Joanna Kubler-Kielb

Poly(γ-d-glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: A potential addition to the anthrax vaccine

Both the protective antigen (PA) and the poly(γ-d-glutamic acid) capsule (γdPGA) are essential for the virulence of Bacillus anthracis. A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-γdPGA. Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines. The nonimmunogenic γdPGA or corresponding synthetic peptides were bound to BSA, recombinant B. anthracis PA (rPA), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA). To identify the optimal construct, conjugates of B. anthracis γdPGA, Bacillus pumilus γdLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of the d or l configuration with active groups at the N or C termini, were bound at 5–32 mol per protein. The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5- to 6-week-old mice. IgG anti-γdPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-γdPGA were elicited by decamers of γdPGA at 10 –20 mol per protein bound to the N- or C-terminal end. High IgG anti-γdPGA levels were elicited by two injections of 2.5 μg of γdPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies. rPA was the most effective carrier. Anti-γdPGA induced opsonophagocytic killing of B. anthracis tox–, cap+. γdPGA conjugates may enhance the protection conferred by PA alone. γdPGA-rPA conjugates induced both anti-PA and anti-γdPGA.

A newly discovered cholesteryl galactoside from Borrelia burgdorferi

Two major glycolipids, which comprise ≈36% of the total lipid mass from Borrelia burgdorferi, the etiological agent of Lyme disease, were investigated. We determined the fatty acid type, sugar identity, anomeric configuration, and substituent type and position. The structures were identified as cholesteryl 6-O-acyl-β-d-galactopyranoside (B. burgdorferi glycolipid 1, BbGL-I), and 1,2-di-O-acyl-3-O-α-d-galactopyranosyl-sn-glycerol (BbGL-II). The major fatty acids were palmitate and oleate. The structures were corroborated by gas–liquid chromatography MS, matrix-assisted laser desorption/ionization time-of-flight spectroscopy, fast atom bombardment MS, detailed NMR spectrometry, and metabolic labeling. This is a previously undescribed demonstration of a cholesteryl galactoside in bacteria. Lipopolysaccharide was not detected in B. burgdorferi. The two glycolipids have several properties suggesting they may function as lipopolysaccharide: both are main components of the bacterial membrane, surface exposed, and have a three-domain structure. BbGL-I elicited specific antibodies in mice and rabbits, and BbGL-II elicited antibodies that reacted with both glycolipids.

Long-lasting and transmission-blocking activity of antibodies to Plasmodium falciparum elicited in mice by protein conjugates of Pfs25

Malaria is a leading cause of morbidity and mortality, estimated to cause >1 million childhood deaths annually. Plasmodium falciparum causes the most severe form of the disease. There is as yet no licensed vaccine for this disease, despite over a half century of research. In this study, we investigated a transmission-blocking vaccine candidate, the ookinete surface protein Pfs25. Antibodies against Pfs25, drawn in during a bite, can block parasite development in the mosquito midgut, preventing transmission to other individuals. Pfs25 is a low-molecular-weight protein, by itself not immunogenic. To increase its immunogenicity, we investigated several methods of conjugating Pfs25 to itself and to other proteins: recombinant Pseudomonas aeruginosa exotoxin A, and ovalbumin, using amide, hydrazone, or thioether linkages. All conjugates were immunogenic and induced booster responses in mice. The scheme to form amide bonds between proteins by using adipic acid dihydrizide as a linker produced the most immunogenic conjugates. Adsorption of the conjugates onto aluminum hydroxide further increased the antibody response. Remarkably, the antibody levels 3 or 7 months after the last injection were significantly higher than those 1 wk after that injection. The observed transmission-blocking activity of immune sera correlated with antibody levels measured by ELISA.

Complete Structures of Bordetella bronchiseptica and Bordetella parapertussis Lipopolysaccharides

The structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these microorganisms to determine the complete carbohydrate structure of the LPS. LPS was analyzed using different chemical degradations, NMR spectroscopy, and mass spectrometry. This identified a novel pentasaccharide fragment that links the O chain to the core in all the LPS studied. In addition, although the O chain of these bacteria was reported as a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-α-galacturonic acid, we discovered that the polymer contains several amidated uronic acids, the number of which varies between strains. These new data describe the complete structure of the LPS carbohydrate backbone for both Bordetella species and help to explain the complex genetics of LPS biosynthesis in these bacteria.

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

Shigellosis, an enteric disease, is on the World Health Organizations 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.

Effect of the nonreducing end of Shigella dysenteriae type 1 O-specific oligosaccharides on their immunogenicity as conjugates in mice

Endemic and epidemic shigellosis, an acute invasive disease of the lower intestines, afflicts millions of people worldwide with an estimated one million fatalities per annum at a low infectious dose. Our approach to vaccine development against Shigella is based on the hypothesis that serum IgG antibodies to the O-specific polysaccharide (O-SP) domains of the LPS of these organisms confer protection to infection. The synthetic oligosaccharides corresponding to the tetrasaccharide repeating unit of the O-SP of Shigella dysenteriae type 1 covalently linked to human serum albumin elicited O-SP-specific IgG in mice. The antibody levels were a function of both the saccharide chain length and their loading on the protein. These synthetic saccharide conjugates elicited significantly higher levels of IgG anti O-SP than conjugates prepared with the O-SP from the bacteria. Here, we evaluated the influence of the nonreducing terminal monosaccharide on the serum antibody response. To this end, we prepared synthetic oligosaccharides comprising hexa- to tridecasaccharide fragments of the native O-SP, having one of the four monosaccharide residues that constitute the repeating unit at their termini and bound them to BSA by a single-point attachment. The conjugates contained an average of 19 saccharide chains per BSA. The synthetic oligosaccharides inhibited the binding of serum raised against whole bacteria to its LPS to a similar extent but lower than the native O-SP. The highest anti-LPS levels were elicited by conjugates having N-acetylglucosamine (10-mer) or galactose residues (7- and 11-mers) at their nonreducing termini.

Additional Conjugation Methods and Immunogenicity of Bacillus anthracis Poly-γ-d-Glutamic Acid-Protein Conjugates

ABSTRACT The capsule of Bacillus anthracis, composed of poly-γ-d-glutamic acid (γDPGA), is an essential virulence factor of B. anthracis. The capsule inhibits innate host defense through its antiphagocytic action. γDPGA is a poor immunogen, but when covalently bound to a carrier protein, it elicits serum antibodies. To identify the optimal construct for clinical use, synthetic γDPGAs of different lengths were bound to carrier proteins at different densities. The advantages of the synthetic over the natural polypeptide are the homogeneous chain length and end groups, allowing conjugates to be accurately characterized and standardized and their chemical compositions to be related to their immunogenicities. In the present study, we evaluated, in addition to methods reported by us, hydrazone, oxime, and thioether linkages between γDPGA and several proteins, including bovine serum albumin, recombinant Pseudomonas aeruginosa exotoxin A, recombinant B. anthracis protective antigen (rPA), and tetanus toxoid (TT). The effects of the dosage and formulation on the immunogenicities of the conjugates were evaluated in mice. All conjugates were immunogenic. The optimal γDPGA chain length of 10 to 15 amino acids and the density, an average of 15 mol γDPGA per mol of protein, were confirmed. The thioether bond was the optimal linkage type, and TT and rPA were the best carriers. The optimal dosage was 1.2 to 2.5 μg of γDPGA per mouse, and adsorption of the conjugates onto aluminum hydroxide significantly increased the antibody response to the protein with a lesser effect on anti-γDPGA levels.

Saccharide/protein conjugate vaccines for Bordetella species: preparation of saccharide, development of new conjugation procedures, and physico-chemical and immunological characterization of the conjugates

Bordetellae are Gram-negative bacilli causing respiratory tract infections of mammals and birds. Clinically important are B. pertussis, B. parapertussis and B. bronchiseptica. B. pertussis vaccines have been successful in preventing pertussis in infants and children. Veterinary vaccines against B. bronchiseptica are available, but their efficacy and mode of action are not established. There is no vaccine against B. parapertussis. Based on the concept that immunity to non-capsulated Gram-negative bacteria may be conferred by serum IgG anti-LPS we studied chemical, serological and immunological properties of the O-specific polysaccharides (O-SP) of B. bronchiseptica and B. parapertussis obtained by different degradation procedures. One type of the B. parapertussis and two types of B. bronchiseptica O-SP were recognized based on the structure of their non-reducing end saccharide; no cross-reaction between the two B. bronchiseptica types was observed. Competitive inhibition assays showed the immunodominance of the non-reducing end of these O-SP. Conjugates of B. bronchiseptica and B. parapertussis O-SP were prepared by two methods: using the anhydro-Kdo residue exposed by mild acid hydrolysis of the LPS or the 2,5-anhydromannose residue exposed by deamination of the core glucosamine of the LPS, for binding to an aminooxylated protein. Both coupling methods were carried out at a neutral pH, room temperature, and in a short time. All conjugates, injected as saline solutions at a fraction of an estimated human dose, induced antibodies in mice to the homologous O-SP. These methodologies can be applied to prepare O-SP-based vaccines against other Gram-negative bacteria.

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.

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.