

Monoclonal IgM antibody protection in mice against infection with an encapsulated strain of Staphylococcus epidermidis.

Passive protective activities of three different classes of monoclonal antibodies in mice against challenge with strain ATCC 31432 (capsular type I) of Staphylococcus epidermidis were examined. Monoclonal IgM antibody passively protected mice against challenge with the homologous strain, whereas monoclonal IgG1 and IgG2b antibodies did not. The protective activity of IgM was absorbed by the cell surface antigen extracted from the homologous strain but not by the antigen from heterologous strains. Rapid reduction of viable cells took place in the peritoneal cavity of mice immunized with monoclonal IgM as early as 6 h after the challenge with the homologous strain. An enzyme-linked immunosorbent inhibition assay showed there was remarkable inhibition with the homologous cell surface antigen but not with heterologous preparations from other strains. Results suggest that in the mouse the major passive protection against the S. epidermidis strain is provided by the IgM antibody to the cell surface antigen.     

Monoclonal antibodies to B.anthracis protective antigen are capable to neutralize and to enhance the anthrax lethal toxin action in vitro

Anthrax belongs to highly dangerous infections of man and animals. No effective treatment methods for pulmonary types of the disease have been yet developed. The existing anthrax vaccines were designed decades ago and need improvement to fit the large-scale vaccination of population. At the same time, the immunological properties of the anthrax vaccine main component, i.e. of the protective agent, have been poorly studied. We obtained, within the present case study, a panel of mouse monoclonal antibodies to the protective agent and investigated the properties of the highest-affine panel representatives. An unusual phenomenon was detected, which is related with enhancement of the anthrax toxin action on the mouse macrophage-like cell-line in presence of the 1F2 monoclonal antibody. The remaining analyzed antibodies, i.e. 6G8 and 6G7, were found to neutralize effectively the toxin action. The enhancing and neutralizing antibodies were proven to be specific to different domains of the protective antigen and to recognize epitopes in its composition. The antibody-mediated enhancement of the anthrax lethal action is a convincing argument for further development of a new-generation anthrax vaccine. Definition of the linear antigen determinants for neutralizing antibodies in the protective antigens is an important step in the development of the next-generation anthrax vaccine.     

Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli

We describe facile isolation of full-length IgG antibodies from combinatorial libraries expressed in E. coli. Full-length heavy and light chains are secreted into the periplasm, where they assemble into aglycosylated IgGs that are captured by an Fc-binding protein that is tethered to the inner membrane. After permeabilizing the outer membrane, spheroplast clones expressing so-called E-clonal antibodies, which specifically recognize fluorescently labeled antigen, are selected using flow cytometry. Screening of a library constructed from an immunized animal yielded several antibodies with nanomolar affinities toward the protective antigen of Bacillus anthracis.     

Prevalence of a Vibrio cholerae 18-kDa antigen in vibrios

Abstract An 18-kDa protein that occurs in Vibrio cholerae has been described as an in vivo and low-iron regulated outer membrane antigen. Monoclonal antibodies which recognized this antigen were protective as passive vaccines in the infant rabbit model of cholera disease. In this study, those monoclonal antibodies were used in three immunological assays for surveillance of various bacteria for the 18-kDa antigen. ELISA, and Western blot assays gave variable results with bacteria or outer membrane preparations. The biodot assay was the most sensitive test, detecting the 18-kDa antigen in 29 of 29 V. cholerae strains, independent of biotype or serotype. A few other Gram-negative bacteria and V. parahaemolyicus reacted weakly with our antibodies and antiserum.     

Immunogenicity of the recombinant Bacillus strains with cloned gene of biosynthesis of protective antigen against Bacillus anthracis

Microbe Russian Anti-Plague Research Institute, Saratov A hybrid plasmid pUB110PA-1 demonstrating stable functioning in the cells of Bacillus strains and containing the gene of biosynthesis of Bacillus anthracis protective antigen was constructed. The recombinant strains surpassing the anthrax vaccinal cultures in the secreted synthesis of the protective antigen were obtained and their immunological efficacy was assessed. A single inoculation of Guinea pigs with the dose of 5 x 107 spores of the recombinant strains imparted efficient protection against B. anthracis challenge. Immune responses were characterized by high indices of immunity and titers of antibodies to the protective antigen. In contrast to the anthrax vaccinal preparations, the gene-engineering strains imposed no residual virulence for BALB/n mice and Guinea pigs.     

Special features of immune response to the lethal toxin of Bacillus anthracis

We and other authors have recently shown that the pattern of the immune response to components of anthrax, the Bacillus anthracis lethal toxin, is complex. In addition to the neutralizing antibodies, the antitoxin antibody pool contains antibodies enhancing the toxin lethal action. We mapped the epitopes in the protective antigen that are responsible for the induction of both antibody types. In this study, we obtained new data on the cytotoxicity of the B. anthracis lethal toxin toward the J774 A.1 cell line in the presence of monoclonal antibodies to various domains of the protective antigen and the lethal factor. The role of the Fc fragment of immunoglobulins in enhancing the lethal toxin action was shown. These results may serve as a basis for the development of a new generation vaccine for anthrax.     

Epitopic and paratopically directed anti-idiotypic factors in the regulation of resistance to murine schistosomiasis mansoni

In this study we investigated aspects of targets and regulatory mechanisms of immunologically mediated resistance to schistosomiasis. The interactions of antigen, monoclonal antibodies (MAb), and anti-idiotypic antibodies were studied by using competitive inhibition ELISA, radioimmunoprecipitation, and direct-binding ELISA techniques. MAb, either protective or nonprotective against challenge with Schistosoma mansoni, recognize either discrete or shared epitopes. MAb that recognize the same specific epitope may or may not express the ability to adoptively transfer resistance to syngeneic recipients. These results suggest that the functional as well as the epitopic specificity must be considered in an evaluation of protective mechanisms. The antibodies also can be characterized by both unique and cross-reacting idiotypic determinants. In addition, a relationship between antigen and anti-idiotypic antibody activity has been demonstrated. The immunologic analogy between antigenic epitopes and anti-idiotypic antibodies has been demonstrated by the ability of these two moieties to reciprocally inhibit the recognition of paratope-associated idiotypes, expressed by the protective MAb. This anti-idiotypic activity can be demonstrated in serum of infected animals. In this study we have identified two specific epitopes related to protection, and we illustrate here the steric relationship between antigen and anti-idiotypic antibody. The presence of idiotypically directed regulatory pathways within actively infected animals suggests that the immune response can be differentially regulated at the clonal level.     

A malaria vaccine based on the polymorphic block 2 region of MSP-1 that elicits a broad serotype-spanning immune response

Polymorphic parasite antigens are known targets of protective immunity to malaria, but this antigenic variation poses challenges to vaccine development. A synthetic MSP-1 Block 2 construct, based on all polymorphic variants found in natural Plasmodium falciparum isolates has been designed, combined with the relatively conserved Block 1 sequence of MSP-1 and expressed in E.coli. The MSP-1 Hybrid antigen has been produced with high yield by fed-batch fermentation and purified without the aid of affinity tags resulting in a pure and extremely thermostable antigen preparation. MSP-1 hybrid is immunogenic in experimental animals using adjuvants suitable for human use, eliciting antibodies against epitopes from all three Block 2 serotypes. Human serum antibodies from Africans naturally exposed to malaria reacted to the MSP-1 hybrid as strongly as, or better than the same serum reactivities to individual MSP-1 Block 2 antigens, and these antibody responses showed clear associations with reduced incidence of malaria episodes. The MSP-1 hybrid is designed to induce a protective antibody response to the highly polymorphic Block 2 region of MSP-1, enhancing the repertoire of MSP-1 Block 2 antibody responses found among immune and semi-immune individuals in malaria endemic areas. The target population for such a vaccine is young children and vulnerable adults, to accelerate the acquisition of a full range of malaria protective antibodies against this polymorphic parasite antigen.     

Virus-like particles as carriers for T-cell epitopes: limited inhibition of T-cell priming by carrier-specific antibodies

Virus-like particles (VLPs) are able to induce cytotoxic T-cell responses in the absence of infection or replication. This makes VLPs promising candidates for the development of recombinant vaccines. However, VLPs are also potent inducers of B-cell responses, and it is generally assumed that such VLP-specific antibodies interfere with the induction of protective immune responses, a phenomenon summarized as carrier suppression. In this study, we investigated the impact of preexisting VLP-specific antibodies on the induction of specific cytotoxic T-cell and Th-cell responses in mice. The data show that VLP-specific antibodies did not measurably reduce antigen presentation in vitro or in vivo. Nevertheless, T-cell priming was slightly reduced by antigen-specific antibodies; however, the overall reduction was limited and vaccination with VLPs in the presence of VLP-specific antibodies still resulted in protective T-cell responses. Thus, carrier suppression is unlikely to be a limiting factor for VLP-based T-cell vaccines.     

Beta-cyclodextrin derivatives that inhibit anthrax lethal toxin

Recently, we demonstrated that simultaneous blocking of bacterial growth by antibiotics and inhibition of anthrax toxin action with antibodies against protective antigen were beneficial for the treatment of anthrax. The present study examined the hypothesis that blocking the pore formed by protective antigen can inhibit the action of anthrax toxin. The potential inhibitors were chosen by a structure-based design using beta-cyclodextrin as the starting molecule. Several beta-cyclodextrin derivatives were evaluated for their ability to protect RAW 264.7 cells from the action of anthrax lethal toxin. Per-substituted aminoalkyl derivatives displayed inhibitory activity and were protective against anthrax lethal toxin action at low micromolar concentrations. These results provide the basis for a structure-based drug discovery program, with the goal of identifying new drug candidates for anthrax treatment.     

Production and characterization of neutralizing monoclonal antibodies that recognize an epitope in domain 2 of Bacillus anthracis protective antigen

Antibodies against the protective antigen (PA) of Bacillus anthracis play a key role in response to infection by this important pathogen. The aim of this study was to produce and characterize monoclonal antibodies (mAbs) specific for PA and to identify novel neutralizing epitopes. Three murine mAbs with high specificity and nanomolar affinity for B. anthracis recombinant protective antigen (rPA) were produced and characterized. Western immunoblot analysis, coupled with epitope mapping using overlapping synthetic peptides, revealed that these mAbs recognize a linear epitope within domain 2 of rPA. Neutralization assays demonstrate that these mAbs effectively neutralize lethal toxin in vitro.     

Immunity to Plasmodium sporozoites: recent advances and applications to field research

The presence of antibodies against Plasmodium falciparum sporozoites in humans living in malaria endemic areas was measured using as antigen the synthetic peptide (NANP)3, which represents the immunodominant region of the circumsporozoite (CS) protein. The results indicate that: i) the production of anti-CS antibodies is unrelated to the presence in the circulation of blood-stage parasites; ii) anti-CS antibodies, raised by natural inoculation, could exert a protective role against natural malaria infection; iii) anti-CS antibodies can be used as indicators of the intensity of malaria transmission.     

Protection of chimpanzees from type B hepatitis by immunization with woodchuck hepatitis virus surface antigen.

Two chimpanzees immunized with woodchuck hepatitis virus (WHV) surface antigen (WHsAg) developed antibodies cross-reactive with hepatitis B virus (HBV) surface antigen (HBsAg). After challenge with HBV, one animal was completely protected and the other experienced a subclinical infection, without evidence of liver disease. Three woodchucks immunized with HBsAg developed antibodies to HBsAg which did not cross-react with WHsAg. After challenge with WHV, all three woodchucks developed typical acute infections with associated hepatic lesions. Serological studies with the cross-reactive antibodies raised in chimpanzees suggested that the protective epitopes of WHsAg were related to the group a specificity of HBsAg. These studies indicated that cross-protective epitopes are shared by HBV and WHV; however, the humoral response to these epitopes can vary among species.     

Evidence that anti-idiotype induced immunity to experimental African trypanosomiasis is genetically restricted and requires recognition of combining site-related idiotopes

The ability of anti-idiotypic (anti-Id) antibodies to immunize mice against African trypanosomiasis independent of antigen has been confirmed. Of three allogeneic anti-Id antibodies raised against three protective monoclonal antibodies, each with specificity for the variant surface antigen of a clone of Trypanosoma rhodesiense, only one (anti-7H11 Id) was effective in immunizing BALB/c mice against homologous challenge. The immunity was associated with the more rapid and enhanced expression of the corresponding Id after infection. The immunity was restricted to mice bearing genes linked to Igh-Ca, which appeared to control expression of this Id both in response to infection and anti-Id treatment. Another Id, 11D5, appeared to be under similar genetic control. Anti-11D5 Id, however, was ineffective in immunizing mice against infection despite inducing high levels of Id bearing molecules before challenge. The immunizing potential of the respective anti-Id antibodies appeared to be related to the relative concentrations of antibodies reactive with idiotopes near to or within the antigen-combining site, which, in turn, determined the relative proportion of Id-bearing clones activated that had antigen binding activity.     

Plasmodium falciparum: production of human antibodies specific for the MSP-3 protein in the Hu-SPL-SCID mouse

Human immunity against Plasmodium falciparum malaria is mediated by IgG antibodies. One of the major targets of protective antibodies is the MSP-3 protein. Anti-MSP-3 human monoclonal antibodies could therefore be valuable for passive immunotherapy, particularly of drug resistant malaria. Human monoclonal antibodies were previously produced in the Hu-SPL-SCID model reconstituted with human splenocytes, immunized by highly immunogenic neo-antigen or a recall antigen. We report here that this model can also be successfully employed to induce human antibody-secreting cells specific of low immunogenicity neo-antigens, such as MSP-3. These cells represent a new and valuable source of human monoclonal anti-malaria antibodies.     

Effectiveness of vaccination with recombinant HpaA from Helicobacter pylori is influenced by host genetic background

Several studies have explored the production and immunogenicity of HpaA as a potential protective antigen against Helicobacter pylori but little is known regarding its protective capabilities. We therefore evaluated the protective efficacy of recombinant HpaA (rHpaA) as a candidate vaccine antigen against H. pylori. To explore the impact of genetic diversity, inbred and outbred mice were prophylactically and therapeutically immunized with rHpaA adjuvanted with cholera toxin (CT). Prophylactic immunization induced a reduction in bacterial colonization in BALB/c and QS mice, but was ineffective in C57BL/6 mice, despite induction of antigen-specific antibodies. By contrast, therapeutic immunization was effective in all three strains of mice. Prophylactic immunization with CT-adjuvanted rHpaA was more effective when delivered via the nasal route than following intragastric delivery in BALB/c mice. However, HpaA-mediated protection was inferior to that induced by bacterial lysate. Hence, protective efficacy is inducible with vaccines containing HpaA, most relevantly shown in an outbred population of mice. The effectiveness of protection induced by HpaA antigen was influenced by host genetics and was less effective than lysate. HpaA therefore has potential for the development of effective immunization against H. pylori but this would probably entail the antigen to be one component of a multiantigenic vaccine.