Transcriptomic signatures were identified in human peripheral blood mononuclear cells (PBMCs) and breast milk lymphocyte (BML) cells induced by trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV) administered after delivery. We performed an RNA-Seq analysis on blood and breast milk samples from a subset of subjects enrolled in a randomized, double-blind controlled study in breastfeeding women who received either intranasal LAIV and intramuscular placebo, or intramuscular TIV and intranasal placebo (LAIV, n = 10 and TIV, n = 6). Differentially expressed genes, gene clusters, and enriched pathways were identified. We observed increased innate immune signaling responses in BML but not in PBMC at Day 28 for the LAIV group. We hypothesize that breastfeeding extended the innate response to LAIV via mucosal immunity. An association between an increased IgG antibody response in TIV vs. LAIV identified in the parent study using ELISA corresponded to IGHG1 immunoglobulin gene expression in Day 28 PBMCs.

Given the local and systemic adverse reactions associated with whole cell pertussis vaccines combined with diphtheria and tetanus toxoids (DTP), acellular pertussis vaccines combined with the same toxoids (DTaP) were developed in the 1990s. In comparison to DTP, DTaP vaccines demonstrated reduced reactogenicity and equivalent or improved immunogenicity and efficacy. However, there has been a resurgence of pertussis disease, particularly in DTaP-vaccinated children, suggesting that immunity wanes more quickly with DTaP vaccination. To elucidate the differences in immune responses to DTP and DTaP vaccines, we employed a systems biology-based strategy to compare global changes in gene expression following primary vaccination with either DTP or DTaP. We used RNA-Seq and ribosome profiling (RP) to identify transcriptional and translational signatures, respectively, in peripheral blood mononuclear cells (PBMCs) collected from 50 infant recipients of DTP or DTaP at two time-points (baseline (pre-vaccination at Day 1) and either Day 2 or 8 post-vaccination). We also used standard serologic methods to assess immunogenicity, and correlated these results with transcriptional and translational signatures. Here, we provide a detailed description of the rationale, experimental design, methodology and enrollment procedures used. Given the technical complexity of our approach, our objective is to fill knowledge gaps, describe key quality metrics and support future publications. In brief, we recovered 4–12 million PBMCs (average 8.9 million) with 99% viability per 2.5 mL blood sample, enabling excellent nucleic acid recovery yields for preparation of high-quality sequencing libraries. In turn, these generated RNA-Seq and RP datasets with sufficient genome coverage breadth and depth to enable differential gene expression analyses, demonstrating the validity of this approach to study pertussis vaccine immunology specifically, and its utility to characterize mechanisms of the human immune response to vaccination generally.

Background Avian influenza viruses pose significant risk to human health. Vaccines targeting the hemagglutinin of these viruses are poorly immunogenic without the use of adjuvants.Methods Twenty healthy men and women (18-49 years of age) were randomized to receive 2 doses of inactivated influenza A/H5N1 vaccine alone (IIV) or with AS03 adjuvant (IIV-AS03) 1 month apart. Urine and serum samples were collected on day 0 and on days 1, 3, and 7 following first vaccination and subjected to metabolomics analyses to identify metabolites, metabolic pathways, and metabolite clusters associated with immunization.Results Seventy-three differentially abundant (DA) serum and 88 urine metabolites were identified for any postvaccination day comparison. Pathway analysis revealed enrichment of tryptophan, tyrosine, and nicotinate metabolism in urine and serum among IIV-AS03 recipients. Increased urine abundance of 4-vinylphenol sulfate on day 1 was associated with serologic response based on hemagglutination inhibition responses. In addition, 9 DA urine metabolites were identified in participants with malaise compared to those without.Conclusions Our findings suggest that tryptophan, tyrosine, and nicotinate metabolism are upregulated among IIV-AS03 recipients compared with IIV alone. Metabolites within these pathways may serve as measures of immunogenicity and may provide mechanistic insights for adjuvanted vaccines.Clinical Trials Registration NCT01573312. Following AS03-adjuvanted influenza A/H5N1 vaccine, enrichment of tryptophan, tyrosine, and nicotinate metabolism pathways was associated with serologic response. More specifically, increased urine abundance of benzoate-metabolism-related 4-vinylphenol sulfate on day 1 postvaccination was highly associated with serologic response.

Filoviruses, including Ebola, Marburg, Sudan, and Taï Forest viruses, are zoonotic pathogens that can cause severe viral hemorrhagic fever and death. Developing vaccines that provide durable, broad immunity against multiple filoviruses is a high global health priority. In this Phase 1 trial, we enrolled 60 healthy U.S. adults and evaluated the safety, reactogenicity and immunogenicity of homologous and heterologous MVA-BN®-Filo and Ad26.ZEBOV prime-boost schedules followed in select arms by MVA-BN®-Filo boost at 1 year (NCT02891980). We found that all vaccine regimens had acceptable safety and reactogenicity. The heterologous prime-boost strategy elicited superior Ebola binding and neutralizing antibody, antibody-dependent cellular cytotoxicity (ADCC), and cellular responses compared to homologous prime-boost. The MVA-BN®-Filo boost administered at 1 year resulted in robust humoral and cellular responses that persisted through 6-month follow-up. Overall, our data demonstrated that a heterologous Ad26.ZEBOV/MVA-BN®-Filo prime-boost was safe and immunogenic and established immunologic memory primed to respond after re-exposure. Clinicaltrials.gov, NCT02891980, registered September 1, 2016.

Introduction Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. The goal of this study was to benchmark key technical and analytical parameters of RNA sequencing (RNA-seq) in the context of a multi-site, double-blind randomized vaccine clinical trial. Methods We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects before and after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two replicate datasets (5 time points for 50 samples each). We evaluated the impact of (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data and established the impact of sequencing depth and gene filtering on transcriptome representation. Lastly, we modeled statistical power to detect DEGs for a range of sample sizes, effect sizes, and sequencing depths. Results and Discussion Our results showed that (i) filtering lowly-expressed genes is recommended to improve fold-change accuracy and inter-site agreement, if possible guided by mRNA spike-ins (ii) read length did not have a major impact on DEG detection, (iii) applying fold-change cutoffs for DEG detection reduced inter-set agreement and should be used with caution, if at all, (iv) reduction in sequencing depth had a minimal impact on statistical power but reduced the identifiable fraction of the PBMC transcriptome, (v) after sample size, effect size (i.e. the magnitude of fold change) was the most important driver of statistical power to detect DEG. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies.

Current seasonal and pre-pandemic influenza vaccines induce short-lived predominantly strain-specific and limited heterosubtypic responses. To better understand how vaccine adjuvants AS03 and MF59 may provide improved antibody responses to vaccination, we interrogated serum from subjects who received 2 doses of inactivated monovalent influenza A/Indonesia/05/2005 vaccine with or without AS03 or MF59 using hemagglutinin (HA) microarrays (NCT01317758 and NCT01317745). The arrays were designed to reflect both full-length and globular head HA derived from 17 influenza A subtypes (H1 to H16 and H18) and influenza B strains. We observed significantly increased strain-specific and broad homo- and heterosubtypic antibody responses with both AS03 and MF59 adjuvanted vaccination with AS03 achieving a higher titer and breadth of IgG responses relative to MF59. The adjuvanted vaccine was also associated with the elicitation of stalk-directed antibody. We established good correlation of the array antibody responses to H5 antigens with standard HA inhibition and microneutralization titers.

ABSTRACT Current seasonal and pre-pandemic influenza vaccines induce short-lived predominantly strain-specific and limited heterosubtypic responses. To better understand how vaccine adjuvants AS03 and MF59 may provide improved antibody responses to vaccination, we interrogated serum from subjects who received 2 doses of inactivated monovalent influenza A/Indonesia/05/2005 vaccine with or without AS03 or MF59 using hemagglutinin (HA) microarrays. The arrays were designed to reflect both full length and globular head HA proteins derived from 17 influenza A subtypes (H1 to H16 and H18) and influenza B strains. We observed significantly increased strain-specific and broad homo- and hetero-subtypic antibody responses with both AS03 and MF59 adjuvanted vaccination with AS03 achieving a higher titer and breadth of IgG responses relative to MF59. Adjuvanted vaccine was also associated with the elicitation of stalk directed antibody. Finally, we established good correlation of the array antibody responses to H5 antigens with standard hemagglutination inhibition and microneutralization titers.

Francisella tularensis (F. tularensis) is an intracellular pathogen that causes a potentially debilitating febrile illness known as tularemia. F. tularensis can be spread by aerosol transmission and cause fatal pneumonic tularemia. If untreated, mortality rates can be as high as 30%. To study the host responses to a live-attenuated tularemia vaccine, peripheral blood mononuclear cell (PBMC) samples were assayed from 10 subjects collected pre- and post-vaccination, using both the 2D-DIGE/MALDI-MS/MS and LC-MS/MS approaches. Protein expression related to antigen processing and presentation, inflammation (PPARγ nuclear receptor), phagocytosis, and gram-negative bacterial infection was enriched at Day 7 and/or Day 14. Protein candidates that could be used to predict human immune responses were identified by evaluating the correlation between proteome changes and humoral and cellular immune responses. Consistent with the proteomics data, parallel transcriptomics data showed that MHC class I and class II-related signals important for protein processing and antigen presentation were up-regulated, further confirming the proteomic results. These findings provide new biological insights that can be built upon in future clinical studies, using live attenuated strains as immunogens, including their potential use as surrogates of protection.

Tularemia is a highly infectious and contagious disease caused by the bacterium Francisella tularensis. To better understand human response to a live-attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated, and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we fully characterized individual lipid species within predominant lipid classes to identify changes in the plasma lipidome during the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid classes. These included 5-hydroxyeicosatetraenoic acid (5-HETE) which is indicative of lipoxygenase activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a dihydroxyeicosatrienoic acid (DHET) by day 7 post-vaccination, shedding light on the kinetics of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 16:1 cholesteryl ester, and other glycerophospholipids; several of these changes in abundance were correlated with serum cytokines and T cell activation. These data provide new insights into alterations in plasma lipidome post-tularemia vaccination, potentially identifying key mediators and pathways involved in vaccine response and efficacy.

Yersinia pestis, the cause of plague, could be weaponized. Unfortunately, development of new vaccines is limited by lack of correlates of protection. We used pre- and post-vaccination sera and peripheral blood mononuclear cells from a flagellin adjuvanted F1/V vaccine trial to evaluate for protective markers. Here, we report for the first time in humans that inverse caspase-3 levels, which are measures of protective antibody, significantly increased by 29% and 75% on days 14 and 28 post-second vaccination, respectively. In addition, there were significant increases in T-cell responses on day 28 post-second vaccination. The strongest positive and negative correlations between protective antibody levels and gene expression signatures were identified for IFNG and ENSG00000225107 genes, respectively. Flagellin/F1/V subunit vaccine induced macrophage-protective antibody and significant CD4 T-cell responses. Several genes associated with these responses were identified that could serve as potential correlates of protection.

The immune response to live-attenuated Francisella tularensis vaccine and its host evasion mechanisms are incompletely understood. Using RNA-Seq and LC–MS on samples collected pre-vaccination and at days 1, 2, 7, and 14 post-vaccination, we identified differentially expressed genes in PBMCs, metabolites in serum, enriched pathways, and metabolites that correlated with T cell and B cell responses, or gene expression modules. While an early activation of interferon α/β signaling was observed, several innate immune signaling pathways including TLR, TNF, NF-κB, and NOD-like receptor signaling and key inflammatory cytokines such as Il-1α, Il-1β, and TNF typically activated following infection were suppressed. The NF-κB pathway was the most impacted and the likely route of attack. Plasma cells, immunoglobulin, and B cell signatures were evident by day 7. MHC I antigen presentation was more actively up-regulated first followed by MHC II which coincided with the emergence of humoral immune signatures. Metabolomics analysis showed that glycolysis and TCA cycle-related metabolites were perturbed including a decline in pyruvate. Correlation networks that provide hypotheses on the interplay between changes in innate immune, T cell, and B cell gene expression signatures and metabolites are provided. Results demonstrate the utility of transcriptomics and metabolomics for better understanding molecular mechanisms of vaccine response and potential host–pathogen interactions.

Doxycycline is a tetracycline-class antimicrobial labeled by the United States (U.S.) Food and Drug Administration for children >8 years of age for many common childhood infections. Doxycycline is not labeled for children ≤8 years of age, due to the association between tetracycline class antibiotics and tooth staining, although doxycycline may be used off-label in severe conditions. Accordingly, there is a paucity of pharmacokinetic (PK) data to guide dosing in children 8 years and younger. We leveraged opportunistically-collected plasma samples after intravenous (IV) and oral doxycycline doses received per standard of care to characterize the PK of doxycycline in children of different ages, and evaluated the effect of obesity and fasting status on PK parameters.We developed a population PK model of doxycycline using data collected from 47 patients 0-18 years of age, including 14 participants ≤8 years. We developed a 1 compartment PK model and found doxycycline clearance to be 3.32 L/h/70 kg and volume to be 96.8 L/70kg for all patients; comparable to values reported in adults. We estimated a bioavailability of 89.6%, also consistent with adult data. Allometrically scaled clearance and volume of distribution did not differ between children 2 to ≤8 years of age and children >8 to ≤18 years of age, suggesting that younger children may be given the same per kg dosing. Obese and fasting status were not selected for inclusion in the final model. Additional doxycycline PK samples collected in future studies may be used to improve model performance and maximize its clinical value.

BACKGROUND:Infection with multiple cytomegalovirus (CMV) strains (mixed infection) was reported in a variety of hosts. As the virus genetic diversity in primary CMV infection and the changes over time remain incompletely defined, we examined CMV diversity and changes in diversity over time in healthy adolescent females who participated in a phase 2 CMV gB/MF59 vaccine trial.METHODS:CMV genetic diversity was determined by genotyping of 5 genes-gB (UL55), gH (UL75), gN (UL73), US28, and UL144-in urine, saliva, and plasma samples from 15 study subjects.RESULTS:At the time of primary infection, 5 of 12 (42%) urine samples had multiple virus strains, and 50% of vaccine recipients were infected with gB1 genotype (vaccine strain). Mixed infection was documented in all 15 subjects within 3 months after primary infection, and the majority had different CMV genotypes in different compartments. Changes in genotypes over time were observed in all subjects.CONCLUSIONS:Infection with multiple CMV genotypes was common during primary infection and further diversification occurred over time. Infection with gB1 genotype in vaccine recipients suggests a lack of strain-specific protection from the vaccine. As only 5 polymorphic genes were assessed, this study likely underestimated the true genetic diversity in primary CMV infection.

Milrinone is a type 3 phosphodiesterase inhibitor used to improve cardiac output in critically ill infants and children. Milrinone is primarily excreted unchanged in the urine, raising concerns for toxic accumulation in the setting of renal dysfunction of critical illness. We developed a population pharmacokinetic model of milrinone using nonlinear mixed‐effects modeling in NONMEM to perform dose‐exposure simulations in children with variable renal function. We included children aged <21 years who received intravenous milrinone per clinical care. Plasma milrinone concentrations were measured using a validated liquid chromatography–tandem mass spectrometry assay (range 1‐5000 ng/mL). We performed dose‐exposure simulations targeting steady‐state therapeutic concentrations of 100‐300 ng/mL previously established in adults and children with cardiac dysfunction. We simulated concentrations over 48 hours in typical subjects with decreasing creatinine clearance (CrCl), estimated using the updated bedside Schwartz equation. Seventy‐four patients contributed 111 plasma samples (concentration range, 4‐634 ng/mL). The median (range) postmenstrual age (PMA) was 3.7 years (0‐18), and median weight (WT) was 13.1 kg (2.6‐157.7). The median serum creatinine and CrCl were 0.5 mg/dL (0.1‐3.1) and 117.2 mL/min/1.73 m2 (13.1‐261.3), respectively. A 1‐compartment model characterized the pharmacokinetic data well. The final model parameterization was: Clearance (L/h) = 15.9*(WT [kg] / 70)0.75* (PMA1.12 / (67.71.12+PMA1.12)*(CrCl / 117)0.522; and Volume of Distribution (L) = 32.2*(WT [kg] / 70). A loading dose of 50 µg/kg followed by a continuous infusion of 0.5 µg/kg/min resulted in therapeutic concentrations, except when CrCl was severely impaired at ≤30 mL/min/1.73 m2. In this setting, a 25 µg/kg loading dose and 0.25 µg/kg/min continuous infusion resulted in therapeutic exposures.

Ketamine is an N‐methyl D‐aspartate receptor antagonist used off‐label to facilitate dissociative anesthesia in children undergoing invasive procedures. Available for both intravenous and intramuscular administration, ketamine is commonly used when vascular access is limited. Pharmacokinetic (PK) data in children are sparse, and the bioavailability of intramuscular ketamine in children is unknown. We performed 2 prospective PK studies of ketamine in children receiving either intramuscular or intravenous ketamine and combined the data to develop a pediatric population PK model using nonlinear mixed‐effects methods. We applied our model by performing dosing simulations targeting plasma concentrations previously associated with analgesia (>100 ng/mL) and anesthesia awakening (750 ng/mL). A total of 113 children (50 intramuscular and 63 intravenous ketamine) with a median age of 3.3 years (range 0.02 to 17.6 years), and median weight of 14 kg (2.4 to 176.1) contributed 275 plasma samples (149 after intramuscular, 126 after intravenous ketamine). A 2‐compartment model with first‐order absorption following intramuscular administration and first‐order elimination described the data best. Allometrically scaled weight was included in the base model for central and peripheral volume of distribution (exponent 1) and for clearance and intercompartmental clearance (exponent 0.75). Model‐estimated bioavailability of intramuscular ketamine was 41%. Dosing simulations suggest that doses of 2 mg/kg intravenously and 8 mg/kg or 6 mg/kg intramuscularly, depending on age, provide adequate sedation (plasma ketamine concentrations >750 ng/mL) for procedures lasting up to 20 minutes.

Abstract Background Influenza A/H5N1 vaccines have been poorly immunogenic. Addition of Adjuvant System 03 (AS03) markedly enhances immune responses, but the mechanisms of this enhancement are unclear. Methods We compared gene expression in peripheral blood mononuclear cells (PBMCs) between recipients of AS03-adjuvanted and unadjuvanted inactivated split-virus H5N1 vaccine on days 1, 3, 7, and 28 postvaccination. We used a systems vaccinology approach to assess functional classifications of differentially expressed (DE) genes between the two vaccine groups, identify DE genes that correlate with serologic responses, and compare these findings with previous cell-specific assessments. Results AS03-adjuvanted vaccine induced the strongest differential gene expression signals on day 1 after vaccination (Figure 1). Multiple innate immune signaling pathways were activated, including the interferon, JAK-STAT, and TNF pathways, and FC gamma receptor (Fc_R) mediated phagocytosis. Immune pathways specific for antigen processing and presentation and influenza A responses were also enriched. Early differential expression of several signal transduction (day 1) and immunoglobulin (day 7) genes were predictive of peak HAI titer (Figure 2). Compared with cell-specific responses, DE gene, and immunologic pathways of PBMCs were most similar to innate immune cell subsets. However, several pathways were unique to PBMCs, and several cell-type-specific pathways, particularly from neutrophils, were absent in PBMCs (Figure 3). Conclusion Transcriptomic analysis of PBMCs after AS03-adjuvanted H5N1 vaccination revealed early differential regulation of multiple innate immune signaling pathways and enrichment of pathways involved in antigen presentation and influenza immune responses. Early expression of several genes was associated with peak HAI responses, suggesting a potential role for application of these signatures in earlier determination of vaccine responses. While PBMC and immune cell-specific results shared key innate immune signals, unique signals were identified by either approach. Disclosures L. Howard, Pfizer: Grant Investigator, Grant recipient. C. B. Creech, Pfizer: Grant Investigator, Research grant. Novartis: Grant Investigator, Research grant. K. Edwards, Novartis: Grant Investigator, Research grant. Novartis: Scientific Advisor, Consulting fee.

BACKGROUND Adjuvant System 03 (AS03) markedly enhances responses to influenza A/H5N1 vaccines, but the mechanisms of this enhancement are incompletely understood. METHODS Using ribonucleic acid sequencing on peripheral blood mononuclear cells (PBMCs) from AS03-adjuvanted and unadjuvanted inactivated H5N1 vaccine recipients, we identified differentially expressed genes, enriched pathways, and genes that correlated with serologic responses. We compared bulk PBMC findings with our previously published assessments of flow-sorted immune cell types. RESULTS AS03-adjuvanted vaccine induced the strongest differential signals on day 1 postvaccination, activating multiple innate immune pathways including interferon and JAK-STAT signaling, Fcγ receptor (FcγR)-mediated phagocytosis, and antigen processing and presentation. Changes in signal transduction and immunoglobulin genes predicted peak hemagglutinin inhibition (HAI) titers. Compared with individual immune cell types, activated PBMC genes and pathways were most similar to innate immune cells. However, several pathways were unique to PBMCs, and several pathways identified in individual cell types were absent in PBMCs. CONCLUSIONS Transcriptomic analysis of PBMCs after AS03-adjuvanted H5N1 vaccination revealed early activation of innate immune signaling, including a 5- to 8-fold upregulation of FcγR1A/1B/1C genes. Several early gene responses were correlated with HAI titer, indicating links with the adaptive immune response. Although PBMCs and cell-specific results shared key innate immune signals, unique signals were identified by both approaches.