Melioidosis is an infectious pathology caused by the tropical bacteria B. pseudomallei, a potential bioterrorism threat. No vaccine is available to fight against melioidosis and thus an intensive antibiotic therapy is necessary for its eradication. An experimental vaccine based on the sugar surface antigens expressed by B. pseudomallei is a promising avenue to follow.
Burkholderia pseudomallei (Bp) is the causal agent of melioidosis. The high infectivity of the bacteria via the human respiratory system combined with high mortality rates and resistance to antibiotics make this category B agent a suitable target for use as a biological warfare by terrorists. Experimental vaccines have been tested in animals, but so far none of them completely suppress the infection. Surface polysaccharides expressed by Bp are able to activate the production of protective antibodies in humans and thus could represent promising subunit vaccine candidates against melioidosis. The main objective of the project was to develop a “glycoconjugate” vaccine, which would be able to mimic the different surface polysaccharides of Bp. The two components vaccine will be built on a sugar unit, obtained by organic synthesis, which was chemically linked to a carrier protein. Such a vaccine, which is able to trigger the long term immune response, has already been commercialized against meningitis for instance. As no bacteria are used in the preparation process, glycoconjugate vaccines are usually free of bacterial contaminants, which is a main advantage over “conventional” vaccine.
Novel methodologies have been developed with the aim of synthesizing functional mimics of the repeating units of the three main surface polysaccharides of Bp, i.e. the capsular polysaccharide, the exopolysaccharide, and the lipopolysaccharide. The developed approaches could be applied to the synthesis of bacterial glycans featuring diverse aglycones. The synthetic sugars were evaluated for their interactions with monoclonal antibodies, which are specific to surface polysaccharides of Bp. In order to do so, enzyme-linked immunosorbent assay, surface plasmon resonance and saturation-transfer difference resonance magnetic nuclear spectroscopy were used to probe the antigen/antibody interactions. The synthetic sugars were functionalized with an aminolinker allowing their covalent coupling with diphtheria toxoid, a clinically approved carrier protein. The glycoconjugates were evaluated for their immunogenicity in mice in the presence of an adjuvant. The mice blood serum were collected and the antibody (IgG) levels were determined using an enzyme-linked immunosorbent assay.
A series of compounds, which mimic the three main surface polysaccharides of Bp and other pathogenic bacteria of the Burkholderia genera, were synthesized using novel methodologies. The oligosaccharides and their protein constructs stand as potential vaccines and diagnostic tools against melioidosis, glanders and Cepacia syndrome. This project has allowed us to start new international collaborations with research groups based in US (Prof. Paul J. Brett, Mary N. Burtnick, and David P. AuCoin) and Italy (Prof. Antonio Molinaro and Alba Silipo).
Bp is a highly virulent bacterium, especially by the respiratory system, multi-resistant to antibiotics, and can kill up to 50% of patients upon infection. No vaccine is currently available to protect against Bp. Each year, more and more people are diagnosed with melioidosis around the world. For these reasons, the discovery of a efficient vaccine against melioidosis is a priority for the French government. The Direction Générale de l’Armement (DGA) is also highly interested in developing such a vaccine. Actually, countries including USA, ex-Soviet Union and Egypt have studied the possibility of using Bp as a biological warfare. Moreover, a number of melioidosis cases have been diagnosed by soldiers and military people on duty in the Asian continent.
The results of this project have been the subject of three publications in J. Org. Chem. (2014, 79, 4615; 2015, 80, 10386; 2016, ASAP) and one publication in ACS Chem. Biol. (2015, 10, 2295). Another important paper is in preparation. Several invited pres
Burkholderia pseudomallei (Bp), the infectious agent of melioidosis, is the cause of important health problems in Thailand and Northern Australia. Because of its high infectivity by inhalation, increased resistance to antibiotics as well as high mortality rate, Bp is considered as a potential bioterrorism threat and biological warfare. The development of a functional vaccine that could protect populations as well as soldiers against melioidosis is thus of capital importance for occidental governments. The outer membrane of Bp is covered by a dense array of polysaccharides (LPS, CPS and EPS), which act as major virulence factors and activate the production of protective antibodies in humans. The main objective of the proposal is the development of a glycoconjugate vaccine against melioidosis based on the covalent liaison between synthetic sugars and a carrier protein. To this end, we propose to devise efficient synthetic routes for the preparation of oligosaccharide mimics of Bp LPS Ag-O, CPS and EPS, functionalized with an aminopentyl linker at the reducing end. Bp CPS oligomers featuring a linear sequence of 2-O-Ac-6-deoxy-beta-D-manno-heptopyranose residues will be synthesized via an intramolecular aglycon delivery approach. En route to the synthesis of Bp EPS mimics, a novel methodology will be developed for the stereoselective formation of beta-D-Kdo linkages. The methoxyphenacyl ester group will be used as a C-1-auxiliary functionality to induce stabilization of the oxonium ion via the alpha-face, hence favouring the coupling on the less hindered beta-face of the Kdo ring. In addition, three hexasaccharides featuring all the possible non-stoichiometric acetylation/methylation pattern of Bp LPS Ag-O will be synthesized through a convergent route based on the judicious combination of two talose donors and one glucose acceptor. The synthesized oligosaccharides will be tested for their antigenicity in mice. Sugars generating the best immunogenic profile will be covalently linked to CRM-197. Conjugation will be performed by taking advantage of the 39 available lysine residues of the carrier protein via either NHS ester or thiophosgene conjugation. Sugars per protein ratio will be determined by SDS-PAGE as well as MALDI-TOF-MS analysis, while 1H NMR and CD spectroscopy will be used to evaluate the protein integrity after modification. BALB/c mice will be immunized with the synthetic glycoconjugates and further challenged with a wild strain of Bp. The immunogenic potential of the glycoconjugate vaccines will be assessed as their ability to increase the life span of infected mice and, hopefully, to protect them against Bp infection.
Institut de Chimie des Milieux et des Matériaux de Poitiers (IC2MP) (Laboratoire public)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Institut de Chimie des Milieux et des Matériaux de Poitiers (IC2MP)
Help of the ANR 187,842 euros
Beginning and duration of the scientific project: September 2012 - 48 Months