The specificity of the immune response to the 23-valent pneumococcal-polysaccharide (PS) vaccine in healthy adults and to a pneumococcal conjugate vaccine in infants was examined by measuring immunoglobulin G (IgG) antibody titers by enzyme-linked immunosorbent assay (ELISA) and the opsonophagocytosis assay. ELISA measures total antipneumococcal IgG titers including the titers of functional and nonfunctional antibodies, while the opsonophagocytosis assay measures only functional-antibody titers. Twenty-four pairs of pre-and post-pneumococcal vaccination sera from adults were evaluated (ELISA) for levels of IgG antibodies against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Twelve of the pairs were also examined (opsonophagocytosis assay) for their functional activities. The correlation coefficients between assay results for most types ranged from 0.75 to 0.90, but the correlation coefficient was only about 0.6 for serotypes 4 and 19F. The specificities of these antibodies were further examined by the use of competitive ELISA inhibition. A number of heterologous polysaccharides (types 11A, 12F, 15B, 22F, and 33A) were used as inhibitors. Most of the sera tested showed cross-reacting antibodies, in addition to those removed by pneumococcal C PS absorption. Our data suggest the presence of a common epitope that is found on most pneumococcal PS but that is not absorbed by purified C PS. Use of a heterologous pneumococcal PS (22F) to adsorb the antibodies to the common epitope increased the correlation between the IgG ELISA results and the opsonophagocytosis assay results. The correlation coefficient improve from 0.66 to 0.92 for type 4 and from 0.63 to 0.80 for type 19F. These common-epitope antibodies were largely absent in infants at 7 months of age, suggesting the carbohydrate nature of the epitope.

Because of the difficulty of conducting efficacy trials of vaccines against group B streptococcus (GBS), the licensure of these vaccines may have to rely on studies that measure vaccine-induced antibody levels that correlate with protection. This study estimates the level of maternal antibody required to protect neonates against early-onset disease (EOD) caused by GBS type Ia. Levels of maternal serum IgG GBS Ia antibodies, measured by ELISAs in 45 case patients (neonates with EOD caused by GBS Ia) and in 319 control subjects (neonates colonized by GBS Ia but without EOD) born at > or =34 weeks gestation were compared. The probability of developing EOD declined with increasing maternal levels of IgG GBS Ia antibody (P = .03). Neonates whose mothers had levels of IgG GBS Ia antibody > or =5 microg/mL had an 88% lower risk (95% confidence interval, 7%-98%) of developing type-specific EOD, compared with those whose mothers had levels < 0.5 microg/mL. A vaccine that induces IgG GBS Ia antibody levels > or =5 microg/mL in mothers can be predicted to confer a high degree of type-specific immunity to EOD to their infants.

The most common infections in primary immune deficiency disease (PIDD) patients involve encapsulated bacteria, mainly Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae (pneumococcus). Thus, it is important to know the titers of Hib-and pneumococcus-specific antibodies that are present in immune globulin (Ig) intravenous (IGIV) preparations used to treat PIDD. In this study, seven IGIV preparations were tested by enzyme-linked immunosorbent assay and opsonophagocytic activity for antibody titers to the capsular polysaccharides of Hib and five pneumococcal serotypes. Differences in Hib-and pneumococcus-specific antibody titer were observed among various IGIV preparations, with some products having higher-or lower-than-average titers. Opsonic activity also varied among preparations. As expected, IgG2 was the most active subclass of both binding and opsonic activity except against pneumococcal serotype 6B where IgG3 was the most active. This study determines antibody titers against capsular polysaccharides of Hib and pneumococcus in seven IGIV products that have been shown to be effective in reducing infections in PIDD patients. As donor antibody levels and manufacturing methods continue to change, it may prove useful from a regulatory point of view to reassess IGIV products periodically, to ensure that products maintain antibody levels that are important for the health of IGIV recipients.

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 (>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.Vaccine-induced protection to Streptococcus pneumoniae (pneumococcus) has been determined through vaccine efficacy trials for both polysaccharide (Ps) vaccines (1, 4, 22) and Psprotein conjugate vaccines (2,5,8). Trials of these pneumococcal (Pnc) vaccine formulations have shown various efficacies for protection depending on the end point being measured and the population being studied. These trials are costly and difficult to perform given the large sample size. In addition, pneumococcus has 90 different capsular serotypes, with the majority of disease being caused by about 30 of these 90 serotypes. Distribution of these serotypes also varies with the geographical region, making the estimation of the burden of disease and the impact of vaccination rather difficult (3, 9, 10).Efforts have been made for the identification and standardization of laboratory correlates of protection that can aid vaccine efficacy trials in the estimation of vaccine-induced protection. Currently, a highly standardized enzyme-linked immunosorbent assay (ELISA) is available (www.vaccine.uab.edu) for the evaluation of infant sera. Several modifications to the protocol described by Quartaert et al. (20) allowed for the measurement of Ps-specific antibodies in children and adults (6,19,18). Adults can have cross-reactive antibodies, which confound the measurements of immunoglobulin G (IgG) antibodies by ELISA, especially if absorption with a nonrelevant serotype is not performed prior to testing (6,7,26). These c...

The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of 40 lactobacillus strains were determined against six antibiotics with broad anaerobic spectra. Penicillin, ampicillin, clindamycin, and cephalothin were the most active inhibitory agents, with 95 to 100% of the strains inhibited at clinically achievable serum levels. However, despite the inhibitory efficacy of these four agents, only 5 to 22% of the isolates were killed at achievable concentrations. MBC:MIC ratios were high, ranging from 30:1 for cephalothin to 266:1 for ampicillin. Cefoxitin and metronidazole were generally ineffective against lactobacilli, with 87.5 to 100% of strains having unachievable MICs and/or MBCs. These findings may partially explain the clinical observations noting the inability to eradicate endocarditic lactobacillemias despite readily achievable MICs.Lactobacilli are being increasingly recognized as important pathogens in human infections (1,3,17,20). An intriguing phenomenon noted in many previously cited cases of lactobacillemia was the therapeutic difficulty in eradicating this organism from deep-seated infected foci, especially the endocardium, despite seemingly appropriate treatment regimens and serum antibiotic concentrations exceeding the minimal inhibitory concentrations (MICs).To investigate this problem of apparent discrepancy between in vitro susceptibility of lactobacilli and poor in vivo therapeutic responses, the MICs and minimal bactericidal concentrations (MBCs) of six antibiotics with broad grampositive anaerobic spectra were determined by a modified broth dilution technique for 40 clinical isolates. MATERLALS AND METHODSIsolation of the lactobacilli. All 40 strains of lactobacilli were clinical isolates from a variety of body sites, including blood, genital tract, oropharynx, ascitic fluid, lymph node, and brain tissue. Ten were obtained from the clinical laboratory of Harbor General Hospital, whereas the remaining 30 organisms were kindly provided by the following investigators: Elizabeth P. Cato, Blacksburg, Va.; Robert E. Weaver, Atlanta, Ga.; Jack P. London, Bethesda, Md.; and M. Elisabeth Sharpe, Reading, England.The lactobacilli were identified by typical appearance on Gram stain and by biochemical reactions according to Bergey's criteria (14). Identification of species was according to classifications of Holdeman and Moore (12). The following strains of lactobacilli were used in this study: Lactobacillus casei (24 strains), L. plantarum (5 strains), L. acidophilus and L. minutis (3 strains each), L. fermentans and L. brevis (2 strains each), and L. leichmanii (1 strain). All of the L. plantarum and L. casei and two of the three L. minutis isolates were facultative anaerobes; the remainder were strict anaerobes.Organisms were maintained in chopped-meat glucose (CMG) broth (Scott Laboratories, Fiskeville, R.I.) and were transferred monthly into fresh media until the susceptibility testing was performed. In the week before testing, one additional transfer was done.Pre...

Eighty-six percent of 707 beta-hemolytic streptococci isolated in a general hospital and excluded by presumptive tests from groups A and D were identified serologically as group B. More than 70% of the group B isolates were from urine cultures, the female genital gract, or newborn infants. Types III and II were the most common group B serotypes from most sources. However, types Ia, Ib, and Ic were more commonly isolated from the respiratory tract than from other sites, and type III was responsible for most serious neonatal infections. All group B streptococci were sensitive in vitro to comparable levels of penicillin G, ampicillin, and cephalothin and were highly resistant to kanamycin. Seventy-two percent were resistant to tetracycline but only 1%-2% to erythromycin, clindamycin, and chloramphenicol. Despite consistent sensitivity to penicillin G, the minimal inhibitory concentrations were significantly higher for group B than for group A streptococci.

An enzyme-linked immunosorbent assay (ELISA) and the antibody concentrations assigned to different pneumococcal capsular polysaccharide types were used to estimate concentrations of antibody to additional pneumococcal types in reference serum 89SF and to confirm assigned antibody values. This was possible because the slopes of curves of antibody binding to all polysaccharide types evaluated (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) were similar. The point estimates for total anti-pneumococcal antibody and immunoglobulin G (IgG) antibody determined by cross-standardization by an ELISA based on use of methylated human serum albumin (mHSA) to improve the efficiency of polysaccharide binding to the ELISA plate differed by less than 40% from those reported by Quataert et al. (Clin. Diagn. Lab. Immunol. 2:590–597, 1995) for types 1, 4, 6B, 7F, 9V, 14, 18C, and 23F. However, large differences were found between the assigned values and those obtained by our mHSA ELISA for types 3 and 19F. The mHSA ELISA and the direct polysaccharide coat ELISA may not measure antibodies to the same epitopes on polysaccharides of types 3 and 19F. The functional importance of these different antibody specificities is being investigated. We have thus confirmed the assigned IgG antibody values for most types by a different method and have extended antibody assignments to several additional types.

We measured the capacity to opsonize Streptococcus pneumoniae serotype 6B and estimated the concentration of immunoglobulin G anti-6B capsular polysaccharide (PS) antibodies in 25 pre- and postimmune sera from adults immunized with a pneumococcal PS vaccine. We first studied two postvaccination serum samples displaying less opsonophagocytic capacity than expected. The majority of anti-6B antibodies in the two samples reacted with the capsular PSs of several unrelated serotypes (2, 4, 9V, 19F, and 23F) and with the lysate of noncapsulated S. pneumoniae bacteria but not with C-PS. The non-type-specific antibodies accounted for at least one-half of anti-6B antibodies in 40% of prevaccination sera and 10% of postvaccination sera from adults. The non-type-specific antibodies could be demonstrated in the enzyme-linked immunosorbent assays (ELISAs) for pneumococcal antibodies to other serotypes (4, 9V, 18C, 19F, and 23F). The nonspecific antibodies appear to bind a contaminant(s) in the current preparations of capsular PS. ELISA for antibodies to pneumococcal capsules may not be serotype specific for some samples.