Driving consistency: CEPI-Centralized Laboratory Network's conversion factor initiative for SARS-CoV-2 clinical assays used for efficacy assessment of COVID vaccines.

To date, thousands of SARS-CoV-2 samples from many vaccine developers have been tested within the CEPI-Centralized Laboratory Network. To convert data from each clinical assay to international standard units, the WHO international standard and the CEPI standard generated by the Medicines and Healthcare products Regulatory Agency were run in multiple facilities to determine the conversion factor for each assay. Reporting results in international units advances global understanding of SARS-CoV-2 immunity and vaccine efficacy, enhancing the quality, reliability, and utility of clinical assay data.

Standardised quantitative assays for anti-SARS-CoV-2 immune response used in vaccine clinical trials by the CEPI Centralized Laboratory Network: a qualification analysis.

BACKGROUND: Accurate quantitation of immune markers is crucial for ensuring reliable assessment of vaccine efficacy against infectious diseases. This study was designed to confirm standardised performance of SARS-CoV-2 assays used to evaluate COVID-19 vaccine candidates at the initial seven laboratories (in North America, Europe, and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN). METHODS: Three ELISAs (pre-spike protein, receptor binding domain, and nucleocapsid), a microneutralisation assay (MNA), a pseudotyped virus-based neutralisation assay (PNA), and an IFN-γ T-cell ELISpot assay were developed, validated or qualified, and transferred to participating laboratories. Immune responses were measured in ELISA laboratory units (ELU) for ELISA, 50% neuralisation dilution (ND50) for MNA, 50% neutralisation titre (NT50) for PNA, and spot-forming units for the ELISpot assay. Replicate assay results of well characterised panels and controls of blood samples from individuals with or without SARS-CoV-2 infection were evaluated by geometric mean ratios, standard deviation, linear regression, and Spearman correlation analysis for consistency, accuracy, and linearity of quantitative measurements across all laboratories. FINDINGS: High reproducibility of results across all laboratories was demonstrated, with interlaboratory precision of 4·1-7·7% coefficient of variation for all three ELISAs, 3·8-19·5% for PNA, and 17·1-24·1% for MNA, over a linear range of 11-30 760 ELU per mL for the three ELISAs, 14-7876 NT50 per mL for PNA, and 21-25 587 ND50 per mL for MNA. The MNA was also adapted for detection of neutralising antibodies against the major SARS-CoV-2 variants of concern. The results of PNA and MNA (r=0·864) and of ELISA and PNA (r=0·928) were highly correlated. The IFN-γ ELISpot interlaboratory variability was 15·9-49·9% coefficient of variation. Sensitivity and specificity were close to 100% for all assays. INTERPRETATION: The CEPI CLN provides accurate quantitation of anti-SARS-CoV-2 immune response across laboratories to allow direct comparisons of different vaccine formulations in different geographical areas. Lessons learned from this programme will serve as a model for faster responses to future pandemic threats and roll-out of effective vaccines. FUNDING: CEPI.

Coronavirus Immunotherapeutic Consortium Database.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seen multiple anti-SARS-CoV-2 antibodies being generated globally. It is difficult, however, to assemble a useful compendium of these biological properties if they are derived from experimental measurements performed at different sites under different experimental conditions. The Coronavirus Immunotherapeutic Consortium (COVIC) circumvents these issues by experimentally testing blinded antibodies side by side for several functional activities. To collect these data in a consistent fashion and make it publicly available, we established the COVIC database (COVIC-DB, https://covicdb.lji.org/). This database enables systematic analysis and interpretation of this large-scale dataset by providing a comprehensive view of various features such as affinity, neutralization, in vivo protection and effector functions for each antibody. Interactive graphs enable direct comparisons of antibodies based on select functional properties. We demonstrate how the COVIC-DB can be utilized to examine relationships among antibody features, thereby guiding the design of therapeutic antibody cocktails. Database URL  https://covicdb.lji.org/.

Safety and immunogenicity of an inactivated recombinant Newcastle disease virus vaccine expressing SARS-CoV-2 spike: Interim results of a randomised, placebo-controlled, phase 1 trial.

Background: Production of affordable coronavirus disease 2019 (COVID-19) vaccines in low- and middle-income countries is needed. NDV-HXP-S is an inactivated egg-based recombinant Newcastle disease virus vaccine expressing the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It's being developed by public sector manufacturers in Thailand, Vietnam, and Brazil; herein are initial results from Thailand. Methods: This phase 1 stage of a randomised, dose-escalation, observer-blind, placebo-controlled, phase 1/2 trial was conducted at the Vaccine Trial Centre, Mahidol University (Bangkok). Healthy males and non-pregnant females, aged 18-59 years and negative for SARS-CoV-2 antibodies, were eligible. Participants were randomised to receive one of six treatments by intramuscular injection twice, 28 days apart: 1 µg, 1 µg+CpG1018 (a toll-like receptor 9 agonist), 3 µg, 3 µg+CpG1018, 10 µg, or placebo. Participants and personnel assessing outcomes were masked to treatment. The primary outcomes were solicited and spontaneously reported adverse events (AEs) during 7 and 28 days after each vaccination, respectively. Secondary outcomes were immunogenicity measures (anti-S IgG and pseudotyped virus neutralisation). An interim analysis assessed safety at day 57 in treatment-exposed individuals and immunogenicity through day 43 per protocol. ClinicalTrials.gov (NCT04764422). Findings: Between March 20 and April 23, 2021, 377 individuals were screened and 210 were enroled (35 per group); all received dose one; five missed dose two. The most common solicited AEs among vaccinees, all predominantly mild, were injection site pain (<63%), fatigue (<35%), headache (<32%), and myalgia (<32%). The proportion reporting a vaccine-related AE ranged from 5·7% to 17·1% among vaccine groups and was 2·9% in controls; there was no vaccine-related serious adverse event. The 10 µg formulation's immunogenicity ranked best, followed by 3 µg+CpG1018, 3 µg, 1 µg+CpG1018, and 1 µg formulations. On day 43, the geometric mean concentrations of 50% neutralising antibody ranged from 122·23 international units per mL (IU/mL; 1 µg, 95% confidence interval (CI) 86·40-172·91) to 474·35 IU/mL (10 µg, 95% CI 320·90-701·19), with 93·9% to 100% of vaccine groups attaining a ≥ 4-fold increase over baseline. Interpretation: NDV-HXP-S had an acceptable safety profile and potent immunogenicity. The 3 µg and 3 µg+CpG1018 formulations advanced to phase 2. Funding: National Vaccine Institute (Thailand), National Research Council (Thailand), Bill & Melinda Gates Foundation, National Institutes of Health (USA).

Interlaboratory Reproducibility of Standardized Hemagglutination Inhibition Assays.

The hemagglutination inhibition (HI) assay is a prominent and commonly accepted method used to determine quantitative antibody titers for influenza virus. However, the reproducibility and consistency of this assay may be affected by several factors, including its reliance on biological reagents that are difficult to standardize, such as red blood cells. This report assesses HI assay performance across three accredited, global laboratories when using test virus and a human serum panel aliquoted and distributed from a centrally located reagent stock. The panel of human sera comprised samples with expected low, medium, and high HI titers against two influenza viruses: A/H1N1/California/07/2009 and B/Victoria/Brisbane/60/2008. HI analysis followed a consensus test protocol. Overall, the HI assay reproducibility within each laboratory was high for both influenza strains, with a within-assay run and intraday precision of 100%. Interlab reproducibility was assessed by comparing the geometric mean titer (GMT) of each sample at each laboratory to the consensus GMT of the sample. A/H1N1 had 100% interlab reproducibility, and none of the individual laboratory GMT values exceeded a 2-fold difference compared to the consensus GMT in any tested sample. B/Victoria had an overall reproducibility of 83%. The results demonstrate that with standardization of key reagents and the use of a common protocol by trained staff, the biologically based HI assay can provide similar results between geographically dispersed laboratories. IMPORTANCE Licensure of influenza vaccines relies on the hemagglutination inhibition (HI) assay as the primary method to determine quantitative functional antibody titers. The HI assay is also widely used for influenza virus surveillance, characterization, and epidemiology studies. However, the HI assay has a notable lack of reproducibility and consistency. If serology results are required from multiple concurrent studies supporting the development and regulatory approval of a product, the testing capacity of any given testing laboratory may be exceeded and data from more than one testing laboratory included in regulatory filings. Thus, understanding the reproducibility of HI assay results over time and between testing laboratories is necessary to support a robust clinical trial serology data set. Our results demonstrate that with standardization of key reagents and use of a common protocol by experienced and trained staff, the biologically based HI assay can provide similar results between geographically dispersed laboratories.

COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models.

Effective treatments against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Monoclonal antibodies have shown promising results in patients. Here, we evaluate the in vivo prophylactic and therapeutic effect of COVA1-18, a neutralizing antibody highly potent against the B.1.1.7 isolate. In both prophylactic and therapeutic settings, SARS-CoV-2 remains undetectable in the lungs of treated hACE2 mice. Therapeutic treatment also causes a reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg-1 one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 shows very strong antiviral activity in the upper respiratory compartments. Using a mathematical model, we estimate that COVA1-18 reduces viral infectivity by more than 95% in these compartments, preventing lymphopenia and extensive lung lesions. Our findings demonstrate that COVA1-18 has a strong antiviral activity in three preclinical models and could be a valuable candidate for further clinical evaluation.

Safety and Immunogenicity of an Inactivated Recombinant Newcastle Disease Virus Vaccine Expressing SARS-CoV-2 Spike: Interim Results of a Randomised, Placebo-Controlled, Phase 1/2 Trial.

BACKGROUND: Production of affordable coronavirus disease 2019 (COVID-19) vaccines in low- and middle-income countries is needed. NDV-HXP-S is an inactivated egg-based Newcastle disease virus vaccine expressing the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It's being developed in Thailand, Vietnam, and Brazil; herein are initial results from Thailand. METHODS: This phase 1 stage of a randomised, dose-escalation, observer-blind, placebo-controlled, phase 1/2 trial was conducted at the Vaccine Trial Centre, Mahidol University (Bangkok). Healthy adults aged 18-59 years, non-pregnant and negative for SARS-CoV-2 antibodies were eligible. Participants were block randomised to receive one of six treatments by intramuscular injection twice, 28 days apart: 1 µg±CpG1018 (a toll-like receptor 9 agonist), 3 µg±CpG1018, 10 µg, or placebo. Participants and personnel assessing outcomes were masked to treatment. The primary outcomes were solicited and spontaneously reported adverse events (AEs) during 7 and 28 days after each vaccination, respectively. Secondary outcomes were immunogenicity measures (anti-S IgG and pseudotyped virus neutralisation). An interim analysis assessed safety at day 57 in treatment-exposed individuals and immunogenicity through day 43 per protocol. ClinicalTrials.gov ( NCT04764422 ). FINDINGS: Between March 20 and April 23, 2021, 377 individuals were screened and 210 were enrolled (35 per group); all received dose one; five missed dose two. The most common solicited AEs among vaccinees, all predominantly mild, were injection site pain (<63%), fatigue (<35%), headache (<32%), and myalgia (<32%). The proportion reporting a vaccine-related AE ranged from 5·7% to 17·1% among vaccine groups and was 2·9% in controls; there was no vaccine-related serious adverse event. The 10 µg formulation's immunogenicity ranked best, followed by 3 µg+CpG1018, 3 µg, 1 µg+CpG1018, and 1 µg formulations. On day 43, the geometric mean concentrations of 50% neutralising antibody ranged from 122·23 IU/mL (1 µg, 95% CI 86·40-172·91) to 474·35 IU/mL (10 µg, 95% CI 320·90-701·19), with 93·9% to 100% of vaccine groups attaining a ≥4-fold increase over baseline. INTERPRETATION: NDV-HXP-S had an acceptable safety profile and potent immunogenicity. The 3 µg and 3 µg+CpG1018 formulations advanced to phase 2. FUNDING: National Vaccine Institute (Thailand), National Research Council (Thailand), Bill & Melinda Gates Foundation, National Institutes of Health (USA).

Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays.

Virus neutralization assays measure neutralizing antibodies in serum and plasma, and the plaque reduction neutralization test (PRNT) is considered the gold standard for measuring levels of these antibodies for many viral diseases. We have developed procedures for the standard PRNT, microneutralization assay (MNA) and pseudotyped virus neutralization assay (PNA) for severe acute respiratory syndrome coronavirus 2. The MNA offers advantages over the PRNT by reducing assay time, allowing increased throughput and reducing operator workload while remaining dependent upon the use of wild-type virus. This ensures that all severe acute respiratory syndrome coronavirus 2 antigens are present, but Biosafety Level 3 facilities are required. In addition to the advantages of MNA, PNA can be performed with lower biocontainment (Biosafety Level 2 facilities) and allows for further increases in throughput. For each new vaccine, it is critical to ensure good correlation of the neutralizing activity measured using PNA against the PRNT or MNA. These assays have been used in the development and licensure of the ChAdOx1 nCoV-19 (AstraZeneca; Oxford University) and Ad26.COV2.S (Janssen) coronavirus disease 2019 vaccines and are critical for demonstrating bioequivalence of future vaccines.

ampG gene of Pseudomonas aeruginosa and its role in ß-lactamase expression.

In enterobacteria, the ampG gene encodes a transmembrane protein (permease) that transports 1,6-GlcNAc-anhydro-MurNAc and the 1,6-GlcNAc-anhydro-MurNAc peptide from the periplasm to the cytoplasm, which serve as signal molecules for the induction of ampC ß-lactamase. The role of AmpG as a transporter is also essential for cell wall recycling. Pseudomonas aeruginosa carries two AmpG homologues, AmpG (PA4393) and AmpGh1 (PA4218), with 45 and 41% amino acid sequence identity, respectively, to Escherichia coli AmpG, while the two homologues share only 19% amino acid identity. In P. aeruginosa strains PAO1 and PAK, inactivation of ampG drastically repressed the intrinsic ß-lactam resistance while ampGh1 deletion had little effect on the resistance. Further, deletion of ampG in an ampD-null mutant abolished the high-level ß-lactam resistance that is associated with the loss of AmpD activity. The cloned ampG gene is able to complement both the P. aeruginosa and the E. coli ampG mutants, while that of ampGh1 failed to do so, suggesting that PA4393 encodes the only functional AmpG protein in P. aeruginosa. We also demonstrate that the function of AmpG in laboratory strains of P. aeruginosa can effectively be inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP), causing an increased sensitivity to ß-lactams among laboratory as well as clinical isolates of P. aeruginosa. Our results suggest that inhibition of the AmpG activity is a potential strategy for enhancing the efficacy of ß-lactams against P. aeruginosa, which carries inducible chromosomal ampC, especially in AmpC-hyperproducing clinical isolates.