Serogroup C Neisseria meningitidis disease epidemiology, seroprevalence, vaccine effectiveness and waning immunity, England, 1998/99 to 2015/16

Helen Findlow1, Helen Campbell2, Jay Lucidarme3, Nick Andrews4, Ezra Linley1, Shamez Ladhani2, Ray Borrow1,3,5 1. Vaccine Evaluation Unit, Public Health England, Manchester Royal Infirmary, Manchester, United Kingdom 2. Immunisation Department, Public Health England, Colindale, London, United Kingdom 3. Meningococcal Reference Unit, Public Health England, Manchester Royal Infirmary, Manchester, United Kingdom 4. Statistics, Modelling, and Economics Department, Public Health England, Colindale, London, United Kingdom 5. University of Manchester, Infection, Immunity & Respiratory Medicine, School of Biological Sciences, Stopford Building, Manchester, United Kingdom


Introduction
Neisseria meningitidis is a major cause of meningitis and septicaemia worldwide. Efforts to control meningococcal disease have been aimed at the development of effective vaccines and subsequent implementation in appropriate immunisation schedules. Twelve different meningococcal serogroups are recognised and serogroup B (MenB) is currently responsible for most cases of invasive meningococcal disease (IMD) in Europe. Many countries, including the United Kingdom (UK), experienced large outbreaks of serogroup C (MenC) disease in the mid-1990s, due mainly to the ST-11 clonal complex (cc11) [1]. In 1999, the UK became the first country to introduce the MenC conjugate (MCC) vaccine in a phased national campaign targeting all those aged less than 18 years over a 12-month period, alongside a routine three-dose infant programme [2]. Vaccine eligibility was later extended up to 24 years of age, although this age group was not actively called for vaccination. Routine use of MCC vaccine in many other European countries followed [3].
As MCC vaccines were licensed on immunogenicity studies alone, without direct evidence of clinical efficacy, comprehensive national surveillance was initiated concurrently in order to monitor the vaccines' impact in a population-based setting [2]. MCC vaccination was associated with rapid and sustained declines in MenC disease across all age groups through direct and indirect (herd/population) protection [3][4][5]. Invasive disease is rare following nasopharyngeal acquisition where the meningococcus can persist for several months before it is cleared; this asymptomatic carriage, especially in older teenagers, is an important reservoir for infection, onward transmission to susceptible individuals and immunity. In the UK, there was a 66% reduction in MenC carriage among [15][16][17][18][19] year-olds within 1 year of MCC vaccine implementation and this reduction was key to establishing indirect protection across the population [6].
The UK MCC immunisation programme has evolved over time (Box) and, from 2013, has included an adolescent MCC vaccine programme to extend direct protection in teenagers and maintain indirect protection in the wider population. In 2015, this vaccine was replaced with the quadrivalent meningococcal conjugate vaccine (MenACWY) to combat a national MenW outbreak. A multi-component, protein-based vaccine against MenB was also implemented in the infant immunisation schedule at the same time [7]. In July 2016, the infant MCC dose was removed because MenC cases were extremely rare in this age group and population protection was likely to be maintained through the adolescent MenACWY programme.

National epidemiology
Public Health England (PHE) conducts enhanced national surveillance of IMD in England. The PHE Meningococcal Reference Unit (MRU) provides a national service for confirming, grouping and characterising invasive meningococcal isolates [11]. The MRU also provides free national Polymerase Chain Reaction (PCR)-testing of clinical samples from patients with suspected IMD; Diagnostic laboratories are required by law to notify PHE when they identify Neisseria meningitidis and samples are requested to be sent to the MRU for confirmation and characterisation. PHE routinely reconciles laboratory-confirmed cases to generate a national dataset [12] and follows up each case for demographic data, vaccination history, clinical presentation and outcomes through Health Protection Teams (HPTs), general practitioners and, for younger cases, hospital clinicians. Details documented on HPZone (a web-based software for public health management of infectious diseases used by HPTs throughout England), were also accessed by PHE to obtain additional information on cases since 2010.

Vaccine effectiveness
Using the national dataset, MCC VE was calculated using the screening method [13] for confirmed MenC cases among MCC-eligible individuals (born since 1 September 1981) in England between 01 January 2000 and 30 June 2016. VE was estimated according to time since vaccination (or, if unvaccinated, since the age at last scheduled dose); analysis included unvaccinated children, those who had completed the recommended primary immunisation course for their birth cohort (three doses for those aged 1 year of less before September 2006), or had received one dose after their first birthday at any age. Effectiveness of the primary plus booster dose was based on cases who had received ≥ 1 primary MCC dose followed by the 12-month Hib/MCC booster. The following formula was used: Where PCV is the proportion of vaccinated MenC cases and PPV is the proportion of vaccinated population (i.e. vaccine coverage, matched to each case based on their age and birth cohort and then averaged). Annual national vaccine coverage by 12 months of age (from April 2001) and by 24 months of age (from April 2002) was used for the latter [14].
Data extracted in May 2016 from The Health Improvement Network (THIN) database [15] for children born from August 1999 to May 2015 was used to estimate partial vaccination proportions for each of the routine schedules (3 + 0, 2 + 1, 1 + 1) by 12 months and 24 months of age. These estimates of partial vaccination were used to adjust the national coverage estimates to give coverage in those who received either no vaccination or the full the schedule. For example, it was estimated that in the three-dose primary cohort, 5.5% received one or two doses by 12 months, so national coverage of 90% for three doses would be adjusted to (90*100)/(100-5.5) = 95.2%, once partial vaccination is excluded.

Molecular characterisation
Typing data and genomes were obtained from the PubMLST Neisseria database and Meningitis Research Foundation (MRF) Meningococcus Genome Library (MGL), to which all MRU isolates from the national dataset have been referred since July 2010 [16,17]. A recent phylogenetic network analysis of the known population structure of MenC-associated cc11 sublineages was re-annotated in the context of the current study since cc11 represented the majority of recent invasive MenC isolates in England [18]. Briefly, all available global cc11 genomes, including 121/122 invasive MenC isolates from England (2010/11-2015/16), and excluding serogroup W-associated lineage 11.1 sublineages (final n = 898 globally; accessed 7 October 16), and two non-cc11 reference genomes (IDs 27778 and 29645; ccs 41/44 and 8, respectively), underwent core genome comparisons (1546 loci) using the PubMLST genome comparator tool. The resulting distance matrices were visualised using SplitsTree4. The invasive MenC cc11 isolates collected in England during 2010/11-2015/16 (inclusive) were highlighted [19].

Serosurvey
Serum samples collected in 2014 were obtained from the PHE Seroepidemiology Unit; a depository of anonymised residual sera from routine diagnostic testing at participating laboratories. Known immunocompromised individuals are excluded from the collection. The age, sex and year of collection were known for individual samples: immunisation status was not known [20].
Serum bactericidal antibody (SBA) assays were performed against the serogroup C target strain, C11 (phenotype C:16: P1.7-1,1) as previously described [21]. The complement source used in the SBA was pooled serum from 3-4 week old rabbits (Pel Freez Biologicals, Arkansas, United States (US)). Titres were expressed as the reciprocal serum dilutions yielding ≥ 50% killing after 60 minutes. The lower limit of detection was a titre of 4. Titres of < 4 were assigned a value of two for geometric mean titre (GMT) analysis. Titres of ≥ 8 were considered protective against MenC disease [22].
Approximately 100 samples were selected from each age-band to fit with different vaccine schedules since MCC introduction and to allow comparison with previous studies. This sample size was selected to achieve reasonable precision around the 95% confidence intervals (CIs) for proportions with SBA titres ≥ 8 and GMTs within the age group of interest.
Since MCC vaccine coverage has been consistently high, the age of the serum donor was used to align the donor with the MenC vaccine schedule they should have been offered and thereby identify which MenC vaccine cohort they belonged to. This then allowed comparison of protection between schedules and over time, using historical data from three previously published similarly designed surveys. These earlier surveys used sera collected from the same Seroepidemiology Unit and tested using the same methodology in the same PHE laboratory during 1996-1999 [8], 2000-2004 [9] and 2009 [10].  Figure  1). Over the last decade, the age distribution has been relatively constant ( Figure 1). However, the proportion of cases among those aged 5-14 years increased from 12% between 2006/07-2010/11 (0.04/100,000) to 17% between 2011/12-2015/16 (0.09/100,000), while the proportion of cases among 1-4 year-olds declined from 11% (0.09/100,000) to 4% (0.05/100,000) over the same period. Cases in those aged 45 years and over (who were not vaccine eligible) were 86% lower in 2015/16 (n = 16) than 1998/99 (n = 111) consistent with a sustained population protection effect. Of the 160 cases, 11 (7%) were living in shared accommodation (three university students, four in care homes or assisted living, four in house share or hostel accommodation). There were six cases in known men who have sex with men (MSM) and eight individuals reported using recreational drugs, were injecting drug users or had alcohol dependency. Nineteen cases (12%) had underlying co-morbidities; heart condition (n = 3), diabetes (n = 2), HIV (n = 6), immunosuppression (two on psoriasis treatment, one with chronic lymphocytic leukaemia, one post liver transplant), chronic respiratory condition (n = 3), liver disease (n = 3). There were three clusters of two cases each in primary schools nationally in 2015; all six children were fully-vaccinated (two-dose primary course with booster).

Vaccine effectiveness
Between

Molecular characterisation
During      (Table 3). In the younger age groups, GMTs and proportion protected were generally lower in 2014 compared with 2009 (Table 3).
The low MenC disease incidence is reassuring and is consistent with continued herd protection in the wider population and high direct protection in those who have been vaccinated. There have been no cases reported among at-risk individuals, currently defined as those with asplenia, splenic dysfunction or complement deficiency. Those born outside the UK (who are less likely to be immunised with MCC, even if eligible on entry), recently travelled and/or socially mixing with these populations accounted for a quarter of all cases, while a significant proportion of the other cases were living in shared or supported accommodation, which is a known risk factor for IMD [23]. An increased risk of IMD in HIV-positive individuals has recently been reported [24], even among those receiving appropriate antiretroviral therapy. Over the past 5 years, six HIVpositive individuals were identified among 160 MenC cases in England, all in the 25-44 year age group, including two who were non-UK born and had recently travelled abroad: Outbreaks of MenC disease in MSM populations have been reported in North America and Europe [25,26]. Whole genome sequencing previously showed that MenC case isolates among MSMs in England, while all belonging to lineage 11.2, were interspersed among sporadic community case isolates and did not form a discrete cluster [27].

B. C.
A) Neighbor-net phylogenetic network based on a comparison of 1,546 core genes among all available cc11 genomes on the PubMLST database, excluding predominantly-serogroup W lineage 11.1 sublineages (final n = 898; accessed 07/10/16). The English meningococcal serogroup C case isolates from 2010/11 to 2015/16 (red dots) were mainly distributed among several closely related lineage 11.1 and lineage 11.2 clusters, respectively. B) Cases due to lineage 11.1 isolates have increased since 2010/11, whilst C) cases due to lineage 11.2 isolates have fluctuated.

Molecular characterisation
Recent MenC cases in England were caused by diverse meningococcal strains, representing at least 10 different clonal complexes over the past 6 years. Most cases, however, were caused by two diversifying cc11 strains, one each from the two recently-resolved major cc11 lineages (lineages 11.1 and 11.2) [27,28]. Our findings warrant continued close monitoring, as lineage 11.1 cases have increased steadily over the past 6 years, while 2015/16 also saw the greatest number of lineage 11.2 cases. In addition to high case fatality rates for cc11 in general, the presence of lineage 11.2 isolates is concerning for several reasons. Evidence of frequent MenB to MenC capsule switching is coupled with poor potential coverage by, and the ability to escape from, several existing and experimental MenB vaccines [18]. Furthermore, distinct lineage 11.2 populations have independently acquired traits that may facilitate urogenital colonisation [29,30]. The acquisition of intact aniA alleles encoding nitrite reductase may enable survival in microanaerobic environments and has been associated with outbreaks of invasive disease among MSM in the US and Europe [30][31][32] and urethritis among heterosexual males in the US [29,30]. The urethritis-associated strain has also lost its ability to express a capsule, a further gonococcal trait that may facilitate adherence to the urogenital mucosa.

Serosurvey
Seroprevalence studies have proven valuable in improving our understanding of population immunity, inform mathematical modelling and can complement disease surveillance to help inform national vaccine policy [8][9][10]. The 2014 serosurvey confirmed the rapid decline in immunity after infant and toddler immunisation (also observed with the 2009 serosurvey) as well as the low proportion of teenagers protected against MenC disease around the time when the adolescent programme was introduced. Similar trends have been reported in the Netherlands, where a large catch-up campaign targeting 1-19 year-olds in 2002 was followed by routine childhood immunisation at 14 months of age only. Here, a recent serosurvey found only 19% of 10 year-olds were protected 9 years after a single MCC dose at 14 months [33]. The successful control of MenC disease in the UK is attributed to the large catch-up campaign in 1999/2000. Teenagers and young adults have the highest nasopharyngeal meningococcal carriage rates and are considered the main source of spread. MCC vaccines not only protect against invasive disease but also against nasopharyngeal acquisition, thereby inducing herd protection across all age groups [4,6]. The current cohort of younger children with poor seroprotection is most likely protected against disease because of this indirect protection.
Most adolescents in 2014 will have received a threedose infant MCC schedule without a booster. The low proportion of teenagers achieving protective antibody thresholds in the 2014 serosurvey indicated there was poor long-term direct protection in the cohort that was only immunised in infancy and ongoing herd protection across the population could be threatened. This observation, predicted after the 2009 serosurvey, helped support the inclusion of MCC in the adolescent programme in 2013 with the aim of providing high antibody concentrations throughout the peak years of carriage [7]. The 2014 serosurvey is already demonstrating higher MCC antibodies among 14-16 year-olds compared with the same age group in the previous serosurvey. The emergency adolescent MenACWY programme in 2015 was implemented to provide direct and indirect protection, not only against MenC but also against the three other serogroups; particularly the emergent hypervirulent MenW strain also belonging to cc11 that was responsible for a quarter of all IMD cases across the UK by 2014/15 [7].

Implications for other countries
In Europe, MenC disease incidence is currently low (< 1/100,000) in countries with and without routine MCC vaccination [3]. Disease in infants remains rare and thus in countries with routine MCC immunisation most cases occur in adults, with a median age of 41 years compared with 22 in countries without MCC vaccination programmes. The recent increase in MenC cases in France highlights the importance of maintaining high vaccine coverage, especially in adolescents, in order to sustain long-term direct and indirect disease control [34]. A number of countries across Africa, Latin America and elsewhere are also experiencing high MenC disease activity [35,36]. Travellers to such countries, including residents visiting family and friends in their home countries, should be appropriately immunised, advised to avoid high-risk behaviours, remain vigilant for symptoms and signs of meningococcal disease and seek immediate medical help if concerned while abroad and when returning to their home countries.

Strengths and limitations
The strength of this study is the consistently high quality of the national surveillance programme with high case ascertainment [12] and active follow-up of cases since 1998/99. This has allowed us to monitor VE in different cohorts over time. Our meningococcal surveillance is complemented with clinical trials, to assess the immunogenicity of different schedules, and serosurveys to monitor population susceptibility. Together, these results have allowed us to adapt the national immunisation programme to provide maximal long-term protection in the most cost-efficient manner. Using the same methodology allows us to monitor population effects over time. A limitation of the serosurvey, however, is that we have no information on the vaccination status of the serum donors or their clinical state. Nonetheless, the large numbers of samples tested, along with the consistently high vaccine coverage nationally has allowed us to analyse the results by birth-cohorts.

Conclusions
We found evidence for rapid waning of immunity following routine infant immunisation, even with a 12-month booster, which was introduced in 2006. Currently, most toddlers (1-4 years) and older children (aged 5-13 years) in England are not seroprotected against MenC disease and are dependent on the indirect protection offered through reduced carriage in young people aged 17-24 years. It is, therefore, reassuring that there is still evidence of herd protection in adults 17 years after mass vaccination of children in 1999. Immunisation of teenagers appears to be key for continued population impact, but also for direct protection before their social behaviour leads to greater exposure, carriage and invasive disease. We have demonstrated high shortterm effectiveness of MCC vaccines in infants and preschool children and the long-term effectiveness of MCC vaccines given to school-aged children and adolescents. These findings have led to several revisions of the UK immunisation schedule to maximise direct and indirect protection. The recent change to emergency MenACWY vaccination for teenagers was brought in to provide more rapid population control for the national MenW outbreak. It should also provide continued direct and population control for MenC and help establish this for MenY disease.
Continued epidemiological and microbiological surveillance will be critical to monitor current programmes which now include newer sub-capsular protein-based vaccines against MenB disease in infants. MenC disease continues to be rare but detailed case-based follow-up has identified certain groups that remain at higher risk for MenC disease despite containment within the wider population, these groups may benefit from targeted vaccination. Stochastic meningococcal outbreaks globally need to be carefully monitored, as well as the recent MenC outbreaks among MSM. High-resolution characterisation is critical to monitor circulating strains and detect known and unknown emerging threats.