A novel immuno-molecular strategy for the detection of Streptococcus pneumoniae serotypes in human cerebrospinal and middle ear fluids.

Streptococcus pneumoniae is one of the most common microorganisms causing acute otitis media (AOM) in children. While bacterial culture of middle ear fluid (MEF) is the gold standard to detect the etiological organisms, several host and pathogen factors impact the survival of the organisms resulting in false negatives. To overcome this limitation, we have developed and validated an innovative multiplex immuno-molecular assay to screen and detect the S. pneumoniae 15-valent pneumococcal conjugate vaccine (PCV15; STs 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) vaccine serotypes in MEF. This novel in vitro approach involves two-step testing. First, the MEF specimens were tested for highly conserved pneumococcal genes, autolysin, lytA, and pneumolysin, ply using direct PCR to identify pneumococcus positive specimens. The pneumococcus positive specimens were screened for the presence of vaccine serotype specific pneumococcal polysaccharides using a 15-plex Pneumococcal Antigen Detection (PAD) assay, with specific capture and detection monoclonal antibodies. Due to the lack of availability of MEF samples, cerebrospinal fluid (CSF) was used as the surrogate matrix for the development and validation of the PCR-PAD assays. The PCR and PAD assays were separately evaluated for sensitivity and specificity. Subsequently, the PCR-PAD assays were cross-validated with human MEF samples (n = 39) which were culture confirmed to contain relevant bacterial strains. The combined PCR-PAD assays demonstrated high rate of agreement 94.9% (95% CI; 82.7, 99.4%) with historical Quellung serotype data of these MEF samples. This novel PCR-PAD assay demonstrates the feasibility of combining molecular and immunological assays to screen and identify PCV15 pneumococcal vaccine serotypes in AOM clinical samples.

Development and Validation of Two Optimized Multiplexed Serologic Assays for the 9-Valent Human Papillomavirus Vaccine Types.

Two multiplex immunoassays are routinely used to assess antibody responses in clinical trials of the 9-valent human papillomavirus (9vHPV) vaccine. The HPV6/11/16/18/31/33/45/52/58 competitive Luminex immunoassay (HPV-9 cLIA) and HPV6/11/16/18/31/33/45/52/58 total immunoglobulin G Luminex immunoassay are used for measurements of immunogenicity. Following their initial validation in 2010, both assays were redeveloped, and several parameters were optimized, including the coating concentration of virus-like particles, type of Luminex microspheres, serum sample and reference standard diluent, reference standard starting dilution and titration series, and vendor and concentration of the phycoerythrin-labeled antibodies. Validation studies evaluated the assay performance parameters, including the intra-assay precision (repeatability), intermediate precision, linearity, relative accuracy, and limits of quantitation. In addition, since maintaining a link to the original assays that were used in trials supporting vaccine licensure is critical, the assays were formally bridged to the previous assay versions by using individual patient sera from a 9vHPV vaccine clinical trial (n = 150 day 1 [prevaccination] samples; n = 100 month 7 [1 month post-last vaccine dose] and n = 100 month 36 [30 months post-last vaccine dose; antibody persistence] samples). The results of the validation studies indicate that both optimized assays are accurate, specific, and precise over their respective quantifiable ranges. There was a strong linear association between the new and previous versions of both assays. Assay serostatus cutoffs for the redeveloped assays were established based on the bridging studies and, for the HPV-9 cLIA, further refined, based on additional data from HPV vaccine clinical studies so as to align the seropositivity rates between assay versions. IMPORTANCE Assay modernization is a key aspect of vaccine life cycle management. Thus, new, reoptimized versions of two 9vHPV immunoassays have been developed and validated for use in ongoing and future HPV vaccine clinical trials. These assays are suitable for use in high-throughput testing for HPV antibodies in serum samples. Bridging to previous versions of the assays allows for the continuous monitoring of immune responses across assay versions, including in immunogenicity studies that involve new populations as well as long-term follow-up studies.

Development and Validation of a Sensitive and Robust Multiplex Antigen Capture Assay to Quantify Streptococcus pneumoniae Serotype-Specific Capsular Polysaccharides in Urine.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia (CAP) in young children, older adults, and those with immunocompromised status. Since the introduction of pneumococcal vaccines, the burden of invasive pneumococcal disease caused by vaccine serotypes (STs) has decreased; however, the effect on the burden of CAP is unclear, potentially due to the lack of testing for pneumococcal STs. We describe the development, qualification, and clinical validation of a high-throughput and multiplex ST-specific urine antigen detection (SSUAD) assay to address the unmet need in CAP pneumococcal epidemiology. The SSUAD assay is sensitive and specific to the 15 STs in the licensed pneumococcal conjugate vaccine V114 (STs 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) and uses ST-specific monoclonal antibodies for rapid and simultaneous quantification of the 15 STs using a Luminex microfluidics system. The SSUAD assay was optimized and qualified using healthy adult urine spiked with pneumococcal polysaccharides and validated using culture-positive clinical urine samples (n = 34). Key parameters measured were accuracy, precision, sensitivity, specificity, selectivity, and parallelism. The SSUAD assay met all prespecified validation acceptance criteria and is suitable for assessments of disease burden associated with the 15 pneumococcal STs included in V114. IMPORTANCE Streptococcus pneumoniae has more than 90 serotypes capable of causing a range of disease manifestations, including otitis media, pneumonia, and invasive diseases, such as bacteremia or meningitis. Only a minority (<10%) of pneumococcal diseases are bacteremic with known serotype distribution. Culture and serotyping of respiratory specimens are neither routine nor reliable. Hence, the serotype-specific disease burden of the remaining (>90%) noninvasive conditions is largely unknown without reliable laboratory techniques. To address this need, a 15-plex urine antigen detection assay was developed and validated to quantify pneumococcal serotype-specific capsular polysaccharides in urine. This assay will support surveillance to estimate the pneumococcal disease burden and serotype distribution in nonbacteremic conditions. Data obtained from this assay will be critical for understanding the impact of pneumococcal vaccines on noninvasive pneumococcal diseases and to inform the choice of pneumococcal serotypes for next-generation vaccines.

Real-time PCR assay as a simple and efficient tool for viral stability study.

Aim: For oncolytic virus trials, regulatory agencies often require pharmaceutical industry to evaluate risks of released viruses from patients to environment. This study was to establish a real-time PCR method to assess viral shedding and viral stability in human urine. Results/methodology: Herein, we describe an incubation of viral drug product in human urine and use of real-time PCR as a simple, efficient and high throughput assay to assess the level and stability of a nonenveloped and single stranded RNA virus. The viral stability issue is critical to the collection, transport, storage and testing of clinical samples. Discussion/conclusion: In summary, this simple method provides useful viral stability information at various temperatures and detergents. A similar approach may apply to other RNA viruses (including SARS-CoV-2).

Development and qualification of a fast, high-throughput and robust imaging-based neutralization assay for respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a common pathogen causing severe respiratory illness in infants and elder adults. The development of an effective RSV vaccine is an important unmet medical need and an area of active research. The traditional method for testing neutralizing antibodies against RSV in clinical trials is the plaque reduction neutralization test (PRNT), which uses 24-well plates and needs several days post infection to develop viral plaques. In this study, we have developed a virus reduction neutralization test (VRNT), which allows the number of RSV infected cells to be automatically counted by an imaging cytometer at one day post infection in 96-well plates. VRNT was found robust to cell seeding density, detection antibody concentration, virus input and infection time. By testing twenty human sera, we have shown good correlation between VRNT50 and PRNT50 titers for multiple RSV strains: A2, Long and 18537 (serotype B). To understand the VRNT performance, eight human serum samples with high, medium and low neutralization titers were selected for VRNT qualification. We have demonstrated that VRNT had good specificity, precision, linearity and relative accuracy. In conclusion, VRNT is a better alternative to PRNT in serum neutralization test for RSV vaccine candidates.

Complexities of clinical assay development and optimization prior to first-in-man immunization trials - a description of immunogenicity assay development for the testing of samples from a phase 1 Alzheimer's vaccine trial.

Immunogenicity is often a critical clinical endpoint in the assessment of vaccines prior to the submission of data to regulatory agencies. As a result, the assays used to measure immunogenicity must be highly characterized, well-controlled, and statistically supported. These goals are not easily attained, however, when the development of the assay must occur prior to the first-in-man studies. Two significant barriers exist in the development of these assays: (1) the lack of experience with the performance of a novel antigen in a clinical assay, and (2) the lack of available proper human clinical samples to create reference standards and assess sample matrices. To help to overcome these obstacles, we employed a screening experimental design to assess assay optimization. Design of experiments (DOE) is a statistical tool that allows for the evaluation of all of the key assay parameters to determine the optimal conditions for the assay, as well as determine if there are any interactions of these parameters on the response of the assay. The multivariate approach that is integral to DOE helps to overcome the lack of experience with the assay reagents by facilitating an understanding of how the variables work together in the performance of the assay. Here, we outline the use of full and fractional factorial DOE in the optimization of a clinical assay on two platforms, Luminex and ELISA, for the measurement of antibodies to the beta-amyloid peptide (Abeta) for a novel first-in-man vaccine program. Both platforms are evaluated in an attempt to determine the assay best suited to the needs of the program. We also describe the specificity experiments performed to further characterize the utility of each assay platform.

Virus Reduction Neutralization Test: A Single-Cell Imaging High-Throughput Virus Neutralization Assay for Dengue.

Vaccine immunogenicity and clinical efficacy are often assessed by the measure of serum-neutralizing antibodies. The present gold standard for detecting neutralizing antibodies against many viruses, including dengue, is the plaque/focus reduction neutralization test (P/FRNT). The FRNT is a cell-based assay that inherits high variability, resulting in poor precision and has lengthy turnaround times. The virus reduction neutralization test (VRNT) is a high-throughput alternative to the standard low-throughput and laborious FRNT. The VRNT is similar to FRNT using unaltered wild-type virus and immunostaining, yet uses imaging cytometry to count virus-infected cells 1 day post-infection, reducing assay time and increasing overall throughput 15-fold. In addition, the VRNT has lowered variability relative to FRNT, which may be explained in part by the observation that foci overlap alters foci count and titer over time, in the FRNT. The ability to count one infected cell, rather than waiting for overlapping foci to form, ensures accuracy and contributes to the precision (7-25% coefficient of variation) and sensitivity of the VRNT. Results from 81 clinical samples tested in the VRNT and FRNT show a clear positive relationship. During sample testing, a 96-well plate edge effect was noted and the elimination of this edge effect was achieved by a simple plate seeding technique. The VRNT is an improvement to the current neutralization assays for its shortened assay time, increased precision and throughput, and an alternative to the P/FRNT.

Optimization and validation of a multiplex, electrochemiluminescence-based detection assay for the quantitation of immunoglobulin G serotype-specific antipneumococcal antibodies in human serum.

Pneumovax 23 consists of a mixture of highly purified capsular polysaccharides (Ps) from 23 of the most prevalent serotypes of Streptococcus pneumoniae. Testing of vaccine immunogenicity has been historically performed on the enzyme-linked immunosorbent assay (ELISA) platform, validated to measure immunoglobulin G (IgG) antibodies to all 23 serotypes included in Pneumovax 23. In order to significantly improve the throughput of this form of testing, we have developed and validated a direct binding electrochemiluminescence (ECL)-based multiplex assay that can measure the antibody response in human serum to eight serotypes within a single microtiter well. The pneumococcal (Pn) ECL assay is based on the Meso Scale Discovery (MSD) technology which utilizes a Sulfo-Tag-labeled anti-human IgG antibody that emits light upon electrochemical stimulation. The Pn ECL assay exhibits a wide dynamic range and provides the ability to read concentrations down to the minimum reported concentration in the Merck ELISA (0.1 microg/ml). Cross-reactivity assessment using type-specific monoclonal antibodies showed no cross talk between antigen spots within a well. By use of the WHO Pn sample reference panel, the results obtained with the Pn ECL assay were compared to the results obtained with the international Pn ELISA. The results for the Pn ECL assay satisfied the WHO-recommended acceptance criterion for concordance for all seven serotypes with published Pn ELISA values, and the overall correlation (r value) across the seven serotypes was 0.994. The MSD methodology has great potential to be extremely useful for simultaneously quantitating IgG responses to several Pn serotypes while conserving serum volumes and laboratory testing time.

Serologic assay to quantify human immunoglobulin G antibodies to the Staphylococcus aureus iron surface determinant B antigen.

A direct binding Luminex assay has been developed and validated for the detection of human immunoglobulin G (IgG) antibodies to the Staphylococcus aureus iron surface determinant B protein (IsdB) in serum following natural infection or immunization with investigational Saccharomyces cerevisiae-derived IsdB-based vaccines. To ensure that IsdB-specific IgG antibodies are measured following immunization with S. cerevisiae-derived IsdB, an Escherichia coli-produced IsdB antigen is used in the assay. The IsdB antigen is covalently conjugated to maleimide microspheres via an engineered carboxy-terminal cysteine residue. Antibody titers are determined in a direct binding format, where the phycoerythrin-labeled monoclonal antibody (HP6043) specific for IgG1 to IgG4 binds to human serum IgG antibodies. Fluorescent signal emitted from bound HP6043 is directly proportional to an individual's antibody levels. A pooled human reference serum from vaccinees with high titers to IsdB is used to generate a 12-point standard curve. The correlation of mean fluorescent intensity (MFI) units to microg/ml of IsdB-specific IgG is made by interpolating the MFI data through a four-parameter curve-fitting algorithm. The assay is sensitive to 1.06 microg/ml with a dynamic range of 2.1 to 10,625 microg/ml. The overall specificity of the assay is >96% and the linearity (parallelism) of the assay is -4% per 10-fold dilution. The total precision of the assay was 16.6% relative standard deviation across three different IsdB antigen lots, three different microsphere lots, two secondary antibody lots, and three different operators. The assay has proven useful for evaluating the immune response following the administration of different dosages and formulations of investigational IsdB-based vaccines.