CD1b presents self and Borrelia burgdorferi diacylglycerols to human T cells.

Lyme disease is a common multisystem disease caused by infection with a tick-transmitted spirochete, Borrelia burgdorferi and related Borrelia species. The monoglycosylated diacylglycerol known as B. burgdorferi glycolipid II (BbGL-II) is a major target of antibodies in sera from infected individuals. Here, we show that CD1b presents BbGL-II to human T cells and that the TCR mediates the recognition. However, we did not detect increased frequency of CD1b-BbGL-II binding T cells in the peripheral blood of Lyme disease patients compared to controls. Unexpectedly, mapping the T cell specificity for BbGL-II-like molecules using tetramers and activation assays revealed a concomitant response to CD1b-expressing APCs in absence of BbGL-II. Further, among all major classes of self-lipid tested, BbGL-II responsive TCRs show strong cross-reactivity to diacylglycerol, a self-lipid antigen with structural similarities to BbGL-II. Extending prior work on MHC and CD1b, CD1c, and CD1d proteins, this study provides evidence for cross-reactive CD1b-restricted T cell responses to bacterial and self-antigens, and identifies chemically defined targets for future discovery of self and foreign antigen cross-reactive T cells.

A T-cell receptor escape channel allows broad T-cell response to CD1b and membrane phospholipids.

CD1 proteins are expressed on dendritic cells, where they display lipid antigens to T-cell receptors (TCRs). Here we describe T-cell autoreactivity towards ubiquitous human membrane phospholipids presented by CD1b. These T-cells discriminate between two major types of lipids, sphingolipids and phospholipids, but were broadly cross-reactive towards diverse phospholipids including phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine. The crystal structure of a representative TCR bound to CD1b-phosphatidylcholine provides a molecular mechanism for this promiscuous recognition. We observe a lateral escape channel in the TCR, which shunted phospholipid head groups sideways along the CD1b-TCR interface, without contacting the TCR. Instead the TCR recognition site involved the neck region phosphate that is common to all major self-phospholipids but absent in sphingolipids. Whereas prior studies have focused on foreign lipids or rare self-lipids, we define a new molecular mechanism of promiscuous recognition of common self-phospholipids including those that are known targets in human autoimmune disease.

Vaccination with Shigella flexneri 2a conjugate induces type 2a and cross-reactive type 6 antibodies in humans but not in mice.

Shigella flexneri (S. flexneri) 6 has emerged as an important cause of shigellosis. Our efficacy study of Shigella sonnei and S. flexneri 2a O-specific polysaccharide (O-SP) conjugates in 1-4year-olds had too few S. flexneri 2a cases for efficacy evaluation but surprisingly showed protection of 3-4year-olds, S. flexneri 2a-recipients, from S. flexneri 6 infection. To investigate this cross-protection antibodies to both Shigella types were investigated in all sera remaining from previous studies. Twenty to 30% of 3-44year-old humans injected with S. flexneri 2a conjugate responded with ≥4-fold increases of IgG anti type 6, p<0.00001. The specificity of these antibodies was shown by inhibition studies. S. flexneri 6 infection of 2 children induced besides S. flexneri 6, also S. flexneri 2a antibodies, at levels of S. flexneri 2a vaccinees. S. flexneri 2a antibodies induced by S. flexneri 6 conjugates could not be studied since no such conjugate was assessed in humans and mice responded almost exclusively to the O-SP of the injected conjugate, with no cross-reactive antibodies. Our results indicate induction of cross-reactive protective antibodies. The O-acetylated disaccharide shared by S. flexneri 6 and 2a O-SPs, is the likely basis for their cross-reactivity. S. flexneri 6 O-SP conjugates, alone and in combination with S. flexneri 2a, merit further investigation for broad S. flexneri protection.

Bacillus anthracis Capsular Conjugates Elicit Chimpanzee Polyclonal Antibodies That Protect Mice from Pulmonary Anthrax.

The immunogenicity of Bacillus anthracis capsule (poly-γ-D-glutamic acid [PGA]) conjugated to recombinant B. anthracis protective antigen (rPA) or to tetanus toxoid (TT) was evaluated in two anthrax-naive juvenile chimpanzees. In a previous study of these conjugates, highly protective monoclonal antibodies (MAbs) against PGA were generated. This study examines the polyclonal antibody response of the same animals. Preimmune antibodies to PGA with titers of >10(3) were detected in the chimpanzees. The maximal titer of anti-PGA was induced within 1 to 2 weeks following the 1st immunization, with no booster effects following the 2nd and 3rd immunizations. Thus, the anti-PGA response in the chimpanzees resembled a secondary immune response. Screening of sera from nine unimmunized chimpanzees and six humans revealed antibodies to PGA in all samples, with an average titer of 10(3). An anti-PA response was also observed following immunization with PGA-rPA conjugate, similar to that seen following immunization with rPA alone. However, in contrast to anti-PGA, preimmune anti-PA antibody titers and those following the 1st immunization were ≤300, with the antibodies peaking above 10(4) following the 2nd immunization. The polyclonal anti-PGA shared the MAb 11D epitope and, similar to the MAbs, exerted opsonophagocytic killing of B. anthracis. Most important, the PGA-TT-induced antibodies protected mice from a lethal challenge with virulent B. anthracis spores. Our data support the use of PGA conjugates, especially PGA-rPA targeting both toxin and capsule, as expanded-spectrum anthrax vaccines.

Toward a new vaccine for pertussis.

To overcome the limitations of the current pertussis vaccines, those of limited duration of action and failure to induce direct killing of Bordetella pertussis, a synthetic scheme was devised for preparing a conjugate vaccine composed of the Bordetella bronchiseptica core oligosaccharide with one terminal trisaccharide to aminooxylated BSA via their terminal ketodeoxyoctanate residues. Conjugate-induced antibodies, by a fraction of an estimated human dose injected into young outbred mice as a saline solution, were bactericidal against B. pertussis, and their titers correlated with their ELISA values. The carrier protein is planned to be genetically altered pertussis toxoid. Such conjugates are easy to prepare, stable, and should add both to the level and duration of immunity induced by current vaccine-induced pertussis antibodies and reduce the circulation of B. pertussis.

Bacillus anthracis cell wall peptidoglycan but not lethal or edema toxins produces changes consistent with disseminated intravascular coagulation in a rat model.

BACKGROUND: Disseminated intravascular coagulation (DIC) appears to be important in the pathogenesis of Bacillus anthracis infection, but its causes are unclear. Although lethal toxin (LT) and edema toxin (ET) could contribute, B. anthracis cell wall peptidoglycan (PGN), not the toxins, stimulates inflammatory responses associated with DIC. METHODS AND RESULTS: To better understand the pathogenesis of DIC during anthrax, we compared the effects of 24-hour infusions of PGN, LT, ET, or diluent (control) on coagulation measures 6, 24, or 48 hours after infusion initiation in 135 rats. No control recipient died. Lethality rates (approximately 30%) did not differ among PGN, LT, and ET recipients (P = .78). Thirty-three of 35 deaths (94%) occurred between 6 and 24 hours after the start of challenge. Among challenge components, PGN most consistently altered coagulation measures. Compared with control at 6 hours, PGN decreased platelet and fibrinogen levels and increased prothrombin and activated partial thromboplastin times and tissue factor, tissue factor pathway inhibitor, protein C, plasminogen activator inhibitor (PAI), and thrombin-antithrombin complex levels, whereas LT and ET only decreased the fibrinogen level or increased the PAI level (P ≤ .05). Nearly all effects associated with PGN infusion significantly differed from changes associated with toxin infusion (P ≤ .05 for all comparisons except for PAI level). CONCLUSION: DIC during B. anthracis infection may be related more to components such as PGN than to LT or ET.

Reinvestigation of the structure of Brucella O-antigens.

O-Specific polysaccharides of Brucella contain two antigenic determinants, called A and M. Most of the strains express epitope A with a small amount of epitope M, whereas Brucella melitensis strain 16 M expresses longer polymer consisting mostly of M-type epitopes. Proposed explanation was that epitope A is defined by 1-2-linked homopolymer of N-formylperosamine (Rha4NFo), while epitope M is a pentasaccharide with four 2- and one 3-substituted Rha4NFo. We reinvestigated both types of structures by 2D NMR and showed that M-epitope is a tetrasaccharide, missing one of the 2-linked Rha4NFo as compared to the previously proposed structure. Polysaccharide from B. melitensis 16 M contains a fragment of 1-2-linked polymer, capped with M-type polymer. Other strains contain one or two M-type units at the non-reducing end of the 1-2-linked O-chain.

The capsular polysaccharide and lipopolysaccharide structures of two carbapenem resistant Klebsiella pneumoniae outbreak isolates.

Carbapenem resistant Klebsiella pneumoniae (CRKP) are isolated with increasing frequency, especially from immunocompromized patients. The capsular polysaccharide (CPS) types of CPKP were not determined. Investigation of two CRKP isolates from a 2011 outbreak at the Clinical Center, the National Institutes of Health, identified a new capsular type shared by the two isolates, similar to K. pneumonia K19 and K34 but structurally different than any published K. pneumoniae CPS repeating unit: The LPS of the two isolates was found to have no O-specific polysaccharide and the chemical structure of the core oligosaccharides agreed with the published data. If this structure type will be prevalent among CPKP isolates, our findings could facilitate rapid diagnosis and help to develop new therapeutic solutions to this antibiotic resistant pathogen.

The study of the core part and non-repeating elements of the O-antigen of Brucella lipopolysaccharide.

Brucella is an animal and human pathogen that expresses several virulence factors required for host cell invasion and intracellular survival. It produces LPS with unusually low toxicity, which hampers the detection of bacteria by the host immune system and thus provides resistance against intracellular antimicrobial mechanisms of the host. By chemical and spectroscopic methods we determined the structure of the LPS core and of a non-repetitive oligosaccharide fragment at the reducing end of the O-specific polysaccharide. These data should be useful for understanding the biological role of the Brucella LPS.

Synthetic oligosaccharides as tools to demonstrate cross-reactivity between polysaccharide antigens.

Escherichia coli O148 is a nonencapsulated enterotoxigenic (ETEC) Gram negative bacterium that can cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. The surface-exposed O-specific polysaccharide (O-SP) of the lipopolysaccharide of this bacterium is considered both a virulence factor and a protective antigen. It is built up of the linear tetrasaccharide repeating unit [3)-α-L-Rhap-(1→2)-α-D-Glcp-(1→3)-α-D-GlcNAcp-(1→3)-α-L-Rhap-(1→] differing from that of the O-SP of Shigella dysenteriae type 1 (SD) only in that the latter contains a D-Galp residue in place of the glucose moiety of the former. The close similarity of the O-SPs of these bacteria indicated a possible cross-reactivity. To answer this question we synthesized several oligosaccharide fragments of E. coli O148 O-SP, up to a dodecasaccharide, as well as their bovine serum albumin or recombinant diphtheria toxin conjugates. Immunization of mice with these conjugates induced anti-O-SP-specific serum IgG antibody responses. The antisera reacted equally well with the LPSs of both bacteria, indicating cross-reactivity between the SD and E. coli O148 O-SPs that was further supported by Western-blot and dot-blot analyses, as well as by inhibition of binding between the antisera and the O-SPs of both bacteria.

Identification of the methyl phosphate substituent at the non-reducing terminal mannose residue of the O-specific polysaccharides of Klebsiella pneumoniae O3, Hafnia alvei PCM 1223 and Escherichia coli O9/O9a LPS.

O-specific polysaccharides of Gram-negative bacteria are synthesized by two different mechanisms: polymerization of the pre-formed O-repeating unit or sequential addition of the monosaccharides to the growing polysaccharide chain. In the second case, growth of the polymer can be further subdivided into two groups depending on the presence or absence of a special monosaccharide or non-sugar substituent that terminates the glycan. A family of polymannose O-polysaccharides provides prototypes for the chain terminating process. Polysaccharides of Klebsiella pneumoniae O3, Hafnia alvei PCM 1223, and Escherichia coli O9 have the same penta-mannose repeating unit. E. coli O9a has tetra-mannose repeat and this structure can be produced by mutants of E. coli O9. The mechanism of biosynthesis of H. alvei 1223 O-polysaccharide has not been reported. Here we show that all above polysaccharides contain the same modification at the non-reducing end; presence of a methyl phosphate group at O-3 of α-mannopyranose, that serves as the signal for termination of the chain elongation.

Conjugation of LPS-derived oligosaccharides to proteins using oxime chemistry.

Conjugates of bacterial polysaccharides covalently bound to a carrier protein are among licensed human vaccines. Immunization of adults and children with these vaccines results in induction of saccharide-specific antibodies composed mainly of the IgG class. Depending on the choice of coupling technique, saccharides can be attached to a protein by either multiple- or single-point attachments. While the first method is suitable for high molecular mass polysaccharides, the second one is beneficial for low-molecular mass compounds such as synthetic carbohydrates or bacterial oligosaccharides obtained by different degradation procedures. This chapter describes a method for coupling low-molecular mass lipopolysaccharide (LPS)-derived oligosaccharides composed of a core or a short O-specific polysaccharide-core fragment (O-SPC) to a carrier protein by a single-point attachment. Conjugation is performed between the carbonyl group of the reducing terminal of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) exposed after acid hydrolyses of LPS and the aminooxy group of a bifunctional linker bound to the protein. This is an efficient reaction that can be carried out quickly and under mild conditions. Conjugates thus prepared using this approach preserve the external nonreducing end of the sugar chain and can induce antibodies to both conjugate components. Consequently, this method is highly suitable for the preparation of LPS-based human vaccines.

Synthesis and antigenicity of BBGL-2 glycolipids of Borrelia burgdorferi, the causative agent of Lyme disease.

Borrelia burgdorferi is the etiological agent for Lyme disease (LD), the most common vector borne disease in the United States. There is no human vaccine against LD currently available. Our approach to a vaccine is based on its surface-exposed glycolipids. One group of these glycolipids termed BBGL-2 consists of 1,2-di-O-acyl-3-O-(α-d-galactopyranosyl)-sn-glycerol congeners having palmitic, oleic, stearic, linoleic, and myristic acids. In order to delineate the immunodominant region(s) of the BBGL-2 components, we embarked on a synthetic project to provide available structurally defined, homogeneous analogs of BBGL-2 that might help identify the best vaccine candidate. The antigenicity of the synthetic glycolipids was examined by dot-blot analysis using mice sera obtained by immunization with killed B. burgdorferi cells, with native BBGL-2 in complete Freund's adjuvant, as well as sera obtained from patients with Lyme disease. We found that the presence of two acyl groups in the glycerol moiety was essential for antigenicity. At least one of these groups must be an oleoyl moiety. Neither the anomeric configuration of the galactose nor the configuration of the glycerol at C-2 was a decisive factor. Based on these findings we designed an 'unnatural' BBGL-2 analog having the structure 3-O-(ß-d-galactopyranosyl)-1,2-di-O-oleoyl-dl-glycerol which is easier and less expensive to synthesize than the other BBGL-2 congeners prepared in this study. This substance proved to be antigenic and is considered a candidate vaccine for Lyme disease.

Oligosaccharide conjugates of Bordetella pertussis and bronchiseptica induce bactericidal antibodies, an addition to pertussis vaccine.

Pertussis is a highly contagious respiratory disease that is especially dangerous for infants and children. Despite mass vaccination, reported pertussis cases have increased in the United States and other parts of the world, probably because of increased awareness, improved diagnostic means, and waning vaccine-induced immunity among adolescents and adults. Licensed vaccines do not kill the organism directly; the addition of a component inducing bactericidal antibodies would improve vaccine efficacy. We investigated Bordetella pertussis and Bordetella bronchiseptica LPS-derived core oligosaccharide (OS) protein conjugates for their immunogenicity in mice. B. pertussis and B. bronchiseptica core OS were bound to aminooxylated BSA via their terminal Kdo residues. The two conjugates induced similar anti-B. pertussis LPS IgG levels in mice. B. bronchiseptica was investigated because it is easier to grow than B. pertussis. Using B. bronchiseptica genetically modified strains deficient in the O-specific polysaccharide, we isolated fractions of core OS with one to five repeats of the terminal trisaccharide, having at the nonreducing end a GlcNAc or GalNAc, and bound them to BSA at different densities. The highest antibody levels in mice were elicited by conjugates containing an average of 8-17 OS chains per protein and with one repeat of the terminal trisaccharide. Conjugate-induced antisera were bactericidal against B. pertussis, and the titers correlated with ELISA-measured antibody levels (r = 0.74). Such conjugates are easy to prepare and standardize; added to a recombinant pertussis toxoid, they may induce antibacterial and antitoxin immunity.

Pre- and postexposure protection against virulent anthrax infection in mice by humanized monoclonal antibodies to Bacillus anthracis capsule.

One of the two essential virulence factors of Bacillus anthracis is the poly-γ-D-glutamic acid (γDPGA) capsule. Five γDPGA-specific antibody antigen-binding fragments (Fabs) were generated from immunized chimpanzees. The two selected for further study, Fabs 11D and 4C, were both converted into full-length IgG1 and IgG3 mAbs having human IgG1 or IgG3 constant regions. These two mAbs had similar binding affinities, in vitro opsonophagocytic activities, and in vivo efficacies, with the IgG1 and IgG3 subclasses reacting similarly. The mAbs bound to γDPGA specifically with estimated binding affinities (K(d)) of 35-70 nM and effective affinities (effective K(d)) of 0.1-0.3 nM. The LD(50) in an opsonophagocytic bactericidal assay was ≈10 ng/mL of 11D or 4C. A single 30-µg dose of either mAb given to BALB/c mice 18 h before challenge conferred about 50% protection against a lethal intratracheal spore challenge by the virulent B. anthracis Ames strain. More importantly, either mAb given 8 h or 20 h after challenge provided significant protection against lethal infection. Thus, these anti-γDPGA mAbs should be useful, alone or in combination with antitoxin mAbs, for achieving a safe and efficacious postexposure therapy for anthrax.

The structure of the Escherichia coli O148 lipopolysaccharide core region and its linkage to the O-specific polysaccharide.

Recently it was demonstrated that Shigella dysenteriae type 1, a cause of severe dysentery epidemics, gained its O-specific polysaccharide (O-SP) from Escherichia coli O148. The O-SPs of these bacteria differ only by a galactose residue in the repeat unit of S. dysenteriae type 1 in place of a glucose residue in E. coli O148. Herein, we analyzed the core structure and its linkage to the O-SP in E. coli O148 LPS. Both were found to be identical to those of S. dysenteriae type 1 structures, further supporting the relatedness of these two bacteria. The following structure of the core with one repeat unit of the O-SP has been assigned (all have d-configuration except l-Rha):

Immunochemical studies of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1 O-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates.

There is no licensed vaccine for the prevention of shigellosis. Our approach to the development of a Shigella vaccines is based on inducing serum IgG antibodies to the O-specific polysaccharide (O-SP) domain of their lipopolysaccharides (LPS). We have shown that low molecular mass O-SP-core (O-SPC) fragments isolated from Shigella sonnei LPS conjugated to proteins induced significantly higher antibody levels in mice than the full length O-SP conjugates. This finding is now extended to the O-SPC of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1. The structures of O-SPC, containing core plus 1-4 O-SP repeat units (RUs), were analyzed by NMR and mass spectroscopy. The first RUs attached to the cores of S. flexneri 2a and 6 LPS were different from the following RUs in their O-acetylation and/or glucosylation. Conjugates of core plus more than 1 RU were necessary to induce LPS antibodies in mice. The resulting antibody levels were comparable to those induced by the full length O-SP conjugates. In S. dysenteriae type 1, the first RU was identical to the following RUs, with the exception that the GlcNAc was bound to the core in the beta-configuration, while in all other RUs the GlcNAc was present in the alpha-configuration. In spite of this difference, conjugates of S. dysenteriae type 1 core with 1, 2, or 3 RUs induced LPS antibodies in mice with levels statistically higher than those of the full size O-SP conjugates. O-SPC conjugates are easy to prepare, characterize, and standardize, and their clinical evaluation is planned.

A bicomponent Plasmodium falciparum investigational vaccine composed of protein-peptide conjugates.

There is yet no licensed vaccine against malaria, a serious human disease affecting mostly children, with an annual death rate of about one million. Plasmodia, the malaria-causing parasites, have two obligatory hosts: mammals or birds, in which they multiply asexually, and mosquitoes with sexual multiplication. The most common and serious type of malaria is caused by Plasmodium falciparum. The circumsporozoite protein (CSP), a major surface antigen of sporozoites, is a protective antigen. A unique feature of P. falciparum CSP is its large central domain composed of over 30 tetrapeptide repeats of Asn-Ala-Asn-Pro (NANP). Several NANP peptide-protein conjugates were tested clinically but elicited a low level of CSP antibodies for a short duration. To provide a CSP-based candidate vaccine, we investigated recombinant CSP and NANP conjugates of various peptide lengths, with different N-terminal amino acids, bound at different ratios to various carrier proteins. Injected into mice, CSP alone and CSP or NANP conjugates induced antibodies with booster responses and were positive by the sporozoite immunofluorescent assay. The use of the mosquito stage P. falciparum ookinete surface protein, Pfs25, cross-linked onto itself as a carrier for NANP, induced in mice high levels of uniquely long-lasting antibodies to both vaccine components with secondary biological activities, that will provide immunity to liver infection by sporozoites and block transmission by mosquitoes.

Immunogenic and adjuvant properties of Haemophilus ducreyi lipooligosaccharides.

Haemophilus ducreyi, the chancroid-causing bacterium, produces lipooligosaccharides (HdLOS) that comprise 5-11 partially sialylated monosaccharides. Subcutaneous immunisation of mice with 5 microg of HdLOS purified from H. ducreyi strains 4438 and 7470 induced high levels of anti-HdLOS IgG. The antibody responses displayed T-cell-independent features, and were dependent upon Toll-like receptor 4/MyD88 signalling pathways as demonstrated using knockout mice. The immunogenicity of HdLOS was found to require the intact lipid A moiety. The specificity studies of the anti-HdLOS antibodies, as revealed by absorption studies, antibody detection in ELISA, and immune thin-layer chromatography, indicated that the majority of the anti-LOS antibodies were specific for the inner core of the HdLOS. Antibodies to HdLOS failed to inhibit LOS induction of TNF-alpha release from human mononuclear cells. The adjuvanticity of HdLOS7470 was assessed in BALB/c mice that were immunised with bovine serum albumin (BSA) with or without the addition of HdLOS. The addition of 5 microg HdLOS resulted in a 10-fold increase in the total anti-BSA IgG antibody level as estimated by ELISA. The highest increase was noted for IgG2b, which contrasted with the predominantly IgG1 subclass response to immunisation with BSA alone, indicating an immunomodulatory activity of the HdLOS.

Synthesis, characterization, and immunogenicity in mice of Shigella sonnei O-specific oligosaccharide-core-protein conjugates.

Shigellosis, an enteric disease, is on the World Health Organization's priority prevention list. In one study, the Shigella sonnei O-specific polysaccharide (O-SP)-protein conjugate showed 72% protection against disease in Israeli army recruits exposed to high rates (8-14%) of infection. The protection was related to vaccine-induced IgG anti-O-SP levels. Synthetic oligosaccharides of Shigella dysenteriae type 1, bound by their reducing ends to a carrier protein ("sun"-type configuration), induced significantly higher antibody levels than the native O-SP bound to protein by multiple-point attachments ("lattice"-type configuration). Attempts to synthesize the S. sonnei O-SP based oligosaccharides were not successful. Here, we describe the isolation, characterization, and conjugation of low-molecular-mass O-SP-core (O-SPC) fragments. The O-SPC fragments were bound by their reducing ends similar to the preparation of the synthetic S. dysenteriae type 1 conjugates. The O-SPC conjugates used oxime linkages between the terminal Kdo residues at the reducing ends of the S. sonnei saccharides and aminooxy linkers bound to BSA or a recombinant diphtheria toxin. The coupling reaction was carried out at a neutral pH and room temperature. IgG antibody levels induced in young outbred mice by the S. sonnei O-SPC conjugates were significantly higher then those elicited by the O-SP conjugates. Accordingly, we propose to evaluate clinically these conjugates.