Only 6 of the 13 meningococcal serogroups commonly cause invasive infections1 and 5 of these serogroups are vaccine preventable. Complement deficient patients have an up to 10,000-fold higher risk of infection with Neisseria meningitidis, and it is estimated that 30%–50% of persons with terminal complement deficiency suffer from more than one infection episode.2,3 As a result, patients with terminal complement deficiency are more susceptible to meningococcal serogroups not included in the current vaccines. Cases of meningitis and septicemia caused by serogroup Z have infrequently been reported.4
The tetravalent polysaccharide vaccine against serogroup A, C, W and Y does not provide cross protection against other serogroups. However, the MenB-4C vaccine consists of purified protein antigens and outer membrane vesicles and appears to induce cross-reactive antibodies against meningococcal serogroup X and even Neisseria gonorrhoeae.5–7 Previous work from our group showed that opsonophagocytic killing of meningococcus serogroup B was improved in blood from complement deficient patients following MenB-4C vaccination.8 Here, we report a case of meningococcus serogroup Z meningitis revealing a complement C8 deficiency and demonstrate that MenB-4C vaccination induces cross-reactive antibodies which increase opsonophagocytic killing of meningococcus serogroup Z.
The study was approved by the ethics committee of the Wilhelmina Children’s Hospital, University Medical Center, Utrecht, the Netherlands. Written informed consent was obtained from the patient and parents.
Whole blood was collected from the patient before the first MenB-4C vaccination and 1 month after the second vaccination into a clot activator tube (BD Diagnostics). Whole blood was centrifuged 1500xg for 5 minutes at 4 °C and serum was stored at −80 °C. To neutralize β-lactam antibiotics, serum was pretreated with 20 µg/mL β-lactamase for 10 min. Serum was heat inactivated at 56 °C for 30 minutes.
Bacterial Growth Conditions
The patients N. meningitidis serogroup Z strain was collected from the Netherlands Reference Laboratory for Bacterial Meningitis at the Amsterdam University Medical Center and grown overnight at 37 °C with 5% CO2 on a GC-agar plate with Isovitalex, suspended in tryptic soy broth and grown to an optical density at 620 nm (OD620) of 0.46 [approximately 5 × 108 colony-forming units (CFU)]. Bacterial stocks were frozen with 15% glycerol at −80 °C.
N. meningitidis serogroup Z bacteria were incubated with 5% serum (C3), or 5% heat-inactivated serum (IgG) diluted in Hank's balanced salt solution + Ca2+/Mg2+ and 0.1% gelatin (Hank's balanced salt solution 3+) for 30 minutes at 37 °C. Surface-bound IgG or complement C3 was determined as previously described for N. meningitidis serogroup B.9
Serum Bactericidal Activity Assay
Serum bactericidal activity titer was determined as previously described for N. meningitidis serogroup B8 but using 30 minutes incubation at 37 °C.
Complement Killing Assay
Bacteria were grown overnight at 37 °C with 5% CO2 on a GC-agar plate with Isovitalex and resuspended in tryptic soy broth to grow to an OD620 = 0.46 and diluted 1000-fold in phosphate-buffered saline (PBS). Ninety-five microliters bacteria were mixed with 5 µL active serum. Samples were incubated in a shaking incubator at 37 °C for 30 minutes while shaking at 250 RPM. CFU counts were determined by plating 10 µL suspension on a GC-agar plate with Isovitalex and incubated overnight at 37 °C.
Whole Blood Killing Assay
The whole blood killing assay was performed as described previously, with some minor adjustments.10 Whole blood was collected in an ethylenediamine tetra-acetic acid tube from one healthy volunteer, washed 3 times with PBS, and the cells were suspended in PBS to 97.5% of the initial blood volume. The bacterial culture with OD620 = 0.46 was washed and diluted 1000-fold in blood. Two and a half microliters active serum was mixed with 97.5 µL blood containing bacteria and was incubated in the 96-well plate in a shaking incubator at 37 °C for 30 minutes while shaking at 250 rpm. CFU counts were determined by plating 10 µL suspension on a GC-agar plate with Isovitalex, which were incubated overnight at 37 °C.
Bacterial Whole-genome Sequencing
The strain was cultivated on a blood agar plate. DNA was isolated using a cetyl trimethylammonium bromide-based method and a library preparation was performed using Nextera Flex (Illumina, San Diego, CA). A 2 × 150 bp paired-end library was sequenced using an Illumina NextSeq500 sequencer (Illumina, San Diego, CA). Raw sequence reads were filtered using fastp (version 0.19.10), genome assembly was performed using SPAdes (version 3.12.0) with settings (-k 21,41,61,81,101;--careful,--only-assembler).
Raw sequence reads were matched against the 11 N. meningitidis serogroup B sequences using KMA (version 1.2.13) by first creating an index with kma_index followed by running kma with option -1t1. A BLASTx was performed to match the genes with the assembly by using BLAST (version 2.2.30+).
Raw sequence reads for factor H binding protein (fHbp) (http://pubmlst.org/neisseria/fHbp/) and Neisserial adhesion A (NadA) (https://pubmlst.org/organisms/neisseria-spp/nada) were sequence typed using PubMLST.org to determine the variant of Fhbp and NadA.
Case Report Recurrent Meningococcal Infections in a Patient With a Type II C8 Deficiency
A 6-year-old girl was evaluated at our outpatient clinic after 2 episodes of invasive meningococcal disease. Otherwise, her previous medical history was unremarkable.
She first presented with a fever and petechiae. There were no signs of meningism or septic shock. Laboratory investigation showed elevated leukocytes of 22.0 × 109/L and a C-reactive protein of 53 mg/L. Blood culture was positive for N. meningitidis, identified as serogroup Z by polymerase chain reaction. Cerebrospinal fluid (CSF) pleocytosis (251 × 106 leukocytes/µL) demonstrated meningitis, but CSF culture was negative. She was treated with intravenous penicillin for 7 days and recovered completely.
One month later, she presented with malaise, abdominal pain, and vomiting. On examination, she had several petechiae but absence of meningism or fever. Laboratory investigation showed no elevated inflammation parameters (leucocytes 8.3 × 109/L; C-reactive protein of 3 mg/L). Given the recent meningococcal infection, intravenous ceftriaxone was started. Blood cultures demonstrated N. meningitidis identified as serogroup C by polymerase chain reaction. CSF culture was negative. She recovered completely. She had been vaccinated with conjugated N. meningitidis serogroup C vaccine 5 years previously during routine childhood vaccinations.
Immune work-up was performed. Immunoglobulin levels were normal (IgG 10.1 g/L; IgA 0.61 g/L and IgM 0.99 g/L). Ultrasound examination of the abdomen demonstrated a normal size spleen. Screening of complement activity showed low CH50 < 10% and low AP50 < 20%. Further evaluation of the complement activity by Sanquin Diagnostic services showed a type 2 C8 deficiency. C8 was present, detected using the Ouchterlony technique, but nonfunctional. After in vitro reconstitution with complement factor C8, classical complement pathway activity was restored. Genetic screening of C8A, C8B and C8G demonstrated a previously described homozygous truncating mutation in exon 3 of C8B (NM_000066.4(C8B):c.271C>T (p.Gln91Ter).11
The girl’s family was screened for complement deficiency. Both parents were heterozygous carriers, with normal complement activity. Her sister, a healthy 10-year-old girl was found to have the same genetic and functional complement deficiency. The sister had received N. meningitidis serogroup C vaccine during routine childhood vaccinations and had no previous history of meningococcal infections. Both girls were living together.
Both the girl and her sister received MenACYW-conjugate and 4CMenB vaccines and were started on amoxicillin prophylaxis. After 2 years, antibiotic prophylaxis was interrupted for our patient, but within 6 months she suffered another meningococcemia episode with mild disease, caused by nontypeable N. meningitidis. She was treated with ceftriaxone and recovered fully.
Assessment of Cross Reactivity
We performed in vitro experiments to determine whether vaccination of this child with MenB-4C increased the killing of N. meningitidis serogroup Z, to assess potential cross protection that could prevent invasive infections by N. meningitidis in a serogroup-independent manner.
Binding of IgG as well as C3 to the bacterial surface of the patient’s N. meningitidis serogroup Z strain was significantly increased after MenB-4C vaccination (Fig. 1A–B), as determined by flow cytometry. Serum bactericidal activity titer was 128 prevaccination and increased to > 512 postvaccination, although no serum killing was observed with the patient’s own serum, confirming the lack of complement-mediated killing (Fig. 1C–D). Increased whole blood killing of N. meningitidis serogroup Z was observed after MenB-4C vaccination, which is consistent with previous results with N. meningitidis serogroup B.8 This indicates that there is cross protection that results in increased clearance of the bacterium (Fig. 1E).
Cross protection could be mediated through homology between the proteins present in the vaccine and the N. meningitidis serogroup Z strain. Homology between the protein antigens in the vaccine and those potentially expressed by the N. meningitidis serogroup Z strain was determined based on bacterial whole-genome sequencing. The homology was 99.5% for Neisserial Heparin Binding Antigen, 99.5% for hemolysin, 94.1% for fHbp, 76.6% for NadA and 79.8% for transferrin binding protein. Since the outer membrane vesicles consist of multiple proteins, we determined homology for Porin B (91.8%), Porin A (95.0%), protein TonB (98.7%) and reduction-modifiable protein from N. meningitidis (100%) because these are the major proteins present in the OMVs.12 The MenZ strain expressed fHbp variant 1, which is very similar to fHbp variant 1.1 that is used in the MenB-4C vaccine. The NadA variant was NadA-2/3.13
Increased recognition by IgG and complement activation could be observed in serum after MenB-4C vaccination of a child with C8 deficiency who experienced a previous N. meningitidis serogroup Z infection. Increased whole blood killing postvaccination was observed, indicating that binding of IgG, complement activation and the presence of immune cells was sufficient to enhance the clearance of N. meningitidis.
Complement deficient patients, particularly those with a terminal pathway deficiency, are susceptible to meningococcal infections. The terminal complement pathway initiates the cleavage of C5 into C5b and C5a, where C5b is essential in the formation of the membrane attack complex resulting in direct lysis of Gram-negative bacteria and C5a release increases chemotaxis and activation of mainly neutrophils. Patients with a primary or acquired complement C5 deficiency can still be susceptible to meningococcal infection after vaccination,14,15 probably because not only complement-mediated bacteriolysis initiated by C5b is lacking but also because neutrophil opsonophagocytic killing is hampered in the absence of C5a.9,16,17 Vaccination of patients with other terminal complement deficiencies (eg, C6–C9) has shown to reduce the incidence of meningococcal disease, as effective opsonophagocytosis helps clear meningococcal infections.8,18
Although the patient did show and effective serum anti-meningococcal IgG increase and increased in vitro clearance of MenZ after vaccination, the patient went through another meningococcal infection two and a half years later due to a nontypeable N. meningitidis strain. Waning of antibodies could potentially explain why the girl encountered another meningococcal infection. This is the reason the CDC’s Advisory Committee on Immunization Practices suggests booster doses for group B meningococcal vaccines in high-risk populations.19 Cross reactivity of MenB-4C vaccination for this nontypeble N. meningitidis strain was not determined.
We recommend vaccinating all children with complement deficiencies with MenB-4C, not only to prevent N. meningitidis serogroup B infections, but also to elicit broader protection against other N. meningitidis serogroups. This illustrates that protein-based vaccines may also have the potency to induce species-wide immunity in for instance Streptococcus pneumoniae or Haemophilus influenzae.
We thank Arie van den Ende, PhD, from the Netherlands Reference Laboratory for Bacterial Meningitis at the Amsterdam University Medical Center for providing the Neisseria meningitidis serogroup Z strain. We thank Dineke Westra, PhD clinical laboratory geneticist for the molecular genetic analysis. We thank Kirsten Huizing, MD pediatrician for initial patient description.
1. Harrison LH, Trotter CL, Ramsay ME. Global epidemiology of meningococcal disease. Vaccine. 2009;27(suppl 2):B51–B63.
2. Skattum L, van Deuren M, van der Poll T, et al. Complement deficiency
states and associated infections. Mol Immunol. 2011;48:1643–1655.
3. Nagata M, Hara T, Aoki T, et al. Inherited deficiency of ninth component of complement: an increased risk of meningococcal meningitis. J Pediatr. 1989;114:260–264.
4. Fijen CA, Kuijper EJ, te Bulte MT, et al. Assessment of complement deficiency
in patients with meningococcal disease in The Netherlands. Clin Infect Dis. 1999;28:98–105.
5. Petousis-Harris H, Radcliff FJ. Exploitation of Neisseria meningitidis Group B OMV Vaccines Against N. gonorrhoeae to Inform the Development and Deployment of Effective Gonorrhea Vaccines. Front Immunol. 2019;10:683.
6. Toneatto D, Pizza M, Masignani V, et al. Emerging experience with meningococcal serogroup B protein vaccines. Expert Rev Vaccines. 2017;16:433–451.
7. Fazio C, Biolchi A, Neri A, et al. Cross-reactivity of 4CMenB vaccine-induced antibodies against meningococci belonging to non-B serogroups in Italy. Hum Vaccin Immunother. 2021;17:2225–2231.
8. van den Broek B, van Els CACM, Kuipers B, et al. Multi-component meningococcal serogroup B (MenB)-4C vaccine induces effective opsonophagocytic killing in children with a complement deficiency
. Clin Exp Immunol. 2019;198:381–389.
9. Langereis JD, van den Broek B, Franssen S, et al. Eculizumab impairs Neisseria meningitidis serogroup B killing in whole blood despite 4CMenB vaccination of PNH patients. Blood Adv. 2020;4:3615–3620.
10. van der Maten E, de Jonge MI, de Groot R, et al. A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood. Sci Rep. 2017;7:42137.
11. Saucedo L, Ackermann L, Platonov AE, et al. Delineation of additional genetic bases for C8 beta deficiency. Prevalence of null alleles and predominance of C–>T transition in their genesis. J Immunol. 1995;155:5022–5028.
12. Tani C, Stella M, Donnarumma D, et al. Quantification by LC-MS(E) of outer membrane vesicle proteins of the Bexsero® vaccine. Vaccine. 2014;32:1273–1279.
13. Ladhani SN, Campbell H, Andrews N, et al. First real world evidence of meningococcal group B vaccine, 4CMenB, protection against meningococcal group W disease; prospective enhanced national surveillance, England [published online ahead of print August 26, 2020]. Clin Infect Dis. doi: 10.1093/cid/ciaa1244.
14. Ladhani SN, Campbell H, Lucidarme J, et al. Invasive meningococcal disease in patients with complement deficiencies: a case series (2008-2017). BMC Infect Dis. 2019;19:522.
15. Parikh SR, Lucidarme J, Bingham C, et al. Meningococcal B vaccine failure with a Penicillin-Resistant strain in a young adult on long-term eculizumab. Pediatrics. 2017;140:e20162452.
16. Nolfi-Donegan D, Konar M, Vianzon V, et al. Fatal nongroupable neisseria meningitidis disease in vaccinated patient receiving eculizumab. Emerg Infect Dis. 2018;24:1561–1564.
17. Konar M, Granoff DM. Eculizumab treatment and impaired opsonophagocytic killing of meningococci by whole blood from immunized adults. Blood. 2017;130:891–899.
18. Platonov AE, Vershinina IV, Kuijper EJ, et al. Long term effects of vaccination of patients deficient in a late complement component with a tetravalent meningococcal polysaccharide vaccine. Vaccine. 2003;21:4437–4447.
19. Mbaeyi SA, Bozio CH, Duffy J, et al. Meningococcal vaccination: recommendations of the advisory committee on immunization practices, United States, 2020. MMWR Recomm Rep. 2020;69:1–41.