C. A. P. Fijen

Assessment of Complement Deficiency in Patients with Meningococcal Disease in the Netherlands

The frequency of complement deficiency in 176 of 7,732 patients with meningococcal disease in the Netherlands from 1959 through 1992 was assessed. Complement deficiency was found in six patients (3%): 3 (7%) of the patients with Neisseria meningitidis serogroup C disease, 1 (2%) of the patients with N. meningitidis serogroup A disease, and 2 (33%) of the patients with infections due to uncommon serogroups and nongroupable strains of N. meningitidis. Of 91 additional patients with meningococcal infections due to uncommon serogroups, 33% also had complement deficiency. Thirty-four of the 36 complement-deficient patients with meningococcal disease who were from 33 families were 5 years of age or older. Twenty-six additional complement-deficient relatives were found. Screening individuals with meningococcal disease due to uncommon serogroups who were 5 years of age or older identified 30 of the 33 complement-deficient families. Only 27% of the complement-deficient relatives had had meningococcal disease. This risk was lower for relatives with properdin deficiency (18%) than for those deficient in the late component of complement (38%). Therefore, pedigree studies are warranted for identifying those complement-deficient persons who require vaccination for meningococcal disease.

Binding of mannan-binding protein to various bacterial pathogens of meningitis

Mannan‐binding protein (MBP), a calcium‐dependent plasma lectin, may play a role in the innate defence against microorganisms. After binding lo carbohydrate structures at the bacterial surface, MBP activates the classical pathway of the complement system. To investigate the binding capacity of MBP to various bacteria associated with meningitis, an assay was developed to study the binding of MBP to bacteria grown in a semisynthetic fluid culture medium. Salmonella montevideo (containing a mannose‐rich lipopolysaccharide (EPS)), used as a positive control strain, showed binding of radiolabelled MBP at a level of 80% compared with binding of MBP to zymosan. Binding of labelled MBP to Salm. montevideo was time‐dependent, temperature‐dependent and saturable. The binding, was inhibited by unlabelled MBP., by mannose and by N‐acetyl‐o‐glucosamine. Among bacterial pathogens often found to cause meningitis, a wide range of MBP binding capacities could be determined. The encapsulated Neisseria meningitidis (representatives from 11 serogroups other than group A were included: n = 22), N. mucosa (n = 1), Haemophilus influenzae type b (n = 10) and Streptococcus agalactiae (n = 5) had a low MBP binding capacity of 21.7% (95% confidence interval (Cl) 3.3–40.1%). Escherichia coli K1 (n =11). Strep, suis (n = 5), Strep, pneumoniae (n = 10) and N. meningitidis scrogroup A (n = 2) showed intermediate MBP binding capacity of 58.4% (95% Cl 40.0–76.8%). A third group consisting of non‐encapsulated Listeria monocytogenes (n = 11), non‐encapsulated H. influenzae (n = 2), non‐encapsulated N. meningitidis (n = 2), N. cinera (n = 1) and N. subflava (n = 1) strains had a high MBP binding capacity of 87.5% (95% CI 62.5–12.5%). The majority of encapsulated pathogens causing bacterial meningitis seem to have a rather low MBP binding capacity.

The role of Fcγ receptor polymorphisms and C3 in the immune defence against Neisseria meningitidis in complement-deficient individuals

Individuals with either a late (C5–9) complement component deficiency (LCCD) or properdin deficiency are at increased risk to develop meningococcal disease, often due to serogroups W135 and Y. Anti‐meningococcal defence in both LCCD persons and properdin‐deficient individuals without bactericidal antibodies depends mainly on phagocytosis. Three types of opsonin receptors are involved in phagocytosis by polymorphonuclear cells (PMN). These represent the polymorphic FcγRIIa (CD32) and FcγRIIIb (CD16b) receptors, and the C3 receptor CR3 (CD11b/CD18). When the distribution of FcγRIIa and FcγRIIIb allotypes was assessed in 15 LCCD and in 15 properdin‐deficient patients with/without previous meningococcal disease, we found the combination of FcγRIIa‐R/R131 with FcγRIIIb‐NA2/NA2 allotypes to be associated with previous meningococcal disease (odds ratio 13·9, Fisher’s test P = 0·036). No such relation was observed in the properdin‐deficient patients. The importance of FcγRIIa allotypes was also demonstrated using in vitro phagocytosis assays. PMN from FcγRIIa‐R/R131 homozygous donors internalized IgG2 opsonized meningococci W135 significantly (P < 0·05) less than PMN from FcγRIIa‐H/H131 donors. When properdin‐deficient serum was tested, it was observed that reconstitution with properdin resulted in enhanced PMN phagocytosis of the W135 meningococci (P = 0·001). This enhanced phagocytosis was parallelled by an increase in C3 deposition onto the opsonized meningococci W135 (r = 0·6568, P = 0·01). We conclude that the occurrence of meningococcal disease in LCCD patients is associated with certain FcγR allotypes. Properdin‐deficient individuals are susceptible to meningococcal disease because of an insufficient C3 deposition on the surface of meningococci, resulting in insufficient phagocytosis.

Protection against meningococcal serogroup ACYW disease in complement-deficient individuals vaccinated with the tetravalent meningococcal capsular polysaccharide vaccine

Individuals with properdin, C3 or late complement component deficiency (LCCD) frequently develop meningococcal disease. Vaccination of these persons has been recommended, although reports on efficacy are scarce and not conclusive. We immunized 53 complement‐deficient persons, of whom 19 had properdin deficiency, seven a C3 deficiency syndrome and 27 had LCCD with the tetravalent (ACYW) meningococcal capsular polysaccharide vaccine. Serological studies were performed in 43 of them. As controls 25 non‐complement‐deficient relatives of the complement‐deficient vaccinees and 21 healthy non‐related controls were vaccinated. Post‐vaccination, complement‐deficient individuals and controls developed a significant immunoglobulin‐specific antibody response to capsular polysaccharides group A, C, Y, W135, but a great individual variation was noticed. Also, the proportion of vaccinees of the various vaccinated groups with a significant increase in bactericidal titre (assayed with heterologous complement) was similar. Opsonization of meningococci A and W135 with sera of the 20 LCCD individuals yielded in 11 (55%) and eight (40%) sera a significant increase of phagocytic activity after vaccination, respectively. Despite vaccination, four complement‐deficient patients experienced six episodes of meningococcal disease in the 6 years post‐vaccination. Four episodes were due to serogroup B, not included in the vaccine. Despite good response to serogroup Y upon vaccination, disease due to serogroup Y occurred in two C8β‐deficient patients, 3.5 and 5 years post‐vaccination. These results support the recommendation to vaccinate complement‐deficient individuals and to revaccinate them every 3 years.

Heterozygous and homozygous factor H deficiency states in a Dutch family

Factor H, a 150‐kD protein, is an important down‐regulating protein of the alternative pathway of the complement system. Presently, only 15 persons, representing seven families, have been described with homozygous factor H deficiency. Deficiency of this protein, inherited as an autosomal recessive trait and resulting in uncontrolled breakdown of C3, results in depletion of components of the alternative pathway (factor B, properdin) and of the terminal pathway (C5), and is associated with the onset of bacterial infections, glomerulonephritis and systemic lupus erythematosus (SLE). The proband of the family in this study suffered from subacute cutaneous lupus erythematosus and had had meningococcal meningitis due to serogroup X. She had a complete factor H deficiency at the protein level as determined by Western blotting. Among 21 relatives of the proband studied, encompassing three generations, 10 had low factor H levels, including the two children of the proband, indicating a heterozygous factor H deficiency state. In serum samples of the proband and 11 relatives prospectively studied, a strong correlation of factor H levels with C3, C3 haemolytic activity, factor B and properdin levels (P<0.0001) was found. Alternative pathway protein levels were significantly lower (Mann–Whitney test; Z values 3.6–2.7) in sera from the four heterozygous relatives studied than in sera from the seven non‐deficient relatives. In addition, a defect of the 37/42‐kD H‐related protein was found in the proband and two of 21 relatives, compared with four of 40 controls. A defect of the 24/29‐kD H‐related protein was present in one of 21 relatives studied and in none of the 40 controls.

Development of antibodies against tetravalent meningococcal polysaccharides in revaccinated complement-deficient patients.

Individuals deficient in C3 or a late complement component are susceptible to recurrent meningococcal infections. Since they experience meningococcal episodes mostly with uncommon meningococcal serogroups, vaccination with a tetravalent vaccine containing A, C, Y and W135 polysaccharides has been suggested. We vaccinated a cohort of two C3 and 17 late complement component‐deficient (LCCD) patients, revaccinated them 7 years later and investigated the development of their IgG antibodies to the capsular polysaccharides of the meningococcal vaccine. Seven years after the first vaccination levels of IgG antibodies declined compared with the levels present at 6 months after the first vaccination, but were still at least four times higher than before vaccination. Levels of antibodies to Y polysaccharide in serum of complement‐deficient patients were rather low but they did not differ significantly from those in serum of healthy non‐related controls (P = 0.07). Three months after the second vaccination IgG antibodies against all polysaccharides increased, exceeding those measured at 6 months after the first vaccination. In the 8 years of observation after the first vaccination two new meningococcal infections with strains related to the vaccine (serogroup Y strains) occurred in two patients, 3.5 and 5 years after the first vaccination. Our findings show that high IgG antibody levels against the tetravalent meningococcal polysaccharide vaccine were reached after revaccination of two C3 and 17 LCCD individuals 7 years after the first vaccination. Whether revaccination should be required within a period shorter than 7 years is discussed, since two vaccinees developed meningococcal disease to vaccine serogroup Y.

Antibody-Dependent Killing of Meningococci by Human Neutrophils in Serum of Late Complement Component-Deficient Patients

Background: Thirty-one Russian patients with late complement component deficiency (LCCD) who had experienced one to five meningococcal infections were immunized with meningococcal polysaccharide vaccine (A + C + W135 + Y) and were followed for 3–8 years. We investigated the potentially protective killing effect of human neutrophils (PMNL) on serogroup A and W135 meningococci. Methods: Meningococci were incubated in LCCD vaccinee sera in the absence or presence of PMNL, and the number of live bacteria (CFU) was determined by plating onto chocolate agar. Results: When meningococci were incubated in the LCCD sera alone, exponential growth of meningococci occurred despite the presence of meningococcal antibodies. After the addition of PMNL, meningococci were inhibited in their growth or even eliminated. Group A or W135 meningococci were killed effectively by PMNL in 80% of the sera which were collected 1 month to 1 year after vaccination compared to only 40% in the prevaccination LCCD sera (p < 0.05). Three years after vaccination 67% of the LCCD sera were still capable of promoting killing (and even 90% after revaccination). The rate of killing correlated with the concentration of serogroup-specific immunoglobulins. In 83% of the 72 LCCD sera with more than 5 µg/ml anti-group A immunoglobulins the killing of group A meningococci was promoted. By contrast, only 21% of 19 samples with lower specific antibody levels showed a PMNL-mediated meningococcal killing (p < 0.05). The same effect was observed for group W135 meningococci. Conclusion: PMNL kill meningococci during incubation in LCCD serum; this effect increases after vaccination and depends on both specific antibody and complement. Protection by vaccination may therefore be caused by an increased killing capacity of PMNL.

The effect of mannan-binding lectin on opsonophagocytosis of Neisseria meningitidis.

Mannan-binding lectin (MBL), an acute phase protein with a structure and a function very similar to that of C1q, is known to act as an opsonin binding to a number of microorganisms. In order to investigate the effect of MBL on the phagocytic killing of meningococci, a serogroup B meningococcal strain (H44/76) and its unencapsulated variant v24, as well as a serogroup A meningococcal strain were opsonized with MBL (purified from normal human plasma at the State Serum Institute, Denmark) and used in a phagocytic killing assay at a density of 7 x 10(3) CFU/ml. Polymorphonuclear cells (PMNs) from one healthy donor were isolated by density gradient centrifugation over Percoll and added to the system (7 x 10(6) cells/ml). In a first set of experiments without addition of serum or complement, no influence of MBL was observed on the killing of any of these strains. Addition of MBL to non-opsonized bacteria of the serogroup A strain did not result in enhanced killing either; on the contrary, the growth of this strain increased significantly when a high MBL concentration (40 micrograms/ml) was used in the presence of PMNs. Further investigations were performed using sera of five individuals with late complement component deficiency (LCCD) and a concomitant MBL deficiency, vaccinated with a tetra-valent (ACYW135) meningococcal capsular polysaccharide vaccine. Pre- and post-vaccination sera (50% final concentration) were tested against a group A strain opsonized or not with MBL. In only one patient was there a moderate increase of killing of the opsonized bacteria after vaccination compared to pre-vaccination serum. Our results suggest that MBL may not play a significant role in the opsonophagocytosis of meningococci, irrespective of its binding to unencapsulated and serogroup A strains.

Inherited complement deficiency in children surviving fulminant meningococcal septic shock

We evaluated the complement system in 29 children (mean age: 4.5 years) who survived fulminant meningococcal septic shock. No terminal complement deficiencies were found. One patient, who experienced the most dramatic disease course, had a decreased haemolytic activity in the haemolytis-in-gel test for the alternative pathway. The properdin concentration in serum of this patient was < 0.1 μg/ml (n = 17.1−27.7 μg/ml). Coagulation studies revealed a heterozygeous type I protein C deficiency as well. He was the only patient with aNeisseria meningitidis group Y infection.

Fulminant meningococcal septic shock in a boy with combined inherited properdin and protein C deficiency.

A 7‐year‐old patient with fulminant septic shock, due to Neisseria meningitidis of the uncommon serogroup Y developed extensive gangrene of the limbs. Multiple amputations were necessary and a pulmonary embolism occurred within 2 days post‐operatively. Complement and haemostatic system studies, done after recovery, showed a complete absence of properdin antigen and a low protein C antigen and activity level in plasma. Defective haemolytic activity in gel by the alternative pathway of complement activation could be restored with purified properdin, indicating a properdin deficiency type 1. Protein C antigen level as well as activity were in agreement with a protein C deficiency type I. The polymerase chain reaction (PCR) product of exon five of the protein C gene showed a substitution of 72Gly by Arg. Both deficiencies were traced among relatives of the patient. Serum of the father of the patients mother was also properdin‐deficient. Microsatellite haplotyping of the X‐chromosome of the patient and his relatives showed that a distinct haplotype cosegregated with the properdin deficiency (Lodscore 2·25; four informative meioses). The protein C type I deficiency was present in the patients mother and her mother and cosegregated with the mutation found. So far as is known, this is the first patient described with combined inherited properdin deficiency and protein C deficiency.