Polymerized Flagellin Research Articles

Crosslinked flagella as a stabilized vaccine adjuvant scaffold

BackgroundEngineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant activity through its TLR5 and inflammasome activation. Moreover, polymerized flagellin filaments can elicit a more robust immunoglobulin response than monomeric flagellin, and the multimeric antigen form can also promote T cell-independent antibody responses. Here, we aim to produce and test a covalently stabilized polymerized flagellar filament, providing additional immune efficacy through stabilization of its polymeric filament structure, as well as stabilization for long-term storage.ResultsComputational modeling of monomer packing in flagellin filaments helped identify amino acids with proximity to neighboring flagella protofilaments. Paired cysteine substitutions were made at amino acids predicted to form inter-monomer disulfide cross-links, and these substitutions were capable of forming flagella when transfected into a flagellin-negative strain of Salmonella enterica subspecies Typhimurium. Interestingly, each paired substitution stabilized different helical conformational polymorphisms; the stabilized filaments lost the ability to transition between conformations, reducing bacterial motility. More importantly, the paired substitutions enabled extensive disulfide cross links and intra-filament multimer formation, and in one of the three variants, permitted filament stability in high acidic and temperature conditions where wild-type filaments would normally rapidly depolymerize. In addition, with regard to potential adjuvant activity, all crosslinked flagella filaments were able to induce wild-type levels of epithelial NF-κB in a cell reporter system. Finally, bacterial virulence was unimpaired in epithelial adherence and invasion, and the cysteine substitutions also appeared to increase bacterial resistance to oxidizing and reducing conditions.ConclusionsWe identified amino acid pairs, with cysteine substitutions, were able to form intermolecular disulfide bonds that stabilized the resulting flagellar filaments in detergent, hydrochloric acid, and high temperatures while retaining its immunostimulatory function. Flagellar filaments with disulfide-stabilized protofilaments introduce new possibilities for the application of flagella as a vaccine adjuvant. Specifically, increased stability and heat tolerance permits long-term storage in a range of temperature environments, as well as delivery under a range of clinical conditions.

Discovery of a proteolytic flagellin family in diverse bacterial phyla that assembles enzymatically active flagella

Bacterial flagella are cell locomotion and occasional adhesion organelles composed primarily of the polymeric protein flagellin, but to date have not been associated with any enzymatic function. Here, we report the bioinformatics-driven discovery of a class of enzymatic flagellins that assemble to form proteolytically active flagella. Originating by a metallopeptidase insertion into the central flagellin hypervariable region, this flagellin family has expanded to at least 74 bacterial species. In the pathogen, Clostridium haemolyticum, metallopeptidase-containing flagellin (which we termed flagellinolysin) is the second most abundant protein in the flagella and is localized to the extracellular flagellar surface. Purified flagellar filaments and recombinant flagellin exhibit proteolytic activity, cleaving nearly 1000 different peptides. With ~ 20,000 flagellin copies per ~ 10-μm flagella this assembles the largest proteolytic complex known. Flagellum-mediated extracellular proteolysis expands our understanding of the functional plasticity of bacterial flagella, revealing this family as enzymatic biopolymers that mediate interactions with diverse peptide substrates.

High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication.

Pseudomonas aeruginosa as an opportunistic pathogen is a significant cause of acute and chronic infections in patients with compromised defenses. This bacterium is motile via a single polar flagellum made of polymerized flagellin subunits differentiated into two major serotypes: A and B. flagellin plays an important role as a virulence factor in the adhesion, colonization and invasion of P. aeruginosa into host epithelial cells. To develop a functional vaccine that can be used in practical application to prevent and treat infection, type B-flagellin was produced as recombinant protein. In this work, the fliC gene was introduced into a pET28a vector and expressed in Escherichia coli BL21 (DE3). The expressed recombinant protein was purified by a modified method without sonication using a HisTrap affinity column. The functional activities of produced flagellin were confirmed by ELISA, western blot analysis, motility inhibition assay and opsonophagocytosis test. The purification process of the type B-flagellin was lead to a high yield. The produced recombinant type B-flagellin showed high biological activity in all of these standard assays. In conclusions, this report provides the new protocol to efficiently obtain the type B-flagellin with high biological activity and immunogenicity. This immunogen can be introduced as an adjuvant or vaccine in the future study.

New possibility for providing protection against urinary tract infection caused by Pseudomonas aeruginosa by non-adjuvanted flagellin ‘b’ induced immunity

In the present study we demonstrated a novel protective role of Pseudomonas aeruginosa PAO1 flagellin ‘b’ in prevention of urinary tract infection (UTI). P. aeruginosa is motile via single polar flagellum made up of polymerized flagellin proteins. It is a serious nosocomial pathogen causing UTIs. Predisposing factors include instrumentation and catheterization which enhance colonization with P. aeruginosa, leading to ascending infection. Hence for a newer, safer and effective approach, the present study focussed on the prophylaxis using bacterial flagellin, isolated and purified (PCR sequencing and MALDI-TOF) from P. aeruginosa PAO1 strain, which triggers immune response [both non-specific and specific (active and passive)] as defense against infection. Administration of flagellin ‘b’ via intraperitoneal route enhanced the clearance of homologous as well as heterologous bacteria (P. aeruginosa uroisolate carrying flagellin ‘a’) in renal tissue, decreased the levels of malondialdehyde (MDA) and reverted structural integrity of renal tissue to near normal in female LACA mice. Immunization suppressed the production of pro-inflammatory cytokines [interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α)] and activated humoral immune response. Anti-flagellin antibodies (quantified by ELISA) helped in the clearance of bacterial load by opsonophagocytosis. Adoptive transfer of antisera also protected mice from PAO1 challenge, indicating protective role of antibodies. In conclusion, this is the first report that describes flagellin as a potential prophylactic agent which downregulates inflammation and curbs UTIs.

Pseudomonas evades immune recognition of flagellin in both mammals and plants.

The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.

Evaluation of Flagella and Flagellin of Pseudomonas aeruginosa as Vaccines

Pseudomonas aeruginosa is a serious pathogen in hospitalized, immunocompromised, and cystic fibrosis (CF) patients. P. aeruginosa is motile via a single polar flagellum made of polymerized flagellin proteins differentiated into two major serotypes: a and b. Antibodies to flagella delay onset of infection in CF patients, but whether immunity to polymeric flagella and that to monomeric flagellin are comparable has not been addressed, nor has the question of whether such antibodies might negatively impact Toll-like receptor 5 (TLR5) activation, an important component of innate immunity to P. aeruginosa. We compared immunization with flagella and that with flagellin for in vitro effects on motility, opsonic killing, and protective efficacy using a mouse pneumonia model. Antibodies to flagella were superior to antibodies to flagellin at inhibiting motility, promoting opsonic killing, and mediating protection against P. aeruginosa pneumonia in mice. Protection against the flagellar type strains PAK and PA01 was maximal, but it was only marginal against motile clinical isolates from flagellum-immunized CF patients who nonetheless became colonized with P. aeruginosa. Purified flagellin was a more potent activator of TLR5 than were flagella and also elicited higher TLR5-neutralizing antibodies than did immunization with flagella. Antibody to type a but not type b flagella or flagellin inhibited TLR5 activation by whole bacterial cells. Overall, intact flagella appear to be superior for generating immunity to P. aeruginosa, and flagellin monomers might induce antibodies capable of neutralizing innate immunity due to TLR5 activation, but solid immunity to P. aeruginosa based on flagellar antigens may require additional components beyond type a and type b proteins from prototype strains.

Salmonella flagellins are potent adjuvants for intranasally administered whole inactivated influenza vaccine

Bacterial flagellins are potent inducers of innate immune responses in the mouse lung because they bind to TLR5 expressed on the apical surfaces of airway epithelial cells. TLR engagement leads to the initiation of a signaling cascade that results in a pro-inflammatory response with subsequent up-regulation of several cytokines and leads to adaptive immune responses. We examined the ability of two soluble flagellins, a monomeric flagellin expressed in Escherichia coli and a highly purified polymeric flagellin directly isolated from Salmonella, to enhance the efficacy of influenza vaccines in mice. Here we demonstrate that both flagellins co-administered intranasally with inactivated A/PR/8/34 (PR8) virus induced robust increases of systemic influenza-specific IgA and IgG titers and resulted in a more comprehensive humoral response as indicated by the increase of IgG2a and IgG2b subclass responses. Groups immunized with the adjuvanted vaccines were fully protected against high dose lethal challenge by homologous virus whereas inactivated PR8 alone conferred only partial protection. Finally we show that shortly after immunization the adjuvanted vaccines induced a dramatic increase in pro-inflammatory cytokines in the lung, resulting in extensive lung infiltration by granulocytes and monocytes/macrophages. Our results reveal a promising perspective for the use of both soluble monomeric and polymeric flagellin as mucosal vaccine adjuvants to improve protection against influenza epidemics as well as a range of other infectious diseases.

Activation of NF-κB-dependent gene expression by Salmonella flagellins FliC and FljB

Bacterial flagellin is recognized by Toll-like receptor (TLR5) and activates NF-κB which leads to the induction of proinflammatory gene expression. Salmonella expresses two flagellin proteins, FliC and FljB. We purified FliC and FljB and examined the ability of the Salmonella flagellins to activate the NF-κB transcription factor in human embryonic kidney cells. We found that FliC and FljB as purified proteins possessed a comparable specific activity for activation of NF-κB-dependent gene expression in HEK293 cells. We also determined the ability of UV-inactivated bacteria, both wild-type and fliC and fljB mutant strains, to activate NF-κB. Wild-type fliC +/ fljB + Salmonella and the fliC +/ fljB − mutant strain were robust activators, whereas the fliC −/ fljB + and flhC − mutant strains were very poor activators. The NF-κB activation capacity of bacterial strains correlated with their flagellin expression level. Finally, Salmonella cell wall-associated polymeric flagellin displayed greatly reduced ability to activate NF-κB compared to purified monomeric flagellin.

GIV004 CagPAI of Helicobacter pylori is important for gastric mucosa differentiation towards ulcer and metaplasia in Mongolian Gerbils

Bacterial flagellins are potent inducers of innate immune responses in the mouse lung because they bind to TLR5 expressed on the apical surfaces of airway epithelial cells. TLR engagement leads to the initiation of a signaling cascade that results in a pro-inflammatory response with subsequent up-regulation of several cytokines and leads to adaptive immune responses. We examined the ability of two soluble flagellins, a monomeric flagellin expressed in Escherichia coli and a highly purified polymeric flagellin directly isolated from Salmonella, to enhance the efficacy of influenza vaccines in mice. Here we demonstrate that both flagellins co-administered intranasally with inactivated A/PR/8/34 (PR8) virus induced robust increases of systemic influenza-specific IgA and IgG titers and resulted in a more comprehensive humoral response as indicated by the increase of IgG2a and IgG2b subclass responses. Groups immunized with the adjuvanted vaccines were fully protected against high dose lethal challenge by homologous virus whereas inactivated PR8 alone conferred only partial protection. Finally we show that shortly after immunization the adjuvanted vaccines induced a dramatic increase in pro-inflammatory cytokines in the lung, resulting in extensive lung infiltration by granulocytes and monocytes/macrophages. Our results reveal a promising perspective for the use of both soluble monomeric and polymeric flagellin as mucosal vaccine adjuvants to improve protection against influenza epidemics as well as a range of other infectious diseases.

Triamcinolone acetonide inhibits lymphocyte differentiation in B cells decorated with artificial antigen receptors

We examined the effects of triamcinolone acetonide (TA) on T cell independent antigen-induced differentiation of human B cells. Purified human B cells artificially decorated with palmitate-conjugated monoclonal IgA antibody specific for 2,4-dinitrophenyl differentiated polyclonally when challenged with optimum concentrations of dinitrophenyl-derivatized polymerized flagellin. This B cell response was reduced by the synthetic corticosteroid TA at a concentration of 10(-6) M. This suggests that TA can inhibit in vitro B lymphocyte differentiation independent of T cells.

Antigen activation of human B lymphocytes bearing artificial antigen receptors

When highly purified human and murine B cells are challenged in vitro with certain so called "T cell-independent" activators such as the polyclonal B cell activator lipopolysaccharide (LPS) or the clonally specific B cell activator dinitrophenyl-conjugated polymerized flagellin (DNP-POL), mouse, but not human, cells differentiate into immunoglobulin-secreting cells. However, results from this study show that DNP-POL can cause human B cell differentiation in a T cell-independent manner when the antigen is concentrated onto the cells via artificially incorporated palmitate-modified anti-DNP mouse IgA molecules. This response is comparable in magnitude to that induced by a T cell-dependent polyclonal B cell activator, pokeweed mitogen, in unfractionated mononuclear cell cultures, suggesting that DNP-POL induced polyclonal B cell differentiation. DNP-POL binding to the artificial receptor molecules on B cells did not cause cellular proliferation, even in unfractionated mononuclear cell populations. These results are similar to those obtained in previous studies using mouse B cells in which the artificial receptor was unable to act as a transmembrane signaling element. From these studies, we conclude that B cells express clonally unrestricted, presumably low-avidity, endogenous receptor for POL, and that signaling through this receptor activates B cell differentiation but not cell proliferation.

Termini of Salmonella flagellin are disordered and become organized upon polymerization into flagellar filament

The terminal regions of Salmonella flagellin are essential for polymerization to form the flagellar filament. It has recently been suggested, on the basis of results from circular dichroism spectroscopy and scanning calorimetry, that these regions are disordered in solution. We report here direct evidence for disorder and mobility in the terminal regions of flagellin using 400 MHz proton nuclear magnetic resonance (n.m.r.) spectroscopy. Comparison of the n.m.r. spectra of monomeric and polymeric flagellin shows that the terminal regions become organized when polymerized to form the filament.

Lateral diffusion of antigen receptors artificially incorporated onto B lymphocytes.

In the companion paper, we have shown that palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) incorporated onto B lymphocytes can bind DNP antigens and that this binding causes polyclonal B cell activation. In this study we use fluorescence photobleaching recovery (FPR) techniques to examine the lateral diffusion and mobile fractions of antigen-receptor complexes on receptor-decorated B cells as functions of antigen concentration and epitope density. Antigens used in this study are DNP conjugates of polymerized flagellin (DNP-POL) and linear dextran of 2 X 10(6) m.w. (DNP-DEX). The diffusion coefficient observed for antigen bound to artificial receptors decreases monotonically with increased antigen dose and epitope density. When the artificial receptor-bearing cells are labeled with either relatively high concentrations of medium epitope density antigen or high epitope density antigen, a large fraction of antigen-receptor complexes become immobile in the time scale of the experiment. We attribute this behavior to extensive receptor cross-linking by antigen. In parallel with these FPR experiments, we examined the effects of antigen concentration and epitope density on the polyclonal humoral response of receptor-decorated B cells. We found that the response is a function of both antigen concentration and epitope density similar to that seen in natural B cells. The combined results of these experiments show that cell activation results when the diffusion coefficient of the antigen-receptor complex ranges between 10 X 10(-11) cm2 sec-1 and 5 X 10(-11) cm2 sec-1. These values represent threefold and sixfold decreases from the diffusion coefficient of antigen-free receptors, respectively. However, when either a high antigen concentration or epitope density causes a large fraction of antigen-receptor complexes to become immobile, B cells become unresponsive not only to the bound antigen, but also to LPS. Results obtained in this study are very similar to those obtained in a study performed with natural antigen-specific B cells. Therefore, for the responding population of receptor-decorated B cells, it is possible that antigens activate and paralyze these B cells by mechanisms similar to those by which antigens regulate normal B cell responses.

Biologic activity of antigen receptors artificially incorporated onto B lymphocytes.

We describe a method for incorporating monoclonal antibody molecules onto viable murine lymphocytes and summarize the biologic activity of these artificial receptors on B cells. Mouse spleen cells incubated overnight with palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) in the presence of deoxycholic acid incorporate about 50,000 antibody molecules per cell. When concentrations of deoxycholate and palmitoyl-protein 315 are carefully controlled, this labeling procedure does not affect the viability or the normal functions of the receptor-decorated cells. The incorporated antibody specifically binds DNP-antigens, although it appears to be unable to communicate directly with internal cellular components. Yet when these receptor-decorated, unprimed cells are challenged with any one of several DNP-antigens, up to 42,000 per 10(6) B cells differentiate into Ig-secreting cells. This response is about 23-fold greater than that induced in normal cell cultures and is of the same magnitude as that induced by the polyclonal B cell activator LPS. This, in addition to the observation that only about 3.6% of receptor-decorated B cells responding to DNP-conjugated polymerized flagellin (DNP-POL) produce hapten-specific antibody, demonstrates that these antigens cause polyclonal B cell differentiation. Normal spleen cells in the presence of DNP-POL and irradiated spleen cells bearing the artificial receptors do not exhibit the polyclonal antibody response. Also, the response of receptor-decorated B cell is blocked by high but nontoxic concentrations of the nonimmunogenic hapten DNP-lysine. These observations demonstrate that the polyclonal B cell response in this system requires the binding of antigen to artificial receptors on functionally viable cells. The polyclonal B cell response to a thymus-dependent antigen DNP-conjugated bovine gamma-globulin (DNP-BGG) requires the presence of the carrier-primed T cells. On the other hand, T cell depletion by anti-Thy-1.2 monoclonal antibody and complement causes only a slight reduction in the number of receptor-decorated B cells that respond to the relatively thymus-independent antigen DNP-POL. This type of phenomenon is also seen with natural antigen-specific B cells. Thus, polyclonal activation of receptor-decorated B cells exhibits the same gross helper cell requirements as antigenic activation of natural antigen-specific B cells. The results of this study are discussed in the context of the role of membrane-bound surface Ig in antigen-dependent B cell activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of potent Salmonella H antisera by immunization with polymeric flagellins

Highly purified polymeric flagellin preparations from 10 different Salmonella serotypes were used to produce specific Salmonella H antisera with high titers by the immunization of rabbits. Antigen emulsions in complete Freund adjuvant were administered at the rate of 50 micrograms per rabbit by multiple intradermal injection. Booster injections were given 110 days after the primary immunization. The immune response was monitored regularly over a period of 200 days. The results showed that the H titers, determined with 125I-labeled antigens, averaged 61,000 +/- 39,000, and the H agglutination titers of 83% of the animals were greater than 40,000. The high titers of the immunized animals persisted for approximately 4 months. The O agglutination titers of the antisera were less than 10 for 9 of the flagellin preparations and ranged from 10 to 320 for the remaining preparation. The antisera obtained were serotype specific after appropriate dilution.

Immunological relationships betweenSalmonella flagellins and between these and flagellins from other species of Enterobacteriaceae

Ten antisera raised in rabbits, against polymeric flagellins from ten different Salmonella serotypes were used to determine the relative cross-activities between salmonella flagellins in monomeric form. The results showed a high degree of cross-reactivity between the antisera (IgG antibodies) and all monomeric flagellins investigated. Consequently, it was possible to detect the ten Salmonella serotypes, after heat depolymerization of flagella using radioimmunometric assay with only one antiserum raised against polymeric flagellin from one serotype. The results also showed that native flagella differed antigenically from repolymerized and monomeric flagellins. This is possibly due to changes in the tertiary structure of sub-units of flagella when depolymerized with acid or heat resulting in unfolding and unmasking of common antigenic determinants. The unfolded and unmasked antigenic determinants were not only common to the Salmonella serotypes investigated, but also to other members of Enterobacteriaceae.

Stimulation of human blood lymphocyte by different polyclonal B cell activators of bacterial and plant origin: production of IgM, IgG and IgA estimated by the ELISA method.

Lymphocytes isolated from peripheral blood of healthy donors were stimulated in vitro with pokeweed mitogen, concanavalin A, flagellin, Nocardia delipidated cell mitogen (NDCM) and heat-killed bacteria Escherichia coli and Actinomyces viscosus. A simple and sensitive technique, enzyme-linked immunosorbent assay (ELISA) was used for the detection of nanogram levels of IgM, IgA and IgC in media from lymphocyte cultures after polyclonal stimulation, Pokeweed mitogen, NDCM and E. coli were shown to stimulate a high production of IgM; after stimulation with A. viscosus a higher production of IgA was detected. No immunoglobulin production was observed after stimulation with polymerized flagellin.

Human interleukin 2 can promote the growth and differentiation of single hapten-specific B cells in the presence of specific antigen.

B lymphocytes specifically reactive to the hapten fluorescein (FLU) were prepared from normal adult murine spleen by the hapten-gelatin affinity procedure. They were placed in 10 microliter of microcultures singly or in small numbers in the absence of any feeder, filler, or accessory cell. The "T cell-independent" antigen FLU-conjugated polymerized flagellin (FLU-POL) or a selected batch of FLU-conjugated Ficoll were used, and these stimulated division and differentiation only in the concomitant presence of lymphokines acting as B-cell growth and differentiation factors (BGDF). It was found that human interleukin 2 (IL-2), prepared by recombinant DNA technology (r-IL-2), was an effective, albeit rather weak, BGDF in this system. When an IL-2-free source of BGDF was used with the antigenic stimulus, addition of r-IL-2 did not augment the response, nor did removal of IL-2 from the crude lymphokine mixture diminish the BGDF activity.

Heterogeneity of Macrophages and Dendritic Cells as Accessory Cells

A number of different antigens was used to define the functional limits of Ia-bearing murine dendritic cells and macrophages in the processing and presentation of antigens for T cell activation. The results show considerable functional overlap as well as differences attributable to known properties of the cells. Thus both cell types could present soluble antigens up to the size of polymeric flagellin (M.W. in millions) about equally well. The nonphagocytic dendritic cells were most effective at inducing mixed leukocyte reactions in accordance with their high constitutive level of Ia expression. On the other hand, splenic macrophages were three to nine times better than dendritic cells at presenting particulate, heat-killed Corynebacterium parvum organisms to T cells lines, and small activated macrophages from bone marrow cultures were three times better again than splenic macrophages. Large activated bone marrow macrophages were not effective antigen presenters probably because of nonspecific suppression. These observations are consistent with the phagocytic and lysosomal activities of macrophages that enable them to ingest and process particulate antigen efficiently. Nevertheless, the capacity of dendritic cells and the dendritic-like line, P388.AD.4, to present particulate bacterial antigens suggests that these cells could either do the processing extracellularly or pick up soluble antigenic moieties shed from the bacteria and antigen processing macrophages. Glutaraldehyde fixation of C. parvum presumably stopped antigen shedding, since it produced a greater reduction of the T cell response with dendritic cells and P388.Ad.4 as presenting cells than with macrophage presenters. Alternatively, the fixation could make the bacteria less “digestible” to dendritic cells than to macrophages. More characterization of the fate of antigens following encounter with accessory cells is necessary to distinguish between these possibilities.

Thymus-independent immunogenicity in vitro of the divalent antigen DNP-polyethylene oxide

Chemically simple and physically well-defined dinitrophenyl derivatives of polyethylene oxide (DNP-PEO) can be prepared in a wide range of forms and sizes. These materials were used to investigate the molecular basis of immunogenicity and the binding of the antigens to membranebound receptors. Both di- and multivalent DNP-PEO activate normal murine B lymphocytes to yield primary anti-DNP antibody response in vitro. The immunogenicity is dependent on the carrier chain length but independent of T cells. Responses comparable to those induced by DNP-conjugated polymerized flagellin are induced by divalent linear materials of medium molecular weights of about 60,000. A highly multivalent material is moderately immunogenic, but at much lower antigen doses than divalent materials. The carrier PEO does not affect B-cell responses to DNP-PEO or T-cell response to succinyl concanavalin A. Moreover, it shows no polyclonal mitogenicity at concentrations as high as 1 mg/ml. Studies of antigen binding to cell surface DNP receptors show that the strongly immunogenic materials of medium molecular weights have an appreciable tendency to bind bivalently and thus potentially to crosslink receptors. The binding of smaller, less immunogenic antigen appears predominantly monovalent.