Human ACE2 Stable Cell Line - HEK293T

Background
This study presents the validation of a pseudovirus neutralization (PNT) assay for detecting SARS-CoV-2 neutralizing antibodies in human serum, supporting its use in evaluating COVID-19 vaccine immunogenicity. The assay demonstrated precision, accuracy, linearity, and specificity for the ancestral strain and Omicron subvariants BA.5 and XBB.1.5, with total geometric coefficient of variation ≤43.4%, relative bias within expected limits, and R² > 0.98. It also correlated strongly with whole-virus neutralization, confirming its reliability for clinical trial sample analysis.
Key Findings
The PNT assay was validated using the ancestral SARS-CoV-2 strain and Omicron subvariants BA.5 and XBB.1.5. Precision testing showed an overall total %GCV of 43.4%, with inter-assay and intra-assay values meeting acceptance criteria. Specificity was demonstrated using pre-pandemic sera and samples from RSV- and influenza-vaccinated individuals, all producing results below the lower limit of quantification. Linearity was confirmed with R² values above 0.98 for all three strains, and the assay was calibrated to the WHO international standard with a conversion factor of 0.214. A strong correlation with a whole-virus microneutralization assay was observed (N=592, Pearson r=0.83, p<0.0001). This high-throughput, BSL-2-compatible pseudovirus assay is a robust tool for measuring neutralizing antibody responses in COVID-19 vaccine clinical trials and can be adapted to newly emerging variants.
Contribution
Creative Biogene supplied the HEK293T/hACE2 cells used in the validated pseudovirus neutralization assay. These cells served as essential permissive host cells for pseudovirus infection and luminescence detection, enabling accurate quantitation of SARS-CoV-2 neutralizing antibody titers in vaccine clinical trial serum samples.

Background
This study investigates how different COVID-19 vaccine platforms affect anti-Spike IgG4 levels and Fc effector function. Repeated mRNA vaccination increased anti-Spike IgG4, which correlated with reduced neutralizing antibodies and surrogate Fc effector functions. Priming with recombinant protein NVX-CoV2373 induced higher IgG1/IgG3, lower IgG4, and stronger neutralizing and effector responses than mRNA. Immune imprinting after mRNA priming was partially overcome by an XBB.1.5 protein vaccine, but not ancestral strains. These findings highlight platform-specific immune priming and its impact on antibody quality.
Key Findings
The study provides first evidence of a negative correlation between increased anti-Spike IgG4 and neutralizing antibody titers, as well as surrogate Fc effector functions such as ADCP, ADCC, and ADCD. Priming with two or three doses of adjuvanted recombinant protein vaccine NVX-CoV2373 followed by a booster resulted in higher IgG1 and IgG3, lower IgG4, and higher neutralizing antibody titers and Fc effector responses compared to mRNA priming. Immune imprinting of anti-S IgG4, neutralizing antibodies, and Fc effector functions was observed after mRNA priming, and this effect was partially overcome by an updated XBB.1.5 protein subunit vaccine, but not by ancestral strain vaccines. The data suggest that vaccine platform and priming regimen shape subsequent antibody responses, with implications for booster vaccination strategies.
Contribution
Creative Biogene provided HEK293T/hACE2 cells used in the pseudovirus neutralization assay, enabling assessment of neutralizing antibody responses.

Background
This paper reports the validation of a pseudovirus neutralization (PNT) assay for quantifying neutralizing antibodies against SARS-CoV-2 Omicron JN.1 and related subvariants (LP.8.1, KP.2, KP.3) in human sera. The assay demonstrated acceptable precision, with total %GCV ≤ 50 in most tested samples, and satisfied criteria for linearity, accuracy, and specificity using matched and heterologous clinical sera. Negative results for pre-pandemic and influenza/RSV-specific samples confirmed its specificity. The validated PNT assay provides a robust BSL-2 compatible tool for evaluating vaccine immunogenicity and cross-reactive antibody responses to emerging JN.1 lineage strains in clinical samples.
Key Findings
Key findings include: (1) The PNT assay was precise for all four Omicron subvariants, with total %GCV ≤ 50 in 87.5–100% of 40 samples per strain; (2) Linearity was confirmed with slopes ranging from 1.041 to 1.213 and R² values of 0.9619–0.9944 across various serum/strain combinations; (3) The assay showed high specificity: all pre-COVID-19 human sera and all influenza/RSV post-vaccination sera tested below LLOQ for SARS-CoV-2; (4) The analytical range was established with LLOQ values of 20–35 and ULOQ values up to 16,775 depending on strain; (5) When applied to clinical trial sera, the assay detected cross-neutralizing antibody responses with no significant differences among JN.1, LP.8.1, KP.2, KP.3, KP.3.1.1, and XEC strains, demonstrating its utility for vaccine immunogenicity assessment and variant surveillance.
Contribution
Creative Biogene (Shirley, NY, USA) supplied the HEK293T/hACE2 cell line used in the pseudovirus neutralization assay. By providing this critical reagent, Creative Biogene enabled the successful execution and validation of the assay, supporting accurate measurement of neutralizing antibodies against SARS-CoV-2 Omicron subvariants.

Background
This article describes the development and validation of a BSL-2 based pseudovirus neutralization assay (PNT) for measuring neutralizing antibodies against SARS-CoV-2 ancestral and emerging Omicron variants, including XBB.1.5, XBB.1.16, and XBB.2.3. The assay employs lentiviral pseudoviruses expressing spike proteins and luciferase reporter that infect 293T/hACE2 cells. It offers a rapid, high-throughput, and cost-effective alternative to BSL-3 live-virus microneutralization. The PNT demonstrated robust precision and linearity, and its results significantly correlated with validated neutralization, anti-spike IgG, and ACE2 inhibition assays. Clinical utility was confirmed using convalescent and NVX-CoV2373-vaccinated sera.
Key Findings
The developed BSL-2 pseudovirus neutralization assay (PNT) reliably quantifies neutralizing antibodies against ancestral, Omicron BA.1/BA.2/BA.5, and XBB subvariants. Optimization included comparing susceptibilities of Vero-E6, A549/hACE2-TMPRSS2, and 293T/hACE2 cells; 293T/hACE2 cells produced highest signals and were chosen. A 250 TCID50/well pseudovirus inoculum and 48-hour endpoint provided optimal performance. The assay demonstrated excellent intra- and inter-assay precision with high, medium, and low titer sera, and linearity with serially diluted serum (R²>0.97). PNT results significantly correlated with a validated neutralization assay (r=0.83), a live-virus microneutralization assay (r=0.93), anti-recombinant spike IgG (r=0.71), and hACE2-binding inhibition (r=0.89). Clinical samples from vaccinated participants revealed increased neutralization breadth against Omicron variants after booster vaccination. Convalescent sera showed comparable neutralizing titers against XBB.1.5, XBB.1.16, and XBB.2.3. This high-throughput, cost-effective assay enables rapid immunogenicity assessment for variant-updated vaccines.
Contribution
Creative Biogene supplied the 293T/hACE2 stable cell line overexpressing human ACE2 receptor. This cell line served as the permissive target for pseudovirus infection in the neutralization assay, providing high luminescent signals and reproducibility. It was essential for developing and validating the rapid BSL-2-based pseudovirus neutralization assay described in this study.

Background
Neutralising antibodies (nAbs) are key correlates of protection against SARS-CoV-2, making their measurement essential for vaccine surveillance. This study evaluated whether dried blood spots (DBS), including fingerstick samples stored on filter paper, can reliably quantify nAb responses compared with paired sera. Samples from acutely infected, convalescent, and vaccinated individuals were tested using SARS-CoV-2 spike pseudotype neutralisation assays. Eluted DBS showed good agreement with paired sera, and nAb stability was maintained after 28 days at room temperature. The findings support DBS as a practical, low-cost, self-collection method for large-scale sero-surveillance and resource-limited settings.
Key Findings
The study demonstrated that filter paper is a viable method for measuring SARS-CoV-2 neutralising antibody (nAb) responses. In paired serum and dried serum spot (DSS) eluates, IC50, IC70, and IC90 values showed strong positive relationships (R² = 0.7432–0.8381, p < 0.0001) and small Bland–Altman biases, indicating high agreement. DSS stored at room temperature for up to 28 days retained comparable neutralising capacity to sera stored at −80°C (p > 0.05). Venous dried blood spot eluates (25 µL and 50 µL) also agreed well with paired sera, though 15 µL overestimated nAb responses due to filter paper saturation issues. Fingerstick dried blood spot eluates gave IC50, IC70, and IC90 values comparable to paired sera, with no significant differences for IC50 or IC90. Overall, DBS sampling can reliably estimate nAb responses, supporting its use for sero-surveillance and pandemic preparedness.
Contribution
Creative Biogene provided the HEK293T/ACE-2 cell line (CSC-RO0641) used to monitor single-round infectious SARS-CoV-2 pseudotype infectivity and to perform sample neutralisation assays. These cells were essential for measuring neutralising antibody responses in dried blood spots and paired sera, supporting the study's comparative validation.