Neisseria meningitidis is a Gram-negative bacterium, also called meningococcus, which asymptomatically colonizes the nasopharyngeal mucosa of 5-10% of the adult population. Colonization mostly induces immunity with protective antibody response, but in rare cases the meningococci can gain access to the circulation and may cause systemic disease such as sepsis and meningitis (1–3). Based on the chemical composition of the capsular polysaccharides, meningococci are divided into 12 serogroups – six of which are reported to cause invasive disease (4, 5). As no immunological memory formation and hyporesponsiveness upon repeated vaccination with unconjugated meningococcal vaccines have been found (6–8), vaccination with conjugated meningococcal capsule vaccines (e.g. MenACWY-TT and MenACWY-CRM₁₉₇) for serogroups A, C, W and Y and with a protein-based vaccine for B meningococci is currently recommended (5).

Killing of N. meningitidis by the complement system membrane attack complex (MAC)-induced bacteriolysis represents a key element of host immune defense against meningococci. Determination of the serum bactericidal antibody activity (SBA) serves as surrogate of meningococcal vaccine efficacy as it measures the MAC-induced killing (9). The SBA assay measures classical pathway (CP) complement-mediated killing by circulating antibodies that induce lysis of meningococci. The dilution of a test serum that results in at least a 50% decrease in colony forming units (CFUs) is defined as a bactericidal titer (10). SBA assays require active intrinsic or exogenous complement, and for serogroup C meningococci SBA titers of either ≥4 or ≥8 have been established as correlates of protection when using human or rabbit complement, respectively (11, 12). Antibody-mediated activation of the CP depends on factors like subclass and avidity of antibody binding, and the accessibility, density and distribution of the target antigen (13, 14). Binding of the C1 complex to the Fc region of the antibodies, which are bound to their target antigen on the bacterial cell surface, initiates CP activation by cleavage of C4 and covalent C4b deposition on the meningococcal cell surface. C4b then recruits C2, which is also cleaved by C1s. The C4b2b complex is a C3 convertase, which cleaves C3 into C3a and C3b, and the generated C3b binds to C4b2b to form a C5 convertase (C4b2b3b). The C5 convertase then cleaves C5 into C5a and C5b. Subsequent interactions between C5b and the other terminal components C6, C7, C8, and C9 form the MAC (C5b-9), which lyses the meningococcal cell by forming pores on the membranes (15, 16).

In the absence of antibodies, alternative complement pathway (AP) activation can also lead to C3b deposition on the meningococcal cell surface, and it also amplifies complement activation initiated by either complement pathway. While the bactericidal efficacy of antibodies directed against abundantly expressed surface antigens like the capsular polysaccharide seems to be unaffected by AP inhibition, antibodies directed against sparsely expressed surface antigens are more dependent on amplification by the AP (17). Activation of the AP can occur directly by C3b or is mediated by the hydrolysis of C3 resulting in the formation of C3(H₂O) that leads to factor B (fB) binding and cleavage of bound fB by factor D (fD) (18). The resulting C3(H₂O)Bb or C3bBb complexes have C3 convertase activity. The importance of the AP for defense against meningococci is reflected by a strong association of invasive meningococcal disease with deficiencies in fD (19–21) and fB (22, 23). Moreover, deficiency in properdin, which stabilizes the AP C3-convertase C3bBb, is associated with a high risk for severe meningococcal disease (24–27) and terminal complement component-deficient individuals have a 5000- to 10 000-fold greater risk of contracting meningococcal diseases (28). Eculizumab, a humanized monoclonal antibody (mAb) is being used to treat patients suffering from atypical hemolytic uremic syndrome (aHUS) and paroxysmal nocturnal hemoglobinuria (PNH). Such individuals have an excessive over-activation of their complement system due to either inherited or acquired genetic mutations or autoantibodies contributing to the pathogenesis. Eculizumab binds C5 and prevents C5 cleavage, which is necessary for the formation of the MAC. By limiting the excessive activation of complement in PNH and aHUS individuals, Eculizumab has proven successful for the treatment of these patients. However, use of Eculizumab is associated with a 1000-2000 fold increased incidence of meningococcal disease, and even after vaccination susceptibility to infection remains substantial (29, 30). Anti-C5 antibodies have black box warnings reflecting their higher infection rate. Currently, several promising drug candidates targeting various other complement proteins in the cascade are in development for the treatment of PNH, aHUS and other complement disorders (16). Here we have investigated the survival of N. meningitidis in sera from individuals before and after vaccination with a conjugate MenACWY vaccine, and together with several drug candidates inhibiting complement components factor B, factor D, C3, C5 and MASP-2.

Treatment with the first complement-targeting drug Eculizumab has shown significant clinical efficacy in patients with PNH and aHUS (15). However, even after meningococcal vaccination, Eculizumab treatment increases susceptibility to invasive meningococcal disease 1000 - 2000-fold (30). As an alternative, a new generation of complement therapeutics, targeting different stages of the complement cascade, is currently in pre-clinical and clinical phases of development. Here we compared the effect of a set of complement inhibitors, including small molecules targeting the AP components fB and fD, a C3 small peptide inhibitor and anti-C5 and anti-MASP-2 mAbs. We investigated the effect of these complement inhibitors on meningococcal killing in the presence of serum from individuals collected before and 2 weeks or 2 months post-immunization with the meningococcal ACWY polysaccharide conjugate vaccine Menveo. After Menveo vaccination, the overall SBA titers against A, C, W and Y meningococci increased for 75% of the serum/serogroup combinations ( Figure 2 ), which positively correlated with the increase of anti-capsular IgG concentrations ( Figure 3 ). SBA titers increased strongly up to an anti-capsular IgG concentration of about 5 µg/mL. Already 2 weeks after the vaccination, 18/20 serum/serogroup combinations had reached SBA titers >4, which are considered to be protective (11, 44).

SBA against meningococci depends on the formation of the MAC (45) and the clinical use of Eculizumab has confirmed the central role of C5 cleavage in meningococcal immunity. Inhibition of C5 cleavage by Eculizumab prevents the generation of C5b, which is the pivotal step in MAC assembly and meningococcal killing. In addition, lack of release of the pro-inflammatory peptide C5a may also affect meningococcal clearance (29, 35). In our study, anti-C5 mAb at a concentration of 15µg/mL completely abrogated meningococcal killing irrespective of serogroup and vaccination status.

As an alternative to C5 inhibition with Eculizumab, several AP-targeted therapeutics, including the fD inhibitor Danicopan (ACH-4471) (46) and the fB inhibitor Iptacopan (LNP023) (38) are currently being evaluated in the clinic, and successful phase 2 clinical trials with both agents against PNH have been recently reported (47, 48). However, reports that fB and fD deficient individuals are known to present with recurring invasive pneumococcal and meningococcal disease, underscores the importance of the AP in immunity against these bacteria (19, 20). The AP is not only continuously undergoing a low-grade antibody-independent activation, but functions also as an amplifier of C3b deposited through the CP or lectin pathway (LP). While inhibition of C5 abrogated killing of meningococci by all sera, the two AP inhibitors affected meningocccal killing only in sera with low anti-capsular IgG titers. Collated data for all subjects and serogroups tested revealed a difference between the pre- and the post-vaccination sera ( Figure 5 ), which was significant, in spite of the fact that vaccination did not elicit in all subjects antibody responses against all serogroups and that some pre-vaccination sera had already SBA titers against certain serogroups. Our data showed, that already two weeks after vaccination, antibody titers were high enough to counteract the inhibition of SBA by the AP inhibitors. Independent of AP amplification, capsule polysaccharide specific antibodies can thus efficiently trigger SBA via the CP alone. In accordance with these results, Granoff et al. found that the fD inhibitor ACH-4471 did not affect meningococcal killing by sera of vaccinated subjects in 6/8 meningococcal isolates (35).

The C3 inhibitor CP-40 tested here, is a small peptide analog of the Compstatin family, which is currently under clinical development for the treatment of age-related macular degeneration (49). A PEGylated version of a C3 inhibitor Pegcetacoplan (APL-2) from the same company has now been approved by the FDA as a new treatment for PNH (50–52). C3 inhibition prevents the formation of both C3 and C5 convertases, and accordingly, vaccination did not restore meningococcal killing in the presence of CP-40. However, for some serum/serogroup combinations, CP-40 did not inhibit bacterial killing completely. The inhibitor binds to the native form of C3 and sterically hinders the binding and cleavage of native C3 by C3 convertases (49). However, the C3 inhibitor does not bind or prevent the spontaneous hydrolysis of C3 into fluid phase C3(H₂O) via the “tick-over” process, nor does it inhibit conformational activation of C3 on surfaces (16, 53, 54). In human blood, C3 is the most abundant complement protein with a concentration of ~2 mg/mL, therefore, allowing the constant availability of a certain amount of hydrolyzed C3. In turn, the AP can directly be activated by the surface-bound C3 or hydrolyzed C3, as C3(H₂O) adopts a structure similar to C3b allowing binding with fB to make a convertase initiating the AP (55). In the presence of high density IgG bound to the target antigen, the immune complexes can bind the C3b formed from cleavage of the hydrolyzed C3 and form a convertase, or form convertases from activation of C3 on the meningococcal surfaces directly (56). Moreover, Rawal and Pangburn showed that the CP C3 convertase C4bC2a is able to cleave C5, but has a lesser affinity for substrate C5 than C3 (57). Despite the low turnover of C5, in conditions with high IgG binding to target antigen, which cleaves C2 and C4 to initiate CP, it is sufficient to generate physiological amounts of MAC. In the clinic, patients with primary C3 deficiencies are at an early age highly susceptible to pyogenic infections, especially Streptococcus pneumoniae. However, a few reports show that these effects subside in adulthood with the development of the adaptive immune system and a higher acquisition of immunoglobulin (55, 58, 59).

An essential serine protease for the activation of the LP is MASP-2. Currently, anti-MASP-2 inhibitors such as Narsoplimab (OMS721) from Omeros is being evaluated as an alternative candidate for the treatment of aHUS. It has had success in the clinic against IgA vasculitis-associated nephritis and thrombotic microangiopathies (42, 60). However, the role of LP in immunity against meningococci has not been clearly defined. Here, SBA experiments with the anti-MASP-2 mAb showed no implication in meningococcal clearance in either pre or post-vaccine sera, indicating that LP may not be essential.

Limitations of the present investigation include that for logistical reasons, we had no access to sera from PNH or aHUS patients immunized with Menveo. Moreover, we did not include results of our analyses with serogroup B isolates (manuscript in preparation) as serogroup B vaccines are multi-protein based, analysis of the immune response is complex and inclusion of data with a serogroup B vaccine would have gone beyond the scope of the present manuscript. Despite the fact that our sample group was relatively small, the sera tested comprised a suitable range of antibody titers to draw firm conclusions.

In summary, our results highlight the advantage of AP inhibitors over C3 and C5 inhibitors as complement therapeutics, as they do not completely prevent terminal pathway activation and meningococcal killing by capsule specific antibodies. Treatment with AP inhibitors should therefore be combined with meningococcal vaccination. In aHUS patients, Gäckler et al. found that one vaccination with an ACWY conjugate vaccine is not sufficient to elicit antibody responses against all four serogroups in the majority of patients (61). A re-vaccination may therefore be advisable.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Ethical approval was obtained from the Ethical Committee of Northwest and Central Switzerland (Ethikkommission Nordwest- und Zentralschweiz (EKNZ), Studie 2018-02341). The patients/participants provided their written informed consent to participate in this study.

EI and LM contributed to the study conceptualization, data acquisition, analysis and data interpretation and drafted the manuscript. GP contributed to study conceptualization and design, data analysis and interpretation and revised the manuscript. AS, CT, NZ, TH, and MK contributed to study conceptualization and design and revised the manuscript. All authors contributed to the article and approved the submitted version.

GP received funding to conduct the study under a research agreement contract between Novartis Pharma AG and the Swiss Tropical and Public Health Institute. The funders initiated study design and decision to implement the SBA assay and compare activity of the tested drugs. The funders had no role in data collection and analysis. GP has received a research grant from Novartis Pharma AG. AS, CT, NZ, TH, and MK are full-time employees of Novartis.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.