Differential effects of therapeutic complement inhibitors on serum bactericidal activity against non-groupable meningococcal isolates recovered from patients treated with eculizumab

Abstract

Eculizumab is licensed for the treatment of paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and generalized myasthenia gravis. By blocking C5, eculizumab inhibits meningococcal serum bactericidal activity (SBA), leaving patients at an approximately 2000-fold higher risk of meningococcal disease than the general population. This increased risk includes invasive disease caused by unencapsulated (non-groupable, NG) strains, which rarely cause invasive disease in normal hosts. ACH-4471 targets complement factor D in the alternative pathway (Online Supplementary Figure S1), and is being developed to treat paroxysmal nocturnal hemoglobinuria and C3 glomerulopathy. Since the terminal pathway remains intact, SBA can occur in the presence of ACH-4471 via the classical and lectin pathways (Online Supplementary Figure S1). In a previous study, eculizumab completely blocked whole blood opsonophagocytic killing of encapsulated serogroup B and C meningococcal strains while blocking factor D with ACH-4471 gave less impairment. Here we describe 18 cases of invasive meningococcal disease in eculizumab recipients reported to the US Centers for Disease Control and Prevention between January 2008 and July 2018 (Online Supplementary Table S1), and the effect of eculizumab and ACH-4471 on SBA against NG meningococcal isolates recovered from eight of these patients. The indications for eculizumab were paroxysmal nocturnal hemoglobinuria in 12 patients, atypical hemolytic uremic syndrome in five, and neuromyelitis optica (Devic disease) in one. Information on antibiotic prophylaxis was available for 14 patients: two (cases 11 and 18) were receiving penicillin at the time of meningococcal disease onset (Online Supplementary Table S1). The small number of patients in the present series receiving prophylactic antibiotics likely reflected lack of an official recommendation in the USA on routine use of antibiotic prophylaxis in this population. The susceptibility of the isolates to penicillin was measured by reference broth microdilution. Isolates from five cases, including both recipients of prophylaxis with the antibiotic, showed intermediate penicillin sensitivity (cases 10, 11, 16, 17 and 18) and one more isolate was penicillin-resistant (case 12, Online Supplementary Table S1). Capsular serogroups were evaluated by slide agglutination, whole genome sequencing and/or polymerase chain reaction. Thirteen of the 17 cases (76%) with a clinical sample or an isolate available for testing were caused by strains that were NG phenotypically (n=12, Table 1) or, for one case where only a clinical specimen was available, by polymerase chain reaction (Table 1). The remaining four were caused by encapsulated serogroup Y isolates (Online Supplementary Table S1). All but one of the NG isolates (case 11, C backbone) showed evidence of disrupted or phase-variable “off” capsular polysaccharide synthesis gene loci (Table 1). For 16 patients there was documentation of receipt of serogroup A, C, W, Y meningococcal quadrivalent polysaccharide or conjugate vaccine, whereas vaccination status for the remaining two patients was unknown. Of the six eculizumab-treated patients with meningococcal disease onset after June 2015, when serogroup B meningococcal vaccine (MenB) was first recommended, one patient had received one dose of MenB and three had received a complete MenB series (Online Supplementary Table S1). The MenB-FHbp (Trumenba®) vaccine contains two recombinant FHbp variants from sub-families A and B, and MenB-4C (Bexsero) contains a sub-family B FHbp and three other meningococcal antigens (recombinant NHba and NadA, and PorA P1.4 in outer membrane vesicles). These antigens are not limited to capsular group B meningococcal strains. Vaccine antigen expression for the 12 available NG isolates was characterized by flow cytometry using mouse antisera to FHbp, NHba, and NadA as previously described. All 12 isolates expressed sub-family A or B FHbp and one or two other MenB-4C antigens (Online Supplementary Figure S2A and S2B). None had PorA P1.4. We measured SBA in sera from eight healthy unvaccinated adults and eight adults vaccinated in previous studies with MenB-4C (n=4) or MenB-FHbp (n=4). Serum selection was based on availability of sufficient volumes for the current studies. One individual, subject 9010, provided sera both before vaccination and 1 month after the MenB-4C dose 2. Sera from the remaining vaccinated subjects were obtained 6 to 12 months after MenB-4C dose 2 or MenB-FHbp dose 3. The SBA assay used early log phase bacteria incubated with a 1:5 dilution of serum with internal complement preserved. Eculizumab (50 μg/mL), ACH-4471 (1 mM), or no inhibitor was added to the reaction and the percent bacterial survival was calculated after incubation at 37°C for 60 min. Against a control serogroup B encapsulated strain (H44/76), all eight unvaccinated sera had SBA titers <1:5 (bacterial survival >50%) (Figure 1A), whereas all eight post-immunization sera had SBA titers ≥1:5 (Figure 1D, G) (P=0.0002, Fisher exact test). Eculizumab completely blocked vaccine-induced SBA (Figure 1D, G). In contrast, ACH-4471 had little or no effect on vaccine-induced SBA against the capsular group B strain (titers ≥1:5 in all 8 vaccinated subjects) (Figure 1D, G). In the absence of an added inhibitor, SBA titers were ≥1:5 against two NG isolates with a disrupted B cps locus (isolate CH891) (Figure 1B, E, H) or phase-variable “off” B cps locus (isolate CH892, Figure 1C, F, I) for all 16 unvaccinated or vaccinated subjects. Eculizumab again completely blocked SBA, whereas ACH-4471 had minimal effect. Similarly, in the absence of a complement inhibitor, three NG isolates with disrupted Y cps loci (Online Supplementary Figure S3), and two NG isolates with capsular null loci (cnl; CH885 and CH896) (Online Supplementary Figure S4) were killed by sera from all 16 unvaccinated or vaccinated adults. Eculizumab completely blocked SBA for all of these isolates and all sera. Blocking the alternative pathway with ACH-4471 had minimal or no effect on SBA against both cnl isolates and two of the three test isolates with disrupted Y capsular loci. Against the third isolate with a disrupted Y capsular locus, SBA titers were <1:5 in the presence of ACH-4471 for five of the eight unvaccinated subjects and one of the eight vaccinated subjects (Online Supplementary Figure S3C, F, and I). The eighth NG test isolate (CH886), with a disrupted E cps locus, was resistant to killing by sera from two of the eight unvaccinated subjects in the absence of added inhibitor (bacterial survival >50%; titers <1:5 ) (Figure 2A, subjects C and E). The isolate was killed by sera from all