Development of a glycoconjugate vaccine against melioidosis – MELIVAC

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 presentations, oral communications and posters have been delivered in connection with this work, including two oral communications at the International Carbohydrate Symposium held in Bangalore in January 2014 and in New Orleans in July 2016.

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.