Synthetic Lipoteichoic Acid Glycans Are Potential Vaccine Candidates to Protect from Clostridium difficile Infections.

Infections with Clostridium difficile increasingly cause morbidity and mortality worldwide. Bacterial surface glycans including lipoteichoic acid (LTA) were identified as auspicious vaccine antigens to prevent colonization. Here, we report on the potential of synthetic LTA glycans as vaccine candidates. We identified LTA-specific antibodies in the blood of C. difficile patients. Therefore, we evaluated the immunogenicity of a semi-synthetic LTA-CRM197 glycoconjugate. The conjugate elicited LTA-specific antibodies in mice that recognized natural LTA epitopes on the surface of C. difficile bacteria and inhibited intestinal colonization of C. difficile in mice in vivo. Our findings underscore the promise of synthetic LTA glycans as C. difficile vaccine candidates.

Synthesis of chlamydia lipopolysaccharide haptens through the use of α-specific 3-iodo-Kdo fluoride glycosyl donors.

A scalable approach towards high-yielding and (stereo)selective glycosyl donors of the 2-ulosonic acid Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) is a fundamental requirement for the development of vaccines against Gram-negative bacteria. Herein, we disclose a short synthetic route to 3-iodo Kdo fluoride donors from Kdo glycal esters that enable efficient α-specific glycosylations and significantly suppress the elimination side reaction. The potency of these donors is demonstrated in a straightforward, six-step synthesis of a branched Chlamydia-related Kdo-trisaccharide ligand without the need for protecting groups at the Kdo glycosyl acceptor. The approach was further extended to include sequential iteration of the basic concept to produce the linear Chlamydia-specific α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo trisaccharide in a good overall yield.

[Glycopolymers of microorganisms: achievements and future research (review)].

This review presents the current literature data on the structure of peptidoglycans, lipopolysaccharides, teichoic acids, the mechanism of biological action of lipopolysaccharides, and the possibility of uising oligosaccharides for creation of glycoconjugate vaccines, as well as promising areas for further research of glycopolymers of microorganisms.

Identification of protective B cell antigens of Legionella pneumophila.

Abs confer protection from secondary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. In this study, we demonstrate that Ab-mediated protection is effective across L. pneumophila serogroups, suggesting that Abs specific for conserved protein Ags are sufficient to mediate this protective effect. We used two independent methods to identify immunogenic L. pneumophila protein Ags, namely, the screening of a λ phage library representing the complete L. pneumophila genome and two-dimensional gel electrophoresis combined with Western blot analysis and protein spot identification by mass spectrometry. A total of 30 novel L. pneumophila B cell Ags were identified, the majority of which are located in or associated with the bacterial membrane, where they are accessible for Abs and, therefore, likely to be relevant for Ab-mediated protection against L. pneumophila. Selected B cell Ags were recombinantly expressed and tested in a vaccination protocol. Mice immunized with either single-protein Ags or an Ag combination showed reduced bacterial titers in bronchoalveolar lavage and lung after L. pneumophila challenge. To determine the clinical relevance of these findings, we tested Legionnaires' disease patient sera for reactivity with the identified L. pneumophila Ags. The recognized Ags were indeed conserved across host species, because Abs specific for all three selected Ags could be detected in patient sera, rendering the identified protein Ags potential vaccine candidates.

The global need for effective antibiotics-a summary of plenary presentations.

To highlight the global need for effective antibiotics and explore possible concerted actions for change, cross-cutting plenary sessions served to frame the program of the conference. These sessions contained presentations on the present state of antibacterial resistance and the availability, the use and misuse of antibiotics. A number of possible actions were discussed, such as rational use of and access to antibiotics from various perspectives. The roles of vaccines and diagnostics were touched upon and followed by in depth discussions on supply-side bottlenecks with their scientific, regulatory and financial challenges. The value chain for research and development (R&D) of antibiotics has to be reengineered if we are to realize the development of much needed new antibiotics. This challenge will require a multitude of actions, some of which are related to changing the financial realities of antibiotics and interventions by global and regional institutions.

Evidence that botulinum toxin receptors on epithelial cells and neuronal cells are not identical: implications for development of a non-neurotropic vaccine.

Botulinum toxin typically interacts with two types of cells to cause the disease botulism. The toxin initially interacts with epithelial cells in the gut or airway to undergo binding, transcytosis, and delivery to the general circulation. The toxin then interacts with peripheral cholinergic nerve endings to undergo binding, endocytosis, and delivery to the cytosol. The receptors for botulinum toxin on nerve cells have been identified, but receptors on epithelial cells remain unknown. The initial toxin binding site on nerve cells is a polysialoganglioside, so experiments were performed to determine whether polysialogangliosides are also receptors on epithelial cells. A series of single mutant and dimutant forms of the botulinum toxin type A binding domain (HC50) were cloned and expressed. One of these (dimutant HC50 A(W1266L,Y1267S)) was shown to have lost its ability to bind nerve cells (phrenic nerve-hemidiaphragm preparation), yet it retained its ability to bind and cross human epithelial monolayers (T-84 cells). In addition, the wild-type HC50 and the dimutant HC50 displayed the same ability to undergo binding and transcytosis (absorption) in a mouse model. The fact that the dimutant retained the ability to cross epithelial barriers but did not possess the ability to bind to nerve cells was exploited to create a mucosal vaccine that was non-neurotropic. The wild-type HC50 and non-neurotropic HC50 proved to be comparable in their abilities to: 1) evoke a circulating IgA and IgG response and 2) evoke protection against a substantial challenge dose of botulinum toxin.

Immunization of Macaca fascicularis against experimental periodontitis using a vaccine containing cysteine proteases purified from Porphyromonas gingivalis.

INTRODUCTION: Periodontitis is a common infectious disease to which Porphyromonas gingivalis has been closely linked, in which the attachment tissues of the teeth and their alveolar bone housing are destroyed. We conducted a study to determine if immunization using a purified antigen could alter the onset and progression of the disease. METHODS: Using the ligature-induced model of periodontitis in Macaca fascicularis, we immunized five animals with cysteine protease purified from P. gingivalis and used an additional five animals as controls. Alveolar bone loss was measured by digital subtraction radiography. RESULTS: Immunization induced high titers of specific immunoglobuin G serum antibodies that were opsonic. Total bacterial load, levels of P. gingivalis in subgingival plaque and levels of prostaglandin E(2) in gingival crevicular fluid were significantly reduced. Onset and progression of alveolar bone loss was inhibited by approximately 50%. No manifestations of toxicity were observed. CONCLUSIONS: Immunization using a purified protein antigen from P. gingivalis inhibits alveolar bone destruction in a ligature-induced periodontitis model in M. fascicularis.

New multi-determinant strategy for a group A streptococcal vaccine designed for the Australian Aboriginal population.

Infection with group A streptococci can result in acute and post-infectious pathology, including rheumatic fever and rheumatic heart disease. These diseases are associated with poverty and are increasing in incidence, particularly in developing countries and amongst indigenous populations, such as Australia's Aboriginal population, who suffer the highest incidence worldwide. Immunity to group A streptococci is mediated by antibodies against the M protein, a coiled-coil alpha helical surface protein of the bacterium. Vaccine development faces two substantial obstacles. Although opsonic antibodies directed against the N terminus of the protein are mostly responsible for serotypic immunity, more than 100 serotypes exist. Furthermore, whereas the pathogenesis of rheumatic fever is not well understood, increasing evidence indicates an autoimmune process. To develop a suitable vaccine candidate, we first identified a minimum, helical, non-host-cross-reactive peptide from the conserved C-terminal half of the protein and displayed this within a non-M-protein peptide sequence designed to maintain helical folding and antigenicity, J14 (refs. 8,9). As this region of the M protein is identical in only 70% of group A streptococci isolates, the optimal candidate might consist of the conserved determinant with common N-terminal sequences found in communities with endemic group A streptococci. We linked seven serotypic peptides with J14 using a new chemistry technique that enables the immunogen to display all the individual peptides pendant from an alkane backbone. This construct demonstrated excellent immunogenicity and protection in mice.

Synthesis and physicochemical and immunological characterization of pneumococcus type 12F polysaccharide-diphtheria toxoid conjugates.

A scheme for the synthesis and purification of conjugates, composed of the type 12F capsular polysaccharide of Streptococcus pneumoniae (Pn12F) and diphtheria toxoid, is described. The scheme is a modification of that described previously for the Vi capsular polysaccharide of Salmonella typhi, a linear homopolymer of N-acetylgalactoseaminouronic acid (S. C. Szu, A. L. Stone, J. D. Robbins, R. Schneerson, and J. B. Robbins, J. Exp. Med. 166:1510-1524, 1986). Pn12F is a branched-chain copolymer composed of a hexasaccharide repeating unit containing an aminouronic acid, N-acetylmannoseaminouronic acid (K. Leontein, B. Lindberg, and J. Lonngren, Can. J. Chem. 59:2081-2085, 1981). Sulfhydryl groups were introduced into Pn12F by forming an amide bond between cystamine and carboxyl groups of N-acetylmannoseaminouronic acid in the presence of a carbodiimide. The disulfide moiety of cystamine was reduced to form the cysteamine derivative of Pn12F which was, in turn, covalently bound to diphtheria toxoid by using the heterobifunctional linker N-succinimidyl-3-(2-pyridylthio)propionate. Unbound, high-molecular-weight Pn12F was removed from the conjugate by hydrophobic interaction chromatography through octyl Sepharose by using n-octyl-beta-D-glucopyranoside as the eluent. In young outbred mice, Pn12F did not elicit detectable serum antibodies. Pn12F-diphtheria toxoid, in contrast, elicited antibodies after two injections and had T-cell-dependent properties as evidenced by a response to priming and by its ability to elicit booster responses. This scheme seems applicable to the synthesis of conjugates with other capsular polysaccharides containing aminouronic acids. Clinical evaluation of Pn12F-diphtheria toxoid conjugates in healthy and in immunocompromised hosts is planned.