A live-attenuated SARS-CoV-2 vaccine candidate with accessory protein deletions

Abstract

We report a live-attenuated SARS-CoV-2 vaccine candidate with (i) re-engineered viral transcription regulator sequences and (ii) deleted open-reading-frames (ORF) 3, 6, 7, and 8 (∆3678). The ∆3678 virus replicates about 7,500-fold lower than wild-type SARS-CoV-2 on primary human airway cultures, but restores its replication on interferon-deficient Vero-E6 cells that are approved for vaccine production. The ∆3678 virus is highly attenuated in both hamster and K18-hACE2 mouse models. A single-dose immunization of the ∆3678 virus protects hamsters from wild-type virus challenge and transmission. Among the deleted ORFs in the ∆3678 virus, ORF3a accounts for the most attenuation through antagonizing STAT1 phosphorylation during type-I interferon signaling. We also developed an mNeonGreen reporter ∆3678 virus for high-throughput neutralization and antiviral testing. Altogether, the results suggest that ∆3678 SARS-CoV-2 may serve as a live-attenuated vaccine candidate and a research tool for potential biosafety level-2 use.

Viral Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Omicron Infection in mRNA-Vaccinated Individuals Treated and Not Treated with Nirmatrelvir-Ritonavir

“…In comparison to non-rebounders, infection in rebounders took longer to suppress to undetectable (Ct≥40) with NMV-r (median 5 vs. 3 days) and viruses from initial infection were more likely to grow in live culture (1/3 vs. 0/4 cultures). Further, we found that sequenced viruses in the rebound group lacked a single nucleotide variation in Orf3a, an accessory gene that is not associated with NMV-r resistance but when deleted, significantly attenuates replicative capacity in animal models 12 . However, given that viruses from 2 of the NMV-r rebounders are epidemiologically linked, the lack of this mutation may not be meaningful.…”

Comparative analysis of the neutralizing activity against SARS-CoV-2 Wuhan-Hu-1 strain and variants of concern: Performance evaluation of a pseudovirus-based neutralization assay

“…Determining neutralization activity poses challenges, as these assays generally rely on live virus replication, requiring a high containment (BSL-3) laboratory, specialized personnel and well-established protocols from the virus isolation and culture to the neutralization assay. To address this issue, a variety of surrogate assays have been proposed, based on enzyme-linked immunosorbent assay (ELISA) test or on live surrogate virus, such as live attenuated SARS-CoV-2 or pseudoviruses (3,(19)(20)(21)(22)(23)(24). ELISA-based test presents advantages such as low cost, speed, and safety but only antibodies that block the RBD/ ACE2 interaction are detected, thus both the neutralizing activity and the detection of non-RBD binding antibodies which may also be neutralizing are missing.…”

“…When 90% inhibition was not observed at the first dilution tested (1:10), the sample was considered not able to neutralize (neutralization titre <10). To standardize the inter-assay procedures, positive control samples showing low (20) and high (160) neutralizing activity were included in each session. In addition, serum from the National Institute for Biological Standards and Control, Blanche Lane, Ridge, Herts, UK (NIBSC) with known neutralization titer (Research reagent for anti-SARS-CoV-2 Ab NIBSC code 20/136) was used as a reference in MNT during the setting-up ad validation of the protocol.…”

Insights into pandemic respiratory viruses: manipulation of the antiviral interferon response by SARS-CoV-2 and influenza A virus

“…Interestingly, compared to the ancestral strains, the Alpha variant of SARS-CoV-2 expresses notably higher levels of Orf6, Orf9b and N proteins, which contributes to the enhanced immunosuppression of this variant and provides evolutionary evidence for the importance of these viral proteins in IFN antagonism [49] . Moreover, profound IFN-mediated attenuation of a recombinant mutant SARS-CoV-2 was only seen when Orf3a was removed in addition to deletion of Orf6, Orf7 and Orf8 [50] , suggesting a major role of Orf3a in manipulating antiviral IFN responses. Additionally, this study underscores the necessity of evaluating the relative contribution of viral proteins to innate immune evasion through the engineering of recombinant viruses using reverse genetics.…”