Development Of Improved Bioprocessing Methods For The Manufacture Of A Vaccine Against Meningococcal Meningitis.

Abstract

Neisseria meningitidis is the causative agent of meningococcal meningitis and septicaemia. Strains of N.meningitidis are conventionally divided according to their polysaccharide capsule and, for the majority of serogroups, there is an effective vaccine available. However, serogroup B produces a polysaccharide capsule based on a sialic acid polymer which is similar in structure to human cell surface polysaccharides and, therefore, cannot be used as the basis for a vaccine against this serogroup. Instead, efforts to produce a protective vaccine have focused on outer membrane protein antigens. Several candidate antigens have been tested and incorporated into commercial vaccines licensed recently. However, both coverage and cost are limiting factors for this new generation of vaccines. This work sought to examine whether alternative methods of antigen processing and presentation could assist in improving meningococcal vaccine efficacy. Virus-Like Particles (VLPs) consist of self-folding and self-assembling polymers of viral capsid proteins. They are able to incorporate heterologous antigens and have been shown to induce protective immunity against several different pathogens. This project used the core protein of Hepatitis B Virus, HBcAg, which forms the viral nucleocapsid and can be readily engineered to incorporate heterologous antigens. Five constructs which incorporated meningococcal outer membrane protein antigens were designed, yielding only three that were amenable to purification. The C-terminus of Factor H binding protein (fHbp), an antigen previously identified by reverse vaccinology, was incorporated into the C-terminus of HBc. This construct was able to elicit reactive IgG antibodies (subclasses IgG1, IgG2a and IgG2b) in mice but low levels of serum bactericidal activity (SBA), which is the accepted correlate of protection for meningococcal vaccines. Examination of the HBc-fHbp construct by cryoelectron microscopy confirmed that the packing of the HBc was unchanged, but showed a lack of density for fHbp, suggesting that the poor level of SBA was attributable to loss of conformational epitopes. Two other constructs were designed which incorporated part of the adhesin NadA into the major immunodominant region of HBc, with and without fHbp at the C-terminus. These constructs were able to elicit reactive IgG antibodies (subclasses IgG1, IgG2a, IgG2b) in mice and also good levels of SBA titres. It is suggested that this improved immune response was attributable to correct folding of conformational epitopes within the antigen. Further evidence was obtained from cryo-electron microscopy analysis, which identified clear density for the inserted NadA antigen. Integral membrane protein antigens- in particular PorA, the major protective antigen within the outer membrane- require stabilisation within a lipophilic formulation. A VLP is not generally a viable choice of platform for display of antigens from this group of proteins, due to the complex folding conditions required. Liposomes were here employed as an alternative display platform to VLPs in the development of a membrane protein refolding protocol that was designed to be more appropriate for scale-up to an industrial level. Previous experiments with liposomes have utilised procedures that rely on expensive or specialist detergents, many of which must also be removed entirely from the end product to be fit for use in humans. The use of human and industry-compatible reagents for the refolding of the major Neisseria antigen, PorA, was here compared to material produced by earlier established methods. It was not possible, within the bounds of these experiments, to exhaust all of the possible combinations of refolding conditions; however, those trialled here, utilising the alternate detergents, were not able to yield correctly refolded material when analysed by circular dichroism, nor was this material immunogenic.

Date of Award	1 Aug 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jeremy Derrick (Supervisor) & Alan Dickson (Supervisor)