Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X.

Neisseria meningitidis is a major cause of bacterial meningitis worldwide, especially in the African meningitis belt, and has a high associated mortality. The meningococcal serogroups A, W, and X have been responsible for epidemics and almost all cases of meningococcal meningitis in the meningitis belt over the past 12 y. Currently no vaccine is available against meningococcal X (MenX). Because the development of a new vaccine through to licensure takes many years, this leaves Africa vulnerable to new epidemics of MenX meningitis at a time when the epidemiology of meningococcal meningitis on the continent is changing rapidly, following the recent introduction of a glycoconjugate vaccine against serogroup A. Here, we report the development of candidate glycoconjugate vaccines against MenX and preclinical data from their use in animal studies. Following optimization of growth conditions of our seed MenX strain for polysaccharide (PS) production, a scalable purification process was developed yielding high amounts of pure MenX PS. Different glycoconjugates were synthesized by coupling MenX oligosaccharides of varying chain length to CRM197 as carrier protein. Analytical methods were developed for in-process control and determination of purity and consistency of the vaccines. All conjugates induced high anti-MenX PS IgG titers in mice. Antibodies were strongly bactericidal against African MenX isolates. These findings support the further development of glycoconjugate vaccines against MenX and their assessment in clinical trials to produce a vaccine against the one cause of epidemic meningococcal meningitis that currently cannot be prevented by available vaccines.

Development of a New Solid-Phase Extraction Base Method for Free Saccharide Content Estimation of Meningococcal Conjugate Vaccines.

GlaxoSmithKline (GSK) is currently developing a fully liquid presentation to ease the administration of the licensed quadrivalent conjugate vaccine (Menveo) against meningococcal serogroup A, C, W, and Y (MenACWY) infections. Herein, we report a new method for determining the free saccharide (FS) content of CRM197-MenACWY conjugated antigens, with the aim of improving accuracy and reproducibility. Mathematical models have been used to support technical knowledge in reducing the need for experimental development. This results in an improved, faster, and platform-based technique for FS separation with one single pretreatment applicable to all antigens of the multivalent meningococcal vaccine.

Bactericidal Immunity to Salmonella in Africans and Mechanisms Causing Its Failure in HIV Infection.

BACKGROUND: Nontyphoidal strains of Salmonella are a leading cause of death among HIV-infected Africans. Antibody-induced complement-mediated killing protects healthy Africans against Salmonella, but increased levels of anti-lipopolysaccharide (LPS) antibodies in some HIV-infected African adults block this killing. The objective was to understand how these high levels of anti-LPS antibodies interfere with the killing of Salmonella. METHODOLOGY/PRINCIPAL FINDINGS: Sera and affinity-purified antibodies from African HIV-infected adults that failed to kill invasive S. Typhimurium D23580 were compared to sera from HIV-uninfected and HIV-infected subjects with bactericidal activity. The failure of sera from certain HIV-infected subjects to kill Salmonella was found to be due to an inherent inhibitory effect of anti-LPS antibodies. This inhibition was concentration-dependent and strongly associated with IgA and IgG2 anti-LPS antibodies (p<0.0001 for both). IgG anti-LPS antibodies, from sera of HIV-infected individuals that inhibit killing at high concentration, induced killing when diluted. Conversely, IgG, from sera of HIV-uninfected adults that induce killing, inhibited killing when concentrated. IgM anti-LPS antibodies from all subjects also induced Salmonella killing. Finally, the inhibitory effect of high concentrations of anti-LPS antibodies is seen with IgM as well as IgG and IgA. No correlation was found between affinity or avidity, or complement deposition or consumption, and inhibition of killing. CONCLUSION/SIGNIFICANCE: IgG and IgM classes of anti-S. Typhimurium LPS antibodies from HIV-infected and HIV-uninfected individuals are bactericidal, while at very high concentrations, anti-LPS antibodies of all classes inhibit in vitro killing of Salmonella. This could be due to a variety of mechanisms relating to the poor ability of IgA and IgG2 to activate complement, and deposition of complement at sites where it cannot insert in the bacterial membrane. Vaccine trials are required to understand the significance of lack of in vitro killing by anti-LPS antibodies from a minority of HIV-infected individuals with impaired immune homeostasis.

Simplified low-cost production of O-antigen from Salmonella Typhimurium Generalized Modules for Membrane Antigens (GMMA).

The Novartis Vaccines Institute for Global Health is developing vaccines using outer membrane particles, known as Generalized Modules for Membrane Antigens (GMMA). These are blebs of outer membrane and periplasm, shed from the surface of living Gram-negative bacteria following the targeted deletion of proteins involved in maintaining the integrity of the inner and outer membranes. The current study investigates the use of GMMA as starting material for extraction of membrane components, focusing on the O-antigen polysaccharide portion of lipopolysaccharide from Salmonella Typhimurium. We show that the amount of O-antigen extracted from GMMA by acid hydrolysis is comparable to the quantity extracted from whole wild type bacteria, but with less protein and DNA contaminants. Compared to conventional purification, GMMA enabled a reduction in the number of purification steps required to obtain the O-antigen polysaccharide with the same purity. Purification processes from GMMA and bacteria were characterised by similar final yields. Use of GMMA as starting material provides the possibility to simplify the purification process of O-antigen, with a consequent decrease in manufacturing costs of O-antigen-based glyconjugate vaccines against Salmonella strains and potentially other Gram-negative bacteria.

Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography.

Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella and so investigate the functionality and diversity of the antibody response to OAg.

O:2-CRM(197) conjugates against Salmonella Paratyphi A.

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM(197), using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM(197) as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.