Meningitis Vaccine Research

The Health Protection Agency (HPA) Meningococcal Reference Unit (MRU) for England and Wales has been based in Manchester since 1978. Originally established to provide phenotypic characterisation of Neisseria meningitidis isolated in laboratories throughout the country, the MRU also pioneered non-culture confirmation by PCR, which has been available across the UK for almost 10 years. Meningococcal bacteria are usually classified based upon serological reactivity of their polysaccharide capsules - serogroups A, B, and C are currently the most common causes of meningococcal disease (bacterial meningitis and septicaemia).

Fig 1: The meningitis system in action

Dr Ray Borrow heads the Vaccine Evaluation Department at the HPA MRU, which played a key role in the UK’s meningococcal serogroup C (Men C) conjugate vaccine trials in the mid-1990s. The UK was the first country to licence the vaccine and has seen an over 90% reduction in serogroup C disease. However, the incidence of Group B has been unaffected by the Men C vaccine and in the UK there has been a rising trend over the last decade.

The Vaccine Evaluation Department is using the Serum Bactericidal Assay (SBA) to: measure functional antibody levels following Men C vaccine; standardise testing as part of multi-centre trials for a serogroup B vaccine; and to analyse serogroup A samples for the World Health Organisation (WHO) Meningitis Vaccine Project (MVP) (http://www.meningvax.org/). Serogroup A meningococcal infection is almost unknown in the UK and Ireland, but is prevalent in sub-Saharan Africa, Pakistan, Nepal, Bhutan and parts of India. The mission of MVP is to eliminate epidemic meningitis as a public heath problem in sub-Saharan Africa.

Serum bactericidal antibody (SBA) activity was first shown in 1969 to correlate with immunity to meningococcal disease^1,2. Induction of complement-dependent bactericidal antibodies after vaccination with meningococcal polysaccharide or protein conjugate vaccines is regarded as acceptable evidence of the potential efficacy of these vaccines³. In 1976, the World Health Organisation (WHO) Expert Committee on Biological Standardisation recommended a SBA assay to satisfy the requirements for the production and release of meningococcal polysaccharide vaccine. Multi-laboratory studies have standardised SBAs for serogroups A and C⁴, and one has just been completed for serogroup B prior to trials of a specific vaccine for New Zealand to address the country's ongoing epidemic⁵ (http://www.immunise.moh.govt.nz/).

The SBA assay works by killing the target meningococcal strain in the presence of a specific antibody and complement (a large group of serum proteins activated in sequence when cells are exposed to a foreign substance). Serial dilutions of vaccinated patient sera are incubated with appropriate target strains and complement. The serum bactericidal titre for each unknown serum is expressed as the reciprocal serum dilution yielding ≥50% killing as compared to the number of viable cells prior to incubation.

Fig 2: Inverted screen image of plate with frame and detected colonies

The 'Tilt' method is used in the assay to plate out the serial dilutions of sera and bacteria on to the surface of the culture medium, usually blood agar, in a standard 90 mm plate. The plate is then tilted through 45 degrees to allow drops to run down the plate and form lanes. Following overnight incubation at 37°C, the number of colonies that have grown in each of the titre lanes are counted using the Sorcerer automatic colony counter from Perceptive Instruments. During the Men C conjugate vaccine trials it became apparent that the previously performed manual colony counting was no longer a valid option for the processing of high-throughput SBA results and the laboratory invested in the labour-saving system to provide the rapid and accurate results required. Up to 64 sera can be analysed per day by a single lab worker.

The colony counter uses a 25 mm fixed focus lens and monochrome CCD camera to obtain a live image of the plate, which is shown on the computer display. The meningococcal colonies are often confluent i.e. overlap with each other, and this was a limitation when considering conventional colony counters as multiple colonies could be counted as single ones. Colony separation algorithms are automatically applied by the Sorcerer system to ensure precise counting with the aid of angled spot illumination to highlight the convex shape of the colonies.

The inverted image of the plate displays highlighted colonies in colour. After counting the first lane, the plate is then moved by hand to position the next lane in the frame, where identical parameters are applied to the counting process. Results are shown on screen after each measurement and appear instantly in a Microsoft Excel workbook. The count data is saved as a Microsoft Excel file for analysis and storage using in-house macros. It is possible to open up an Excel workbook containing sample codes, dilution factors etc. and instruct Sorcerer to carry out measurements directly. Perceptive Instruments' colony counters have been used in the Manchester laboratory for the 'Tilt' method since 1997 and have also proved invaluable in laboratories in Norway, New Zealand, Niger, and the USA as part of the global effort to develop effective meningitis vaccines.

Even with the advent of molecular diagnostic tests such as DNA amplification and sequencing from isolates and clinical specimens, SBA remains the gold standard for monitoring meningococcal vaccine efficacy and automated colony counting continues to play a crucial role in a process that spans a wide range of international projects.

Meningococci are usually classified by the serological activity of their polysaccharide capsules. Most cases of meningococcal disease in the UK are caused by serogroup B for which there is no available vaccine. Most remaining cases are due to serogroup C for which an effective vaccine was introduced in 1999. Serogroup A meningococcal infection is prevalent in sub-Saharan Africa and a new vaccine against this serogroup is currently being trialled in Africa.

The development and evaluation of new meningococcal vaccines is an ongoing process particularly for serogroups A and B. Perceptive Instruments colony counting systems are used in laboratories in England, Finland, The Netherlands, Norway, New Zealand, Niger and the USA to evaluate the immunogenicity of these vaccines.

The colony counter uses a 25 mm fixed focus lens and monochrome CCD camera to obtain a live image of the plate, which is shown on the computer display. The meningococcal colonies often overlap with each other but accurate enumeration is facilitated by using angled spot illumination which highlights their convex shape.

Automatic colony separation algorithms are also applied. Fig 2 shows the inverted image of the plate showing colonies as dark objects with a coloured detection overlay. After counting the first lane, the plate is then moved by hand to position the next lane in the frame to ensure consistent, optimised illumination.

The count data is saved as a Microsoft Excel file for the necessary data processing and storage to be performed. The colony counter has been used in the laboratory for the tilt method since 1997 and has proved to be accurate, reliable and labour saving.

References

1. Gotschlich E.C., Goldschneider I, and Artenstein M.S (1969). Human immunity to the meningococcus IV. Immunogenicity of serogroup A and serogroup C polysaccharides in human volunteers. J. Exp. Med. 129, 1367-1384.

2. Goldschneider I, Gotschlich E.C, and Artenstein M.S (1969). Human immunity to the meningococcus II. Development of natural immunity. J. Exp. Med. 129, 1327-1348.

3. Goldschneider I, Gotschlich E.C, and Artenstein M.S (1969). Human immunity to the meningococcus I. Development of natural immunity. J. Exp. Med. 129, 1307-1326.

4. Maslanka S.E., Gheesling L.L., LiButti D.E. et al. (1997). Standardization and a multilaboratory comparison of Neisseria meningitides serogroup A and C serum bactericidal assays. Clin. Diag. Lab. Immunol. 4, 156-167

5. Borrow R, Aaberge I.S., Santos G et al. (2005) Interlaboratory standardization of the measurement of serum bactericidal activity using human complement against meningococcal serogroup B, strain 44/76-SL, before and after vaccination with the Norwegian MenBvac OMV vaccine. Clin. Diag. Lab. Immunol. 12, 970-976.

Meningitis Vaccine Research

References

Useful links