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Neisseria meningitidis (Nm), or meningococcus, is the only natural host of which we are aware. The carriage rate among healthy people is 10%-15%. Nm only resides on the mucous membranes of the human nasopharynx and it is a commensal and an important cause of invasive meningitis and septicemia.
Most Nm strains have a capsule. Capsule polysaccharide is an acidic linear or branched polymer composed of oligosaccharide repeating units. Serogroup typing is based on immunologically distinct capsule polysaccharides. There are 13 serogroups, of which six (A, B, C, W, X and Y) are responsible for almost all invasive disease. Antigenic variability in the outer membrane proteins (OMPs) (PorA and PorB) and LOS further differentiates strains into serotypes and subtypes.
About 30%-70% of Nm colonizing the nasopharynx cannot be serogrouped and are called non-groupable strains. These strains either agglutinate with two or more diagnostic antisera simultaneously or do not agglutinate with any. The genetic mechanisms behind non-groupable strains include point mutations or slipped-strand mispairing in capsule genes, insertion sequences (IS) disrupting capsule gene transcription or translation, or a complete lack of capsule synthesis genes, known as capsule null locus (cnl) strains. Formation of non-groupable strains often involves capsule switching or loss, enabling the bacteria to evade vaccine-induced immunity.
Figure 1. Schematic illustration of Neisseria meningitidis outer membrane proteins involved in colonization
(Source: Pizza M, et al. 2015)
Nm adheres to and colonizes the epithelial cells of the human nasopharynx, then invades the bloodstream and eventually the nervous system.
Figure 2. Overview of Neisseria meningitidis transmission, carriage state, invasion and virulence factors of the meningococcal outer membrane
(Source: Caugant DA, et al. 2020)
Adhesion and colonization of the nasopharynx by Nm are prerequisites for bacterial infection and disease. This colonization can be asymptomatic or cause local inflammation, involving multiple mechanisms. Typically, Nm adhesion is mainly mediated by type IV pili. In addition to adhering to epithelial cells, type IV pili also play a role in Nm adhesion to vascular endothelial cells, bacterial aggregate formation, bacterial motility, and exchange of genetic material. Type IV pili are composed of more than 20 proteins. The major structural protein of type IV pili is PilE (Pilus E) which makes up the backbone, while PilQ makes up the channel.
The bacteria adhere to nasopharyngeal epithelial cells, cross the mucosal barrier into the bloodstream, and tightly bind to brain microvascular endothelial cells, causing cortical spots that allow bacteria to cross the blood-brain barrier. This entire process is mediated by type IV pili. The pili proteins bind to two endothelial cell receptors, CD147 and β2-adrenergic receptor. The binding of pili proteins to CD147 is associated with bacterial adherence, whereas the binding to the β2-adrenergic receptor elicits endothelial cell signaling and activation, leading to bacterial invasion of the brain vascular endothelium and damage to the blood-brain barrier. The process is associated with the formation of the complex between CD147 and β2-adrenergic receptor and molecular rearrangement of the hydrophobic end of the PilE protein.
The capsule of Nm is an important virulence factor related to bacterial adhesion, colonization, and blood survival. The capsule is crucial for maintaining the balance between Nm and the human body. When bacteria adhere, high capsule expression is actually unfavorable for bacterial adhesion and colonization. The capsule polysaccharide synthesis genes and cell membrane surface transport genes are located in a single chromosomal region called the capsule polysaccharide gene cluster (cps).
Except for groups A and X, the CPS structures of groups B, C, W135, and Y are all sialic acid derivatives. Group A CPS consists of repeating units of N-acetylmannosamine linked by phosphate groups. Group X CPS is composed of phosphate groups linked to N-acetylglucosamine. The repeating units of groups B and C CPS contain only sialic acid, differing in their glycosidic bonds. In W135 and Y group CPS molecules, sialic acid is linked to either galactose or glucose through α-1,4 glycosidic bonds to form repeating units—W135 group links to galactose while Y group links to glucose.
The capsule helps Nm survive in blood. Capsule expression can be regulated through on/off phase variation. During pathogenesis, capsule expression is upregulated, inhibiting cell adhesion and biofilm formation while promoting bacterial entry into the bloodstream. During Nm carriage, capsule expression is downregulated or absent, facilitating adhesion. To adapt to the host environment, Nm exhibits highly variable phenotypes. Studies have found that different serogroup strains of Nm can undergo capsule switching, which can generate new epidemic pathogenic clones and serves as a mechanism for bacteria to escape vaccine immunity.
The initial development of vaccines against Nm focused on purified polysaccharide vaccines. But polysaccharides are thymus-independent antigens (TI) that only elicit antibody production by mature B2 cells and do not induce B1 cell responses. As TI antigens, polysaccharides are unable to induce immunoglobulin (Ig) class switching (e.g. from IgM to IgG or IgA) or production of high-affinity antibodies during primary immune responses. Although CPS vaccines have been shown to be clinically effective in adult populations, infant and young children (i.e. those at greatest risk of Nm infection) have not fully developed B2 cells, and have poor immune responses to CPS vaccines, and provide little to no immune protection.
As vaccine development advanced, the new approach of covalently linking polysaccharides to protein carriers was quickly applied to Nm vaccine research. Meningococcal polysaccharide conjugate vaccines chemically attach extracted Nm capsule polysaccharides (CPS) to a protein carrier. Because CPS is by itself a thymus-independent antigen that elicits a weak immune response, conjugating it to a protein carrier transforms it into a thymus-dependent antigen (TD), against which a much more effective immune response is generated. In contrast to unmodified CPS vaccines, meningococcal polysaccharide conjugate vaccines may be given not only to children over 2 years old and adults, but also for routine immunization of infants under 2 years old.
Table 1. Comparison of the properties of plain CPS and CPS protein-conjugated vaccines
| Plain CPS vaccine | Conjugated CPS (glycoconjugate) vaccine |
| T-cell-independent antigen | T-cell-dependent antigen |
| Safe and immunogenic in adults and older children | Safe and immunogenic (all age groups) |
| Not immunogenic in children<2 years of age, the age group at highest risk of meningococcal infection | Induces immune responses in infants (<2 years of age) |
| Induced protection is serogroup specific | Induced protection is serogroup specific |
| IgM antibodies induced | Enables affinity maturation of B-cell response to generate IgG |
| SBA responses are short lived | SBA responses are longer lived |
| Adjuvant enhancement of antibody responses does not occur | Antibody responses can be augmented with adjuvants |
| No induction of immunological memory | Induces immunological memory |
| Repeated CPS vaccination induces hyporesponsiveness |
(Source: Christodoulides M, et al. 2017)
Polysaccharide vaccines or polysaccharide-protein conjugate vaccines based on the capsule polysaccharides of Nm serogroups A, C, W, and Y have been widely used. However, there is still some debate regarding the use of group B Nm capsule polysaccharides in vaccine development. It is generally believed that group B capsule polysaccharides have low immunogenicity and are less able to induce high-quality protective antibodies. The composition of group B capsule polysaccharide is also structurally related to polysialic acid that is found on the surface of some human glycoproteins, including neural cell adhesion molecules. Antibodies generated against group B capsule polysaccharide are able to cross-react with these neural molecules, and this could lead to autoimmune disease. For this reason, the approach taken with vaccines against group B meningococcus has primarily been to develop protein-based vaccines, either using outer membrane vesicle (OMV) vaccines or recombinant protein vaccines.
The group B OMV vaccine, also called a "customized" vaccine, is made from outer membrane vesicles extracted from the predominant group B strains circulating in different countries. The main antigen in OMV vaccines is the PorA protein, which varies specifically between vaccines. The PorA antigen in OMV vaccines induces effective bactericidal activity only against strains with homologous PorA types, showing weak activity against other PorA variants. This limits the broad applicability of OMV vaccines.
An ideal group B vaccine protein antigen should cover as many circulating B strains as possible. The method of discovering antigens based on genome analysis to develop new vaccines is called reverse vaccinology, which was then applied to group B vaccine development. Researchers used computer analysis of the group B Nm genome and identified 600 potential vaccine candidate antigens. After selection, 350 candidates were expressed in E. coli, purified, and used to immunize mice. The experiments found 29 proteins that could induce antibody production in mice.
Next, the bactericidal activity of mouse immune serum was tested in the presence of complement. Candidate antigens were ranked based on these results, followed by studies on bactericidal titers, sequence conservation among group B Nm strains, expression levels in different bacterial isolates, and homology or absence in human proteins. Through this screening process, three candidate antigens were selected: Factor H-binding protein (fHBP), Neisseria heparin binding antigen (NHBA), and Neisseria adhesion A (NadA). These were combined with OMV to create the quadrivalent group B meningococcal vaccine.
Improved X-ray crystallography, nuclear magnetic resonance imaging, and electron microscopy now permit atomic level study of individual antigens or complexes of antigens with protective antibodies. This permits structure-based, precise modification of antigens (structural vaccinology). In recent years, structural vaccinology has also been applied to group B Nm vaccine research.
Nm's factor H-binding protein (fHBP) is a key virulence factor that binds to the complement regulatory protein factor H. fHBP is a Neisseria-specific surface lipoprotein composed of two antiparallel barrel-shaped domains connected by a five-amino-acid linker. There are about 300 amino acid variants of fHBP, divided into three variants: V1, V2, and V3. Based on protein diversity, fHBP is classified into two subfamilies, A and B.
Using the 3D structure of fHBP, researchers identified amino acids in epitopes that determine its immunogenicity against group B Nm. They found that amino acids in variant epitope regions do not overlap, meaning an epitope from one variant can be inserted into another variant to create an improved antigen.
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