Development and validation of a 6-plex Luminex-based assay for measuring human serum antibodies to group B streptococcus capsular polysaccharides.

Six serotypes (Ia, Ib, II, III, IV, and V) cause nearly all group B streptococcal (GBS) disease globally. Capsular polysaccharide (CPS) conjugate vaccines aim to prevent GBS disease, however, licensure of a vaccine would depend on a standardized serological assay for measuring anti-CPS IgG responses. A multiplex direct Luminex-based immunoassay (dLIA) has been developed to simultaneously measure the concentration of serum IgG specific for the six prevalent GBS CPS serotypes. Assay validation was performed using serum samples obtained from human subjects vaccinated with an investigational 6-valent GBS CPS conjugate vaccine. Results for the assay are expressed as IgG concentrations (µg/mL) using a human serum reference standard composed of pooled sera from vaccinated subjects. The lower limits of quantitation (LLOQ) for all serotypes covered in the 6-plex GBS IgG dLIA fell within the range of 0.002-0.022 µg/mL IgG. Taken together, the 6-plex GBS IgG dLIA platform is specific for the six GBS serotypes included in Pfizer's investigational vaccine, has a wide dilution adjusted assay range, and is precise (<18.5% relative standard deviation) for all serotypes, and, therefore, is suitable for quantitatively measuring vaccine-induced or naturally acquired serotype-specific anti-CPS IgG responses against GBS.

Interlaboratory comparison of a multiplex immunoassay that measures human serum IgG antibodies against six-group B streptococcus polysaccharides.

Measurement of IgG antibodies against group B streptococcus (GBS) capsular polysaccharide (CPS) by use of a standardized and internationally accepted multiplex immunoassay is important for the evaluation of candidate maternal GBS vaccines in order to compare results across studies. A standardized assay is also required if serocorrelates of protection against invasive GBS disease are to be established in infant sera for the six predominant GBS serotypes since it would permit the comparison of results across the six serotypes. We undertook an interlaboratory study across five laboratories that used standardized assay reagents and protocols with a panel of 44 human sera to measure IgG antibodies against GBS CPS serotypes Ia, Ib, II, III, IV, and V. The within-laboratory intermediate precision, which included factors like the lot of coated beads, laboratory analyst, and day, was generally below 20% relative standard deviation (RSD) for all six serotypes, across all five laboratories. The cross-laboratory reproducibility was < 25% RSD for all six serotypes, which demonstrated the consistency of results across the different laboratories. Additionally, anti-CPS IgG concentrations for the 44-member human serum panel were established. The results of this study showed assay robustness and that the resultant anti-CPS IgG concentrations were reproducible across laboratories for the six GBS CPS serotypes when the standardized assay was used.

Phase 3 Safety and Immunogenicity Study of a Three-dose Series of Twenty-valent Pneumococcal Conjugate Vaccine in Healthy Infants and Toddlers.

BACKGROUND: Global pediatric immunization programs with pneumococcal conjugate vaccines (PCVs) have reduced vaccine-type pneumococcal disease, but a substantial disease burden of non-PCV serotypes remains. METHODS: This phase 3, randomized (1:1), double-blind study evaluated safety and immunogenicity of 20-valent PCV (PCV20) relative to 13-valent PCV (PCV13) in healthy infants. Participants received 2 infant doses and a toddler dose of PCV20 or PCV13, with diphtheria-tetanus-acellular pertussis combination vaccine at all doses and measles, mumps, rubella and varicella vaccines at the toddler dose. Primary pneumococcal immunogenicity objectives were to demonstrate noninferiority (NI) of PCV20 to PCV13 for immunoglobulin G geometric mean concentrations after infant and toddler doses and percentages of participants with predefined serotype-specific immunoglobulin G concentrations after infant doses. Safety endpoints included local reactions, systemic events and adverse events. RESULTS: Overall, 1204 participants were vaccinated (PCV20, n = 601; PCV13, n = 603). One month after the toddler dose, 19/20 serotypes met NI for immunoglobulin G geometric mean concentrations; serotype 6B narrowly missed NI [PCV20/PCV13 geometric mean ratio: 0.57 (2-sided 95% confidence interval: 0.48-0.67); NI criterion: lower 2-sided 95% confidence interval >0.5]. Sixteen/twenty serotypes met NI for ≥1 primary objective after 2 infant doses. PCV20 induced robust opsonophagocytic activity, and boosting responses were observed for all vaccine serotypes, including those missing statistical NI. The safety/tolerability profile of PCV20 was like that of PCV13. CONCLUSIONS: PCV20 3-dose series in infants was safe and elicited robust immune responses. Based on these results and PCV13 experience, PCV20 3-dose series is expected to be protective for all 20 vaccine serotypes. NCT04546425.

A Phase Three Study of the Safety and Immunogenicity of a Four-dose Series of 20-Valent Pneumococcal Conjugate Vaccine in Healthy Infants.

BACKGROUND: The 20-valent pneumococcal conjugate vaccine (PCV20) was developed to extend pneumococcal disease protection beyond 13-valent PCV (PCV13). METHODS: This phase 3, double-blind study conducted in the United States/Puerto Rico evaluated PCV20 safety and immunogenicity. Healthy infants were randomized to receive a 4-dose series of PCV20 or PCV13 at 2, 4, 6 and 12-15 months old. Objectives included demonstrating noninferiority (NI) of PCV20 to PCV13 immunoglobulin G (IgG) geometric mean concentrations after doses 3 and 4 and percentages of participants with predefined IgG concentrations after dose 3, with 7 additional PCV20 serotypes compared with the lowest result among vaccine serotypes in the PCV13 group. Safety assessments included local reactions, systemic events, adverse events, serious adverse events and newly diagnosed chronic medical conditions. RESULTS: Overall, 1991 participants were vaccinated (PCV20, n = 1001; PCV13, n = 990). For IgG geometric mean concentrations 1 month after both doses 3 and 4, all 20 serotypes met NI criteria (geometric mean ratio lower 2-sided 95% confidence interval > 0.5). For percentages of participants with predefined IgG concentrations after dose 3, NI (percentage differences lower 2-sided 95% confidence interval > -10%) was met for 8/13 matched serotypes and 6/7 additional serotypes; 4 serotypes missed the statistical NI criterion by small margins. PCV20 also elicited functional and boosting responses to all 20 serotypes. The safety profile of PCV20 was similar to PCV13. CONCLUSION: A 4-dose series of PVC20 was well tolerated and elicited robust serotype-specific immune responses expected to help protect infants and young children against pneumococcal disease due to the 20 vaccine serotypes. Clinical trial registration: NCT04382326.

A phase 3 randomized study to evaluate safety and immunogenicity of 20-valent pneumococcal conjugate vaccine in healthy Japanese infants.

OBJECTIVES: Safety and immunogenicity evaluation of a 4-dose series with 20-valent pneumococcal conjugate vaccine (PCV20). METHODS: This phase 3, double-blind study randomized healthy Japanese infants to receive 4 doses (3 infant doses, 1 toddler dose) of PCV20 by subcutaneous (SC) or intramuscular (IM) injection or 13-valent PCV (PCV13) SC. A primary immunogenicity objective was to demonstrate noninferiority of PCV20 SC to PCV13 SC for percentages of participants meeting predefined serotype-specific immunoglobulin G concentrations 1 month after Dose 3. The 7 additional PCV20 serotypes were compared with the lowest vaccine serotype result in the PCV13 group. Safety and tolerability were assessed as the primary safety objective. RESULTS: Overall, 668 participants were randomized (PCV20 SC, n = 226; PCV13 SC, n = 224; PCV20 IM, n = 218). The primary noninferiority objective for PCV20 SC to PCV13 SC was met for 11/13 matched and 5/7 additional serotypes. Additional data showed PCV20 SC and IM elicited robust functional opsonophagocytic activity and boosting responses to all 20 vaccine serotypes. PCV20 had a similar safety/tolerability profile to PCV13, although local reactions were less frequent with PCV20 IM. CONCLUSIONS: A 4-dose series of PCV20 SC or IM elicited immune responses expected to be protective against all 20 serotypes in Japanese infants. NCT04530838.

Safety and immunogenicity of a multivalent pneumococcal conjugate vaccine given with 13-valent pneumococcal conjugate vaccine in healthy infants: A phase 2 randomized trial.

Use of pneumococcal conjugate vaccines (PCVs) has led to substantial reductions in the global burden of pediatric pneumococcal disease. Expansion of serotype coverage has been achieved by increasing PCV valency, but this may carry the potential risk of antibody interference. A complementary 7-valent PCV (cPCV7) including polysaccharide conjugates from 7 non-13-valent (PCV13) serotypes was developed to potentially complement PCV13-mediated protection and expand serotype coverage. This study evaluated cPCV7 and PCV13 coadministered in separate limbs or separated in time in infants. This phase 2, multicenter, open-label study included 512 infants randomized 1:1:1 to receive cPCV7 coadministered with PCV13 at ages 2, 4, 6, and 12 months (cPCV7 Coadministered); cPCV7 given at ages 3, 5, 7, and 13 months, 3‒5 weeks after PCV13 (cPCV7 Separated); or PCV13 at ages 2, 4, 6, and 12 months followed by a single supplemental dose of cPCV7 at 13 months (PCV13 Control). Safety evaluations included local reactions, systemic events, and adverse events. Serotype-specific immunoglobulin G concentrations and opsonophagocytic activity titers were assessed. The safety profile of cPCV7 was similar to that of PCV13. cPCV7 was well-tolerated in infants when coadministered with or given separately from PCV13. Robust and functional immune responses for all cPCV7 serotypes were observed in both cPCV7 groups. No immunologic interference was observed for either the cPCV7 or PCV13 serotypes with coadministration. A single cPCV7 dose induced immune responses in toddlers. These findings support potential coadministration of a complementary PCV to supplement protection provided by existing PCVs.Trial registration: ClinicalTrials.gov, NCT03550313.

A randomized, open-label, phase 3 study evaluating safety and immunogenicity of 13-valent pneumococcal conjugate vaccine in Chinese infants and children under 6 years of age.

Streptococcus pneumoniae causes a considerable disease burden among children in China. Many isolates exhibit antimicrobial resistance but are often serotypes covered by the 13-valent pneumococcal conjugate vaccine (PCV13). Because the approved infant immunization schedule in China allows PCV13 vaccination only for those 6 weeks to 15 months of age, this phase 3 study was conducted to evaluate PCV13 immunogenicity and safety in unvaccinated older infants and children. Eligible participants were stratified by age into four cohorts: Cohort 1 (n = 125), 6 weeks-2 months; Cohort 2 (n = 354), 7-<12 months; Cohort 3 (n = 250), 1 -<2 years; Cohort 4 (n = 207), 2-<6 years. Cohort 1 received PCV13 at ages 2, 4, and 6 months; older cohorts were randomized 2:1 to PCV13 or Haemophilus influenzae type b (Hib) vaccine using age-appropriate schedules. Within-group immune responses were assessed by immunoglobulin G (IgG) concentrations and opsonophagocytic activity (OPA) titers. Safety evaluations included solicited reactogenicity events and adverse events (AEs). IgG geometric mean concentrations and OPA geometric mean titers for all 13 PCV13 serotypes increased for all participants vaccinated with PCV13, but not those vaccinated with Hib. Immune responses in Cohorts 2-4 were generally comparable with those in Cohort 1 (the infant series) for most serotypes. PCV13 was well tolerated across cohorts, with reported AEs consistent with expectations in these age groups; no new safety signals were identified. These results suggest that PCV13 administered as a catch-up regimen to infants and children 7 months-<6 years of age in China will effectively reduce vaccine-type pneumococcal disease in this population. NCT03574389.

Advances towards licensure of a maternal vaccine for the prevention of invasive group B streptococcus disease in infants: a discussion of different approaches.

Group B streptococcus (Streptococcus agalactiae, GBS) is an important cause of life-threatening disease in newborns. Pregnant women colonized with GBS can transmit the bacteria to the developing fetus, as well as to their neonates during or after delivery where infection can lead to sepsis, meningitis, pneumonia, or/and death. While intrapartum antibiotic prophylaxis (IAP) is the standard of care for prevention of invasive GBS disease in some countries, even in such settings a substantial residual burden of disease remains. A GBS vaccine administered during pregnancy could potentially address this important unmet medical need and provide an adjunct or alternative to IAP for the prevention of invasive GBS disease in neonates. A hurdle for vaccine development has been relatively low disease rates making efficacy studies difficult. Given the well-accepted inverse relationship between anti-GBS capsular polysaccharide antibody titers at birth and risk of disease, licensure using serological criteria as a surrogate biomarker represents a promising approach to accelerate the availability of a GBS vaccine.

A phase 3, multicenter, single-arm, open-label study to assess the safety, tolerability, and immunogenicity of a single dose of 13-valent pneumococcal conjugate vaccine in Japanese participants aged 6-64 years who are considered to be at increased risk of pneumococcal disease and who are naive to pneumococcal vaccines.

BACKGROUND: This open-label, single-arm, phase 3 study evaluated safety and immunogenicity of the 13-valent pneumococcal conjugate vaccine (PCV13) in pneumococcal vaccine-naive Japanese individuals aged 6-64 years at increased risk of pneumococcal disease (PD). METHODS: Participants received 1 PCV13 dose. Reactogenicity events were recorded for 7 days (individuals aged 6- to 17-year-old) or 14 days (individuals aged 18 to 64 years old) postvaccination. Adverse events (AEs) were collected for 1 month postvaccination. Opsonophagocytic activity (OPA) and anticapsular immunoglobulin G (IgG) geometric mean concentrations (GMCs) were measured for vaccine serotypes before and 1 month postvaccination. Post hoc analyses compared immunogenicity in participants categorized as at-risk (immunocompetent but having chronic medical conditions associated with increased PD risk) or high-risk (immunocompromised due to diseases/conditions and/or medications). RESULTS: 206 participants aged 6- to 17-year-old (n = 53) and 18 to 64 years old (n = 153) completed the study. Reactogenicity events were generally mild to moderate in severity. AEs were reported in 16% (33/206) of participants; 1.0% (2/206) were severe. Six AEs were vaccine-related; most were associated with local reactions. No serious AEs occurred. Circulating antibody levels for all 13 serotypes increased postvaccination. OPA geometric mean fold rises (GMFRs) from prevaccination to 1 month postvaccination were 5.5-61.7; lower limits of the 2-sided, 95% CI were > 1 for all serotypes. IgG GMFRs were consistent with OPA analyses. In post hoc analyses, 55.8% (115/206) and 44.2% (91/206) of participants were categorized as at risk and at high risk of PD, respectively; OPA GMFRs from prevaccination to 1 month postvaccination were 3.9-635.1, with lower limits of the 2-sided 95% CIs > 1 for all 13 serotypes across these risk groups; IgG GMFRs were consistent with OPA analyses. CONCLUSIONS: PCV13 was well tolerated and immunogenic in Japanese individuals aged 6-64 years considered at increased risk of PD. Results were broadly comparable with past PCV13 studies in other Japanese and non-Japanese populations. Registration number: NCT03571607; JapicCTI-184024.

Safety and Immunogenicity of a 20-valent Pneumococcal Conjugate Vaccine in Healthy Infants in the United States.

BACKGROUND: The development and widespread use of pneumococcal conjugate vaccines (PCVs) substantially reduced the global burden of pneumococcal disease. Expanding the serotypes covered by PCVs may further reduce disease burden. A 20-valent PCV (PCV20) has been developed to add coverage for 7 additional serotypes (8, 10A, 11A, 12F, 15B, 22F and 33F) to those in the existing 13-valent PCV (PCV13). This phase 2 study evaluated the safety, tolerability and immunogenicity of PCV20 in healthy US infants. METHODS: In this randomized, active-controlled, double-blind study, 460 infants were randomized 1:1 to receive a 4-dose series of either PCV20 or PCV13 at 2, 4, 6 and 12 months of age. Solicited local reactions and systemic events, adverse events (AEs) and serious AEs were recorded. Immunogenicity was assessed by measuring serotype-specific IgG concentrations and opsonophagocytic activity titers at 1 month after Dose 3, before Dose 4 and 1 month after Dose 4. RESULTS: Of 460 infants, 82.8% completed the 1-month visit after Dose 4. Local reactions and systemic events were mostly mild to moderate in severity and similar between the PCV20 and PCV13 groups. Treatment-related AEs were uncommon, with no related serious AEs or deaths reported. IgG and opsonophagocytic activity responses elicited by PCV20 were robust and demonstrated a booster response after Dose 4. CONCLUSIONS: Administration of PCV20 in US infants was well tolerated, with a safety profile similar to PCV13, and induced robust serotype-specific immune responses. These findings support continued development of PCV20 in the pediatric population.

Safety and immunogenicity of a novel hexavalent group B streptococcus conjugate vaccine in healthy, non-pregnant adults: a phase 1/2, randomised, placebo-controlled, observer-blinded, dose-escalation trial.

BACKGROUND: Group B streptococcus (GBS) is a major cause of invasive disease in young infants. Infants born to women with sufficient pre-existing anti-GBS capsular IgG antibodies are at reduced risk of GBS disease, making maternal immunisation a potential strategy for prevention. We aimed to assess the safety and immunogenicity of a novel hexavalent (serotypes Ia, Ib, II, III, IV, and V) GBS conjugate vaccine (GBS6). METHODS: This phase 1/2, placebo-controlled, observer-blinded, dose-escalation trial, was done at four clinical research centres in the USA (Kentucky, Georgia, and two sites in Utah). Healthy, non-pregnant adults aged 18-49 years were randomly assigned using an interactive, web-based response technology system. Within each dose group (low, medium, or high), participants in sentinel cohorts were randomly assigned 2:2:1 and expanded cohort participants were randomly assigned 4:4:1 to receive GBS6 with aluminium phosphate (AlPO4), GBS6 without AlPO4, or placebo (saline control). One 0·5 mL dose of either saline placebo or 5 µg capsular polysaccharide per serotype in the low-dose group, 10 µg capsular polysaccharide per serotype in the medium-dose group, or 20 µg capsular polysaccharide per serotype in the high-dose group was administered by intramuscular injection into the deltoid muscle on day 1. The primary outcome was safety up to 6 months after vaccination, including the proportion of sentinel cohort participants with clinical laboratory abnormalities at 1 week, the proportion of all participants reporting solicited local reactions, systemic events, or use of antipyretic or pain medication within 14 days, adverse events up to 1 month, and medically attended or serious adverse events up to 6 months. The secondary outcome was GBS immunogenicity (serotype-specific IgG geometric mean concentrations at 1 month). This study is registered with ClinicalTrials.gov, NCT03170609. FINDINGS: Between June 5, 2017, and June 25, 2018, 365 participants were randomly assigned and 364 (52 in each dose group) were vaccinated and included in the safety analysis. Unsolicited adverse events were reported by 15 (29%) participants in the 5 µg with AlPO4 group, 13 (25%) in the 5 µg without AlPO4 group, 22 (42%) in the 10 µg with AlPO4 group, 12 (23%) in the 10 µg without AlPO4 group, 25 (48%) in the 20 µg with AlPO4 group, 21 (40%) in the 20 µg without AlPO4 group, and 20 (38%) in the placebo group. The most common unsolicited adverse events were in the system organ class of infections and infestations in any dose or formulation of GBS6 (ranging from six [12%] in the 10 µg without AlPO4 group to 15 [29%] in the 20 µg with AlPO4 group and placebo group). Three participants reported at least one serious adverse event during the study, one each in the 5 µg GBS6 with AlPO4 group (diabetic ketoacidosis, two events; resolved), 10 µg GBS6 with AlPO4 group (died by suicide), and 20 µg GBS6 with AlPO4 group (metrorrhagia; resolved). None of these serious adverse events were considered related to the vaccine. 11 of the 365 participants were excluded from the evaluable immunogenicity population, including one participant who did not receive the vaccine, and ten who at 1 month after vaccination were withdrawn for various reasons. GBS serotype-specific IgG geometric mean concentrations increased by 1 week after vaccination for all GBS6 groups, peaked at 2 weeks, stabilised by 1 month, and declined gradually but remained higher than placebo at 6 months. INTERPRETATION: GBS6 was well tolerated in healthy adults and elicited robust immune responses for all dose levels and formulations that persisted 6 months after vaccination. This study supports further evaluation of GBS6 in pregnant women. FUNDING: Pfizer.

Qualification and Clinical Validation of an Immunodiagnostic Assay for Detecting 11 Additional Streptococcus pneumoniae Serotype-specific Polysaccharides in Human Urine.

BACKGROUND: Identifying Streptococcus pneumoniae serotypes by urinary antigen detection (UAD) assay is the most sensitive way to evaluate the epidemiology of nonbacteremic community-acquired pneumonia (CAP). We first described a UAD assay to detect the S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, covered by the licensed 13-valent S. pneumoniae conjugate vaccine. To assess the substantial remaining pneumococcal disease burden after introduction of several pneumococcal vaccines, a UAD-2 assay was developed to detect 11 additional serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F) in individuals with radiographically confirmed CAP. METHODS: The specificity of the UAD-2 assay was achieved by capturing pneumococcal polysaccharides with serotype-specific monoclonal antibodies, using Luminex technology. Assay qualification was used to assess accuracy, precision, and sample linearity. Serotype positivity was based on cutoffs determined by nonparametric statistical evaluation of urine samples from individuals without pneumococcal disease. The sensitivity and specificity of the positivity cutoffs were assessed in a clinical validation, using urine samples obtained from a large study that measured the proportion of radiographically confirmed CAP caused by S. pneumoniae serotypes in hospitalized US adults. RESULTS: The UAD-2 assay was shown to be specific and reproducible. Clinical validation demonstrated assay sensitivity and specificity of 92.2% and 95.9% against a reference standard of bacteremic pneumonia. In addition, the UAD-2 assay identified a S. pneumoniae serotype in 3.72% of nonbacteremic CAP cases obtained from hospitalized US adults. When combined with bacteremic CAP cases, the proportion of pneumonias with a UAD-2 serotype was 4.33%. CONCLUSIONS: The qualified/clinically validated UAD-2 method has applicability in understanding the epidemiology of nonbacteremic S. pneumoniae CAP and for assessing the efficacy of future pneumococcal conjugate vaccines that are under development.

Immunogenicity and Safety of the 13-Valent Pneumococcal Conjugate Vaccine Administered in a 3 + 1 versus 2 + 1 Dose Schedule Among Infants in China.

BACKGROUND: 13-valent pneumococcal conjugate vaccine (PCV13) was licensed in China based on immunologic noninferiority to 7-valent PCV (PCV7). As part of the noninferiority study, immunogenicity and safety of PCV13 administered as a 3- or 2-dose infant series followed by a toddler dose were examined in healthy Chinese infants. METHODS: Infants (42- to 77-days-old) were randomized to a 3-dose PCV13 or PCV7 infant series administered double-blind at 3, 4 and 5 months or PCV13 administered open-label at 2, 4 and 6 months and a 2-dose open-label series at 3 and 5 months; all subjects received a toddler dose (12 months). Serotype-specific immunoglobulin G (IgG) concentrations were measured 1 month after the infant series and before and after the toddler dose. Opsonophagocytic activity (OPA) was measured in a subset of subjects at each time point. Safety was evaluated. RESULTS: One month after the infant series, serotype-specific immune responses (IgG ≥ 0.35 µg/mL) were similar for the 2- versus 3-dose schedules, except for serotype 6B, which was significantly lower in the 2-dose group [70.1% in the PCV13 (3, 5 + 12 mo) group vs. 93.2% in the PCV13 (3, 4, 5 + 12 mo) group and 94.7% in the PCV13 (2, 4, 6 + 12 mo) group]. IgG geometric mean concentrations and OPA geometric mean titers trended numerically higher with 3- versus 2-dose schedules. No significant differences in immunogenicity were observed between the 3- versus 2-dose schedules after the toddler dose. PCV13 was well-tolerated across all schedules. CONCLUSIONS: PCV13 administered as a 3- or 2-dose infant series followed by a toddler dose was immunogenic and well tolerated in healthy Chinese infants and likely protective against PCV13 serotypes; immune responses with a 2-dose schedule were lower for some serotypes.

Evaluation of a Validated Luminex-Based Multiplex Immunoassay for Measuring Immunoglobulin G Antibodies in Serum to Pneumococcal Capsular Polysaccharides.

This article describes the results of a study designed to bridge the World Health Organization (WHO) pneumococcal enzyme-linked immunosorbent assay (ELISA) platform to the validated Luminex-based 13-plex direct immunoassay (dLIA) platform developed by Pfizer, Inc. Both assay platforms quantify serotype-specific serum IgG antibodies (in micrograms per milliliter) against an international reference standard serum. The primary goal of this study was to determine if the dLIA is a suitable replacement for the ELISA to support clinical vaccine studies that include the evaluation of immune responses to serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. Serum samples were selected from four pivotal 13-valent pneumococcal conjugate vaccine (13vPnC; Prevnar 13) clinical trials on the basis of their serotype-specific IgG concentrations by ELISA. In these studies, subjects were immunized either with 13vPnC or with 7-valent pneumococcal conjugate vaccine (7vPnC; Prevnar). There were 1,528 of 1,574 selected samples with sufficient remaining volume for reanalysis in the dLIA. A comparison of assay results from the dLIA and ELISA platforms showed clear and robust linear quantitative relationships across all 13 serotypes. In addition, lower IgG antibody concentrations in preimmunization samples were measured in the dLIA, thus allowing better differentiation between preimmunization and low-titer postimmunization samples. Overall, the results showed that the established population-level protective threshold IgG concentration, 0.35 µg/ml of serotype-specific serum IgG antibodies, is appropriate for use for data generated using the dLIA platform developed by Pfizer, Inc., for 10 serotypes: serotypes 1, 3, 4, 6A, 7F, 9V, 14, 18C, 19F, and 23F. On the basis of the extensive bridging analyses, however, the use of dLIA cutoff values of 0.23, 0.10, and 0.12 µg/ml is recommended for serotypes 5, 6B, and 19A, respectively. This adjustment will ensure that the consistency of the established population-level protective threshold IgG concentration is maintained when switching from the ELISA to the dLIA platform. The results of this bridging study demonstrate that the 13-plex dLIA platform is a suitable replacement for the WHO reference ELISA platform.IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.

Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides.

A Luminex-based direct immunoassay (dLIA) platform has been developed to replace the standardized pneumococcal enzyme-linked immunosorbent assay platform. The multiplex dLIA simultaneously measures the concentration of serum immunoglobulin G (IgG) antibodies specific for pneumococcal capsular polysaccharide (PnPS) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The assay uses poly-l-lysine (PLL)-conjugated PnPS, chemically coupled to spectrally distinct Luminex microspheres. Assay validation experiments were performed using residual human serum samples obtained from 13-valent pneumococcal conjugate vaccine (13vPnC) clinical studies. Assay results are expressed as IgG antibody concentrations in micrograms per milliliter using the international reference serum, 007sp. The lower limit of quantitation (LLOQ) for all serotypes covered in the 13-plex dLIA fell within the range of 0.002 to 0.038 µg/ml serum IgG. The difference between the lower limit and upper limit of the assay range was >500-fold for all serotypes, and assay variability was <20% relative standard deviation (RSD) for all serotypes. IgG antibody measurements were shown to be serotype-specific (some cross-reactivity was observed only between the structurally related serotypes 6A and 6B as well as 19A and 19F), and no interference was observed between the serotypes when the assay was performed in the 13-plex format compared to the singleplex assays. The 13-plex dLIA platform developed by Pfizer Inc. generates up to 143 test results in a single 96-well plate and is a suitable replacement of the enzyme-linked immunosorbent assay (ELISA) platform for evaluating vaccine clinical trials.IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.

A randomized study of fever prophylaxis and the immunogenicity of routine pediatric vaccinations.

OBJECTIVE: Prophylactic antipyretic use during pediatric vaccination is common. This study assessed whether paracetamol or ibuprofen prophylaxis interfere with immune responses to the 13-valent pneumococcal conjugate vaccine (PCV13) given concomitantly with the combined DTaP/HBV/IPV/Hib vaccine. METHODS: Subjects received prophylactic paracetamol or ibuprofen at 0, 6-8, and 12-16 h after vaccination, or 6-8 and 12-16 h after vaccination at 2, 3, 4, and 12months of age. At 5 and 13months, immune responses were evaluated versus responses in controls who received no prophylaxis. RESULTS: After the infant series, paracetamol recipients had lower levels of circulating serotype-specific pneumococcal anticapsular immunoglobulin G than controls, reaching significance (P<0.0125) for 5 serotypes (serotypes 3, 4, 5, 6B, and 23F) when paracetamol was started at vaccination. Opsonophagocytic activity assay (OPA) results were similar between groups. Ibuprofen did not affect pneumococcal responses, but significantly (P<0.0125) reduced antibody responses to pertussis filamentous hemagglutinin and tetanus antigens after the infant series when started at vaccination. No differences were observed for any group after the toddler dose. CONCLUSIONS: Prophylactic antipyretics affect immune responses to vaccines; these effects vary depending on the vaccine, antipyretic agent, and time of administration. In infants, paracetamol may interfere with immune responses to pneumococcal antigens, and ibuprofen may reduce responses to pertussis and tetanus antigens. The use of antipyretics for fever prophylaxis during infant vaccination merits careful consideration. ClinicalTrials.gov identifier: NCT01392378https://clinicaltrials.gov/ct2/show/NCT01392378?term=NCT01392378&rank=1.

Circulating Antibody 1 and 2 Years After Vaccination With the 13-Valent Pneumococcal Conjugate Vaccine in Preterm Compared With Term Infants.

BACKGROUND: Premature infants have lower short-term immune responses to vaccination than term infants, but patterns of antibody persistence in preterm infants over longer periods are not well established. This study assessed the persistence of antibody response to the 13-valent pneumococcal conjugate vaccine (PCV13) in formerly preterm versus term infants. METHODS: In total, 100 preterm and 100 term infants received PCV13 with routine vaccines at ages 2, 3, 4 and 12 months. Serotype-specific anticapsular immunoglobulin G (IgG)-binding antibodies and opsonophagocytic activity were determined 1 and 2 years after the last PCV13 dose. RESULTS: At 1 and 2 years after the last vaccination (toddler dose), IgG geometric mean concentrations (GMCs) for all serotypes had declined from levels measured 1 month after the toddler dose but remained above pretoddler dose levels. IgG GMCs were significantly lower in preterm than term subjects for a majority of serotypes at both follow-up time points. IgG GMCs increased in both groups for some serotypes from the 1-year to 2-year follow-up, whereas others declined. Opsonophagocytic activity results supported the IgG results. CONCLUSIONS: The routine (3 + 1) vaccination schedule is likely to offer long-term protection against invasive pneumococcal disease in preterm infants and should be initiated regardless of gestational age or weight at birth, without delay of the toddler dose.

Long-term antibody persistence study (3 years after last dose) of the 7-valent pneumococcal conjugate vaccine in young children in China.

BACKGROUND: In a previous study, Chinese infants were vaccinated with 7-valent pneumococcal conjugate vaccine (PCV7) ⩾7days before routine diphtheria, tetanus, and acellular pertussis vaccine (DTaP); PCV7 administered concomitantly with DTaP (PCV7+DTaP); or DTaP alone. This study examined antibody persistence at a single time point 3years after the last vaccination. METHODS: Children who participated in the prior PCV7 study were eligible to participate. A single blood sample was drawn at enrollment. Immunoglobulin G (IgG) geometric mean concentrations (GMCs) specific to the PCV7 serotypes and percentages of subjects with IgG ⩾0.35µg/mL were compared for subjects receiving PCV7 versus PCV7+DTaP (concomitant) and for PCV7 or PCV7+DTaP (concomitant) versus DTaP alone. IgG concentrations at 3years after the last vaccination were also compared with those after the infant series and toddler dose. RESULTS: Three years after the last vaccination with PCV7 or PCV7+DTaP (concomitant), IgG GMCs for most PCV7 serotypes were lower than after the infant series or toddler dose but remained above prevaccination concentrations. IgG GMC were similar between the PCV7 and PCV7+DTaP (concomitant) groups for 5 out of 7 serotypes but serotypes 4 and 19F were significantly lower in the PCV7+DTaP (concomitant) recipients. Three years after the last vaccination, IgG GMCs were significantly higher for 6 of 7 PCV7 serotypes among those receiving PCV7 or PCV7+DTaP (concomitant) compared with recipients of DTaP alone. Among subjects receiving DTaP alone, serotype-specific antibody concentrations were significantly higher for all serotypes 3years after the last vaccination compared with after the infant series. CONCLUSION: Three years after PCV7 vaccination, serotype-specific antibodies were lower than after the primary infant series but higher than prevaccination levels and higher among subjects who received PCV7 compared with those who did not. The immune response was comparable in children who received PCV7 with and without concomitant DTaP. CLINICAL TRIAL REGISTRATION: NCT01298544.

Immunogenicity and Safety of 13-valent Pneumococcal Conjugate Vaccine Compared With 7-valent Pneumococcal Conjugate Vaccine Among Healthy Infants in China.

BACKGROUND: Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine (PCV13) were compared with 7-valent pneumococcal conjugate vaccine (PCV7) in Chinese infants. METHODS: Healthy infants aged 2 months were randomized to a double-blind 3-dose infant series plus 1 toddler dose of PCV7 or PCV13 at 3, 4, 5 and 12 months or open-label PCV13 at 2, 4, 6 and 12 months. Serotype-specific immunoglobulin G (IgG) binding and functionality were measured 1 month after the infant series and after the toddler dose. Local reactions, systemic events and adverse events were assessed postvaccination. RESULTS: For the 7 common serotypes, serotype-specific binding IgGs elicited by PCV13 were noninferior to PCV7 after the 3-dose infant series; functional antibodies were comparable. For the 6 additional serotypes, PCV13 recipients had significantly higher serotype-specific IgGs and functional antibodies than PCV7 recipients after the infant series. The toddler dose boosted the immune response. Local reactions and systemic events were mild in severity and similar across groups. No new safety signals were identified. CONCLUSIONS: For the 7 common serotypes, serotype-specific binding IgG after 2 different 3-dose regimens of PCV13 were noninferior to PCV7 responses. PCV13 recipients had significantly higher immune responses to the 6 additional serotypes. PCV13 is expected to provide pneumococcal disease protection comparable to PCV7 for the common serotypes and further protection against disease caused by the 6 additional serotypes. Safety of PCV7 and PCV13 was comparable.

Safety, tolerability, and immunogenicity of 7-valent pneumococcal conjugate vaccine in older infants and young children in China who are naive to pneumococcal vaccination: Results of a phase 4 open-label trial.

BACKGROUND: This postlicensure study was conducted to assess immunogenicity and safety of PCV7 catch-up regimens in previously unvaccinated older infants and young children in China. METHODS: Healthy children 121 days to <72 months were grouped by age and immunized with 1 of 4 PCV7 dosing regimens. Serotype-specific IgG geometric mean concentrations (GMCs) and percentage of subjects with IgG≥0.35µg/mL were assessed before vaccination and 1 and 12 months postvaccination. The incidence of clinically important adverse events (AEs) and serious AEs (SAEs), AEs leading to study withdrawal, and protocol-related AEs were assessed throughout the study. RESULTS: Prevaccination serotype-specific GMCs were generally low in subjects <24 months; the majority of children 24 to <72 months had IgG concentrations ≥0.35 µg/mL. One month postvaccination, GMCs were similar across groups for the 7 PCV serotypes, ranging from 3.95 to 13.02 µg/mL; the highest antibody levels were observed for serotype 14. Regardless of dosing regimen, >90% of subjects had IgG≥0.35 µg/mL for each PCV serotype. At 12-month follow-up, IgG GMCs ranged from 0.65 to 5.19, and all remained above prevaccination IgG GMC; >70% of subjects had IgG≥0.35 µg/mL. Older children generally had the most robust immune response both at 1 month postvaccination and during 12-month follow-up. PCV7 was well tolerated. Pyrexia, which was mild to moderate in severity, was the most common AE. Two subjects reported SAEs (n=4), and there was 1 study withdrawal; none of these were considered treatment related. CONCLUSION: In China, PCV7 catch-up vaccinations given to older infants and young children naive to pneumococcal vaccines resulted in a robust immune response to all serotypes; this response persisted after 1 year. PCV7 was well tolerated in Chinese infants and children.