(T1430-01-09) An Orthogonal Bioanalytical Approach to Assess the Immunogenicity and Efficacy of Covid-19 Vaccines

Purpose: This project aimed to develop and validate an orthogonal approach to assess the efficacy of Covid-19 vaccines. The approach consisted of cell-based and non-cell-based assays on singleplex and multiplex platforms. A secondary objective was to evaluate if the PRNT assay, Gold Standard assays for SARS-COV2 immunogenicity, which involves live virus handling, can be replaced with surrogate and pseudovirion-based methods, thus adding biological safety and efficiency to the vaccine development process.

Methods: Four methods were developed and validated as per ICH Q2R1 guidelines.
1) Surrogate Virus Neutralizing Antibody Assay:
Assay platform: ECL/MSD
Assay principle: Neutralization of the binding between sulfotag RBD and ACE2 ECD
2) Pseudovirion Based Neutralizing Antibody Assay
Assay platform: Cell based-Firefly luciferase-CMV promoter based
Assay principle: Neutralization of the interaction of SARS-CoV-2 spike protein Pseudotyped Lentivirus with ACE2 receptor on recombinant ACE2 HEK293 cells required for transduction of the Pseudovirion into the cells.
3) T-cell assay for Cell-Mediated Immune Response (CMI) against SARS-COV2
Assay platform: T cell-based functional assay and Luminex
Assay principle: T cells expression upon PepTivator® SARS- CoV-2 stimulation for cytokine markers
4) T-cell assay for Cell-Mediated Immune Response (CMI) against SARS-COV2
Assay platform: Cell-based and Flow Cytometer
Assay principle: T cells expression upon PepTivator® SARS- CoV-2 stimulation for CD25+, CD38+ PD-1 (T cell activation)

Results: Vaccine development involves clinical testing of the vaccine for safety and efficacy. Subject samples dosed with the vaccine in these trials should be evaluated for immunogenicity and T-cell mediated immunity responses at regular intervals. As was in the case of the COVID-19 pandemic, speed to market is essential during the testing phase. The methods were established for a fit to purpose for vaccine testing and added a higher throughput to the process by eliminating the need to handle live virus assays for the determination of neutralization. Across development and validation, samples from pre-Covid individuals (including PBMCs) COVID-19-infected and COVID-19-vaccinated individuals were tested. A clear correlation was observed between the surrogate neutralizing antibody assay, the pseudovirion-based neutralizing antibody assay, and the (Gold Standard) Plaque Reduction Neutralization Test, PRNT assay was observed for induction of neutralizing antibodies in infected as well as vaccinated individuals. The sensitivity and specificity were in the range of 85-100% for both assays. Our assays for post-exposure T-cell function demonstrated induction of cell surface markers –CD25+ and CD38+, IFN-gamma, IL-2 (43), IL-6, TNF-alpha, which have been reported to be some of the primary determinations for cell-mediated immune responses to SARS-COV2. Validation parameters such as Accuracy, Precision, Linearity, Assay Cut Point, limit of blank, Sensitivity, Specificity, Selectivity, Robustness and Stability were studied for all four assays. As per assay requirements, reference standards were prepared from COVID-19 infected serum pools and the same were routinely qualified. Pre-covid serum samples were used as negative controls to ensure a clear distinction between signal and noise.

Conclusion: The assays were developed and validated in a GLP-certified laboratory. The methods were validated as per ICH Q2R1 guidelines. Calibrations and standardization of internal reference standards were done using the NIBSC/WHO International Working Standard. The establishment of orthogonal methods and technical and GLP compliance of the established assays make them helpful for the clinical testing of SARS-COV2 vaccines. GLP-compliant systems and processes for all four assays in one lab ensure lab-to-lab sample transfers are unnecessary. The data can be submitted to global regulators.

References: 1. Myler et al., Anti-drug Antibody Validation testing and Reporting Harmonisation.
2. Sholukh et al. Evaluation of Cell-Based and Surrogate SARS-CoV-2 Neutralization Assays. J Clin Microbiol. 2021
3. Ni et al., Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity in COVID-19 Convalescent Individuals, Immunity 2020
4. Sara De Biasi1 et al., Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with COVID-19 pneumonia, Nature Communications 2020
5. ICH Q2R1 guidelines for analytical method validation


Concordance between pseudovirion NAb assay and PRNT assay


Expression of cytokine biomarkers in vaccinated individuals and peptivator-stimulated samples


Covid-19 specific antigen peptivator activation of T-Cells

Acknowledgements: This work was funded by the National Biopharma Mission, BIRAC, DBT, Government of India, under the project BT/CS0008/CS/02/20.