One of the principal challenges for both meningococcal vaccine developers and regulators is the development of a suitable strategy for the evaluation of functional antibody responses to new protein-based vaccine candidates. Most virulent Neisseria meningitidis isolates belong to one of five serogroups (A, B, C, Y and W-135) that are defined by the immunochemistry of their capsular polysaccharides. With the exception of group B, vaccines based on these capsular antigens have been available for many years, offering protection to those over the age of 2 years who are at particular risk of disease. More recently, with the development of conjugate vaccine technology, vaccines offering protection to infants and providing herd immunity have also been introduced into immunization programs worldwide. The poor immunogenicity of group B polysaccharide, even when conjugated to a carrier protein, and its similarity to glycosylated structures on human cells has caused vaccine developers to look to protein antigens for the solution to the problem of group B disease. As a result, vaccine candidates based on detergent extracted outer membrane vesicles (OMVs) and on purified protein antigens are at an advanced stage of clinical development Citation[1].
The comparatively low burden of group B meningococcal disease makes the conventional assessment of vaccine efficacy in placebo-controlled protection studies unrealistic. Instead, regulatory approval of new vaccines will be based on an immunological correlate of protection. The bactericidal activity of antibody and complement plays an important role in protecting the host against meningococcal disease Citation[2,3]. Evidence from a number of efficacy studies of OMV vaccines suggests that the level of serum bactericidal antibody (SBA) is a reliable surrogate for predicting the effectiveness of a protein-based meningococcal vaccine Citation[3]. A consultation involving world leaders in meningococcal vaccines, held in Atlanta in 2005, concluded that the use of SBA level as a surrogate for protection is appropriate in the assessment of candidate vaccine efficacy for regulatory approval Citation[4].
In common with other assays that measure functional antibody responses, the meningococcal SBA assay is complex and difficult to standardize. In essence, it consists of incubating dilutions of serum from vaccinees with an exogenous complement source and the target meningococcal cells, and then determining the number of surviving bacteria after a fixed period. Predictably, most problems encountered with the assay are associated with its most variable components, the meningococcal isolates and the source of complement. A major challenge for those using the assay is to match appropriate isolates with a complement source so as to obtain reliable SBA titers while maintaining a high level of bacterial survival in controls lacking serum from vaccinees.
Meningococcal isolates are genetically and antigenically diverse, reflecting the complex population structure of this organism Citation[5,6]. Thus, an assessment of the breadth of coverage of a protein-based vaccine candidate against a range of isolates forms an essential part of its clinical evaluation. A combination of accumulating mutations and horizontal genetic exchange has given rise to a level of antigenic diversity among protein antigens that appears to be too extensive to test comprehensively in SBA assays. This raises the questions of which and how many isolates need to be included in such studies. In general, variants of a particular protein antigen fall into broad families with similar amino acid sequences. Differences within families consist of small numbers of amino acid changes localized within epitopes. In addition, the phenotypic expression of meningococcal surface proteins is often tightly controlled; many proteins are phase-variable, are the products of contingency genes or are regulated at the transcription level in response to environmental stimuli Citation[7–10]. Thus, not only the choice of isolates but also how they are grown and maintained in the laboratory can have an impact on the results of SBA assays. Differences in the behavior of genetically similar and even identical isolates can be explained by such phenotypic differences Citation[11,12].
Since meningococci have evolved specific mechanisms to mitigate the impact of human complement on their survival, such as, for example, the expression of a complement factor H-binding protein Citation[13,14], only human serum is an acceptable source of complement for the clinical evaluation of candidate vaccines. The meningococcus has a commensal relationship with man, and most adults experience several episodes of meningococcal carriage during their life Citation[5]. As a result, human serum usually contains antibodies to meningococcal antigens and is likely to be inherently bactericidal for some isolates, even in the absence of additional serum antibodies from vaccinees. Typically, serum from an individual is only useful as a complement source in conjunction with one or a limited number of meningococcal isolates, and the range of isolates that can be used differs from one complement source to another. Taken together with the amount of complement required for large clinical studies, this complexity tends to confound attempts to demonstrate the breadth of coverage offered by vaccine candidates to antigenically diverse isolates using a single complement source.
Despite these challenges, there is reason to be optimistic that scientifically robust strategies, acceptable to licensing authorities, can be developed for the evaluation of these vaccines. In the absence of clinical evidence of protection, the SBA assay will remain at the core of such strategies, but the composition of the strain panels employed has to reflect the scientific question being addressed. For example, the simplest question is whether a given antigen elicits an SBA response. This can be tested with a single isolate expressing the homologous variant of the antigen. By contrast, a panel of isolates has to be used to assess more complex issues such as the breadth of vaccine coverage against diverse invasive isolates, or the impact of differences in antigen amino acid sequence and expression level on potential protection.
To provide evidence for the prospective breadth of coverage of a new vaccine, the panels of isolates have to consist of organisms that are typical of those responsible for the majority of cases of invasive disease. Given the limited supply of suitable complement, these panels cannot be extensive and there should be a rational approach to their design. Fortunately, the molecular epidemiology of the meningococcus is well-understood and provides a good basis for this process. The ability to cause meningococcal disease is limited to a comparatively small number of clonal complexes, the so-called ‘hyperinvasive lineages’, each expressing a characteristic repertoire of antigen variants Citation[5]. Antigenic structuring in meningococcal populations limits the repertoire of variants associated with particular clonal complexes and thereby offers a way to restrict the number of representatives of each hyperinvasive lineage required to assess vaccine coverage. For example, the clonal complex ST-269, which is commonly associated with invasive infections, typically expresses one of two PorA variants (P1.22,9 or P1.19-1,15-11), the F5–1 variant of FetA and variant B44 (also known as 1.15) of factor H-binding protein Citation[15]. Thus, the majority of ST-269 disease could theoretically be represented by as few as two isolates. The hyperinvasive lineages and their association with particular repertoires of antigen variants are stable over many years, suggesting that the composition of well-designed panels of isolates would not have to be changed substantially for long periods. Although atypical variants of antigens arise frequently, they do not persist in meningococcal populations and therefore do not warrant inclusion among isolates used to address the issue of coverage.
When assessing the impact of specific differences between antigens, for instance, differences in amino acid sequence or level of expression, there is a strong case for using an isogenic panel of strains that only differ from one another in the antigen gene of interest. This should ensure, as far as possible, that all other strain-related factors that might affect the SBA titer remain constant in the assay. This approach has been used to compare the bactericidal activity of sera from clinical studies against different variants of the PorA antigen Citation[16].
Although the level of SBA in serum is the key surrogate for protection and its measurement a critical part of vaccine evaluation, there remains much that can be learned from antibody-binding studies, thereby reducing the requirement for a large supply of complement. The capacity of antibody to mediate bactericidal activity is largely dependent upon four factors: the class and subclass of the antigen-specific immunoglobulin; antibody concentration; antibody avidity; and the density of target epitopes on the bacterial surface. These data can realistically be acquired for much larger collections of isolates and used to support SBA data obtained with limited panels of isolates.
The vaccine developers whose products have been in clinical trials have adopted some aspects of the approaches outlined here into their current vaccine evaluation strategies. It is anticipated that their data will form a sound basis for regulatory decisions on vaccine effectiveness. However, to date, each has used their own distinct panels of meningococcal isolates. Looking to the future, a standard panel of isolates will be required when comparing the effectiveness of these so-called ‘MenB’ vaccines (the protein antigen components are in fact present among meningococci of all serogroups) in head-to-head trials such as, for example, noninferiority studies. Arguably, this panel should be assembled and maintained by an independent laboratory.
Financial & competing interests disclosure
The author holds a number of patents for meningococcal vaccine developments for which no royalties are received. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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