PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-11 (11)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  HEPTAVALENT PNEUMOCOCCAL CONJUGATE VACCINE IMMUNOGENICITY IN VERY-LOW-BIRTH-WEIGHT, PREMATURE INFANTS 
Background
The heptavalent pneumococcal-CRM197 conjugate vaccine (PCV-7) has been incompletely studied in very-low-birth-weight (VLBW, ≤1500 grams) infants.
Objective
To assess PCV-7 immunogenicity in VLBW, premature infants. We hypothesized that the frequency of post-vaccine antibody concentrations ≥0.15 µg/mL would vary directly with birth weight.
Methods
This was a multi-center observational study. Infants 401–1500 grams birth weight and <32 0/7 weeks gestation, stratified by birth weight, were enrolled from 9 NICHD Neonatal Research Network centers. Infants received PCV-7 at 2, 4 and 6 months after birth and had blood drawn 4–6 weeks following the third dose. Antibodies against the 7 vaccine serotypes were measured by enzyme-linked immunosorbent assay.
Results
Of 369 enrolled infants, 244 completed their primary vaccine series by 8 months and had serum obtained. Subjects were 27.8 ± 2.2 (mean ± standard deviation) weeks gestation and 1008 ± 282 grams birth weight. Twenty-six percent had bronchopulmonary dysplasia and 16% had received postnatal glucocorticoids. Infants 1001–1500 grams birth weight were more likely than those 401–1000 grams to achieve antibody concentrations ≥0.15 µg/mL against the least two immunogenic serotypes (6B: 96% v. 85%, P = 0.003 and 23F: 97% v. 88%, P = 0.009). In multiple logistic regression analysis, lower birth weight, postnatal glucocorticoid use, lower weight at blood draw and Caucasian race were each independently associated with antibody concentrations <0.35 µg/mL against serotypes 6B and/or 23F.
Conclusion
When compared with larger premature infants, infants weighing ≤1000 grams at birth have similar antibody responses to most, but not all, PCV-7 vaccine serotypes.
doi:10.1097/INF.0b013e3181d264a6
PMCID: PMC2949965  PMID: 20234331
Infant, premature; infant, very low birth weight; pneumococcal vaccines; immunization; vaccines
2.  Modified Opsonization, Phagocytosis, and Killing Assays To Measure Potentially Protective Antibodies against Pneumococcal Surface Protein A 
Clinical and Vaccine Immunology : CVI  2013;20(10):1549-1558.
The standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface protein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant α-helical or proline-rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels of antibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonal antibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibodies to PspA.
doi:10.1128/CVI.00371-13
PMCID: PMC3807198  PMID: 23925886
3.  The 7-valent pneumococcal conjugate vaccine elicits cross-functional opsonophagocytic killing responses to Streptococcus pneumoniae serotype 6D in children 
BMC Infectious Diseases  2013;13:474.
Background
We investigated the immune response to serogroup 6 with the opsonophagocytic killing assay (OPKA) in children aged 12–23 months of age after immunization with the 7-valent pneumococcal conjugate vaccine (PCV7) containing serotype 6B.
Methods
Blood samples were obtained from 59 children who had blood sampling for medical examination. Immunization status against PCV7 was confirmed by immunization records and samples were categorized according to immunization status into a booster, primary, or control group. The OPKA was performed for serotypes 6A, 6B, 6C, and 6D.
Results
Subjects with no previous PCV7 immunization history showed opsonic activity for serogroup 6 in 5-30% (according to serotype). In subjects vaccinated with a 3-dose primary series, 81% showed opsonic activity for serotypes 6B and 6D, and 29% showed opsonic activity for serotypes 6A and 6C. Among subjects vaccinated with a booster dose, all subjects had opsonic activity against serotype 6B. Subjects in the booster group with opsonic activity against serotypes 6A, 6C, and 6D were 100%, 78%, and 89%, respectively.
Conclusions
In subjects aged 12–23 months, an immune response is elicited after a primary series of immunizations with PCV7 for serotypes 6B and 6D, and a booster dose enhances a cross reactive immune response against serotypes 6A, 6C and 6D.
doi:10.1186/1471-2334-13-474
PMCID: PMC3852776  PMID: 24112237
Streptococcus pneumoniae; Heptavalent pneumococcal conjugate vaccine; Pneumococcal polysaccharide type 6
4.  Long-term Comparative Immunogenicity of Protein Conjugate and Free Polysaccharide Pneumococcal Vaccines in Chronic Obstructive Pulmonary Disease 
The Food and Drug Adminstration recently approved a diphtheria-conjugated pneumococcal polysaccharide vaccine for adults, although its long-term immunogenicity is unknown. We report that, in patients with moderate to severe chronic obstructive pulmonary disease, conjugate vaccination elicits a superior immune response to free-polysaccharide vaccine that persists for >2 years.
Background. Although the 23-valent pneumococcal polysaccharide vaccine (PPSV23) protects against invasive disease in young healthy persons, randomized controlled trials in chronic obstructive pulmonary disease (COPD) have demonstrated no benefit in the intention-to-treat population. We previously reported that the 7-valent diphtheria-conjugated pneumococcal polysaccharide vaccine (PCV7) is safe and induced greater serotype-specific immunoglobulin G (IgG) and functional antibody than did PPSV23 1 month after vaccination. We hypothesized that these advantages would persist at 1 and 2 years.
Methods. One hundred eighty-one patients with moderate to severe COPD were randomized to receive PPSV23 (n = 90) or PCV7 (1.0 mL; n = 91). We measured IgG by enzyme-linked immunosorbent assay and assessed functional antibody activity by a standardized opsonophagocytosis assay, reported as a killing index (OPK). We determined differences in IgG and OPK between vaccine groups at 1 and 2 years.
Results. Relative to PPSV23, PCV7 induced greater OPK at both 1 and 2 years for 6 of 7 serotypes (not 19F). This response was statistically greater for 5 of 7 serotypes at 1 year and 4 of 7 at 2 years. Comparable differences in IgG were observed but were less often statistically significant. Despite meeting Centers for Disease Control and Prevention criteria for PPSV23 administration, almost 50% of individuals had never been vaccinated. No differences in the frequency of acute exacerbations, pneumonia, or hospitalization were observed.
Conclusions. PCV7 induces a greater functional antibody response than PPSV23 in patients with COPD that persists for 2 years after vaccination. This superior functional response supports testing of conjugate vaccination in studies examining clinical end points.
Clinical Trials Registration: NCT00457977.
doi:10.1093/cid/cis513
PMCID: PMC3491850  PMID: 22652582
5.  Pneumococcal polysaccharide vaccine at 12 months of age produces functional immune responses 
Background
Infections with Streptococcus pneumoniae (pneumococcus) are a cause of significant child mortality in the world. Pneumococcal glycoconjugate vaccines are expensive and provide limited serotype coverage. The 23-valent pneumococcal polysaccharide vaccine (Pneumovax®) may provide wider serotype coverage but is reported to be weakly immunogenic in children under 2 years of age. We have previously reported that Pneumovax® administered to healthy 12 month old Fijian infants elicits significant serotype-specific IgG responses. However, the functional capacity of these responses in 12 month old infants is not known.
Objective
To assess the functional, serotype-specific immune response of 12 month old infants following immunization with Pneumovax®.
Methods
Functional responses of 12 month old infants were assessed using the opsonophagocytic and antibody avidity assay against 8 serotypes and 23 serotypes, respectively.
Results
71% of infants produced strong opsonophagocytic activity against 4 of 8 serotypes and 30% produced high avidity serotype-specific IgG antibodies to 10 of 23 serotypes at 2 weeks post- Pneumovax®. Responses were protective for most serotypes that cause disease in western countries while responses to most of the epidemiologically relevant serotypes for developing countries were low.
Conclusion
This is the first comprehensive study evaluating the functional antibody response to Pneumovax® in 12-month old infants. Pneumovax® induced functional antibody responses to several serotypes causing disease in Western countries but induced poorer responses to serotypes that are responsible for the majority of disease in developing countries. Pneumovax® may be of benefit in some populations but further studies are required before this can be recommended in developing countries.
doi:10.1016/j.jaci.2011.11.043
PMCID: PMC3318993  PMID: 22305678
pneumococcal polysaccharide vaccine; antibody; opsonophagocytosis; avidity; function; serotype; Pneumovax®; 23vPPV
6.  Impaired Function of Antibodies to Pneumococcal Surface Protein A but Not to Capsular Polysaccharide in Mexican American Adults with Type 2 Diabetes Mellitus 
The goal of the study was to determine baseline protective titers of antibodies to Streptococcus pneumoniae surface protein A (PspA) and capsular polysaccharide in individuals with and individuals without type 2 diabetes mellitus. A total of 561 individuals (131 individuals with diabetes and 491 without) were screened for antibodies to PspA using a standard enzyme-linked immunosorbent assay (ELISA). A subset of participants with antibodies to PspA were retested using a WHO ELISA to determine titers of antibodies to capsular polysaccharide (CPS) (serotypes 4, 6B, 9V, 14, 18C, 19A, 19F, and 23F). Functional activity of antibodies was measured by assessing their ability to enhance complement (C3) deposition on pneumococci and promote killing of opsonized pneumococci. Titers of antibodies to protein antigens (PspA) were significantly lower in individuals with diabetes than controls without diabetes (P = 0.01), and antibodies showed a significantly reduced complement deposition ability (P = 0.02). Both antibody titers and complement deposition were negatively associated with hyperglycemia. Conversely, titers of antibodies to capsular polysaccharides were either comparable between the two groups or were significantly higher in individuals with diabetes, as was observed for CPS 14 (P = 0.05). The plasma specimens from individuals with diabetes also demonstrated a higher opsonophagocytic index against CPS serotype 14. Although we demonstrate comparable protective titers of antibodies to CPS in individuals with and individuals without diabetes, those with diabetes had lower PspA titers and poor opsonic activity strongly associated with hyperglycemia. These results suggest a link between diabetes and impairment of antibody response.
doi:10.1128/CVI.00268-12
PMCID: PMC3428395  PMID: 22761295
7.  Development of a Fourfold Multiplexed Opsonophagocytosis Assay for Pneumococcal Antibodies against Additional Serotypes and Discovery of Serological Subtypes in Streptococcus pneumoniae Serotype 20 
Opsonophagocytic killing assays (OPAs) are important in vitro surrogate markers of protection in vaccine studies of Streptococcus pneumoniae. We have previously reported the development of a 4-fold multiplexed OPA (MOPA) for the 13 serotypes in Prevnar 13. Because new conjugate vaccines with increased valence are being developed, we developed 4-fold MOPAs for an additional 13 serotypes: serotypes 6C and 6D, plus the 11 serotypes contained in Pneumovax but not in Prevnar 13. A high level of nonspecific killing (NSK) was observed for three serotypes (10A, 15B, and 33F) in multiple batches of baby rabbit complement. The NSK could be reduced by preadsorbing the complement with encapsulated, as well as unencapsulated, pneumococcal strains. The MOPA results compared well with the results of single-serotype OPA for all serotypes except for serotype 3. For serotype 3, the results obtained from the MOPA format were ∼40% higher than those of the single-serotype format. Interassay precision of MOPA was determined with 5 serum samples, and the coefficient of variation was generally <30% for all serotypes. MOPA was also specific for all serotypes except for serotype 20; i.e., free homologous polysaccharide (PS), but not unrelated PS, could completely and efficiently inhibit opsonization. However, serotype 20 PS from ATCC could efficiently inhibit opsonization of one serotype 20 target strain but not three other type 20 target strains even at a high (>80 mg/liter) PS concentration. This suggests the presence of serologic heterogeneity among serotype 20 strains.
doi:10.1128/CVI.00086-12
PMCID: PMC3370448  PMID: 22518015
8.  Active Immunization with Pneumolysin versus 23-Valent Polysaccharide Vaccine for Streptococcus pneumoniae Keratitis 
Current immunization strategies directed at the polysaccharide capsule offer no protection against pneumococcal keratitis. Active immunization with a protein virulence factor such as pneumolysin may be a more effective approach for significantly reducing the corneal damage associated with pneumococcal keratitis.
Purpose.
The purpose of this study was to determine whether active immunization against pneumolysin (PLY), or polysaccharide capsule, protects against the corneal damage associated with Streptococcus pneumoniae keratitis.
Methods.
New Zealand White rabbits were actively immunized with Freund's adjuvant mixed with pneumolysin toxoid (ψPLY), Pneumovax 23 (PPSV23; Merck, Whitehouse Station, NJ), or phosphate-buffered saline (PBS), before corneal infection with 105 colony-forming units (CFU) of S. pneumoniae. Serotype-specific rabbit polyclonal antisera or mock antisera were passively administered to rabbits before either intravenous infection with 1011 CFU S. pneumoniae or corneal infection with 105 CFU of S. pneumoniae.
Results.
After active immunization, clinical scores of corneas of the rabbits immunized with ψPLY and Freund's adjuvant were significantly lower than scores of the rabbits that were mock immunized with PBS and Freund's adjuvant or with PPSV23 and Freund's adjuvant at 48 hours after infection (P ≤ 0.0010), whereas rabbits immunized with PPSV23 and Freund's adjuvant failed to show differences in clinical scores compared with those in mock-immunized rabbits (P = 1.00) at 24 and 48 hours after infection. Antisera from rabbits actively immunized with PPSV23 and Freund's adjuvant were nonopsonizing. Bacterial loads recovered from infected corneas were higher for the ψPLY- and PPSV23-immunized rabbits after infection with WU2, when compared with the mock-immunized rabbits (P ≤ 0.007). Conversely, after infection with K1443, the ψPLY-immunized rabbits had lower bacterial loads than the control rabbits (P = 0.0008). Quantitation of IgG, IgA, and IgM in the sera of ψPLY-immunized rabbits showed high concentrations of PLY-specific IgG. Furthermore, anti-PLY IgG purified from ψPLY-immunized rabbits neutralized the cytolytic effects of PLY on human corneal epithelial cells. Passive administration of serotype-specific antisera capable of opsonizing and killing S. pneumoniae protected against pneumococcal bacteremia (P ≤ 0.05), but not against keratitis (P ≥ 0.476).
Conclusions.
Active immunization with pneumococcal capsular polysaccharide and Freund's adjuvant fails to produce opsonizing antibodies, and passive administration of serotype specific opsonizing antibodies offers no protection against pneumococcal keratitis in the rabbit, whereas active immunization with the conserved protein virulence factor PLY and Freund's adjuvant is able to reduce corneal inflammation associated with pneumococcal keratitis, but has variable effects on bacterial loads in the cornea.
doi:10.1167/iovs.10-6968
PMCID: PMC3302430  PMID: 22039231
9.  Low-Cost, High-Throughput, Automated Counting of Bacterial Colonies 
Research involving bacterial pathogens often requires enumeration of bacteria colonies. Here we present a low-cost, high-throughput colony counting system consisting of colony counting software and a consumer-grade digital camera or document scanner. We demonstrate that this software, called “NICE” (NIST's Integrated Colony Enumerator), can count bacterial colonies as part of a high-throughput multiplexed opsonophagocytic killing assay (MOPA) used to characterize pneumococcal vaccine efficacy. The results obtained with NICE correlate well with the results obtained from manual counting, with a mean difference of less than 3 %. NICE is also rapid; it can count colonies from multiple reaction wells within minutes and export the results to a spreadsheet for data processing. As this program is freely available from NIST, NICE should be helpful in bacteria colony enumeration required in many microbiological studies, and in standardizing colony counting methods.
doi:10.1002/cyto.a.20864
PMCID: PMC2909336  PMID: 20140968
Colony counting; pneumoniae; bacteria; vaccine; software; imaging; MOPA
10.  Multilaboratory Comparison of Streptococcus pneumoniae Opsonophagocytic Killing Assays and Their Level of Agreement for the Determination of Functional Antibody Activity in Human Reference Sera ▿ †  
Antibody-mediated killing of Streptococcus pneumoniae (pneumococcus) by phagocytes is an important mechanism of protection of the human host against pneumococcal infections. Measurement of opsonophagocytic antibodies by use of a standardized opsonophagocytic assay (OPA) is important for the evaluation of candidate vaccines and required for the licensure of new pneumococcal conjugate vaccine formulations. We assessed agreement among six laboratories that used their own optimized OPAs on a panel of 16 human reference sera for 13 pneumococcal serotypes. Consensus titers, estimated using an analysis-of-variance (ANOVA) mixed-effects model, provided a common reference for assessing agreement among these laboratories. Agreement was evaluated in terms of assay accuracy, reproducibility, repeatability, precision, and bias. We also reviewed four acceptance criterion intervals for assessing the comparability of protocols when assaying the same reference sera. The precision, accuracy, and concordance results among laboratories and the consensus titers revealed acceptable agreement. The results of this study indicate that the bioassays evaluated in this study are robust, and the resultant OPA values are reproducible for the determination of functional antibody titers specific to 13 pneumococcal serotypes when performed by laboratories using highly standardized but not identical assays. The statistical methodologies employed in this study may serve as a template for evaluating future multilaboratory studies.
doi:10.1128/CVI.00370-10
PMCID: PMC3019776  PMID: 21084458
11.  Development and Validation of a Fourfold Multiplexed Opsonization Assay (MOPA4) for Pneumococcal Antibodies 
Clinical and Vaccine Immunology  2006;13(9):1004-1009.
Opsonophagocytic killing assays (OPAs) are essential for developing and improving pneumococcal vaccines. There is a need for a high-throughput, reliable, standardized, and fully characterized OPA for pneumococcal antibodies. To meet the need, we have developed and characterized a fourfold multiplexed OPA (MOPA4) against 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) of pneumococci. Thirteen target bacteria were made resistant to only one of the following antibiotics: optochin, streptomycin, spectinomycin, and trimethoprim. Following optimization of assay conditions, accuracy of MOPA4 was determined by testing 30 sera from old adults in the MOPA4 and the single-serotype assays. The opsonization titers obtained with both assays agreed well (r2 > 0.95). Although 22 (out of 390; ∼6%) results differed more than twofold, the differences were not reproducible. The assay was specific: preabsorbing test sera with homologous polysaccharide (PS) completely abrogated opsonic activity, but a pool of unrelated PS (5 μg/ml of each) had no effect. Intra- and interassay coefficients of variation were 10 and 22%, respectively. MOPA4 results were unaffected by having different target pneumococcal serotypes in each assay group. Also, HL60 cell-to-bacteria ratios could be varied twofold without affecting the results. We conclude that MOPA4 is sensitive, accurate, specific, precise, and robust enough for large-scale clinical studies. Furthermore, MOPA4 should allow evaluation of multivalent pneumococcal vaccines with the limited volume of serum typically available from young children.
doi:10.1128/CVI.00112-06
PMCID: PMC1563573  PMID: 16960111

Results 1-11 (11)