Correlation between serum bactericidal activity against Neisseria meningitidis serogroups A, C, W-135 and Y measured using human versus rabbit serum as the complement source (original) (raw)
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Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 2015
PsA-TT (MenAfriVac) is a conjugated polysaccharide vaccine developed to eliminate group A meningococcal disease in Africa. Vaccination of African study participants with 1 dose of PsA-TT led to the production of anti-A polysaccharide antibodies and increased serum bactericidal activity measured using rabbit complement (rSBA). Bactericidal responses measured with human complement (hSBA) are presented here. Sera collected before and at 28 days and 1 year after vaccination with either PsA-TT or quadrivalent polysaccharide vaccine (PsACWY) from a random, age-distributed 360-subject subset of the Meningitis Vaccine Project study of PsA-TT in Africans aged 2-29 years were tested for hSBA. Geometric mean titer, fold-rise, and threshold analyses were compared between vaccine groups and age groups. hSBA, rSBA, and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) results were compared and assay correlation and agreement determined. hSBA responses to PsA-TT were substantially h...
Immunobiology, 2016
Polysaccharide conjugate vaccines are available to prevent disease caused by Neisseria meningitidis serogroups A, C, W and Y. Meningococcal vaccine efficacy is assessed in clinical trials using serum bactericidal assays (SBAs). Baby rabbit serum is usually used as the source of complement for SBAs as it lacks endogenous bactericidal activity compared to human serum. Previous studies have shown that SBA activities determined with rabbit (rSBA) and human (hSBA) complement correlate poorly, possibly due to different interactions between antibody subclasses and the complement source, and species-specific interaction of Neisseria meningitidis with complement regulators. The aim of this project was to investigate the mechanisms responsible for differential bactericidal activities of human and rabbit complement against Neisseria meningitidis. The serum concentration of polysaccharide-specific antibody subclasses was measured following vaccination with quadrivalent meningococcal polysaccharide vaccines; data showed that the concentration of polysaccharide-specific IgG1 antibody correlated most significantly with hSBA titres whereas the concentration of polysaccharide-specific IgM antibody correlated most significantly with rSBA titres. The interaction of human IgM and IgG subclasses with human and rabbit complement was compared at the level of C1q and C3 using both binding and functional assays. These data define important differences in the ability of human antibody subclasses to fix human and rabbit complement. Specifically, polysaccharide-specific IgM contributes significantly more to bactericidal titres in rSBAs compared to hSBAs. As a consequence, rSBAs produce misleadingly high titres in individuals with large IgM responses to vaccination. Using a series of pathway-specific inhibitors, it was shown that the alternative pathway contributes significantly more to the bactericidal activity of rabbit complement towards Neisseria meningitidis than human complement. This project provides significant insight into the difficulties and challenges associated with the interpretation of rSBA data, enhances the understanding of antibody responses to meningococcal vaccines and will support improvements in the development and testing of meningococcal vaccines.
Clinical and Vaccine Immunology, 2005
There is currently no standardized serum bactericidal antibody (SBA) assay for evaluating immune responses to meningococcal outer membrane vesicle or protein vaccines. Four laboratories, Manchester Health Protection Agency (MC HPA), New Zealand Institute of Environmental Science and Research Limited (NZ ESR), Norwegian Institute of Public Health (NIPH), and Chiron Vaccines (Chiron), measured SBA titers in the same panel of human sera (n ؍ 76) from laboratory staff (n ؍ 21) vaccinated with MenBvac. Blood samples were collected prevaccination, prior to each of the three doses of MenBvac given at 6-week intervals, and 6 weeks following the third dose. Initial results showed a number of discrepancies in results between the four participating laboratories. The greatest effect on titers appeared to be due to differences among laboratories in the maintenance of the meningococcal serogroup B test strain, 44/76-SL. A repeat study was conducted using the same frozen isolate (meningococcal serogroup B test strain 44/76-SL), freshly distributed to all four laboratories. Using SBA titers from the tilt method for all samples, and using MC HPA as the comparator, the results were as follows for NZ ESR, NIPH, and Chiron, respectively, using log 10 titers: correlation coefficients (r) were 0.966, 0.967, and 0.936; intercepts were 0.08, 0.15, and 0.17; and slopes were 0.930, 0.851, and 0.891. In both prevaccination and postvaccination samples from 15 subjects assayed by all four laboratories, similar increases in SBA (fourfold or greater) were observed (for 11, 11, 9, and 9 subjects for MC HPA, NZ ESR, NIPH, and Chiron, respectively), and similar percentages of subjects with SBA titers of >4 prevaccination and 6 weeks following each dose were found. The SBA assay has been harmonized between the four different laboratories with good agreement on seroconversion rates, n-fold changes in titers, and percentages of subjects with SBA titers of >4.
Clinical and Vaccine Immunology, 2014
ABSTRACTA meningococcal group A polysaccharide (PS) conjugate vaccine (PsA-TT) has been developed for African countries affected by epidemic meningitis caused byNeisseria meningitidis. Complement-mediated serum bactericidal antibody (SBA) assays are used to assess protective immune responses to meningococcal vaccination. Human complement (hC′) was used in early studies demonstrating antibody-mediated protection against disease, but it is difficult to obtain and standardize. We developed and evaluated a method for sourcing hC′ and then used the SBA assay with hC′ (hSBA) to measure bactericidal responses to PsA-TT vaccination in 12- to 23-month-old African children. Sera with active complement from 100 unvaccinated blood donors were tested for intrinsic bactericidal activity, SBA titer using rabbit complement (rSBA), and anti-group A PS antibody concentration. Performance criteria and pooling strategies were examined and then verified by comparisons of three independently prepared hC′...
Infection and Immunity, 2010
Certain complement defects are associated with an increased propensity to contract Neisseria meningitidis infections. We performed detailed analyses of complement-mediated defense mechanisms against N. meningitidis 44/76 with whole blood and serum from two adult patients who were completely C2 or C5 deficient. The C5-deficient patient and the matched control were also deficient in mannose-binding lectin (MBL). The proliferation of meningococci incubated in freshly drawn whole blood was estimated by CFU and quantitative DNA real-time PCR. The serum bactericidal activity and opsonophagocytic activity by granulocytes were investigated, including heat-inactivated postvaccination sera, to examine the influence of antimeningococcal antibodies. The meningococci proliferated equally in C2-and C5-deficient blood, with a 2 log 10 increase of CFU and 4-to 5-log 10 increase in DNA copies. Proliferation was modestly decreased in reconstituted C2-deficient and control blood. After reconstitution of C5-deficient blood, all meningococci were killed, which is consistent with high antibody titers being present. The opsonophagocytic activity was strictly C2 dependent, appeared with normal serum, and increased with postvaccination serum. Serum bactericidal activity was strictly dependent on C2, C5, and high antibody titers. MBL did not influence any of the parameters observed. Complement-mediated defense against meningococci was thus dependent on the classical pathway. Some opsonophagocytic activity occurred despite low levels of antimeningococcal antibodies but was more efficient with immune sera. Serum bactericidal activity was dependent on C2, C5, and immune sera. MBL did not influence any of the parameters observed.
International Archives of Allergy and Immunology, 2003
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 Ìg/ml antigroup A immunoglobulins the killing of group A meningococci was promoted. By contrast, only 21% of 19 samples with lower specific antibody levels showed a PMNLmediated 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.
Clinical and Vaccine Immunology, 2011
Functional anti-N. meningitidis serogroup A (MenA) activity in human serum is detected by serum bactericidal assay (SBA), using either rabbit (rSBA) or human (hSBA) complement, with F8238 as the recommended MenA SBA target strain. However, the F8238 strain may not be optimal for this purpose because, as we show here, it expresses the L11 immunotype, whereas most MenA invasive strains express the L(3,7)9 or L10 immunotype. Moreover, SBA results may be strain dependent, because immunotypes differ in their sensitivity to complement, emphasizing the need to choose the most appropriate strain. Sera from random subsets of infants, toddlers, children, and adolescents in clinical trials of MenA conjugate vaccines were tested by rSBA using strains 3125 (L10) and F8238 (L11). In unvaccinated subjects from all age groups, the percentages of seropositive samples (rSBA-MenA titer, >1:8) was lower using strain 3125 than using strain F8238. However, in toddlers and adolescents immunized with a conjugate MenA vaccine, the percentages of seropositive samples generally were similar using either strain in the rSBA. In two studies, sera also were tested with hSBA. Using hSBA, the differences in the percentages of seroprotective samples (hSBA-MenA titer, >1:4) between strains 3125 and F8238 was less apparent, and in contrast with rSBA, the percentage of seroprotective samples from unvaccinated subjects was slightly higher using strain 3125 than using strain F8238. In adults vaccinated with plain MenA polysaccharide, the percentage of seroprotective samples was higher using strain 3125 than with strain F8238, and the vaccine response rates using strain 3125 were better aligned with the demonstrated efficacy of MenA vaccination. In conclusion, SBA results obtained using the MenA L10 3125 strain better reflected vaccine-induced immunity. . Sequence typing of strain 3125 (performed by the Health Protection Agency, United Kingdom) revealed that it belongs to ST-7776 within the ST-1 clonal complex (serotype NT, serosubtype P1.5,2), and it has been characterized in the present study as immunotype L10. F8238 belongs to the ST-5 clonal complex (serotype 4.21, serosubtype P1.20,9), and it has been characterized as immunotype L11.
Clinical and Diagnostic Laboratory Immunology
A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strain F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was ؎1 dilution; interlaboratory reproducibility was ؎2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r ؍ 0.86; P < 0.001; slope ؍ 0.5) and serogroup C (n ؍ 0.86; P < 0.001; slope ؍ 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.