Quantifying the impact of pre-vaccination titre and vaccination history on influenza vaccine immunogenicity.

Epidemiological studies suggest that heterogeneity in influenza vaccine antibody response is associated with host factors, including pre-vaccination immune status, age, gender, and vaccination history. However, the pattern of reported associations varies between studies. To better understand the underlying influences on antibody responses, we combined host factors and vaccine-induced in-host antibody kinetics from a cohort study conducted across multiple seasons with a unified analysis framework. We developed a flexible individual-level Bayesian model to estimate associations and interactions between host factors, including pre-vaccine HAI titre, age, sex, vaccination history and study setting, and vaccine-induced HAI titre antibody boosting and waning. We applied the model to derive population-level and individual effects of post-vaccine antibody kinetics for vaccinating and circulating strains for A(H1N1) and A(H3N2) influenza subtypes. We found that post-vaccine HAI titre dynamics were significantly influenced by pre-vaccination HAI titre and vaccination history and that lower pre-vaccination HAI titre results in longer durations of seroprotection (HAI titre equal to 1:40 or higher). Consequently, for A(H1N1), our inference finds that the expected duration of seroprotection post-vaccination was 171 (95% Posterior Predictive Interval[PPI] 128-220) and 159 (95% PPI 120-200) days longer for those who are infrequently vaccinated (<2 vaccines in last five years) compared to those who are frequently vaccinated (2 or more vaccines in the last five years) at pre-vaccination HAI titre values of 1:10 and 1:20 respectively. In addition, we found significant differences in the empirical distributions that describe the individual-level duration of seroprotection for A(H1N1) circulating strains. In future, studies that rely on serological endpoints should include the impact of pre-vaccine HAI titre and prior vaccination status on seropositivity and seroconversion estimates, as these significantly influence an individual's post-vaccination antibody kinetics.

Superior immunogenicity of mRNA over adenoviral vectored COVID-19 vaccines reflects B cell dynamics independent of anti-vector immunity: Implications for future pandemic vaccines.

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. The aim of this study was to validate whether immunogenicity differs for adenoviral vectored (AdV) versus mRNA vaccines against SARS-CoV-2, and to investigate how anti-vector immunity and B cell dynamics modulate immunogenicity. We enrolled SARS-CoV-2 infection-naïve health care workers who had received two doses of either AdV AZD1222 (n = 184) or mRNA BNT162b2 vaccine (n = 274) between April and October 2021. Blood was collected at least once, 10-48 days after vaccine dose 2 for antibody and B cell analyses. Median ages were 42 and 39 years, for AdV and mRNA vaccinees, respectively. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p < 0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine, which reflected greater B cell expansion and targeting of the RBD rather than an attenuating effect of anti-vector antibodies. ClinicalTrials.gov Identifier: NCT05110911.

Comparative B cell and antibody responses induced by adenoviral vectored and mRNA vaccines against COVID-19.

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. However, adenoviral vectored (AdV) vaccines may be less immunogenic than mRNA vaccines against SARS-CoV-2. We assessed anti-spike and anti-vector immunity among infection-naïve Health Care Workers (HCW) following two doses of AdV (AZD1222) versus mRNA (BNT162b2) vaccine. 183 AdV and 274 mRNA vaccinees enrolled between April and October 2021. Median ages were 42 and 39 years, respectively. Blood was collected at least once, 10-48 days after vaccine dose 2. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p<0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human Adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine due to greater B cell expansion and targeting of the RBD. Pre-existing AdV vector cross-reactive antibodies were boosted following AdV vaccination but had no detectable effect on immunogenicity.

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study.

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.

Pathways linked to unresolved inflammation and airway remodelling characterize the transcriptome in two independent severe asthma cohorts.

BACKGROUND AND OBJECTIVE: Severe asthma (SA) is a heterogeneous disease. Transcriptomic analysis contributes to the understanding of pathogenesis necessary for developing new therapies. We sought to identify and validate mechanistic pathways of SA across two independent cohorts. METHODS: Transcriptomic profiles from U-BIOPRED and Australian NOVocastrian Asthma cohorts were examined and grouped into SA, mild/moderate asthma (MMA) and healthy controls (HCs). Differentially expressed genes (DEGs), canonical pathways and gene sets were identified as central to SA mechanisms if they were significant across both cohorts in either endobronchial biopsies or induced sputum. RESULTS: Thirty-six DEGs and four pathways were shared across cohorts linking to tissue remodelling/repair in biopsies of SA patients, including SUMOylation, NRF2 pathway and oxidative stress pathways. MMA presented a similar profile to HCs. Induced sputum demonstrated IL18R1 as a shared DEG in SA compared with healthy subjects. We identified enrichment of gene sets related to corticosteroid treatment; immune-related mechanisms; activation of CD4+ T cells, mast cells and IL18R1; and airway remodelling in SA. CONCLUSION: Our results identified differentially expressed pathways that highlight the role of CD4+ T cells, mast cells and pathways linked to ongoing airway remodelling, such as IL18R1, SUMOylation and NRF2 pathways, as likely active mechanisms in the pathogenesis of SA.

An altered sputum macrophage transcriptome contributes to the neutrophilic asthma endotype.

BACKGROUND: Neutrophilic asthma (NA) is a clinically important asthma phenotype, the cellular and molecular basis of which is not completely understood. Airway macrophages are long-lived immune cells that exert important homeostatic and inflammatory functions which are dysregulated in asthma. Unique transcriptomic programmes reflect varied macrophage phenotypes in vitro. We aimed to determine whether airway macrophages are transcriptomically altered in NA. METHODS: We performed RNASeq analysis on flow cytometry-isolated sputum macrophages comparing NA (n = 7) and non-neutrophilic asthma (NNA, n = 13). qPCR validation of RNASeq results was performed (NA n = 13, NNA n = 23). Pathway analysis (PANTHER, STRING) of differentially expressed genes (DEGs) was performed. Gene set variation analysis (GSVA) was used to test for enrichment of NA macrophage transcriptomic signatures in whole sputum microarray (cohort 1 - controls n = 16, NA n = 29, NNA n = 37; cohort 2 U-BIOPRED - controls n = 16, NA n = 47, NNA n = 57). RESULTS: Flow cytometry-sorting significantly enriched sputum macrophages (99.4% post-sort, 44.9% pre-sort, p < .05). RNASeq analysis confirmed macrophage purity and identified DEGs in NA macrophages. Selected DEGs (SLAMF7, DYSF, GPR183, CSF3, PI3, CCR7, all p < .05 NA vs. NNA) were confirmed by qPCR. Pathway analysis of NA macrophage DEGs was consistent with responses to bacteria, contribution to neutrophil recruitment and increased expression of phagocytosis and efferocytosis factors. GSVA demonstrated neutrophilic macrophage gene signatures were significantly enriched in whole sputum microarray in NA vs. NNA and controls in both cohorts. CONCLUSIONS: We demonstrate a pathophysiologically relevant sputum macrophage transcriptomic programme in NA. The finding that there is transcriptional activation of inflammatory programmes in cell types other than neutrophils supports the concept of NA as a specific endotype.

Transcriptomics of biopsies identifies novel genes and pathways linked to neutrophilic inflammation in severe asthma.

BACKGROUND: Severe asthma is a complex disease. Transcriptomic profiling has contributed to understanding the pathogenesis of asthma, especially type-2 inflammation. However, there is still poor understanding of non-type-2 asthma, and consequently, there are limited treatment options. OBJECTIVE: The aim of this study was to identify differentially expressed genes (DEGs) and pathways in endobronchial biopsies associated with inflammatory phenotypes of severe asthma. METHODS: This cross-sectional study examined endobronchial biopsies from 47 adults with severe asthma (neutrophilic asthma (NA) n = 9, eosinophilic asthma (EA) n = 22 and paucigranulocytic asthma (PGA) n = 16) and 13 healthy controls (HC). RNA was extracted and transcriptomic profiles generated (Illumina Humanref-12 V4) and analysed using GeneSpring GX14.9.1. Pathway identification using Ingenuity Pathway Analysis. RESULTS: NA had the most distinct profile, with signature of 60 top-ranked DEGs (FC >±2) including genes associated with innate immunity response, neutrophil degranulation and IL-10 signalling. NA presented enrichment to pathways previously linked to neutrophilic inflammation; dendritic cell maturation, Th1, TREM1, inflammasome, Th17 and p38 MAPK, as well as novel links to neuroinflammation, NFAT and PKCθ signalling. EA presented similar transcriptomic profiles to PGA and HC. Despite the higher proportion of bacterial colonization in NA, no changes were observed in the transcriptomic profiles of severe asthma culture positive compared with severe asthma culture negative. CONCLUSIONS & CLINICAL RELEVANCE: NA features a distinct transcriptomic profile with seven pathways enriched in NA compared to EA, PGA and HC. All those with severe asthma had significant enrichment for SUMOylation, basal cell carcinoma signalling and Wnt/ß-catenin pathways compared to HC, despite high-dose inhaled corticosteroids. These findings contribute to the understanding of mechanistic pathways in endobronchial biopsies associated with NA and identify potential novel treatment targets for severe asthma.

Six gene and TH2 signature expression in endobronchial biopsies of participants with asthma.

BACKGROUND: Both the six gene signature (6GS: CPA3, DNASE1L3, CLC, IL1B, ALPL, and CXCR2) and T-helper 2 signature (TH2S: CLCA1, SERPINB2, and POSTN) are proposed as biomarkers in the identification of inflammatory phenotypes of asthma in induced sputum and epithelial brushings, respectively. The aim of this study was to explore patterns of gene expression of known signatures, 6GS and TH2S in endobronchial biopsies. METHODS: This was an exploratory cross-sectional study of gene expression in endobronchial biopsies of 55 adults with asthma and 9 healthy controls (HC). The expression of the 6GS and TH2S was determined by quantitative polymerase chain reaction. Correlations with clinical and cellular characteristics were performed, and receiver operating characteristic was utilized to assess signatures' ability to predict asthma from HC and inflammatory phenotypes. RESULTS: Gene expression of DNASE1L3 (P = .045) was upregulated in asthma compared with HC, and IL1B (P = .017) was upregulated in neutrophilic asthma compared with non-neutrophilic asthma. In asthma, the expression of CPA3 was negatively associated with ICS daily dose (r = -.339; P = .011), IL1B expression was positively associated with bronchial lavage fluid (BLF) total cell count (r = .340; P = .013) and both CLC and POSTN expression were associated with lymphocytes percentage in BLF (r = -.355, P = .009; r = -.300, P = .025, respectively). Both 6GS (area under curve [AUC] = 86.3%; P = .017) and TH2S (AUC = 72.7%; P = .037) could significantly predict asthma from HC. In addition, 6GS can identify neutrophilic (AUC = 93.2%; P = .005) and TH2S identifies eosinophilic (AUC = 62.7%; P = .033) asthma. CONCLUSIONS AND CLINICAL RELEVANCE: There was increased expression of DNASE1L3 in asthma and IL1B in neutrophilic asthma. These results show similar upregulated patterns of expression in two genes of the 6GS in endobronchial biopsies, previously identified in sputum. The upregulation of DNASE1L3 and IL1B suggests that common mechanisms may be at play throughout the airway.