This study aims to examine the N-terminal-domain (NTD) of the SARS-CoV-2 Spike protein that has evolved to evade the humoral immune response. We achieve this by employing Deep Mutational Scanning (DMS), utilizing an attenuated SARS-CoV-2ΔORF3-8 virus containing deletions of all accessory genes (ORF3,6,7 and 8) to construct a comprehensive viral library with mutations in the NTD region. Generation of NTD mutant virus libraries allows the examination of the early evolution of SARS-CoV-2 in a complex vaccine and convalescent immunity landscape and potentially future-proof SARS-CoV-2 antigen design for vaccination.
We RT-PCR amplified cDNA fragments from the QLD02 isolate into a plasmid vector and assembled into a full-length infectious clone of QLD02ΔORF3-8 from individual fragments by in vitro DNA ligation. Deep sequencing of fragments and assembled product indicate the successful cloning and assembly of amplified fragments. Utilising the assembled product, we successfully generated viral RNA from the DNA template by in vitro RNA transcription.
For constructing a diverse mutagenized DMS-NTD plasmid library, a set of tiling primers with NNK/MNN nucleotides was utilised to mutate each NTD codon. We successfully constructed and validated the sub-cloned DMS library by employing three sequencing methods: Sanger sequencing of individual clones, long-read Nanopore sequencing and low input Illumina sequencing. All three methods indicate generation of diverse mutants across the NTD domain. Sanger sequencing revealed that no individual clones contained wild-type NTD mutants and had, on average, 2.75 mutations/clone.
Our future aims entail incorporating this NTD-DMS library fragment into attenuated SARS-CoV-2ΔORF3-8 RNA and transfecting it into VeroE6ACE2/hTMPRSS2 cells to recover the viral library, all of which will be performed in a PC3 facility. The recovered viral DMS library will be screened against a panel of sera obtained from COVID-19-vaccine-immunised and COVID-19-infected individuals to identify escape mutations.