A High-throughput <em>Shigella</em>-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

University of Alabama at Birmingham

Currently available pneumococcal vaccines were examined for contamination by pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA). PspA could be detected in some (but not all) lots of 23-valent polysaccharide vaccine. Many lots of pneumococcal vaccines, including the heptavalent conjugate vaccine, were found to elicit small (but variable) antibody responses to PspA, PsaA, or both. The degree of contamination was highly variable, and this should be considered in pneumococcal vaccine evaluations or when capsular polysaccharide is used for pneumococcal antibody assays.

Immunogenic protein contaminants in pneumococcal vaccines.

For evaluating pneumococcal vaccines, the opsonophagocytic killing assay (OPKA) is useful as a supplement to the pneumococcal antibody enzyme-linked immunosorbent assay (ELISA). However, evaluations of pneumococcal vaccines require the determination of antibody responses to 7 to 11 serotypes, and the OPKA is tedious to perform and requires more serum than the ELISA. Consequently, the OPKA is infrequently used for evaluating pneumococcal vaccines. To overcome these limitations, we have developed a simple multiplexed (double-serotype) OPKA by using antibiotic-resistant pneumococci for nine serotypes. Serotype 6B, 9V, 19A, and 23F strains were made streptomycin resistant, and serotype 4, 6A, 14, 18C, and 19F strains were made optochin resistant. The multiplexed OPKA was the same as the single-serotype OPKA except for two changes. First, the target bacteria were a mixture of one streptomycin-resistant strain and one optochin-resistant strain. Second, the surviving bacteria of each serotype were enumerated by plating on Todd-Hewitt agar plates with yeast extract and an agar overlay containing the appropriate antibiotics and 2,3,5-triphenyl tetrazolium chloride. The performance of the multiplexed OPKA was evaluated by analyzing 28 serum samples from adults immunized with a 23-valent polysaccharide vaccine by using the single-serotype OPKA and the multiplexed OPKA. The multiplexed OPKA was specific for the desired serotypes. The multiplexed and conventional OPKAs had comparable assay sensitivities and produced results that were highly correlated (r(2) values ranging from 0.92 to 0.98) for all nine serotypes. A simple modification of the conventional OPKA produces a multiplexed assay that greatly reduces effort, reagents, and the necessary amount of serum.

Efficiency of a pneumococcal opsonophagocytic killing assay improved by multiplexing and by coloring colonies.

We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotype-specific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy.

Rapid multiplex assay for serotyping pneumococci with monoclonal and polyclonal antibodies.

We have undertaken a structural assessment of Streptococcus pneumoniae 11A polysaccharide as well as two clinical isolates related to 11A. The clinical isolates were labeled 11Aalpha and 11Abeta. The result of our experiments is a revision to the old structure for S. pneumoniae 11A polysaccharide. The new structure differs from the old structure in both the primary connectivities and acetylation pattern. We also show that 11A contains an acetylglycerol-PO4 moiety, a substitution that is heretofore unknown in the bacterial polysaccharide literature. The two clinical isolates were also structurally characterized. 11Aalpha was determined to be identical to 11A. 11Abeta is a new serotype, which differs from 11A in the absence of the acetylation of the glycerol-PO4 moiety and a different acetylation pattern of the saccharides. Thus, we propose that the acetylglycerol is the structural basis for 11Aalpha and 11Abeta subtypes.

Structure of the capsular polysaccharide of pneumococcal serotype 11A reveals a novel acetylglycerol that is the structural basis for 11A subtypes.

We have discovered a novel bacterial polysaccharide structural element, 3-O-acetylglycerol, in the Streptococcus pneumoniae ST11A polysaccharide: This moiety was elucidated through a combination of homonuclear and heteronuclear 1D and 2D NMR experiments using (1)H, (13)C, and (31)P in various combinations. The 3-O-acetylglycerol moiety is substoichiometrically O-acetylated in ST11A; yet, key connectivities that unequivocally show O-acetylation at the glycerol are provided by the long-range correlations from the acetate methyl groups to the glycerol in the (1)H-(13)C HMBC spectrum. Additionally, we clarify the (1)H-(31)P assignments previously presented.

Identification of 3-O-acetylglycerol, a novel structural element in bacterial polysaccharides.

To determine the prevalence of serotypes of Streptococcus pneumoniae responsible for pneumonia with pleural effusion, we determined the capsular polysaccharide (PS) type directly on 49 pleural fluid specimens collected from pediatric patients during 2007 to 2009 with laboratory-confirmed pneumococcal pneumonia by using monoclonal antibodies and a multiplex, bead array immunoassay. Because the fluids had to be heated to remove nonspecific reactivity before being tested in the immunoassay and type 19A PS is heat labile, the pleural fluid samples were also tested for serotype 19A capsule gene locus by PCR. Use of the multiplex immunoassay combined with type-specific 19A PCR allowed for serotype determination on 40 of 49 pleural fluids. Pneumococcal pneumonia with pleural effusion was associated with a limited number of serotypes, with types 1, 3, 7F/A, and 19A accounting for 75% of the typeable cases. The concentration of capsular PS in the pleural fluids was often greater than 1 μg/ml and sufficient to inhibit the opsonic capacity of sera from individuals who had received the 23-valent pneumococcal PS vaccine. Based on the serotypes observed before and after introduction of the 7-valent pneumococcal conjugate vaccine, the recently licensed 13-valent pneumococcal conjugate vaccine may reduce the incidence of pneumonia with pleural effusions.

Pneumococcal serotypes causing pneumonia with pleural effusion in pediatric patients.

To confirm the effect of 7-valent pneumococcal conjugate vaccine (PCV7), pneumococcal nasopharyngeal (NP) carriage was compared between vaccinated (3 + 1 doses PCV7) and non-vaccinated children. Vaccinated subjects were recruited from highly vaccinated regions (≥ 60%), Seoul and Incheon whereas control subjects were recruited from Jeju Island where vaccination rates are low (< 15%). NP swabs were obtained from 400 children aged 18-59 months. Serotype and antibiotic susceptibility was analyzed. Pneumococcal carriage rate was 18.0% (36/200) and 31.5% (63/200) for the vaccinated and control group, respectively. Among those vaccinated, 41.7% (15/36) of the serotypes were vaccine-related type (VRT: 6A, 6C, 19A) with the most common serotype 6C. The next common type was non-typable/non-capsule 30.6% (11/36) followed by non-vaccine type 16.7% (6/36) and vaccine type (VT) serotypes were found in only 11.1% (4/36). In contrast, 52.4% (33/63) of the isolates in the control group were VT. Resistance rates for penicillin and erythromycin were lower in the vaccine group (vaccine vs control; penicillin 45.2% vs 71.4%, erythromycin 74.2% vs 90.5%, P < 0.05). Multi-drug resistance was also lower in vaccinated subjects (vaccine vs control; 45.2% vs 69.8%, P < 0.05). PCV7 reduces carriage in VT which leads to replacement of pneumococci by antibiotic susceptible VRT or non-vaccine type strains.

Nasopharyngeal pneumococcal carriage of children attending day care centers in Korea: comparison between children immunized with 7-valent pneumococcal conjugate vaccine and non-immunized.

Streptococcus pneumoniae expresses more than 90 capsule types, and currently available pneumococcal vaccines are designed to provide serotype-specific protection. Consequently, serotyping of pneumococcal isolates is important for determining the serotypes to be included in pneumococcal vaccines and to monitor their efficacy. Yet serotyping of pneumococcal isolates has remained a significant technical challenge. By multiplexing many assays, we have now developed a simple yet comprehensive serotyping assay system that can not only identify all known pneumococcal serotypes but also subdivide nontypeable (NT) isolates into those with or without the conventional capsule locus. We have developed this assay system to require only six key reagents: two are used in one multiplex inhibition-type immunoassay, and four are required in two multiplex PCR-based assays. The assay system is largely automated by a seamless combination of monoclonal antibody-based and PCR-based multiplex assays using the flow cytometric bead array technology from Luminex. The assay system has been validated with a panel of pneumococci expressing all known pneumococcal serotypes and was found to be easily transferable to another laboratory.

Development of an automated and multiplexed serotyping assay for Streptococcus pneumoniae.

The divergent epidemiological behavior of Streptococcus pneumoniae serotypes suggests that serotype-specific features such as capsule O-acetylation influence the propensity of a strain to cause invasive pneumococcal disease (IPD). We hypothesize that innate host factors mediate the observed negative association between IPD and the serotype 11A (ST11A) capsule O-acetyltransferase gene, wcjE.

Low invasiveness of pneumococcal serotype 11A is linked to ficolin-2 recognition of O-acetylated capsule epitopes and lectin complement pathway activation.

Streptococcus pneumoniae clinical isolates were recently described that produced capsular polysaccharide with properties of both serotypes 6A and 6B. Their hybrid serological property correlated with mutations affecting the glycosyltransferase WciP, which links rhamnose to ribitol by an α(1-3) linkage for serotypes 6A and 6C and an α(1-4) linkage for serotypes 6B and 6D. The isolates had mutations in the triad residues of WciP that have been correlated with enzyme specificity. The canonical triad residues of WciP are Ala192-Ser195-Arg254 for serotypes 6A and 6C and Ser192-Asn195-Gly254 for serotypes 6B and 6D. To prove that the mutations in the triad residues are responsible for the hybrid serotype, we introduced the previously described Ala192-Cys195-Arg254 triad into a 6A strain and found that the change made WciP bispecific, resulting in 6A and 6B repeat unit expression, although 6B repeat unit production was favored over production of 6A repeat units. Likewise, this triad permitted a 6C strain to express 6C and 6D repeat units. With reported bispecificity in WciN, which adds either glucose or galactose as the second sugar in the serogroup 6 repeat unit, the possibility exists for a strain to simultaneously produce all four serogroup 6 repeat units; however, when genes encoding both bispecific enzymes were introduced into a 6A strain, only 6A, 6B, and 6D repeat units were detected serologically. Nonetheless, this may be the first example of a bacterial polysaccharide with three different repeat units. This strategy of expressing multiple repeat units in a single polymer is a novel approach to broadening vaccine coverage by eliminating the need for multiple polysaccharide sources to cover multiple serogroup members. 

Genetic, biochemical, and serological characterization of a new pneumococcal serotype, 6H, and generation of a pneumococcal strain producing three different capsular repeat units.

 is an important cause of diarrhea worldwide, with serotypes  2a,  3a, and  demonstrating epidemiological prevalence. Many development efforts are focused on  lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization with  vaccines containing LPS can elicit antibodies capable of killing  in a serotype-specific manner. Thus, to facilitate  vaccine development, we have developed a serum bactericidal assay (SBA) specific for three  serotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies against  2a,  3a, and  Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. The  SBA, combined with a reference serum, should facilitate the development of  vaccines across the field. is an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effective  vaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuring -specific functional antibodies  We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitate  vaccine development.

Jigui Yu

University of Alabama at Birmingham

A High-throughput Shigella-specific Bactericidal Assay