Humoral and cellular immune responses after pandemic and seasonal influenza vaccination in humans

Vaccination is the most effective prophylaxis against epidemic and pandemic influenza. Annual seasonal influenza vaccination is recommended for high-risk groups such as younger children <5 years old and occupational workers such as frontline healthcare workers (HCW). In 2009, a novel A/H1N1 influenza virus emerged causing the first pandemic of the 21st century. The AS03-adjuvanted inactivated monovalent A/H1N1 pandemic vaccine was rapidly deployed prior to the peak of pandemic in Bergen. HCW were prioritized for the first round of the vaccination, during the autum of 2009 to maintain the integrity of the healthcare system. The A(H1N1)pdm09 strain was subsequently included in seasonal trivalent inactivated influenza vaccines (TIVs) from 2010/2011 until 2016/2017. The trivalent Live Attenuated Influenza Vaccine (LAIV) was licensed for seasonal use in Europe in 2012 and it is recommended for children 2-17 years old. We conducted two vaccine clinical trials using licensed influenza vaccines. In the first clinical trial we evaluated both the immediate and durable humoral and cellular immune responses in HCW vaccinated with the AS03-adjuvanted pandemic vaccine and subsequent annual seasonal TIVs. In the second clinical trial we investigated in depth the follicular helper T (TFH) cell and antibody responses elicited by LAIV in children and adults. We reported that the pandemic vaccine induced rapid homologous and cross-reactive T cell, B cell and antibody responses against the A(H1N1)pdm09 strain and pre-2009 seasonal influenza A/H1N1 strains. We observed that the baseline A(H1N1)pdm09-specific immune responses significantly increased from 2009 to 2013 and were maintained at high levels after 3–4 repeated vaccinations. Collectively our data from the HCW study provide the immunological evidence for continuing annual influenza vaccination policy in adults. Furthermore, we demonstrated that LAIV induced significant increase in influenza specific systemic and local antibody responses against the three vaccine strains tested as early as day 14 post-vaccination. We also showed that LAIV elicited potent and rapid influenza specific TFH cell responses in children. Our LAIV results provide valuable insights into the immunogenicity of LAIV in different age groups with variable levels of pre-existing immunity.Doktorgradsavhandlin

Single dose vaccination of the ASO3-adjuvanted A (H1N1)pdm09 monovalent vaccine in health care workers elicits homologous and cross-reactive cellular and humoral responses to H1N1 strains

Healthcare workers (HCW) were prioritized for vaccination during the 2009 influenza A(H1N1)pdm09 pandemic. We conducted a clinical trial in October 2009 where 237 HCWs were immunized with a AS03-adjuvanted A(H1N1)pdm09 monovalent vaccine. In the current study, we analyzed the homologous and cross-reactive H1N1 humoral responses using prototype vaccine strains dating back to 1977 by the haemagglutinin inhibition (HI), single radial hemolysis SRH), antibody secreting cell (ASC) and memory B cell (MBC) assays. The cellular responses were assessed by interferon-γ (IFN-γ) ELISPOT and by intracellular staining (ICS) for the Th1 cytokines IFN-γ, interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). All assays were performed using blood samples obtained prior to (day 0) and 7, 14 and 21 d post-pandemic vaccination, except for ASC (day 7) and ICS (days 0 and 21). Vaccination elicited rapid HI, SRH and ASC responses against A(H1N1)pdm09 which cross reacted with seasonal H1N1 strains. MBC responses were detected against the homologous and seasonal H1N1 strains before vaccination and were boosted 2 weeks post-vaccination. An increase in cellular responses as determined by IFN-γ ELISPOT and ICS were observed 1–3 weeks after vaccination. Collectively, our data show that the AS03-adjuvanted A(H1N1)pdm09 vaccine induced rapid cellular and humoral responses against the vaccine strain and the response cross-reacted against prototype H1N1 strains dating back to 1977

Assay harmonization and use of biological standards to improve the reproducibility of the hemagglutination inhibition assay: A FLUCOP collaborative study

The hemagglutination inhibition (HAI) assay is an established technique for assessing influenza immunity, through measurement of antihemagglutinin antibodies. Improved reproducibility of this assay is required to provide meaningful data across different testing laboratories. This study assessed the impact of harmonizing the HAI assay protocol/reagents and using standards on interlaboratory variability. Human pre- and postvaccination sera from individuals (n = 30) vaccinated against influenza were tested across six laboratories. We used a design of experiment (DOE) method to evaluate the impact of assay parameters on interlaboratory HAI assay variability. Statistical and mathematical approaches were used for data analysis. We developed a consensus protocol and assessed its performance against in-house HAI testing. We additionally tested the performance of several potential biological standards. In-house testing with four reassortant viruses showed considerable interlaboratory variation (geometric coefficient of variation [GCV] range of 50% to 117%). The age, concentration of turkey red blood cells, incubation duration, and temperature were key assay parameters affecting variability. Use of a consensus protocol with common reagents, including viruses, significantly reduced GCV between laboratories to 22% to 54%. Pooled postvaccination human sera from different vaccination campaigns were effective as biological standards. Our results demonstrate that the harmonization of protocols and critical reagents is effective in reducing interlaboratory variability in HAI assay results and that pools of postvaccination human sera have potential as biological standards that can be used over multiple vaccination campaigns. Moreover, the use of standards together with in-house protocols is as potent as the use of common protocols and reagents in reducing interlaboratory variability.publishedVersio

Attack rates amongst household members of outpatients with confirmed COVID-19 in Bergen, Norway: A case-ascertained study

Background Households studies reflect the natural spread of SARS-CoV-2 in immunologically naive populations with limited preventive measures to control transmission. We hypothesise that seropositivity provides more accurate household attack rates than RT-PCR. Here, we investigated the importance of age in household transmission dynamics. Methods We enroled 112 households (291 participants) in a case-ascertained study in Bergen, Norway from 28th February to 4th April 2020, collecting demographic and clinical data from index patients and household members. SARS-CoV-2-specific antibodies were measured in sera collected 6–8 weeks after index patient nasopharyngeal testing to define household attack rates. Findings The overall attack rate was 45% (95% CI 38–53) assessed by serology, and 47% when also including seronegative RT-PCR positives. Serology identified a higher number of infected household members than RT-PCR. Attack rates were equally high in children (48%) and young adults (42%). The attack rate was 16% in asymptomatic household members and 42% in RT-PCR negative contacts. Older adults had higher antibody titres than younger adults. The risk of household transmission was higher when the index patient had fever (aOR 3.31 [95% CI 1.52–7.24]; p = 0.003) or dyspnoea (aOR 2.25 [95% CI 1.80–4.62]; p = 0.027) during acute illness. Interpretation Serological assays provide more sensitive and robust estimates of household attack rates than RT-PCR. Children are equally susceptible to infection as young adults. Negative RT-PCR or lack of symptoms are not sufficient to rule out infection in household members.publishedVersio

Differential expression of PPP1R12A transcripts, including those harbouring alternatively spliced micro-exons, in placentae from complicated pregnancies

Introduction Placenta-associated pregnancy complications, including pre-eclampsia (PE) and intrauterine growth restriction (IUGR) are conditions postulated to originate from initial failure of placentation, leading to clinical sequelae indicative of endothelial dysfunction. Vascular smooth muscle aberrations have also been implicated in the pathogenesis of both disorders via smooth muscle contractility and relaxation mediated by Myosin Light Chain Phosphatase (MLCP) and the oppositional contractile action of Myosin Light Chain Kinase. PPP1R12A is a constituent part of the MLCP complex responsible for dephosphorylation of myosin fibrils. We hypothesize that alternative splicing of micro-exons result in isoforms lacking the functional leucine zipper (LZ) domain which may give those cells expressing these alternative transcripts a tendency towards contraction and vasoconstriction. Methods Expression was determined by qRT-PCR. Epigenetic profiling consisted of bisulphite-based DNA methylation analysis and ChIP for underlying histone modifications. Results We identified several novel transcripts with alternative micro-exon inclusion that would produce LZ- PPP1R12A protein. qRT-PCR revealed some isoforms, including the PPP1R12A canonical transcript, are differentially expressed in placenta biopsies from PE and IUGR samples compared to uncomplicated pregnancies. Discussion We propose that upregulation of PPP1R12A expression in complicated pregnancies may be due to enhanced promoter activity leading to increased transcription as a response to physiological stress in the placenta, which we show is independent of promoter DNA methylation

Reduced cognitive deficits after FLASH irradiation of whole mouse brain are associated with less hippocampal dendritic spine loss and neuroinflammation

Aim To evaluate the impact of ultra-rapid FLASH mouse whole brain irradiation on hippocampal dendritic spines and neuroinflammation, factors associated with cognitive impairment after brain irradiation. Methods We administered 30 Gy whole brain irradiation to C57BL6/J mice in sub-second (FLASH) vs. 240 s conventional delivery time keeping all other parameters constant, using a custom configured clinical linac. Ten weeks post-irradiation, we evaluated spatial and non-spatial object recognition using novel object location and object recognition testing. We measured dendritic spine density by tracing Golgi-stained hippocampal neurons and evaluated neuroinflammation by CD68 immunostaining, a marker of activated microglia, and expression of 10 pro-inflammatory cytokines using a multiplex immunoassay. Results At ten weeks post-irradiation, compared to unirradiated controls, conventional delivery time irradiation significantly impaired novel object location and recognition tasks whereas the same dose given in FLASH delivery did not. Conventional delivery time, but not FLASH, was associated with significant loss of dendritic spine density in hippocampal apical dendrites, with a similar non-significant trend in basal dendrites. Conventional delivery time was associated with significantly increased CD68-positive microglia compared to controls whereas FLASH was not. Conventional delivery time was associated with significant increases in 5 of 10 pro-inflammatory cytokines in the hippocampus (and non-significant increases in another 3), whereas FLASH was associated with smaller increases in only 3. Conclusion Reduced cognitive impairment and associated neurodegeneration were observed with FLASH compared to conventional delivery time irradiation, potentially through decreased induction of neuroinflammation, suggesting a promising approach to increasing therapeutic index in radiation therapy of brain tumors

Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.publishedVersio

Live attenuated influenza vaccine in children induces b-cell responses in tonsils

Background. Tonsils play a key role in eliciting immune responses against respiratory pathogens. Little is known about how tonsils contribute to the local immune response after intranasal vaccination. Here, we uniquely report the mucosal humoral responses in tonsils and saliva after intranasal live attenuated influenza vaccine (LAIV) vaccination in children. Methods. Blood, saliva, and tonsils samples were collected from 39 children before and after LAIV vaccination and from 16 agematched, nonvaccinated controls. Serum antibody responses were determined by a hemagglutination inhibition (HI) assay. The salivary immunoglobulin A (IgA) level was measured by an enzyme-linked immunosorbent assay. Antibody-secreting cell (ASC) and memory B-cell (MBC) responses were enumerated in tonsils and blood. Results. Significant increases were observed in levels of serum antibodies and salivary IgA to influenza A(H3N2) and influenza B virus strains as early as 14 days after vaccination but not to influenza A(H1N1). Influenza virus-specific salivary IgA levels correlated with serum HI responses, making this a new possible indicator of vaccine immunogenicity in children. LAIV augmented influenza virus-specific B-cell responses in tonsils and blood. Tonsillar MBC responses correlated with systemic MBC and serological responses. Naive children showed significant increases in MBC counts after LAIV vaccination. Conclusions. This is the first study to demonstrate that LAIV elicits humoral B-cell responses in tonsils of young children. Furthermore, salivary IgA analysis represents an easy method for measuring immunogenicity after vaccination

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.publishedVersio

A rapid antibody screening haemagglutination test for predicting immunity to SARS-CoV-2 variants of concern

Background: Evaluation of susceptibility to emerging SARS-CoV-2 variants of concern (VOC) requires rapid screening tests for neutralising antibodies which provide protection. Methods: Firstly, we developed a receptor-binding domain-specific haemagglutination test (HAT) to Wuhan and VOC (alpha, beta, gamma and delta) and compared to pseudotype, microneutralisation and virus neutralisation assays in 835 convalescent sera. Secondly, we investigated the antibody response using the HAT after two doses of mRNA (BNT162b2) vaccination. Sera were collected at baseline, three weeks after the first and second vaccinations from older (80–99 years, n = 89) and younger adults (23–77 years, n = 310) and compared to convalescent sera from naturally infected individuals (1–89 years, n = 307). Results: Here we show that HAT antibodies highly correlated with neutralising antibodies (R = 0.72–0.88) in convalescent sera. Home-dwelling older individuals have significantly lower antibodies to the Wuhan strain after one and two doses of BNT162b2 vaccine than younger adult vaccinees and naturally infected individuals. Moverover, a second vaccine dose boosts and broadens the antibody repertoire to VOC in naïve, not previously infected older and younger adults. Most (72–76%) older adults respond after two vaccinations to alpha and delta, but only 58–62% to beta and gamma, compared to 96–97% of younger vaccinees and 68–76% of infected individuals. Previously infected older individuals have, similarly to younger adults, high antibody titres after one vaccination. Conclusions: Overall, HAT provides a surrogate marker for neutralising antibodies, which can be used as a simple inexpensive, rapid test. HAT can be rapidly adaptable to emerging VOC for large-scale evaluation of potentially decreasing vaccine effectiveness.publishedVersio