Fluorescent Multivalent Opsonophagocytic Assay for Measurement of Functional Antibodies to Streptococcus pneumoniae

We developed fluorescent mono- and multivalent opsonophagocytic assays (fOPA and fmOPA, respectively) specific for seven Streptococcus pneumoniae serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F). Bacterial survival was quantitated with alamar blue, a fluorescent metabolic indicator. Both fOPA and fmOPA allow for determination of viability endpoints for up to seven serotypes with high levels of agreement to the reference method. The fmOPA eliminates colony counting, reduces serum volume, and produces results in 1 day.     

Approach to Validating an Opsonophagocytic Assay for Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) polysaccharide serotype-specific antibodies that have opsonophagocytic activity are considered a primary mechanism of host defense against pneumococcal disease. In vitro opsonophagocytic assays (OPAs) with antibody and complement to mediate opsonophagocytic killing of bacteria have been designed and developed as an adjunct to the standardized serum immunoglobulin G antipneumococcal capsular polysaccharide enzyme immunoassay to assess the effectiveness of pneumococcal vaccines. OPA presents challenges for assay standardization and assay precision due to the multiple biologically active and labile components involved in the assay, including human polymorphonuclear leukocytes or cultured effector cells, bacteria, and complement. Control of these biologically labile components is critical for consistent assay performance. An approach to validating the performance of the assay in accordance with International Conference for Harmonization guidelines, including its specificity, intermediate precision, accuracy, linearity, and robustness, is presented. Furthermore, we established parameters for universal reagents and standardization of the use of these reagents to ensure the interlaboratory reproducibility and validation of new methodologies.     

Method for Simultaneous Measurement of Antibodies to 23 Pneumococcal Capsular Polysaccharides

We describe a fluorescent covalent microsphere immunoassay (FCMIA) method for the simultaneous (multiplexed) measurement of immunoglobulin G (IgG) antibodies to 23 pneumococcal capsular polysaccharide (PnPS) serotypes present in the pneumococcal polysaccharide vaccine (PPV23) licensed by the Food and Drug Administration, i.e., PnPSs 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. In addition, the assay incorporates an internal control that allows for contemporaneous evaluation of the effectiveness of pneumococcal cell wall polysaccharide (C-PS) preadsorption and a second control of PnPS 25 (which is not present in any polysaccharide or conjugate vaccine), which can be used to evaluate interassay reproducibility (useful for pre- versus postvaccination studies). The FCMIA was standardized with U.S. reference antipneumococcal serotype standard serum 89S-2. Preadsorption of 89S-2 with each PnPS and C-PS yielded homologous inhibition for serotypes 1, 6B, 9N, 9V, 11A, 12F,14, 15B, 18C, 19A, 19F, 20, 22F, 25, and 33F; heterologous inhibition for serotypes 9V, 10A, 11A, 12F, 15B, 17F, 20, and 23F; and neither homologous nor heterologous inhibition for serotypes 2, 3, 4, and 5. The minimum detectable concentrations for the 24 multiplexed (PnPS and C-PS) FCMIAs ranged from 20 pg/ml for PnPS 3 to 600 pg/ml for PnPS 14. The PnPS FCMIA method has numerous benefits over enzyme-linked immunosorbent assays commonly used to measure anti-PnPS-specific IgG levels, including increased speed, smaller sample volumes, equivalent or better sensitivity, and increased dynamic range.     

Specificities and Opsonophagocytic Activities of Antibodies to Pneumococcal Capsular Polysaccharides in Sera of Unimmunized Young Children

An enzyme immunoassay (EIA) for antibodies to pneumococcal capsular polysaccharides (Pnc PSs) detects in some cases antibodies that are cross-reactive within different Pnc PSs. Recently, it has been suggested that for detection of only serotype-specific antibodies, EIA can be modified by removing cross-reactive antibodies by absorption with an irrelevant PS, e.g., the type 22F PS. The opsonophagocytosis assay measures the functional activities of antibodies in vitro, and the results of that assay correlate with in vivo protection better than measurement of the antibody concentration by EIA. We compared these different methods for measuring antibodies to type 1, 6B, 11A, 14, 19F, and 23F Pnc PSs in the sera of unimmunized young children who had been monitored for pneumococcal carriage, acute otitis media, and acquisition of antibodies to Pnc PSs from 2 to 24 months of age. Serum samples with antibody increases after contact with a pneumococcus of a homologous serotype contained specific antibodies and often had opsonophagocytic activity (OPA) (20 of 46). In samples with antibody increases from children who had not had contact with a pneumococcus of a homologous serotype, the antibodies found to be type specific by conventional EIA were usually cross-reactive and infrequently had OPA (10 of 68). When type 22F PS absorption was used in the EIA, most of the false antibody increases were eliminated, but most of the true antibody increases were still detected and the association between the antibody concentration detected by EIA and OPA was improved. However, there were serotype-dependent differences in the frequency of OPA. Use of absorption with a heterologous PS in EIA should be encouraged, and both the specificity of EIA and the sensitivity of opsonophagocytic assays should be further evaluated and improved.     

M-Ficolin Binds Selectively to the Capsular Polysaccharides of Streptococcus pneumoniae Serotypes 19B and 19C and of a Streptococcus mitis Strain

The three human ficolins (H-, L-, and M-ficolins) and mannan-binding lectin are pattern recognition molecules of the innate immune system mediating activation of the lectin pathway of the complement system. These four human proteins bind to some microorganisms and may be involved in the resolution of infections. We investigated binding selectivity by examining the binding of M-ficolin to a panel of more than 100 different streptococcal strains (Streptococcus pneumoniae and Streptococcus mitis), each expressing distinct polysaccharide structures. M-ficolin binding was observed for three strains only: strains of the pneumococcal serotypes 19B and 19C and a single S. mitis strain expressing a similar polysaccharide structure. The bound M-ficolin, in association with MASP-2, mediated the cleavage of complement factor C4. Binding to the bacteria was inhibitable by N-acetylglucosamine, indicating that the interaction with the bacterial surface takes place via the fibrinogen-like domain. The common N-acetylmannosamine residue present in the structures of the four capsular polysaccharides of group 19 is linked via a phosphodiester bond. This residue is apparently not a ligand for M-ficolin, since the lectin binds to two of the group 19 polysaccharides only. M-ficolin bound strongly to serotype 19B and 19C polysaccharides. In contrast to those of serotypes 19A and 19F, serotype 19B and 19C polysaccharides contain an extra N-acetylmannosamine residue linked via glycoside linkage only. Thus, this extra residue seems to be the M-ficolin ligand. In conclusion, we were able to demonstrate specific binding of M-ficolin to some capsular polysaccharides of the opportunistic pathogen S. pneumoniae and of the commensal bacterium S. mitis.     

Specific Capsular Polysaccharide of Type 45 Streptococcus pneumoniae (American Type 72)

The specific capsular polysaccharide of type 45 Streptococcus pneumoniae was isolated in pure form by chemical and chromatographic methods and found to be a high-molecular-weight, glycosidically linked polymer of a hexasaccharide repeating unit composed of D-galactose (2 mol), L-rhamnose (1 mol), N-acetyl-D-glucosamine (1 mol), N-acetyl-D-galactosamine (1 mol), N-acetyl-L-fucosamine (1 mol), and phosphate (1 mol).     

Specific Capsular Polysaccharide of Type 46 Streptococcus pneumoniae (American Type 73)

The specific capsular polysaccharide of type 46 Streptococcus pneumoniae (American type 73) was isolated in pure form and shown to be a high-molecular-weight, glycosidically linked polymer composed of d-galactose (2 mol), N-acetyl-d-glucosamine (1 mol), N-acetyl-d-galactosamine (1 mol), and N-acetyl-l-fucosamine (1 mol).     

A Flow Cytometric Opsonophagocytic Assay for Measurement of Functional Antibodies Elicited after Vaccination with the 23-Valent Pneumococcal Polysaccharide Vaccine

Opsonophagocytosis is the primary mechanism for clearance of pneumococci from the host, and the measurement of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. We developed a semiautomated flow cytometric opsonophagocytosis assay using HL-60 granulocytes as effector cells and nonviable 5,6-carboxyfluorescein, succinimidyl ester-labeled Streptococcus pneumoniae (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) as bacterial targets. The flow cytometric opsonophagocytosis assay was highly reproducible (for 87% of repetitive assays the titers were within 1 dilution of the median titer) and serotype specific, with ≥97% inhibition of opsonophagocytic titer by addition of homologous serotype-specific polysaccharide. In general, opsonophagocytic titers were not significantly inhibited by the presence of either heterologous pneumococcal polysaccharide or penicillin in the serum. The flow cytometric assay could reproducibly measure functional antibody activity in prevaccination (n = 28) and postvaccination (n = 36) serum specimens from healthy adult volunteers vaccinated with the 23-valent pneumococcal polysaccharide vaccine. When compared with a standardized manual viable opsonophagocytic assay, a high correlation (r = 0.89; P ≤ 0.01) was found between the two assays for the seven serotypes tested. The flow cytometric assay is rapid (~4 h) with high throughput (~50 serum samples per day per technician) and provides a reproducible measurement of serotype-specific functional antibodies, making it a highly suitable assay for the evaluation of the immune responses elicited by pneumococcal vaccines.     

Multilaboratory evaluation of a viability assay for measurement of opsonophagocytic antibodies specific to the capsular polysaccharides of Streptococcus pneumoniae

Opsonophagocytosis is a correlate of protection that measures the functional activity of vaccine-induced antibodies. A standardized opsonophagocytosis assay (OPA) should be used as part of the evaluation of current and future pneumococcal (Pnc) polysaccharide (Ps)-based vaccines. We enrolled five laboratories to evaluate a previously standardized viability OPA. Each laboratory was provided with a detailed OPA protocol, seven target Pnc strains (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F), two quality control sera and 12 paired sera (blinded) from adult donors who received one dose of the 23-valent Pnc Ps vaccine. Laboratories sent their results to the Centers for Disease Control and Prevention for analysis. Sera were tested in duplicate (single run), and the results were averaged to yield a single OPA titer (> or = 50% killing) for each serum sample. The percentage of sera within one or two dilutions of the calculated median OPA titer was determined for each laboratory and for each serotype. In general, laboratories were capable of detecting OPA titers within one or two dilutions of the median for at least 75 and 88%, respectively, of the sera tested. The level of agreement with the median OPA titers varied depending on the participating laboratory (overall agreement = 0.8 [99% confidence interval = 0.75 to 0.85]). All OPA median titers reported for quality control sera were within one dilution of the expected titer. We conclude that this OPA can be done in multiple laboratories with a high degree of interlaboratory reproducibility.     

Antibodies Specific for the Hia Adhesion Proteins of Nontypeable Haemophilus influenzae Mediate Opsonophagocytic Activity

The Hia autotransporter proteins are highly immunogenic surface adhesins expressed by nontypeable Haemophilus influenzae (NTHI). The objective of our study was to assess the opsonophagocytic activity of anti-Hia antibodies against homologous and heterologous NTHI. A segment of the hia gene that encodes a surface-exposed portion of the H. influenzae strain 11 Hia protein was cloned into a pGEMEX-2 expression vector. Escherichia coli JM101 was transformed with the resulting pGEMEX-Hia BstEII del recombinant plasmid, and recombinant fusion protein was recovered. An immune serum against recombinant GEMEX-Hia (rGEMEX-Hia)-mediated killing of the homologous NTHI strain 11 at a 1:160 titer and five heterologous Hia-expressing strains at titers of ≥1:40. Immune serum did not mediate killing of two Hia-knockout strains whose hia genes were inactivated but did mediate killing of one knockout strain at a high titer after the strain was transformed with a plasmid containing the hia gene. Immune serum did not mediate killing of HMW1/HMW2-expressing NTHI strains, which do not express the Hia adhesin. However, when two representative HMW1/HMW2-expressing strains were transformed with the plasmid containing the hia gene, they expressed abundant Hia and were susceptible to killing by the immune serum. Immune serum did not mediate killing of HMW1/HMW2-expressing strains transformed with the plasmid without the hia gene. Our results demonstrate that the Hia proteins of NTHI are targets of opsonophagocytic antibodies and that shared epitopes recognized by such antibodies are present on the Hia proteins of unrelated NTHI strains. These data argue for the continued investigation of the Hia proteins as vaccine candidates for the prevention of NTHI disease.Otitis media remains a significant health problem for children in this country and elsewhere in the world (10, 11). Most children in the United States have had at least one episode of otitis by their third birthdays, and one-third have had three or more episodes (34). In addition to the short-term morbidity and costs of this illness, the potential for delay or disruption of normal speech and language development in children with persistent middle ear effusions is a subject of considerable concern (33, 41). Experts in the field have strongly recommended that efforts be made to develop safe and effective vaccines for the prevention of otitis media in young children (20). Although the total prevention of disease will be a difficult goal to achieve, the prevention of even a portion of cases would be beneficial, given the magnitude and costs of the problem.Bacteria, usually in pure culture, can be isolated from middle ear exudates in approximately two-thirds of the cases of acute otitis media (16, 35). Streptococcus pneumoniae is the most common bacterial pathogen recovered in all age groups, with isolation rates commonly ranging from 35% to 40% (16, 35). Nontypeable Haemophilus influenzae (NTHI) is the second-most-common bacterium recovered and accounts for 20% to 30% of the cases of acute otitis media and a larger percentage of the cases of chronic and recurrent disease (26). Interestingly, since the introduction of the pneumococcal conjugate vaccine as part of the regular childhood vaccine schedule, NTHI has become an even more common cause of acute and recurrent middle ear disease, often surpassing S. pneumoniae in the frequency of recovery from middle ear specimens (12, 26). Many different antigens have been suggested as possible NTHI vaccine candidates (1, 3, 18, 29, 30, 42). Outer membrane proteins appear to be the principal targets of bactericidal and protective antibodies (22), and as a group, they have been the major focus of vaccine development efforts. Table ​Table11 summarizes the relevant characteristics of some of the leading vaccine candidates currently under active investigation.

TABLE 1.

Potential vaccine antigens of NTHI

Thermodynamics and Density of Binding of a Panel of Antibodies to High-Molecular-Weight Capsular Polysaccharides

The interaction between antipolysaccharide (anti-PS) antibodies and their antigens was investigated by the use of isothermal titration calorimetry to determine the thermodynamic binding constant (K), the change in the enthalpy of binding (ΔH), and the binding density (N) to high-molecular-weight PSs. From these values, the change in the entropy of binding (ΔS) was calculated. The thermodynamic parameters of binding to high-molecular-weight capsular PSs are reported for two monoclonal antibodies (MAbs) with different specificities for meningococcal serogroup C PS, five MAbs specific for different pneumococcal serotypes, and the Fab fragments of two antipneumococcal MAbs. The K values were in the range of 10⁶ to 10⁷ M⁻¹, and these values were 1 to 2 orders of magnitude greater than the previously reported K values derived from antibody-oligosaccharide interactions. The ΔH associated with binding was favorable for each MAb and Fab fragment. The ΔS associated with binding was also generally favorable for both the MAbs and the Fab fragments, with the exception of the anti-serotype 14 MAb and its Fab fragment. N provides information regarding how densely MAbs or Fabs can bind along PS chains and, as expressed in terms of monosaccharides, was very similar for the seven MAbs, with an average of 12 monosaccharides per bound MAb. The value of N for each Fab was smaller, with five or seven monosaccharides per bound Fab. These results suggest that steric interactions between antibody molecules are a major influence on the values of N of high-affinity MAbs to capsular PSs.     

Streptococcus pneumoniae Resistance to Complement-Mediated Immunity Is Dependent on the Capsular Serotype

Streptococcus pneumoniae strains vary considerably in the ability to cause invasive disease in humans, and this is partially associated with the capsular serotype. The S. pneumoniae capsule inhibits complement- and phagocyte-mediated immunity, and differences between serotypes in these effects on host immunity may cause some of the variation in virulence between strains. However, the considerable genetic differences between S. pneumoniae strains independent of the capsular serotype prevent an unambiguous assessment of the effects of the capsular serotype on immunity using clinical isolates. We have therefore used capsular serotype-switched TIGR4 mutant strains to investigate the effects of the capsular serotype on S. pneumoniae interactions with complement. Flow cytometry assays demonstrated large differences in C3b/iC3b deposition on opaque-phase variants of TIGR4(−)+4, +6A, +7F, and +23F strains even though the thicknesses of the capsule layers were similar. There was increased C3b/iC3b deposition on TIGR4(−)+6A and +23F strains compared to +7F and +4 strains, and these differences persisted even in serum depleted of immunoglobulin G. Neutrophil phagocytosis of the TIGR4(−)+6A and +23F strains was also increased, but only in the presence of complement, showing that the effects of the capsular serotype on C3b/iC3b deposition are functionally significant. In addition, the virulence of the TIGR4(−)+6A and +23F strains was reduced in a mouse model of sepsis. These data demonstrate that resistance to complement-mediated immunity can vary with the capsular serotype independently of antibody and of other genetic differences between strains. This might be one mechanism by which the capsular serotype can affect the relative invasiveness of different S. pneumoniae strains.The important Gram-positive pathogen Streptococcus pneumoniae has an extracellular polysaccharide capsule that inhibits complement activity, neutrophil phagocytosis, and bacterial killing by neutrophil extracellular traps (19, 23, 25, 26, 29, 31), as well as having major effects on bacterial interactions with the epithelium (8, 25, 26, 29, 31, 37). As a consequence, the capsule is essential for virulence (6, 38). Different strains of S. pneumoniae can express capsules with different structures, depending on the type of monosaccharide units and their bonds within the polysaccharide chain, the enzymes for the synthesis of which are encoded by genes within a specific locus in the genome (5, 27, 30). The different types of capsules are divided into 91 capsular serotypes. Although most S. pneumoniae strains can cause disease in humans, the ability to cause invasive infections (septicemia and meningitis) varies up to 60-fold between strains and is closely associated with the capsular serotype (4, 12). Some serotypes (e.g., 1, 4, 5, 7, and 14) are overrepresented among invasive disease isolates compared to the frequency of their isolation as nasopharyngeal commensals, while other capsular serotypes only rarely cause invasive disease despite being common nasopharyngeal commensals (4, 12, 15).The mechanisms causing capsular serotype-dependent variation in virulence are largely unknown but could reflect differences between the abilities of strains of different serotypes to inhibit host immune responses. Potentially, strains expressing capsular serotypes that strongly inhibit immunity could be more likely to establish invasive infection than strains with capsular serotypes that weakly inhibit host immunity, and this hypothesis is partially supported by existing experimental data. The virulence of different capsular serotypes varies markedly in mouse models of infection, but as there is only a weak relationship between virulence in mice and invasive potential in humans, the clinical relevance of these findings is unclear (1, 7, 9, 33). Because of the central role of complement and phagocytosis for systemic immunity to S. pneumoniae (11, 20, 45, 46), differences in the effects of different capsular serotypes on complement activity or phagocytosis are strong candidates for explaining why the serotype can affect virulence. Indeed, existing data show that resistance to complement activity and phagocytosis varies between strains with different capsular serotypes (18, 28, 46). However, in general, these studies have not controlled for strain phase variation or for noncapsular genetic variation between strains. S. pneumoniae has two main phase variants, opaque with an increased capsule thickness and transparent with a thinner capsule but increased expression of some surface proteins, such as PspC, that can affect complement activity (24, 31). Differences in phase variation between strains could therefore affect complement susceptibility. Furthermore, there is considerable genetic variation between S. pneumoniae strains independent of the capsular serotype. Only 60% of gene clusters are common to all S. pneumoniae strains, and the genome content differs by 8 to 10%, on average, between any two strains (10, 13, 16, 17). This genetic variation is partially linked to the capsular serotype (http://www.mlst.net/), and hence, the relationship between the capsular serotype and invasiveness could be due to noncapsular genetic variation rather than direct effects of the capsule.To overcome strain genetic variation confounding the assessment of capsular serotype interactions with the immune system, the capsular loci of one strain can be replaced with the capsular loci from another, creating otherwise isogenic strains expressing different capsular serotypes (29, 35, 43). Data obtained using capsular serotype-switched strains have shown that expression of capsular serotype 3 reduced complement deposition on a previously serotype 2 strain (2), increased the virulence in mice of an originally serotype 6B strain, and conversely decreased the virulence of a serotype 5 strain (22). Furthermore, a recent study demonstrated variations in resistance to neutrophil killing of unopsonized bacteria between capsular serotype-switched strains expressing different capsular serotypes and correlated reduced sensitivity to neutrophil killing with increased prevalence of that capsular serotype as a nasopharyngeal commensal (39). These studies have established the principle that the capsular serotype can affect the complement sensitivity, neutrophil killing, and virulence of S. pneumoniae independently of the strain background. However, as yet, there are only limited data on the effects of different capsular serotypes on complement-dependent immunity to S. pneumoniae and a more detailed assessment is required to help understand why a strain''s capsular serotype is linked to its invasive potential.We have used opaque- and transparent-phase variants of TIGR4 S. pneumoniae strains modified to express different capsular serotypes, two representative of relatively invasive capsular serotypes (4 and 7F) and two representative of less invasive serotypes (6A and 23F), to assess capsular serotype-dependent effects on immunity. We have investigated the effects of the capsular serotype on opsonization of S. pneumoniae with the complement-derived opsonins C3b and iC3b, as well as on neutrophil phagocytosis and virulence in a mouse model of septicemia.     

Latex Agglutination Test for Measurement of Antibodies to Meningococcal Polysaccharides

A latex agglutination test employing serogroup-specific meningococcal polysaccharides as the antigen has been developed. The test has been used to measure serological responses in patients with meningococcal disease, in meningococcal carriers, and in volunteers who received meningococcal vaccines. It has been shown to be a sensitive and highly specific test for the detection of group-specific meningococcal antibody. The advantages of standardized particles and antigens which are simple to prepare and stable make this assay feasible for many nonspecialized or small laboratories.     

Inclusion Fluorescent-Antibody Test as a Screening Assay for Detection of Antibodies to Chlamydia pneumoniae

A study was conducted to determine the ability of the inclusion immunofluorescence assay (inclusion IFA) to act as a screening test to detect samples with antibodies to Chlamydia pneumoniae; microimmunofluorescence (MIF) was used as the “gold standard.” In addition, the inclusion IFA was compared using HEp-2 cells infected with either C. pneumoniae CM-1 or Chlamydia trachomatis serovar E. A total of 331 serum samples representing a range of MIF titers were evaluated. The sensitivities of the inclusion IFA for detecting samples with C. pneumoniae MIF titers of ≥16 were 96.9 and 74.8% with C. pneumoniae- and C. trachomatis-infected cells, respectively. For samples with an elevated C. pneumoniae MIF titer of ≥512, the sensitivities of the C. pneumoniae- and C. trachomatis-based inclusion IFA were 97.0 and 8.8%, respectively. These results suggest that the inclusion IFA is not a genus-specific test, as evidenced by the failure of the C. trachomatis-infected cells to detect a significant number of samples with C. pneumoniae antibodies. Samples that had elevated C. pneumoniae inclusion IFA and MIF titers but that were found negative (titer, <16) by the C. trachomatis inclusion IFA were further tested by an in vitro neutralization assay for functional antibodies that might not have been detected by the serological assays. The in vitro neutralization results corroborated the serological results in that all seven sera tested had a neutralization titer for C. pneumoniae (range, 20 to 225), while all but one failed to have any effect on the infectivity of C. trachomatis serovar E. While the C. pneumoniae inclusion IFA had a high sensitivity for detecting chlamydial antibodies, depending on whether it was used as a screening test for detecting samples with low (≥16) or elevated (≥512) MIF titers, its specificity ranged from 53.4 to 77.1%. In conclusion, the inclusion IFA with C. pneumoniae-infected cells was best suited as a sensitive screening test for identifying specimens with elevated MIF titers (those associated with a possible acute infection with C. pneumoniae).     

Prevalence of Specific Antibodies to Chlamydia pneumoniae in Korea

To clarify the endemic status of Chlamydia pneumoniae in Korea, the incidence of antibodies in 564 serum samples from healthy individuals, patients with respiratory infection, and cord blood specimens was evaluated. We conclude that C. pneumoniae infection is highly endemic in Korea and that this infection is associated with acute respiratory diseases.     

Evaluation of Streptococcus pneumoniae Type XIV Opsonins by Phagocytosis-Associated Chemiluminescence and a Bactericidal Assay

The relative roles of serum factors required for opsonization of type XIV Streptococcus pneumoniae were investigated by means of luminol-enhanced chemiluminescence (CL), bactericidal, and immunofluorescence assays employing adult sera containing high (>1,000 ng of antibody nitrogen per ml) or low (<200 ng of antibody nitrogen per ml) antibody concentrations as determined by radioimmunoassay. Specific antibody concentration correlated directly with both total and heat-labile CL activity (P < 0.005) and with the bactericidal index (P < 0.05) at a serum concentration of 10%. The importance of specific antibody as an opsonin was confirmed by the abolition of CL activity and immunoglobulin immunofluorescence observed after absorption of heated sera with type XIV pneumococcal cells and by the dose response in CL and bactericidal activity observed with the addition of immunoglobulin G to hypogammaglobulinemic serum. A role for the classical complement pathway in opsonization was indicated by significantly greater CL integrals for high-antibody sera than for low-antibody sera depleted of factor D and by the bactericidal activity noted for untreated, but not magnesium ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid-chelated low-antibody sera. The alternative pathway contributed more than half of the CL activity of both high- and low-antibody sera. However, after magnesium ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid chelation, only sera with high antibody concentrations or agammaglobulinemic serum reconstituted with immunoglobulin G with high specific antibody levels supported significant bactericidal activity. Therefore, type-specific antibody and complement promote opsonization of type XIV S. pneumoniae, and this may occur via either complement pathway. These results suggest that CL is a suitable tool to delineate serum factors and their contribution to opsonization, but results must be related to other functional assays.     

Comparative Evaluation of Profiles of Antibodies to Mycobacterial Capsular Polysaccharides in Tuberculosis Patients and Controls Stratified by HIV Status

Despite the complexity of tuberculosis (TB) serology, antibodies (Abs) remain attractive biomarkers for TB. Recent evidence of a mycobacterial capsule that consists mainly of the polysaccharides arabinomannan (AM) and glucan provides new options for serologic targets. For this study, Ab responses to AM and glucan for 47 U.S. TB patients (33 HIV negative [HIV⁻], 14 HIV positive [HIV⁺]), 42 healthy controls, and 38 asymptomatic HIV⁺ controls were evaluated by enzyme-linked immunosorbent assays (ELISAs). The results were compared with Ab responses to the mycobacterial glycolipid cell wall antigen lipoarabinomannan (LAM) and to the proteins malate synthase (MS) and MPT51. We found that the main immunoglobulin (Ig) isotype response to polysaccharides was IgG, predominantly of subclass IgG2. IgG responses to AM were significantly higher for HIV⁻ and HIV⁺ TB cases than for controls (P, <0.0001 and <0.01, respectively); significantly higher for HIV⁻ than for HIV⁺ TB cases (P, <0.01); and significantly higher in sputum smear-positive than smear-negative patients in both HIV⁻ and HIV⁺ cases (P, 0.01 and 0.02, respectively). In both TB groups, titers of Ab to glucan were significantly lower than titers of Ab to AM (P, <0.0001). IgG responses to AM and MS or to AM and MPT51 did not correlate with each other in HIV⁻ TB patients, while they correlated significantly in HIV⁺ TB patients (P, 0.01 and 0.05, respectively). We conclude that Ab responses to AM could contribute to the serodiagnosis of TB, especially for HIV⁻ TB patients. This study also provides new and important insights into the differences in the profiles of Abs to mycobacterial antigens between HIV⁻ and HIV⁺ TB patients.