key: cord-0885690-trubxmjn authors: Liu, Yang; Liu, Jianying; Xia, Hongjie; Zhang, Xianwen; Zou, Jing; Fontes-Garfias, Camila R.; Weaver, Scott C.; Swanson, Kena A.; Cai, Hui; Sarkar, Ritu; Chen, Wei; Cutler, Mark; Cooper, David; Muik, Alexander; Sahin, Ugur; Jansen, Kathrin U.; Xie, Xuping; Dormitzer, Philip R.; Shi, Pei-Yong title: BNT162b2-Elicited Neutralization against New SARS-CoV-2 Spike Variants date: 2021-05-12 journal: N Engl J Med DOI: 10.1056/nejmc2106083 sha: cd5d6bd286ab9e93b3cd4e61b2fde558043d40c7 doc_id: 885690 cord_uid: trubxmjn nan Materials and Methods 2 Figure Table S1 . PRNT50 values of twenty BNT162b2-immunized sera against USA-WA1/2020 and variant SARS-CoV-2 7 Recombinant SARS-CoV-2s with variant spike mutations ( Figure S1 ) were engineered into the genetic background of an infectious cDNA clone of clinical isolate USA-WA1/2020. 1 The spike mutations were introduced into the infectious cDNA clone using PCR-based mutagenesis as previously described. 2 Figure S2 ). The complete sequences of spike genes from the P1 viruses were verified by Sanger sequencing to ensure no undesired changes. A detailed protocol for the mutagenesis of SARS-CoV-2 has been recently reported. 3 For determining the specific infectivity of each virus preparation, the P1 stocks were quantified for their genomic RNA content and plaque-forming units (PFU) by RT-qPCR and plaque assay on Vero E6 cells, respectively. The methods for RT-qPCR and plaque assay have been reported previously. 4 Genomic RNA to PFU ratios (genomes/PFU) were calculated to indicate the specific infectivity of each virus preparation. were collected from BTN162b2-immunized human participants as illustrated in Figure S3 . The set of twenty sera, which has also been used in previous studies of SARS-CoV-2 neutralization by BNT162b2-elicited sera, was selected from available samples drawn 14 days or one month after two doses of 30 µg BNT162b2 were administered to participants in the phase 1 portion of the C4591001 clinical trial. 2, 5, 6 The sera were selected to represent a wide range of neutralization participants in the study. A conventional 50% plaque-reduction neutralization test (PRNT50) was performed to measure the serum-mediated virus suppression as reported previously. 5 The values in the graph represent the means with 95% confidence intervals. A non-parametric Mann-Whitney test was used to determine significant differences between the variants and USA-WA1/2020. P values were adjusted using the Bonferroni correction to account for multiple comparisons. Differences were considered significant if p < 0.05; n.s., no statistical difference. Figure S4 . BNT162b2 immunization scheme and serum collection. *The data for USA-WA1/2020 are from two independent experiments; the others are from one experiment each. For each independent experiment, the individual PRNT50 value is the geometric mean of duplicate plaque assay results; no differences were observed between the duplicate assays. Restriction of Zika Virus by Host Innate Immunity Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera Engineering SARS-CoV-2 using a reverse genetic system Spike mutation D614G alters SARS-CoV-2 fitness Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates Neutralizing Activity of BNT162b2-Elicited Serum A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation We thank the Pfizer-BioNTech clinical trial C4591001 participants, from whom the postimmunization human sera were obtained. We thank the many colleagues at Pfizer and BioNTech who developed and produced the BNT162b2 vaccine candidate. We thank LarissaFalcao, Parinati Kharel, and Frank Gillett for gene synthesis.