MICA: Does phase variation contribute to virulence and immune evasion of Neisseria meningitidis?

Many bacterial pathogens colonise and persist in our bodies as harmless 'commensals' that do not cause disease. These bacteria persist even though our bodies produce antibodies against exposed structures on their surface. In this proposal, we aim to improve our understanding of how bacteria avoid these antibodies and whether these avoidance mechanisms influence the ability of bacteria to cause disease.

Neisseria meningitidis is a bacterium that is a major cause of meningitis and blood-poisoning but is found in 10-30% of people (i.e. 'carriers') as a harmless commensal of the upper respiratory tract. This bacterial species is present on nasal tissues and at the back of the throat where it can persist for up to twelve months despite the generation of antibodies against surface antigens of the bacteria. One way these bacteria avoid deleterious effects of antibodies is by changing the structure or amounts of these surface antigens. These changes are controlled by repetitive DNA tracts, which mutate at high frequencies resulting in alterations in the expression of genes that make these surface antigens. We have shown that persistence of N. meningitidis in the upper respiratory tract is facilitated by high frequencies of mutations in these repetitive DNA tracts causing reductions in expression of some of the surface antigens. As many of these surface antigens help the bacteria to invade tissues and to survive killing by cells and molecules present in blood, this finding has led us to speculate that reductions in expression of surface antigens, due to mutations in repetitive DNA, affects the ability of N. meningitidis to cause blood poisoning and meningitis. We will test this hypothesis by comparing the repetitive DNA tracts in bacterial isolates from patients and carriers. We will also test whether reductions in expression of specific antigens during persistent carriage of N. meningitidis is correlated with the presence of antibodies against that surface antigen.

We will use samples from previous studies in volunteers from whom bacterial isolates, serum and saliva extracts were obtained at three or more time points over a six to twelve month time period. Similarly we will use pre-existing collections of N. meningitidis isolates from disease cases. The repetitive DNA tracts of multiple genes will be analysed by sizing of DNA fragments spanning the repetitive region so that we can count the numbers of repeats and then use these numbers to determine whether a gene is expressed. We will examine if a gene changes as the bacteria persist within a carrier and if the genes are in different states in the disease isolates as compared to those of carriers. The amounts of antibodies will be measured using a microassay. For this, protein products of genes will be purified, linked to fluorescent microspheres and incubated with serum. This will result in specific binding of antibodies to the proteins on the microspheres. Antibodies will then be detected with a different fluorescent tag and quantified in a fluorescence detection machine.

We anticipate two major outcomes. Firstly, evidence of whether repetitive DNA produces a particular pattern of gene expression that enables N. meningitidis to cause disease. Identification of a 'virulence-associated' pattern would lead to production of novel preventive measures (i.e. vaccines containing these virulence determinants) or management of disease-cases (i.e. identification of sources of infections and rational decisions on when to provide preventive therapeutic treatment to contacts). Secondly, a demonstration that antibodies are driving reductions in expression of N. meningitidis surface antigens in carriers. This finding will indicate that vaccines containing surface antigens could reduce persistence of disease-causing N. meningitidis strains on mucosal surfaces of carriers and would be important for assessing whether Bexsero (the new MenB vaccine) can prevent spread of disease-causing strain.

Asymptomatic carriage of Neisseria meningitidis (Nm) in the upper respiratory tract is frequent and is associated with the induction of antibodies against surface antigens. Multiple genes of meningococci undergo phase variation due to mutations in simple sequence repeat (SSR) tracts. Recently, we have shown that persistent carriage of Nm is associated with reductions in expression of outer membrane proteins (OMP) due to changes in SSR tracts. Serum samples from these carriers were found to contain antibodies specific for PorA, a major OMP, and bactericidal antibodies against the capsule. We hypothesize that antigen-specific antibodies select for variants with reduced expression of phase-variable determinants and that these variants have a reduced capacity to cause disease. Expression states of multiple phase-variable genes will be analysed in comparable sets of Nm disease and carriage isolates. Data sets will include isolates from a longitudinal Novartis-sponsored carriage study and the phase-variable genes required for synthesis of O-linked glycans. Repeat numbers of phase-variable genes will be determined by dideoxy Sanger sequencing and PCR-based GeneScan assays of DNA extracts from isolates. Statistical tests will be performed to determine whether Nm disease requires a specific pattern of phase-variable gene expression and if persistent isolates have an expression pattern indicative of a reduced capacity to cause disease. Antibody levels for allelic variants of OMPs and for different glycan structures will be measured using bioplex assays, which detect binding of antibodies to purified, recombinant proteins linked to fluorescently-labelled microspheres. Bactericidal antibody levels will be measured using strains expressing target antigens but having mismatches in other antigens. Statistical testing will determine whether reductions in antigen expression are due to selection by specific antibodies.

This research application addresses the specificity of immune responses to Neisseria meningitidis ('the meningococcus), a major cause of bacterial meningitis, and how this organism causes disease. This study is likely to be pursued as introduction of a new meningococcal vaccine is implemented and, indeed, will utilise samples intended to test the effectiveness of this vaccine. The major beneficiaries will be the wider public as we aim to uncover basic principles and mechanisms associated with disease and disease-prevention for bacterial pathogens. Other beneficiaries will be the companies, government agencies and charities with an interest in vaccine development, implementation and monitoring. Further benefits will accrue from impacts on the skills of the early researchers directly involved in the project but also from colleagues, students and members of the public addressed by our planned outreach activities.

Pathogenicity in N. meninigitidis is thought to be polygenic. Our study will determine whether specific patterns of phase-variable gene expression contribute to disease phenotypes and, if they exist, generate tests to rapidly and reproducibly detect such patterns. These tests could then be employed to identify sources of disease cases/outbreaks and to improve targeting of prophylactic treatments to prevent further cases. Vaccine development would also be influenced by identification of novel vaccine targets for inclusion in second-generation meningococcal vaccines. Additionally, our methodologies could be utilized to test whether phase variation enables meningococci to escape the immune responses generated by the recently licensed meningococcal serogroup B vaccine, Bexsero, during both asymptomatic carriage and in disease cases attributable to suspected vaccine escape. The latter is particularly important as three of the vaccine antigens are directly or indirectly influenced by phase variation.

Two key components of vaccine efficacy are whether or not a vaccine elicits herd protection and the frequency of evasion of immune responses elicited by vaccination. Our study will indicate whether adaptive immune responses to outer membrane proteins (OMPs) drive changes in the expression of these surface antigens thereby providing an indication of how protein-based vaccines may impact on asymptomatic carriage and of the potential for immune escape. Most importantly, we will generate reagents and methodologies useful for detecting and evaluating the immune responses to OMPs as elicited by natural carriage and vaccination. A critical aspect of these tests may be an ability to identify allele-specific immune responses and hence an ability to distinguish responses elicited by a vaccine as opposed to the disease strain, an aspect of potential importance in evaluating potential cases of vaccine escape.

A demonstration of glycan-specific antibodies in serum samples and of an impact of these responses on glycan structure would provide proof-in-principle that glycans of the O-glycosylation system have utility as vaccine antigens. These findings could be exploited to generate novel vaccines against the pathogenic neisserial species and other organisms with similar glycan structures.

Many of the methodologies and reagents described herein will become available during the lifetime of the grant and could be exploited immediately for characterising disease cases and aspects related to implementation of the Bexsero vaccine such as evaluating potential cases of vaccine-escape. Findings related to novel vaccine targets will have a 5- to 10-year implementation curve. Most importantly there is potential for a significant impact on the effectiveness of the services offered by Public Health England and on the policies implemented by the Department of Health in relation to meningococcal vaccination with direct effects on the nation's health and quality of life.