Background:The isolation of capsular polysaccharides from pathogenic bacteria for vaccine production is cost-intensive. Results: We describe the cloning, recombinant expression, and functional characterization of three enzymes from Neisseria meningitidis serogroup A that facilitate in vitro synthesis of the capsule polymer. Conclusion:The study presents a novel basis for efficient vaccine production. Significance: Economic vaccine production is prerequisite to combat meningococcal diseases.

Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. A key feature in pathogenicity is the capsular polysaccharide (CPS) that prevents complement activation and thus supports bacterial survival in the host. Twelve serogroups characterized by immunologically and structurally different CPSs have been identified. Meningococcal CPSs elicit bactericidal antibodies and consequently are used for the development of vaccines. Vaccination against the epidemiologically most relevant serogroups was initially carried out with purified CPS and later followed by conjugate vaccines which consist of CPS covalently linked to a carrier protein. Of increasing importance in the African meningitis belt is NmX for which no vaccine is currently available. Here, we describe the molecular cloning, recombinant expression and purification of the capsule polymerase (CP) of NmX called CsxA. The protein expressed with N- and/or C-terminal epitope tags was soluble and could be purified to near homogeneity. With short oligosaccharide primers derived from the NmX capsular polysaccharide (CPSX), recombinant CsxA produced long polymer chains in vitro that in immunoblots were detected with NmX-specific antibodies. Moreover, the chemical identity of in vitro produced NmX polysaccharides was confirmed by NMR. Besides the demonstration that the previously identified gene csxA encodes the NmX CP CsxA, the data presented in this study pave the way for the use of the recombinant CP as a safe and economic way to generate the CPSX in vaccine developmental programs.

serogroups A and X are among the leading causes of bacterial meningitis in the African meningitis belt. Glycoconjugate vaccines, consisting of an antigenic carrier protein coupled to the capsular polysaccharide of the bacterial pathogen, are the most effective strategy for prevention of meningococcal disease. However, the distribution of effective glycoconjugate vaccines in this region is limited by the high cost of cultivating pathogens and purification of their capsular polysaccharides. Moreover, chemical approaches to synthesize oligosaccharide antigens have proven challenging. In the current study, we present a chemoenzymatic approach for generating tailored oligosaccharide fractions ready for activation and coupling to the carrier protein. In a first step, the elongation modes of recombinant capsular polymerases from serogroups A (CsaB) and X (CsxA) were characterized. We observed that CsaB is a distributive enzyme, and CsxA is a processive enzyme. Sequence comparison of these two family proteins revealed a C-terminal extension in CsxA, which conferred processivity because of the existence of a second product-binding site. Deletion of the C-terminal domain converted CsxA into a distributive enzyme, allowing facile control of product length by adjusting the ratio of donor to acceptor sugars. Solid-phase fixation of the engineered capsular polymerases enabled rapid production of capsular polysaccharides with high yield and purity. In summary, the tools developed here provide critical steps toward reducing the cost of conjugate vaccine production, which will increase access in regions with the greatest need. Our work also facilitates efforts to study the relationship between oligosaccharide size and antigenicity.

Studies on the polymerization mode of Neisseria meningitidis serogroup X capsular polymerase CsxA recently identified a truncated construct that can be immobilized and used for length controlled on-column production of oligosaccharides. Here, we combined the use of a synthetic acceptor bearing an appendix for carrier protein conjugation and the on-column process to a novel chemo-enzymatic strategy. After protein coupling of the size optimized oligosaccharide produced by the one-pot elongation procedure, we obtained a more homogeneous glycoconjugate compared to the one previously described starting from the natural polysaccharide. Mice immunized with the conjugated fully synthetic oligomer elicited functional antibodies comparable to controls immunized with the current benchmark MenX glycoconjugates prepared from the natural capsule polymer or from fragments of it enzymatically elongated. This pathogen-free technology allows the fast total in vitro construction of predefined bacterial polysaccharide fragments. Compared to conventional synthetic protocols, the procedure is more expeditious and drastically reduces the number of purification steps to achieve the oligomers. Furthermore, the presence of a linker for conjugation in the synthetic acceptor minimizes manipulations on the enzymatically produced glycan prior to protein conjugation. This approach enriches the methods for fast construction of complex bacterial carbohydrates.

Rotaviruses are a major cause of severe infantile gastroenteritis in humans and animals worldwide, producing a childhood mortality exceeding 650 000 annually.[1] Mapping host cell glycan-virus interactions to define a viral glycointeractome is invaluable in providing new directions for the discovery of novel broad-spectrum drugs and vaccines. In that context we have recently reported the first NMR-based structural analysis of the interaction of GD1a (1) and GM1 (2) ganglioside glycans with recombinantly expressed rotaviral surface lectin VP8* from two distinct rotavirus strains. [2] In that study we demonstrated the absolute requirement for sialic acid (Sia) and identified other subterminal carbohydrates, such as galactose (Gal), in host cell glycan-virus recognition mediated through the rotavirus VP8*. In the present work we have addressed a gap in our initial study and contribute to rotavirus glycobiology [3][4][5] with another important ganglioside rotavirus receptor, GM3. Furthermore, employing intact, infectious rotavirus particles, we also present our investigation on the influence of multiple copies of VP8* (higher protein valency) and trypsin activation [6,7] of virus on glycan recognition. Thus, herein we report saturation transfer difference (STD) NMR and cell-based experiments that reveal novel structural and functional insight into the interactions of the ganglioside GM3 glycan (3, a-GM3, Neu5Aca(2,3)Galb(1,4)Glc) with Rhesus rotavirus (RRV) particles.STD NMR spectroscopy [8,9] is an ideal tool to study the interaction between virus particles and ligands because the broad NMR signal linewidth of virions enables saturation without affecting ligand signals. A very limited number of STD NMR spectroscopic studies using intact virions or viruslike particles (VLPs), such as human rhinovirus, [10] H5-containing avian influenza VLPs, [11] and rabbit hemorrhagic disease VLPs, [12] have been published. A particular advantage of using whole virions or virus-like particles is that any contribution of the viral capsid environment in protein organization and function will be taken into account. This methodology provides a more biologically relevant model for the study of interactions between the virus and the host cell glycans.The 1 H NMR spectrum of 3 (Figure 1 a) and the STD NMR spectra of 3 when bound to RRV particles not treated with trypsin (Figure 1 b) and trypsin-activated (Figure 1 c) clearly reveals that RRV particles bind to 3. Very strong STD NMR signals for the methyl protons of the N-acetyl group (NHAc, d = 1.95 ppm) are observed, and both trypsin-treated and untreated RRV particles bind to 3. Furthermore, clear STD NMR signals are also observed for both the axial (H3 ax ) and the equatorial protons (H3 eq ) of Neu5Ac. A detailed comparison of these STD NMR spectra (Figure 1 b and c) also reveals that the binding epitope of 3 when bound to trypsintreated and untreated RRV particles is similar, if not identical. This important observation clearly suggests that trypsin activation of rotavirus particles i...