106 research outputs found
The formulation of artificial reference standards for use within the ELISPOT assay
No full textWhether to assess the functionality of equipment or as a determinate for the accuracy of assays, reference standards are essential for the purposes of standardisation and validation. The ELISPOT assay, developed over thirty years ago, has emerged as a leading immunological assay in the development of novel vaccines for the assessment of efficacy. However, with its widespread use, there is a growing demand for a greater level of standardisation across different laboratories. One of the major difficulties in achieving this goal has been the lack of definitive reference standards. This is partly due to the ex vivo nature of the assay, which relies on cells being placed directly into the wells. Thus, the aim of this thesis was to produce an artificial reference standard using liposomes, for use within the assay. Liposomes are spherical bilayer vesicles with an enclosed aqueous compartment and therefore are models for biological membranes. Initial work examined pre-design considerations in order to produce an optimal formulation that would closely mimic the action of the cells ordinarily placed on the assay. Recognition of the structural differences between liposomes and cells led to the formulation of liposomes with increased density. This was achieved by using a synthesised cholesterol analogue. By incorporating this cholesterol analogue in liposomes, increased sedimentation rates were observed within the first few hours. The optimal liposome formulation from these studies was composed of 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Chol) and brominated cholesterol (Brchol) at a 16:4:12 µMol ratio, based on a significantly higher (p<0.01) sedimentation (as determined by a percentage transmission of 59 ± 5.9 % compared to the control formulation at 29 ± 12 % after four hours). By considering a range of liposome formulations ‘proof of principle’ for using liposomes as ELISPOT reference standards was shown; recombinant IFN? cytokine was successfully entrapped within vesicles of different lipid compositions, which were able to promote spot formation within the ELISPOT assay. Using optimised liposome formulations composed of phosphatidylcholine with or without cholesterol (16 µMol total lipid) further development was undertaken to produce an optimised, scalable protocol for the production of liposomes as reference standards. A linear increase in spot number by the manipulation of cytokine concentration and/or lipid concentrations was not possible, potentially due to the saturation that occurred within the base of wells. Investigations into storage of the formulations demonstrated the feasibility of freezing and lyophilisation with disaccharide cryoprotectants, but also highlighted the need for further protocol optimisation to achieve a robust reference standard upon storage. Finally, the transfer of small-scale production to a medium lab-scale batch (40 mL) demonstrated this was feasible within the laboratory using the optimised protocol
Humoral immune responses to ChAdOx1 nCoV-19 in children
No full textCOVID-19 vaccination of children and young people is recommended in some countries. Data on immune responses induced by COVID-19 vaccines in children have been reported less widely than for adults.
COV006 was a phase II, single-blind, randomised controlled trial of ChAdOx1 nCoV-19 in healthy participants 6-17 years of age. Children were assigned to four groups to receive two intramuscular doses of 5x1010 viral particles of ChAdOx1 nCoV-19 or control, 28 or 84 days apart. Study groups were stratified by age (6-11 years and 12-17 years). Due to restrictions in the use of ChAdOx1 nCoV-19 in people under the age of 30, introduced during the study, only participants 12-17 years of age who were randomised to the 28-day interval received their vaccines at the intended interval. The remaining participants received their vaccines at a 112-day interval. The primary outcome was safety, and the secondary outcome was immunogenicity. Exploratory immunology studies were also conducted.
Between February and April 2021, 262 participants were randomised into the study. No serious adverse events related to ChAdOx1 nCoV-19 were recorded. 80% of participants (169 of 210 ChAdOx1 nCoV-19 recipients) reported at least one solicited local or systemic side effect in the first seven days after vaccination. Pain and tenderness were the most common local side effects. Among seronegative participants, anti-SARS-CoV-2 IgG titres were higher in participants 12-17 years of age who received their vaccinations at a 112-day interval compared with 28-day interval, measured at 28 days after the second dose (geometric means 1963 EU/ml, [95% CI 1575-2448] versus 1194 EU/ml [95% CI 908-1568])) and highest in younger participants 6-11 years of age who received their vaccine at a 112-day interval (geometric mean 2310 EU/ml [95% CI 1700-3138]). Significant boosting of betacoronavirus titres was measured after the first dose of ChAdOx1 nCoV-19 in children but not adults over 18 years of age (ANOVA, p<0.05). Children 6-11 years of age generated higher titres of SARS-CoV-2 spike-specific IgG1 than older children and adults (ANOVA, p=0.02). SARS-CoV-2 spike-specific FcgR 2a binding correlated most closely with psuedoneutralising antibody titres (Spearman, r =0.89)
T-cell mediated immunity after AZD1222 vaccination: A polyfunctional spike-specific Th1 response with a diverse TCR repertoire
No full textAZD1222 (ChAdOx1 nCoV-19), a replication-deficient simian adenovirus-vectored vaccine, has demonstrated safety, efficacy, and immunogenicity against coronavirus disease 2019 (COVID-19) in clinical trials and real-world studies. We characterized CD4+ and CD8+ T-cell responses induced by AZD1222 vaccination in peripheral blood mononuclear cells (PBMCs) from 280 unique vaccine recipients aged 18-85 years who enrolled in the phase 2/3 COV002 trial. Total spike-specific CD4+ T cell helper type 1 (Th1) and CD8+ T-cell responses were significantly increased in AZD1222-vaccinated adults of all ages following two doses of AZD1222. CD4+ Th2 responses following AZD1222 vaccination were not detected. Furthermore, AZD1222-specific Th1 and CD8+ T cells both displayed a high degree of polyfunctionality in all adult age groups. T-cell receptor (TCR) β sequences from vaccinated participants mapped against TCR sequences known to react to SARS-CoV-2 revealed substantial breadth and depth across the SARS-CoV-2 spike protein for the AZD1222-induced CD4+ and CD8+ T-cell responses. Overall, AZD1222 vaccination induced a robust, polyfunctional Th1-dominated T-cell response, with broad CD4+ and CD8+ T-cell coverage across the SARS-CoV-2 spike protein.One Sentence Summary: Polyfunctional CD4+ and CD8+ T-cell responses are elicited against the SARS-CoV-2 spike protein following vaccination with AZD1222.</p
Homologous & heterologous COVID-19 vaccine priming schedules: an analysis of systemic & mucosal immunity
No full textMost two dose vaccination schedules, including UK-licensed SARS-CoV2 vaccinations, are licensed as homologous schedules. At the point of UK emergency licensure of the first two SARS-CoV2 vaccines, global demand for vaccine outstripped supply. National immunisation programmes faced logistical challenges to ensure sufficient doses of the same vaccine could be distributed to each person in the manufacturer-recommended timeframe.
Two randomised control trials (Com-COV1 and Com-COV2) assessed safety, tolerability and non-inferiority of humoral immunogenicity of heterologous priming schedules using vaccines available to the UK immunisation program – AstraZeneca, Pfizer, Moderna and Novavax.
Heterologous schedules are more reactogenic than their equivalent homologous schedules, however, all schedules are tolerable and without safety concern.
Not all heterologous schedules are non-inferior to their relevant homologous schedule in terms of serum anti-SARS-CoV2 spike binding IgG, but they do always achieve a robust immunological response at least as large as the least immunogenic schedule studied (homologous AstraZeneca), which has proven efficacy against both severe disease and symptomatic infection.
Differences between vaccine platforms are also qualitative with clear differences in the capability of each schedule to produce neutralising responses. T-cell responses differ markedly with the greatest responses seen in those receiving heterologous schedules with AstraZeneca as the first dose.
Increasing priming interval from one to three months resulted in a modest increase in antibody response and a decrease in cellular response.
Mucosal responses were evaluated as an exploratory endpoint. There was no evidence that mucosal anti-SARS-CoV2 spike IgA was induced by intramuscular vaccination. There is evidence that nasal mucosal responses can occur in the absence of systemic responses, possibly due to asymptomatic mucosal SARS-CoV2 exposure. Existing mucosal IgA responses against seasonal coronaviruses may confer some degree of protection against SARS-CoV2 infection.
These results support the use of heterologous schedules as viable alternatives to homologous schedules and support flexibility in priming interval. They have informed UK and global immunisation policy. There is a need for alternative vaccination strategies, such as mucosally delivered vaccines, which induce mucosal responses that may reduce transmission
Safety and humoral immunogenicity of the ChAdOx1 nCoV-19 vaccine administered as a fourth dose booster following two doses of ChAdOx1 nCoV-19 and a third dose of BNT162b2 (COV009) : a prospective cohort study
No full textObjectives: evaluation of the safety and humoral immunogenicity of ChAdOx1 nCoV-19 as a fourth dose booster in individuals who have had two initial doses of the vaccine and a third dose of BNT162b2. Methods: COV009 is a safety follow-up study of volunteers enroled in the pivotal pre-licensure ChAdOx1 nCoV-19. In this sub-study, 149 eligible participants were given a fourth dose of ChAdOx1 nCoV-19. Primary outcomes were reactogenicity, safety, and humoral immunogenicity. Anti-spike IgG and pseudo-neutralising antibody against multiple variants were measured from pre-first dose to 28 days post-second and post-fourth dose (third dose samples were unavailable). Results: a fourth dose of ChAdOx1 nCoV-19 had an acceptable safety profile with no vaccine-related serious adverse events. Humoral responses against various SARS CoV-2 variants post-fourth dose were significantly increased compared with the responses after the second dose (7- to 9-fold increase for anti-spike IgG responses across variants, all p<0.05). Those with lower antibody levels prior to the 4th dose had stronger responses to a 4th dose booster. Seropositivity by anti-nucleocapsid, or higher antibody responses pre-fourth dose correlated with lower infection risks six months thereafter (OR: 0.16, 95% CI: 0.05, 0.50).Conclusions: the ChAdOx1 nCoV-19 fourth dose is well tolerated and boosts humoral immunity; this was evident as an increased humoral response across multiple variants of concern. These data support its use as a booster dose against SARS-CoV-2 infection.</p
Safety and immunogenicity of the ChAdOx1 nCoV-19 (AZD1222) vaccine in children aged 6-17 years: a preliminary report of COV006, a phase 2 single-blind, randomised, controlled trial.
No full textBACKGROUND: Vaccination of children and young people against SARS-CoV-2 is recommended in some countries. Scarce data have been published on immune responses induced by COVID-19 vaccines in people younger than 18 years compared with the same data that are available in adults. METHODS: COV006 is a phase 2, single-blind, randomised, controlled trial of ChAdOx1 nCoV-19 (AZD1222) in children and adolescents at four trial sites in the UK. Healthy participants aged 6-17 years, who did not have a history of chronic respiratory conditions, laboratory-confirmed COVID-19, or previously received capsular group B meningococcal vaccine (the control), were randomly assigned to four groups (4:1:4:1) to receive two intramuscular doses of 5 × 1010 viral particles of ChAdOx1 nCoV-19 or control, 28 days or 84 days apart. Participants, clinical investigators, and the laboratory team were masked to treatment allocation. Study groups were stratified by age, and participants aged 12-17 years were enrolled before those aged 6-11 years. Due to the restrictions in the use of ChAdOx1 nCoV-19 in people younger than 30 years that were introduced during the study, only participants aged 12-17 years who were randomly assigned to the 28-day interval group had received their vaccinations at the intended interval (day 28). The remaining participants received their second dose at day 112. The primary outcome was assessment of safety and tolerability in the safety population, which included all participants who received at least one dose of the study drug. The secondary outcome was immunogenicity, which was assessed in participants who were seronegative to the nucleocapsid protein at baseline and received both prime and boost vaccine. This study is registered with ISRCTN (15638344). FINDINGS: Between Feb 15 and April 2, 2021, 262 participants (150 [57%] participants aged 12-17 years and 112 [43%] aged 6-11 years; due to the change in the UK vaccination policy, the study terminated recruitment of the younger age group before the planned number of participants had been enrolled) were randomly assigned to receive vaccination with two doses of either ChAdOx1 nCoV-19 (n=211 [n=105 at day 28 and n=106 at day 84]) or control (n=51 [n=26 at day 28 and n=25 at day 84]). One participant in the ChAdOx1 nCoV-19 day 28 group in the younger age bracket withdrew their consent before receiving a first dose. Of the participants who received ChAdOx1 nCoV-19, 169 (80%) of 210 participants reported at least one solicited local or systemic adverse event up to 7 days following the first dose, and 146 (76%) of 193 participants following the second dose. No serious adverse events related to ChAdOx1 nCoV-19 administration were recorded by the data cutoff date on Oct 28, 2021. Of the participants who received at least one dose of ChAdOx1 nCoV-19, there were 128 unsolicited adverse events up to 28 days after vaccination reported by 83 (40%) of 210 participants. One participant aged 6-11 years receiving ChAdOx1 nCoV-19 reported a grade 4 fever of 40·2°C on day 1 following first vaccination, which resolved within 24 h. Pain and tenderness were the most common local solicited adverse events for all the ChAdOx1 nCoV-19 and capsular group B meningococcal groups following both doses. Of the 242 participants with available serostatus data, 14 (6%) were seropositive at baseline. Serostatus data were not available for 20 (8%) of 262 participants. Among seronegative participants who received ChAdOx1 nCoV-19, anti-SARS-CoV-2 IgG and pseudoneutralising antibody titres at day 28 after the second dose were higher in participants aged 12-17 years with a longer interval between doses (geometric means of 73 371 arbitrary units [AU]/mL [95% CI 58 685-91 733] and 299 half-maximal inhibitory concentration [IC50; 95% CI 230-390]) compared with those aged 12-17 years who received their vaccines 28 days apart (43 280 AU/mL [95% CI 35 852-52 246] and 150 IC50 [95% CI 116-194]). Humoral responses were higher in those aged 6-11 years than in those aged 12-17 years receiving their second dose at the same 112-day interval (geometric mean ratios 1·48 [95% CI 1·07-2·07] for anti-SARS-CoV-2 IgG and 2·96 [1·89-4·62] for pseudoneutralising antibody titres). Cellular responses peaked after a first dose of ChAdOx1 nCoV-19 across all age and interval groups and remained above baseline after a second vaccination. INTERPRETATION: ChAdOx1 nCoV-19 is well tolerated and immunogenic in children aged 6-17 years, inducing concentrations of antibody that are similar to those associated with high efficacy in phase 3 studies in adults. No safety concerns were raised in this trial. FUNDING: AstraZeneca and the UK Department of Health and Social Care through the UK National Institute for Health and Care Research
Variation in hospital admission in febrile children evaluated at the Emergency Department (ED) in Europe: PERFORM, a multicentre prospective observational study
No full textObjectives Hospitalisation is frequently used as a marker of disease severity in observational Emergency Department (ED) studies. The comparison of ED admission rates is complex in potentially being influenced by the characteristics of the region, ED, physician and patient. We aimed to study variation in ED admission rates of febrile children, to assess whether variation could be explained by disease severity and to identify patient groups with large variation, in order to use this to reduce unnecessary health care utilization that is often due to practice variation. Design MOFICHE (Management and Outcome of Fever in children in Europe, part of the PERFORM study, www.perform2020.org), is a prospective cohort study using routinely collected data on febrile children regarding patient characteristics (age, referral, vital signs and clinical alarming signs), diagnostic tests, therapy, diagnosis and hospital admission. Setting and participants Data were collected on febrile children aged 0–18 years presenting to 12 European EDs (2017–2018). Main outcome measures We compared admission rates between EDs by using standardised admission rates after adjusting for patient characteristics and initiated tests at the ED, where standardised rates >1 demonstrate higher admission rates than expected and rates <1 indicate lower rates than expected based on the ED patient population. Results We included 38,120 children. Of those, 9.695 (25.4%) were admitted to a general ward (range EDs 5.1–54.5%). Adjusted standardised admission rates ranged between 0.6 and 1.5. The largest variation was seen in short admission rates (0.1–5.0), PICU admission rates (0.2–2.2), upper respiratory tract infections (0.4–1.7) and fever without focus (0.5–2.7). Variation was small in sepsis/meningitis (0.9–1.1). Conclusions Large variation exists in admission rates of febrile children evaluated at European EDs, however, this variation is largely reduced after correcting for patient characteristics and therefore overall admission rates seem to adequately reflect disease severity or a potential for a severe disease course. However, for certain patient groups variation remains high even after adjusting for patient characteristics
Persistence of the immune response after two doses of ChAdOx1 nCov-19 (AZD1222): 1 year of follow up of two randomised controlled trials
No full textThe trajectory of immune responses following the primary dose series determines the decline in vaccine effectiveness over time. Here we report on maintenance of immune responses during the year following a two-dose schedule of ChAdOx1 nCoV-19/AZD1222, in the absence of infection, and also explore the decay of antibody after infection. Total spike-specific IgG antibody titres were lower with two low doses of ChAdOx1 nCoV-19 vaccines (two low doses) (P = 0.0006) than with 2 standard doses (the approved dose) or low dose followed by standard dose vaccines regimens. Longer intervals between first and second doses resulted in higher antibody titres (P < 0.0001); however, there was no evidence that the trajectory of antibody decay differed by interval or by vaccine dose, and the decay of IgG antibody titres followed a similar trajectory after a third dose of ChAdOx1 nCoV-19. Trends in post-infection samples were similar with an initial rapid decay in responses but good persistence of measurable responses thereafter. Extrapolation of antibody data, following two doses of ChAdOx1 nCov-19, demonstrates a slow rate of antibody decay with modelling, suggesting that antibody titres are well maintained for at least 2 years. These data suggest a persistent immune response after two doses of ChAdOx1 nCov-19 which will likely have a positive impact against serious disease and hospitalization
Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial.
No full textBACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. METHODS: We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18-55 years with no history of laboratory confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 × 1010 viral particles or MenACWY as a single intramuscular injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque reduction neutralisation assay [PRNT50]; a microneutralisation assay [MNA50, MNA80, and MNA90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. FINDINGS: Between April 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p<0·05). There were no serious adverse events related to ChAdOx1 nCoV-19. In the ChAdOx1 nCoV-19 group, spike-specific T-cell responses peaked on day 14 (median 856 spot-forming cells per million peripheral blood mononuclear cells, IQR 493-1802; n=43). Anti-spike IgG responses rose by day 28 (median 157 ELISA units [EU], 96-317; n=127), and were boosted following a second dose (639 EU, 360-792; n=10). Neutralising antibody responses against SARS-CoV-2 were detected in 32 (91%) of 35 participants after a single dose when measured in MNA80 and in 35 (100%) participants when measured in PRNT50. After a booster dose, all participants had neutralising activity (nine of nine in MNA80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R2=0·67 by Marburg VN; p<0·001). INTERPRETATION: ChAdOx1 nCoV-19 showed an acceptable safety profile, and homologous boosting increased antibody responses. These results, together with the induction of both humoral and cellular immune responses, support large-scale evaluation of this candidate vaccine in an ongoing phase 3 programme. FUNDING: UK Research and Innovation, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research (NIHR), NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and the German Center for Infection Research (DZIF), Partner site Gießen-Marburg-Langen
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