Studying antibody composition after infection with Neisseria lactamica
Dr Nigel Saunders, Brunel University
Dr Saunders and his team will use extremely sensitive laboratory techniques to identify new antigens, which may be able to stimulate an immune response.
With previously available sampling methods, the concentrations of antibodies to some minor bacterial antigens were too low to be measured, but Dr Saunders and his team have developed a new method which is up to 10,000 times more sensitive and, therefore, will measure the antibodies previously missed. Using this method, the team hope to identify lesser known antigens involved in stimulating immunity against Neisseria lactamica, since immunity against this bacterium also protects from invasive Neisseria meningitidis infection.
The research team plan to use the new technology to investigate blood samples from current clinical trials, delving deeper into them than was previously possible, and hopefully discovering new antigens that were previously too small to detect.
This research will generate a tool to study meningococcal immunity and vaccine responses in detail. Furthermore, it may yield new, previously unknown, vaccine candidates that are able to stimulate immunity against Neisseria meningitidis.
Completion of the Protein Bank
The research project has achieved a major breakthrough that has the potential to impact the field of research. A major aim was to prepare surface protein antigens that could be used to generate the microarray (see below). This was a technically challenging undertaking, given the number of proteins proposed. In addition, issues over solubility hampered the production of protein, but in looking for a solution, Dr Saunders found that, by generating the proteins solubilised and purified in urea, there was a greater stability than found in the original approach. All the relevant proteins have been successfully manufactured, and the new technique has the potential for broad application. Essentially, it means that scientists can now work with proteins that are poorly soluble and form solid inclusions (proteins), opening up this technique to the most problematic antigen targets, such as the immunologically important surface proteins. It also means that the proteins can be stored and handled at room temperature in a form which is far more stable than would otherwise be the case, and thus have to be produced less frequently and more easily. These properties extend the shelf-life of the microarrays and make them easier to use. Importantly, these proteins and the arrays can be distributed to other groups without the need for special transport conditions and storage. This is an important attribute because it means that data can be generated by many other laboratories working on vaccines.
Development of a New Micoarray
The microarray system uses a physical template onto which the individual protein antigens (see above) are ‘spotted’ and onto which clinical or laboratory animal serum samples can be applied. If the serum contains antibodies that specifically recognise any of the Meningitis B protein antigens, this will be shown by an increase in colour reaction (a positive reaction). A key objective of Dr Saunders’ study was to therefore use the protein antigen microarray strategy to begin to determine those proteins that can induce cross-protective immunity to N. meningitidis after natural colonisation and also after natural colonisation or vaccination with the non-pathogen N. lactamica. Essentially, the microarray could be used to identify, by screening large numbers of clinical serum samples, those antigens that were commonly recognised. In theory, these commonly-recognised Meningitis B antigens would be able, if used in a vaccine, to provide protection against a wide variety of different meningococci.
However, before screening a large number of clinical samples, it was important to validate the microarray detection system. The system is reproducible and works with only one microlitre of serum and can measure responses to 90 proteins at the same time, which considerably increases the speed at which samples can be tested. This is a significant achievement, given that researchers usually work with far fewer proteins, sometimes even just single proteins, when analysing serum samples, and often using larger volumes of sera as well. In addition, Dr Saunders’ microarray is also 10,000 times more sensitive than existing methods and, therefore, will detect proteins previously missed.
Using the New Microarray
As was previously mentioned, the microarray platform needs to be validated before it can be used for screening large numbers of human clinical samples. Dr Saunders has done the preliminary hybridisations, i.e. reacted serum with the microarray, and the early results show features of the anti-neisserial response that are genuinely novel, and potentially functionally important. Provisionally, it would appear that the antibody profiles of different individuals may have different patterns of immune reactivity, thus showing inter-individual differences in antibodies directed against Meningitis B organisms. An example of the microarray ‘in action’ is shown in the figure below: the panels show the reaction of two different serum samples. On the left, the circles surround two Meningitis B proteins (shown as stars) that have specifically reacted with serum (the sample has been tested in triplicate) whereas on the right we can see one Meningitis B protein (shown as a single star and the sample tested also tested in triplicate)’.
.jpg "Working with the protein array printers")
Page Updated: 27.01.12
Outcomes will be shown here once the project is complete.
Microserological determination of N. lactamica induced cross-protective meningococcal immunity
Dr Nigel Saunders, Brunel University
Dr Saunders and his team will use extremely sensitive laboratory techniques to identify new antigens, which may be able to stimulate an immune response.
The antigens that generate protective immunity following immunisation, or infection by pathogens or commensal colonisation are unknown. This has been experimentally intractable due to the limits of sensitivity of assays such as ELISA and Western blotting. Dr Saunders and his team have solved problems with detection by using protein microarrays that are 10,000 times more sensitive than previous methods. Protein microarray methods were refined, developed and tested to yield a new, highly sensitive ‘dendrimer-based’ detection system. This is capable of detecting resting antibodies in donated sera against highly dilute and denatured fractionated bacterial proteins.
To-date no project has systematically determined the antigen-specific responses that comprise either, the naturally Outer Membrane Vesicle vaccine, or Neisseria lactamica-induced protective response.
The team plan to generate a meningococcal antigen protein microarray, and use this to assess naturally occurring and protective immunity as measured and induced in vaccine trials using Neisseria lactamica.
The responses will be interpreted in the context of various assays as correlates of protection. The team will be using the natural host response to determine the antigens that are most important and effective within both live and component vaccines.
By defining the targets of the cross-strain and cross-species protective antibodies, the team will obtain key information relevant for the production of component vaccines, improved Outer Membrane Vesicle vaccines, and for the characterisation of the protective immune reaction developed in response to natural exposure and vaccination.
The purpose of this project is to establish a new tool that will be used:
i. to characterise reactions in response to Neisseria lactamica Outer Membrane Vesicle vaccines
ii. to compare this with the targets of immunity following natural infection, and
iii. to monitor and compare responses to any future vaccines.
One of the two protein array printers
The protein clone set and establishment / production of the test proteins is essentially complete. This excludes two proteins that could not be produced in decent yields, but as these were surface proteins and were not strong vaccine candidates, their omission does not affect the objectives of the project. As expected, there were substantial solubility issues and challenges producing some of the proteins that were not expressing. These were predominantly surface proteins with multiple trans-membrane domains. This presented real issues, the solution of which presents a significant breakthrough for this and related projects, as well as ultimately improving the performance of the microarray.
The ‘solution’ was to generate these proteins solubilised and purified in urea. A protocol was subsequently developed to print and re-fold these proteins on the microarray surface, using progressive reductions in urea concentrations. Originally, this protocol was established using green fluorescent protein, and re-generation of its native fluorescence. Parallel studies were then performed on the re-folding and antigenic features of the proteins using control sera from laboratory donors with high anti-meningococcal killing.
To compare the performance of the proteins that were soluble and those that had to be generated in urea the proteins that were soluble were processed in both ways. It was expected that the urea-processed samples would perform less well (but adequately), but the result was better than this. The performance of the proteins printed following re-folding was actually better and more consistent than those that were produced, stored, and printed in their non-denatured state. Notably, the team were able to measure immune responses to several proteins that were originally soluble following urea printing and re-folding, that did not generate detectable signals using the original approach. This is interpreted to reflect that there is a greater stability of the proteins while they are in urea and are less prone to chemical and auto-proteolytic damage. Also, the hybridisation to the sera occurs immediately after the proteins have been re-folded, so there is little time for degradation prior to serological detection.
This means that researchers can now work with proteins that are poorly soluble and form inclusions, opening up this technique to the most problematic targets (and essentially the immunologically important surface proteins). Furthermore, it means that the proteins can be stored and handled at room temperature in a form which is far more stable than would otherwise be the case, and thus have to be produced less frequently and more easily. The proteins printed onto the microarrays have a longer half-life (at room temperature) because the proteins remain in the protected / denatured state prior to re-folding during the processing / serological detection protocol, and perform well up to a week which makes their use easier, and means that they could be distributed to other groups in the future. This approach also generates data for a larger number of targets than any previously used.
Preliminary hybridisations have been performed with results which show features of the anti-neisserial response which are genuinely novel, and potentially functionally important. These will be established in detail in the last phase of the project, but the primary point is that there are substantial inter-individual differences in the cross-protective targets for which antibodies are present. These include both probably protective and potentially blocking antibody targets, and the spectrum and relative abundance of these different targets and types of response have never been something that could be addressed previously. This is illustrated below, with the sample on the left including a number of common reactive components plus clear sample-specific responses to two proteins (marked with white stars, in triplicate) in the serum sample assessed on the left, and one protein in serum assessed on the right.
What the above illustrates is remaining issue of ‘tearing’, which occurs during the manual processing of these protein microarrays. This has been addressed by changing the print buffers, which has been facilitated by the change to printing denatured proteins, and by the acquisition of an automated processing station in which the team can now process the arrays for the re-folding and serological assessment in two consecutive protocols. It is now possible to generate cleaner / quantitative data for these responses.
Page Updated: 27.01.12
Outcomes will be shown here once the project is complete.
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