Development and validation of a robust multiplex serological assay to quantify antibodies specific to pertussis antigens.

Despite wide spread vaccination, the public health burden of pertussis remains substantial. Current acellular pertussis vaccines comprise upto five Bordetella pertussis (Bp) antigens. Performing an ELISA to quantify antibody for each antigen is laborious and challenging to apply to pediatric samples where serum volume may be limited. We developed a microsphere based multiplex antibody capture assay (MMACA) to quantify antibodies to five pertussis antigens; pertussis toxin, pertactin, filamentous hemagglutinin and fimbrial antigens 2/3, and adenylate cyclase toxin in a single reaction (5-plex) with a calibrated reference standard, QC reagents and SAS® based data analysis program. The goodness of fit (R2) of the standard curves for five analytes was ≥0.99, LLOQ 0.04-0.15 IU or AU/mL, accuracy 1.9%-23.8% (%E), dilutional linearity slopes 0.93-1.02 and regression coefficients r2 = 0.91-0.99. MMACA had acceptable precision within a median CV of 16.0%-22.8%. Critical reagents, antigen conjugated microsphere and reporter antibody exhibited acceptable (<12.3%) lot-lot variation. MMACA can be completed in <3 h, requires low serum volume (5µL/multiplex assay) and has fast data turnaround time (<1 min). MMACA has been successfully developed and validated as a sensitive, specific, robust and rugged method suitable for simultaneous quantification of anti-Bp antibodies in serum, plasma and DBS.

Impact of maternally derived pertussis antibody titers on infant whole-cell pertussis vaccine response in a low income setting.

BACKGROUND: Maternal vaccines against pertussis are not yet recommended in the developing world. Besides unclear burden estimates, another concern is that transplacental transfer of maternal pertussis antibodies could result in attenuation of the immune response to whole cell pertussis (DTwP) primary vaccination series in infants. This study was taken up to determine whether higher levels of maternal pertussis antibodies attenuate immune response of infants to DTwP vaccination series given at 6-10-14 weeks of age. METHODOLOGY: A total of 261 pregnant women and their infants from four low-income settlements in Karachi, Pakistan were enrolled in this study. The study endpoints were infant antibody titers for Pertussis toxin (PTx), Filamentous hemagglutinin antigen (FHA), Pertactin (PRN) and Fimbriae type 2/3 (FIM) - from birth through 18 weeks of age. Cord blood or pre-vaccine pertussis antibody titers indicate the concentration of maternal antibodies transferred to infants. Linear regression models were used to determine the association between higher maternal antibody titers and infant immune response to DTwP vaccine. Geometric Mean Ratio (GMR) was calculated as the ratio of infant antibody titers at specified time points against the maternal antibody titers at the time of delivery. RESULTS: At eighteen weeks of age, the adjusted ß regression coefficient for PTx was 0.06 (95% CI: -0.49-0.61), FHA 0.02 (95% CI: -0.26 -0.29), PRN 0.02 (95%CI -0.38- 0.43), and FIM 0.17 (95%CI: -0.21-0.54). Among infants who received at least two doses of DTwP vaccine, higher maternal antibody titers did not have any attenuating effect on infant post-immunization antibody titers against all four pertussis antigens. CONCLUSION: Maternal pertussis antibodies did not attenuate infant's immune response to pertussis antigens in DTwP primary vaccine given at 6-10-14 weeks of age.

Type I Vs. Type II Cytokine Levels as a Function of SOD1 G93A Mouse Amyotrophic Lateral Sclerosis Disease Progression.

Amyotrophic Lateral Sclerosis (ALS) is a fatal motoneuron disease that is characterized by the degradation of neurons throughout the central nervous system. Inflammation have been cited a key contributor to ALS neurodegeneration, but the timeline of cytokine upregulation remains unresolved. The goal of this study was to temporally examine the correlation between the varying levels of pro-inflammatory type I cytokines (IL-1ß, IL-1α, IL-12, TNF-α, and GFAP) and anti-inflammatory type II cytokines (IL-4, IL-6, IL-10) throughout the progression of ALS in the SOD1 G93A mouse model. Cytokine level data from high copy SOD1 G93A transgenic mice was collected from 66 peer-reviewed studies. For each corresponding experimental time point, the ratio of transgenic to wild type (TG/WT) cytokine was calculated. One-way ANOVA and t-tests with Bonferonni correction were used to analyze the data. Meta-analysis was performed for four discrete stages: early, pre-onset, post-onset, and end stage. A significant increase in TG cytokine levels was found when compared to WT cytokine levels across the entire SOD1 G93A lifespan for majority of the cytokines. The rates of change of the individual cytokines, and type I and type II were not significantly different; however, the mean fold change of type I was expressed at significantly higher levels than type II levels across all stages with the difference between the means becoming more pronounced at the end stage. An overexpression of cytokines occurred both before and after the onset of ALS symptoms. The trend between pro-inflammatory type I and type II cytokine mean levels indicate a progressive instability of the dynamic balance between pro- and anti-inflammatory cytokines as anti-inflammatory cytokines fail to mediate the pronounced increase in pro-inflammatory cytokines. Very early immunoregulatory treatment is necessary to successfully interrupt ALS-induced neuroinflammation.