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Meningitis vaccines consist of mixtures of complex high-molecular-weight polysaccharides from four sero groups that can be difficult to characterise, but a new capillary zone electrophoresis method offers a simple and rapid way to confirm and quantify the different components. Meningitis is a killer disease that affects people of all ages, but is more likely to strike infants and children. It strikes quickly and can progress from early symptoms to death in as little as 24 hours. Of the two main types of the disease, viral and bacterial meningitis, the viral form is nasty but is not life threatening. Bacterial meningitis is the lethal form, accounting for around 171,000 deaths worldwide each year, according to the WHO . The fatality rates are around 5-10% in industrialised countries and greater in the developing world. The disease is an inflammation of the meninges, the membranes that cover the brain and spinal cord, and is brought about by everyday bacteria and viruses that are normally associated with other illnesses. Bacteria like Escherichia coli, Group B Streptococcus and Listeria monocytogenes and viruses such as enteroviruses (that usually bring on sore throats, colds and flu-like illnesses), mumps, measles and herpes are typical agents. Once they spread into the bloodstream, usually via the gastrointestinal tract, they can enter the meninges and bring on an infinitely more serious condition. One of the most common bacterial agents is Neisseria meningitides. The bacterium currently has 13 known sero groups , of which A, B, C, W135 and Y cause the most meningitis infections. However, there are effective vaccines to immunise people at risk. The major meningococcal vaccine in the US protects against four of these five groups, but there is no known vaccine to protect against type B. The vaccine contains mixtures of native capsular polysaccharides from groups A, C, W135 and Y, each one having a molecular mass 200,000 or greater. They comprise either unbranched homopolymers of mannosamine phosphate (group A) or sialic acid (C), or heteropolymers of sialic acid with galactose (W165) or with glucose (Y). Quality control of these vaccines is difficult, involving separation of the polysaccharides followed by quantitation. In order to circumvent the acid digestion step associated with several current methods, US researchers have developed a simple and rapid method based on free-solution capillary zone electrophoresis (CZE). David Lamb and colleagues from Aventis Pasteur, renamed sanofi pasteur in January 2005, described their technique in Anal. Biochem. 2005 . Vaccine powders were dissolved in water or dilute sodium chloride and injected directly into a bare silica capillary. A separation buffer consisting of sodium phosphate and sodium borate in aqueous methanol brought about clear separation of the 4 sero groups within 30 minutes. Even the closely related W135 and Y groups were separated with no overlap. It had been thought that the resolution of these two types was based on differential binding with borate but good separation was still observed in a phosphate solution in the absence of borate, so another separation mechanism, as yet undefined, must be at play. When the separation conditions such as temperature and ionic strength were varied, the predicted behaviour was observed, illustrating the robustness of the method. For example, increasing the temperature from 20 to 40 °C brought the 4 peaks closer together, although still separated, but higher temperatures introduced some distortion of peak shape. Apart from this range of temperatures, a range of ionic strengths of the buffers could also be accommodated. Polysaccharide quantification by CZE with UV detection showed good agreement with two wet chemistry methods based on the colorimetric measurement of sialic acid or phosphorus determination (for sero group A). Bearing in mind that the wet methods require preliminary hydrolysis of the polysaccharides with strong acids at elevated temperatures, the CZE method is preferable. Several tests for the sero groups based on the use of monospecific antibodies also exist but the antisera are expensive to purchase or are costly and time consuming to prepare. In addition, cross-reactivity between some sera prepared against W135 and Y can confuse quantification. So the CZE method offers several advantages over existing methods, including speed, cost, complexity and sample preparation and is an excellent alternative for the determination of meningococcal polysaccharides.
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Article by Steve Down |
 Infants under one year of age have the greatest incidence of meningitis  If the rash does not fade when pressed with a glass, it is a good indication of meningococcal septicaemia. Image: courtesy Meningitis Research Foundation
 Photomicrograph of Neisseria meningitidis recovered from the urethra of an asymptomatic male. Image: Courtesy CDC Public Health Image Library
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