Identifying proteins on meningitis B bacteria that are important in stimulating protective immune responses
Professor John Heckels, University of Southampton
Professor Heckels and his team investigated blood samples from individuals, before and after meningitis B infection, to identify new antigens involved in the development of natural immunity. To read about Professor Heckels' current research entitled "Evaluating proteins on Meningitis B bacteria for use in a vaccine".
The introduction of the Meningitis type C (MenC) vaccine into the UK immunisation program resulted in a dramatic decline in cases of meningitis and septicaemia caused by serogroup C meningococci. Professor Heckels recognised that the development of an effective vaccine for prevention of serogroup B meningococcal (Men B) infection was now the most important goal for meningitis research.
It is known that most people develop immunity to meningococcal infection naturally. This happens when individuals become colonised and carry meningococci in their nasopharynx. Around 10% of the population carry the meningococcal bacteria, which normally live harmlessly in the back of the nose and throat. For some reason, in certain cases the bacteria go beyond colonisation and cross over into the bloodstream to cause serious and life-threatening disease.
Professor Heckels’ team collected blood samples from students before and after they became carriers of the bacteria which, as explained by Professor Heckels, “is an extremely valuable resource which can be used to identify which antigens are associated with the production of protective immunity as these would then be attractive candidates for the production of a vaccine.”
Professor Heckels and his research team had blood samples from individuals before and after they became carriers of Meningitis type B (MenB) and planned to use this resource:
i. to identify the antigens that are associated with the production of protective immunity by looking at the changes that have occurred in the sera of individuals before and after infection
ii. to manufacture these antigens in the laboratory andiii. to test them for their ability to produce protective antibodies
Such antigens would be attractive candidates for the production of a vaccine designed to prevent serogroup B meningococcal infections.
Through this project, new candidates for a vaccine against MenB were identified, which could ultimately lead to a vaccine to protect from the disease.
This project is complete - see outcomes for more detail
During Years 1 & 2 of a project funded by MenUK, the researchers used a biochemical technique called immunoproteomics to identify the meningococcal proteins that reacted specifically with sera from colonised individuals who subsequently developed protective immunity to meningococci. This protection was judged by the ability of their serum to kill meningococci in the gold-standard serum bactericidal activity (SBA) assay. They were able to identify 43 proteins that were potentially associated with the development of SBA, not only against the strain that colonised the individual but also against other different strains. This suggested that certain proteins could induce antibodies that potentially could kill a broad range of different meningococci.
During Year 3, the researchers prioritised antigens from this list for further studies by excluding proteins that they and others had already worked on (e.g. PorA, PorB, Opa and Opc) and by focusing initially on proteins that were present on the outer surface of the meningococcus and had not been investigated before. They identified 7 such proteins (designated A-G), and work was then directed towards making pure proteins by recombinant DNA technology and using these as experimental vaccines to generate antibodies in animals. Preparation of the recombinant proteins was technically challenging and some proteins were easier to make than others. Thus, the team has finished the analysis of protein F, which was the easiest protein to make and showed that it can induce antibodies that kill the homologous strain, but not different strains. They also have generated antibodies to recombinant proteins A and G and showed that these reacted with the homologous strain. This leaves 4 proteins (B, C, D and E) in various stages of preparation and testing by the team.
In summary, the team successfully identified in this project novel meningococcal proteins with the potential for use as potential Meningitis B vaccines, either singly or more likely combined as a ‘cocktail’. The researchers predict that such vaccines will provide protection against a broad range of different MenB strains and further studies are proposed to validate the findings.
Identification of meningococcal antigens associated with development of cross-reactive immunity following colonisation and infection
Professor John Heckels, University of Southampton
Professor Heckels and his team investigated blood samples from individuals, before and after meningitis B infection, to identify new antigens involved in the development of natural immunity. To read about Professor Heckels' current research entitled "Evaluating proteins on Meningitis B bacteria for use in a vaccine".
The development of an effective vaccine for prevention of serogroup B meningococcal infection is one of the most important goals for current meningitis research. So far, most vaccines shown to have demonstrable protective effect in humans were based on outer membrane vesicles and the protection, as measured by bactericidal activity, was restricted to strains of the same serogroup. However, there is now one other vaccine candidate that has shown promising results in ongoing clinical trials.
In previous studies of meningococcal carriage and infection, the researchers collected matched sera and strains from individuals and determined levels of bactericidal activity against both homologous and heterologous serogroup B meningococcal strains.
A major goal for the development of effective vaccines against serogroup B meningococci is to identify antigens that are capable of inducing a cross-protective immune response against a wide range of strains.
Studies on natural immunity to meningococci and the immune responses of individuals immunised with Outer Membrane Vesicle vaccines, combined with the fact that repeat meningococcal infections are extremely rare in people with fully developed complement systems, suggest that cross-reactive antigens exist but, because of the limits of previous technology, they have not yet been identified.
The researchers planned to utilise the sensitive methods now available, in combination with the well characterised panel of human sera to investigate the antigens associated with development of serum bactericidal activity against serogroup B meningococci and to use this information to investigate the vaccine potential of the promising cross-reactive antigens.
The researchers planned to take advantage of the availability of matched sera taken before and after infection to identify antigens that are associated with the development of bactericidal activity against heterologous serogroup B strains.
The team then aimed to produce the antigens free from other meningococcal components and use them for immunisation. Consequently, they would then be able to identify potential vaccine antigens that had the ability to induce cross-protective immunity to a wide range of serogroup B strains. Such antigens would be attractive candidates, either singly or in combination, for the production of a vaccine designed to prevent serogroup B meningococcal infections.
This project is complete - see outcomes for more detail
YEAR 1
During Year 1 the team developed and refined the methodology for the immunoproteomic study. For each serum sample, OM were subjected to simultaneous 2-D electrophoresis in triplicate. One gel was stained with silver to produce a reference gel. The two unstained 2-D gels were electroblotted onto PVDF membranes and both membranes were stained with the MemCodeTM Protein Stain and scanned to produce an accurate map of spot positions. The membrane stain was then reversed then incubated with serum, at a standard dilution, taken from the volunteer pre-colonisation and post-colonisation. Immunoreactivity was determined using peroxidase-labelled anti-human Ig.
YEAR 2
During Year 2 the team applied this methodology to their resource of strains and sera. Matched sera from individuals from before and after colonisation were subjected to immunoproteomic analysis. Each sample was tested against (1) the homologous strain(s) colonising that individual, (2) the other heterologous serogroup B strains isolated during the study and (3) heterologous strain MC58 for which the genome sequence is available. Membranes were then scanned and the profile of immunoreactive proteins matched to the spot position on the stained membrane and hence to the reference gel of the strain being investigated. The signal intensities of individual antigen reactions were compared and scored. The acquisition of meningococcal carriage was associated with by raised bactericidal antibody activity and was always accompanied by increased immunoreactivity visible on the immunoblots. This development of an immune response was reflected both in a greater number of spots and increased intensity of existing spots. The number, range and relative intensities of immune-reactions to meningococcal proteins varied between students and between strains and included both homologous and heterologous reactivity. In order to focus on cross reacting immune responses the reactivity with strain MC58 was analysed in detail. Twenty seven discrete immunoreactive spots could be detected and these were identified. Protein spots that showed increases in immunoreactivity as well as spots showing high levels of pre-colonisation reactivity were excised from the stained reference gel, subjected to mass spectrometry, and identified by comparison with the MC58 genome and the NCBI databases. This resulted in the identification of 43 proteins potentially associated with the development of SBA against both homologous and heterologous strains. The list of protein immunogens generated included both well-established protein antigens such as PorA, PorB and OpC, as well as novel antigens.
YEAR 3
During Year 3 the team prioritised antigens for further studies based on the relative frequency with which they are associated with the development of heterologous bactericidal activity, the degree of conservation between strains (as revealed by comparison of the available meningococcal genome sequences) and levels of expression. The team also excluded the OM proteins PorA, PorB, Opa and Opc, which they had already studied extensively. They selected 7 proteins (designated A-G) for further study using the same techniques. Firstly the gene of interest was amplified from strain MC58 by PCR. The gene was then cloned into the pRSETA expression vector, transformed into E. coli and the resulting colonies were screened for the presence of the insert. After determination of optimal conditions the protein was expressed and purified by affinity chromatography. The resulting protein was then refolded to a native conformation in artificial membranes (liposomes) or by using detergent to produce protein micelles (proteasomes). The formulations were then used for immunisation of mice, the sera were collected and tested for their ability to induce bactericidal killing of strain MC58.
The team have completed analysis of protein F and shown that it can induce antibodies bactericidal for the homologous strain, but these did not show activity against the heterologous strains tested. They have also generated murine and rabbit antibodies against proteins A and G and these react with the homologous strain. These now need testing for serum bactericidal activity against homologous and heterologous strains. This leaves 4 proteins (B, C, D and E) in various stages of expression and purification. Further work on these proteins will be the subject of a new project application to Meningitis UK.
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