We used whole-genome sequence based analyses to provide a macro-regional overview of the circulating and emerging invasive meningococcal strains in meningitis belt countries between 2011 and 2016. Using phylogenetic analysis, we provide information regarding the evolutionary relationships of the invasive lineages of the meningitis belt and their inter-country transmission. Notably, we identified geographically distinct strains within CC11, CC5, and CC181 that were circulating and expanding in Central and West African countries during this time period. We also build on past findings describing the primary lineage of CC11 NmW in the meningitis belt, showing the diversity within this lineage, and how it has spread across the region. Our work provides a comprehensive phylogeny for the emerging CC10217, including outbreak and epidemic isolates from Nigeria and Niger as well as invasive isolates collected from Mali and Burkina Faso. For CC181, our results show that at least two strains of this clonal complex were circulating during the time period, each of which appear to be expanding in distinct regions.

While epidemics of serogroup A have been largely eliminated from the meningitis belt, other serogroups have risen as major causes of meningitis in the region. Understanding the regional spreading and molecular characteristics of these strains is critical for implementing effective surveillance strategies and for developing vaccines targeting these strains.

With an estimated population of 400 million, the meningitis belt of sub-Saharan Africa, a region stretching from the coast of Senegal to Ethiopia, holds the highest reported meningitis incidence rate globally [1]. The majority of meningococcal epidemics have historically been caused by N. meningitidis serogroup A (NmA), but the incidence has been substantially reduced since the introduction of a serogroup A conjugate vaccine (MACV, MenAfriVac™) in 2010 [3]. Since then, major causes of meningococcal meningitis in the region have shifted to other serogroups, such as serogroup W (NmW) [4], serogroup X (NmX) [5], and more recently, serogroup C (NmC) [6]. Of note, NmC, prior to recent years, had been very rare and had not caused large outbreaks in the region since 1979 [7].

Between 2011 and 2016, over half (62%; n = 3309) of the confirmed meningococcal meningitis cases reported to the World Health Organization (WHO) in meningitis belt countries were NmW [8]. This included an epidemic in Burkina Faso in 2012 where 2353 confirmed meningitis cases were reported; 1451 (61%) of which were confirmed to be NmW [4]. These NmW cases primarily belonged to clonal complex 11 (CC11), a hyper-invasive lineage typically associated with NmC or NmW and less commonly B or Y [9]. Studies revealed that the epidemic strain shares a common ancestor with the outbreak strain collected in 2000 during the Hajj to Saudi Arabia [10].

While NmW has been the dominant cause of meningococcal meningitis in the region since 2010, NmC has also been a major contributor, accounting for 12.5% (n = 666) of the cases reported to the WHO between 2011 and 2016 [8]. In 2013 and 2014, NmC outbreaks occurred in Nigeria and were caused by a sequence type (ST) that had not been previously detected (ST-10217) [11]. In 2015, ST-10217 was responsible for an epidemic in Niger; phylogenetic analysis of 81 ST-10217 isolates from this epidemic compared with global NmC isolates from eight different clonal complexes showed that the Niger ST-10217 isolates formed a distinct cluster [6].

Along with NmW and NmC, NmX has remained a consistent cause of meningococcal meningitis in the region, contributing 7.6% (n = 406) of the confirmed cases reported to the WHO between 2011 and 2016 [8]. In 2006, NmX was responsible for outbreaks in Niger, comprising 51% of 1139 confirmed meningococcal meningitis cases [12]. Shortly after, sporadic cases and outbreaks of NmX were reported in 2007 in Togo and in 2010 and 2012 in Burkina Faso [5]. All of these reported NmX cases were identified to be ST-181 belonging to clonal complex 181 (CC181).

As continued epidemics and outbreaks of non-A meningococcal serogroups occur in the region, there is a growing need for the use of multivalent conjugate vaccines. In addition to MenAfriVac, several other polysaccharide conjugate vaccines are available, but many are currently not affordable for most African countries [13]. Two protein-based meningococcal vaccines targeting N. meningitidis serogroup B (NmB) are approved for use in the United States and Europe: MenB-FHbp (Trumenba) [14] and MenB-4C (Bexsero) [15]. Although NmB is rarely found in the meningitis belt, previous studies have demonstrated the potential cross-protective effect of these vaccines on diverse strains [16], including those from the meningitis belt [17], raising interest in assessing the potential protection offered on other meningococcal serogroups.

Proper understanding of the major and emerging causes of meningococcal meningitis in the region is critical for guiding surveillance strategies and vaccine development. We used whole-genome sequencing (WGS) to characterize these strains and to determine their phylogenetic relationships, regional spread, and allelic variants of interest.

We provide a genomic insight into the changing epidemiological landscape of meningococcal strains in the meningitis belt in the period 2011–2016. This follows the introduction of MACV to the region in 2010 and the subsequent vaccination campaigns throughout several countries in the meningitis belt. Prior to the introduction of MACV, during the years 2004–2010, NmA CC5 isolates dominated with 74.8% of the isolates collected, while NmW CC11 isolates represented 8.8% and NmX CC181 isolates accounted for 6.2% [28]. Post-MACV introduction, NmW CC11 isolates represented 60% of the collection, while NmA CC5 isolates only constituted only 12.5% of the collected isolates and were collected in the earlier years of the study (2011−2013) from regions or individuals that had not yet received vaccination. Furthermore, NmX CC181 remained fairly constant throughout the two time-periods, accounting for 8.6% of the isolates collected in 2011–2016. Finally, the emerging NmC CC10217 isolates constituted 17.5% of our isolate collection, and were collected only in the latter years (2013–2016) of the study.

CC11 was predominant and was collected from nine of 11 countries included in the study. Retchless et al. [10] described four major clades (I,II,III,IV) containing NmW CC11 isolates, with isolates from Burkina Faso 2011–2012 and Mali 2012 falling into subclade IVa within clade IV. Our results corroborate their findings of the primary lineage (subclade IVa) expanding throughout the meningitis belt sharing a common ancestor with the 2000 Hajj-related outbreak strain. Our time-based molecular clock phylogeny suggests that this common ancestor emerged between 1998 and 1999, and diverged into two clades: clade 1.1 contained the Hajj-related outbreak strain and did not appear to continue expanding in the region, while clade 1.2 continued to expand throughout the belt. We also identified a sub-lineage (sub-clade 2.1) which has primarily been expanding throughout Central African countries such as the Central African Republic and Chad. Clade 3 dates back to a common ancestor estimated to have been introduced into the region between 2004 and 2005, which appears to be the source for the main strains circulating throughout West Africa. This clade shows the diversity present in West Africa during the Burkina Faso 2012 epidemic, with evidence of inter-country spreading and introduction events of these strains across the countries of West Africa.

Along with CC11, CC10217 has emerged as a major contributor to meningitis in the region. Kretz et al. [6] hypothesized that comparisons between ST-10217 invasive isolates and a nongroupable, ST-9367 (6/7 MLST alleles shared with ST-10217) carriage isolate from Burkina Faso 2012 may illuminate the transition of this strain from carriage to invasive. Brynildsrud et al. [29] performed this comparison and identified the acquisition of virulence factors in the invasive strain, providing evidence for the origins of this epidemic prone strain. In our results, the isolates associated with the Niger 2015 epidemic were found in one clade which formed two distinct subclades in the phylogeny, and also contained two isolates from Nigeria 2015. As cases of meningococcal meningitis caused by CC10217 were first reported during outbreaks in Nigeria 2013, the presence of Nigeria isolates in the clade containing Niger 2015 isolates strongly suggests Nigeria as the transmission source of the Niger 2015 epidemic strain. This finding is consistent with the geographical range of this ST described by Brynildsrud et al. [29]. Additionally, the Nigeria isolates formed several distinct clades, potentially indicating that several strains from this sequence type have diverged in the country. Furthermore, our results show that the Niger and Nigeria strains were more closely related to each other than those from Mali or Burkina Faso. While the Mali isolates harbor a novel ST (ST-12446), the Burkina Faso isolate shares the same ST as the rest of the collection (ST-10217) while still being divergent from the rest of the collection, possibly indicating a new strain. The divergence of ST-10217 in Nigeria, along with the emergence of ST-12446 in Mali, conveys the risk of the continued genetic evolution of this virulent clonal complex.

While NmA CC5 isolates were included in our study, these strains have not caused major outbreaks in the region since the introduction of the MACV. Our results suggest that there were several strains circulating within Chad during the 2011–2013 time period. Furthermore, a sub-lineage of CC5 NmA derived from ST-7 and comprised of ST-2859 has been expanding throughout most West African countries between the early 2000s and 2011. These results are consistent with the study by Lamelas et al. [30], wherein the researchers concluded that ST-2859 strains most likely emerged in Africa from ST-7 meningococci a few years prior to 2003.

Agnememel et al. [31] showed the presence of two main sub-lineages for CC181 African isolates, one of which contained historical (1990s) and more recent (since 2006) isolates, while the other contained only more recent isolates (since 2006). Our work expands on this finding by showing the spread of these sub-lineages throughout the region. Within our collection of CC181 isolates, we identified two major clades, one of which contained 2011–2013 Chad isolates while the other contained 2011–2015 West African isolates from Burkina Faso, Mali, Ivory Coast, Togo and Benin. As clade I only consisted of historical Niger and recent Chad isolates, there is not enough data to suggest that this strain is circulating throughout Central Africa, as it may also be a strain primarily circulating in Chad. Additional sampling of CC181 isolates from neighboring countries will provide greater resolution on this topic. Regardless, our results suggest at least two major strains of CC181 expanding in certain geographical regions of the meningitis belt. Finally, the presence of historical Niger isolates in both clades I and II suggests that Niger may have been a transmission source for the introduction of these strains to the region.

Our vaccine antigen composition results suggest that the protein-based vaccines Trumenba and Bexsero may offer protection against some of these strains. While none of the FHbp variants identified matched the Bexsero variant 1.1 target, we identified several strains with FHbp variants within family 1, and studies examining the antigenicity of other subvariant targets within this family have previously shown promising results, specifically against NmX CC181 isolates [17]. Furthermore, serum bactericidal assays on diverse FHbp sequences raised against Trumenba sera have suggested a broad range of protection against various strains [16].

Previously-circulating strains of NmX CC181 and NmW CC11, as well as emerging strains of NmC from CC10217, have risen as major causes of meningococcal meningitis in the meningitis belt. While our results corroborate the effectiveness of MACV, they also stress the importance of continued meningococcal molecular surveillance in the region. Understanding the strains involved in clonal expansion throughout the region is critical for guiding these surveillance strategies and informing implementation of future vaccines. Our results convey the importance of developing affordable multivalent conjugate vaccines targeting these emerging strains.

We would like to acknowledge the Meningitis Research Foundation, GAVI, and the CDC's Office of Advanced Molecular detection for supporting this work. We would also like to acknowledge the clinical and public health labs of the following countries for providing isolates that were used in this study: Benin, Burkina Faso, Cameroon, Central African Republic, Chad, Guinea, Ivory Coast, Mali, Niger, Nigeria, and Togo. We also acknowledge Adam C. Retchless and Sandeep J. Joseph for their technical discussions. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. The funder of the study had no role in study design, data collection, data analysis, data interpretation, writing of the report or the decision to submit the paper for publication. The corresponding author had full access to the study data and had final responsibility for the decision to submit to publication.

NT, DAC, MKT, OBB, EH and XW all contributed to the study design, data generation and data interpretation. ND, AED were involved in the data generation. RO, SO, KG, BMNL, SD were involved in collecting and sharing isolates that were used in the study. NT prepared the manuscript draft with contributions from DAC, MKT, OBB, LMF and XW. XW supervised the study. All authors critically reviewed the manuscript and gave approval for publication.

Dr. Caugant reports grants from Meningitis Research Foundation during the conduct of the study; all other authors have nothing to disclose.