ENINGOCOCCAL DISEASE caused predominantly by Neisseria meningitidis serogroups A, B, and C occurs predominantly in young children and remains a substantial cause of morbidity and mortality worldwide. 1,2 In addition to causing endemic disease globally, meningococci, unlike other encapsulated bacteria, cause epidemics. Serogroup B epidemics, problematic in Norway and throughout much of Latin America in the 1980s and 1990s, 1 have recently emerged in New Zealand 3 and the United States. [4][5][6] Response to serogroup B epidemics, unlike serogroup A and C epidemics, is difficult because existing serogroup B vaccines have not been shown to be efficacious on an international scale. [7][8][9][10] Quadrivalent meningococcal polysaccharide vaccine is efficacious against meningococcal disease caused by the A, C, W-135, and Y serogroups. [11][12][13] Serogroup B polysaccharide antigen, however, is poorly immunogenic in humans, 14,15 and the elicitation of antibodies to serogroup B polysaccharide antigen is of concern because this antigen is present in human neonatal neural tissue. 16,17 Therefore, alternative Author Affiliations are listed at the end of this article.
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.
Host protection against pneumococcal disease is primarily mediated by phagocytosis. We developed and standardized an opsonophagocytic assay using HL-60 cells (human promyelocytic leukemia cells). Fifty-five serum samples were analyzed for the presence of functional antibody against seven pneumococcal serogroups or serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by using differentiated HL-60 cells (granulocytes) and peripheral blood leukocytes (PBLs). Six of the 55 serum samples were from unvaccinated adult volunteers, 31 serum samples were from adults who received one dose of the 14-valent or the 23-valent polysaccharide vaccine, and 18 serum samples were from 16-month-old infants who received four doses of an investigational 7-valent polysaccharide-protein conjugate vaccine. The results of an opsonophagocytic assay with HL-60 cells correlated highly with those of an assay with PBLs as effector cells (median r for seven serotypes ؍ 0.87; P < 0.01). Opsonophagocytic titers were compared with the immunoglobulin G antibody concentrations determined by enzyme-linked immunosorbent assay (ELISA). The r values for serogroups or serotypes 4
Serum bactericidal activity (SBA) and ELISA antibody levels elicited by two efficacious serogroup B meningococcal vaccines were measured in a controlled trial involving 408 15- to 20-year-olds. Subjects were given two doses at a 6-week interval of a serogroup B or control vaccine. Response was defined as > or = 4-fold rise in antibody level. After two doses of the Finlay Institute (Havana) vaccine at 12 months, the proportions of SBA and ELISA responders were not different from those of the control group (15% and 17% [vaccine] vs. 13% and 9% [control], P > .05). After two doses of the National Institute of Public Health (Oslo) vaccine, there were more SBA and ELISA responders than in the control group (47% and 34% [vaccine] vs. 10% and 1% [control]) or the Finlay Institute vaccine group (P < .05 for both). SBA and ELISA may be insensitive correlates for protective efficacy for some outer membrane protein-based serogroup B meningococcal vaccines.
Botulism is a potentially fatal paralytic disease caused by the action of botulinum neurotoxin (BoNT) on nerve cells. There are 7 known serotypes (A-G) of BoNT and up to 40 genetic variants. Clostridium botulinum strain IBCA10-7060 was recently reported to produce BoNT serotype B (BoNT/B) and a novel BoNT, designated as BoNT/H. The BoNT gene (bont) sequence of BoNT/H was compared to known bont sequences. Genetic analysis suggested that BoNT/H has a hybrid-like structure containing regions of similarity to the structures of BoNT/A1 and BoNT/F5. This novel BoNT was serologically characterized by the mouse neutralization assay and a neuronal cell-based assay. The toxic effects of this hybrid-like BoNT were completely eliminated by existing serotype A antitoxins, including those contained in multivalent therapeutic antitoxin products that are the mainstay of human botulism treatment.
An infant mouse assay system for assessment of protective concentrations of human serum pneumococcal anticapsular antibodies is described. Passive immunization of anticapsular antibodies was evaluated for protection of infant mice challenged with Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 18C, and 23A, with bacteremia as an end point. Protection was defined as no detectable bacteremia in 70% of mice 48 h after challenge. Type-specific anticapsular concentrations required for protection varied with serotype (=0.05 to >0.4 microg/mL). Across serotypes, there was no significant correlation between human IgG concentration in mouse serum and protection from bacteremia or between IgG concentration and opsonophagocytic titer. Significant correlation (r=.84, P<.001) was observed between opsonophagocytic titer of human IgG antibody in mouse sera and protection from bacteremia. Thus, protective concentrations of anticapsular antibodies against bacteremia are serotype dependent. Opsonophagocytosis is a better predictor of in vivo protective capacity of pneumococcal anticapsular antibodies than are ELISA IgG antibody concentrations.
A unique strain of Clostridium botulinum (IBCA10-7060) was recently discovered which produces two toxins; botulinum neurotoxin (BoNT) serotype B and a novel BoNT reported as serotype H. Previous molecular assessment showed that the light chain (LC) of the novel BoNT most resembled the bont of the light chain of known subtype F5 while the C-terminus of the heavy chain (HC) most resembled the binding domain of serotype A. We evaluated the functionality of both toxins produced in culture by first incorporating an immunoaffinity step using monoclonal antibodies to purify BoNT from culture supernatants and tested each immune-captured neurotoxin with full-length substrates vesicle-associated membrane protein 2 (VAMP-2), synaptosomal-associated protein 25 (SNAP-25), syntaxin, and shortened peptides representing the substrates. The BoNT/B produced by this strain behaved as a typical BoNT/B, having immunoaffinity for anti-B monoclonal antibodies, and cleaving both full length VAMP-2 and a peptide based on the sequence of VAMP-2 in the expected location. As expected, there was no activity towards SNAP-25 or syntaxin. The novel BoNT demonstrated immunoaffinity for anti-A monoclonal antibodies, but did not cleave SNAP-25 as expected for BoNT/A. Instead, the novel BoNT cleaved VAMP-2 and VAMP-2-based peptides in the same location as BoNT/F5. This is the first discovery of a single botulinum neurotoxin with BoNT/A antigenicity and BoNT/F light chain function. This work suggests that the newly reported serotype H may actually be a hybrid of previously known BoNT serotype A and serotype F, specifically subtype F5.
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