The in vivo pathogenicity, transmissibility, and fitness of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron (B.1.1.529) variant are not well understood. We compared these virological attributes of this new variant of concern (VOC) with those of the Delta (B.1.617.2) variant in a Syrian hamster model of COVID-19. Omicron-infected hamsters lost significantly less body weight and exhibited reduced clinical scores, respiratory tract viral burdens, cytokine and chemokine dysregulation, and lung damage than Delta-infected hamsters. Both variants were highly transmissible through contact transmission. In noncontact transmission studies Omicron demonstrated similar or higher transmissibility than Delta. Delta outcompeted Omicron without selection pressure, but this scenario changed once immune selection pressure with neutralizing antibodies—active against Delta but poorly active against Omicron—was introduced. Next-generation vaccines and antivirals effective against this new VOC are therefore urgently needed.

SARS-CoV-2 nsp3 is essential for viral replication and host responses. The SARS-unique domain (SUD) of nsp3 exerts its function through binding to viral and host proteins and RNAs. Herein, we show that SARS-CoV-2 SUD is highly flexible in solution. The intramolecular disulfide bond of SARS-CoV SUD is absent in SARS-CoV-2 SUD. Incorporating this bond in SARS-CoV-2 SUD allowed crystal structure determination to 1.35 Å resolution. However, introducing this bond in SARS-CoV-2 genome was lethal for the virus. Using biolayer interferometry, we screened compounds directly binding to SARS-CoV-2 SUD and identified theaflavin 3,3’-digallate (TF3) as a potent binder, K_d 2.8 µM. TF3 disrupted the SUD-guanine quadruplex interactions and exhibited anti-SARS-CoV-2 activity in Vero E6-TMPRSS2 cells with an EC₅₀ of 5.9 µM and CC₅₀ of 98.5 µM. In this work, we provide evidence that SARS-CoV-2 SUD harbors druggable sites for antiviral development.

The global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become more serious because of the continuous emergence of variants of concern (VOC), thus calling for the development of broad-spectrum vaccines with greater efficacy. Adjuvants play important roles in enhancing the immunogenicity of protein-based subunit vaccines. In this study, we compared the effect of three adjuvants, including aluminum, nanoparticle manganese and MF59, on the immunogenicity of three protein-based COVID-19 vaccine candidates, including RBD-Fc, RBD and S-trimer. We found that the nanoparticle manganese adjuvant elicited the highest titers of SARS-CoV-2 RBD-specific IgG, IgG1 and IgG2a, as well as neutralizing antibodies against infection by pseudotyped SARS-CoV-2 and its Delta variant. What is more, the nanoparticle manganese adjuvant effectively reduced the viral load of the authentic SARS-CoV-2 and Delta variant in the cell culture supernatants. These results suggest that nanoparticle manganese, known to facilitate cGAS-STING activation, is an optimal adjuvant for protein-based COVID-19 subunit vaccines.

Enterovirus A71 (EV-A71) infection is a major cause of hand, foot, and mouth disease (HFMD), which may be occasionally associated with severe neurological complications. There is currently a lack of treatment options for EV-A71 infection. The Raf-MEK-ERK signaling pathway, in addition to its critical importance in the regulation of cell growth, differentiation, and survival, has been shown to be essential for virus replication. In this study, we investigated the anti-EV-A71 activity of vemurafenib, a clinically approved B-Raf inhibitor used in the treatment of late-stage melanoma. Vemurafenib exhibits potent anti-EV-A71 effect in cytopathic effect inhibition and viral load reduction assays, with half maximal effective concentration (EC₅₀) at nanomolar concentrations. Mechanistically, vemurafenib interrupts both EV-A71 genome replication and assembly. These findings expand the list of potential antiviral candidates of anti-EV-A71 therapeutics.

Live attenuated vaccines against SARS-CoV-2 activate all phases of host immunity resembling a natural infection and they block viral transmission more efficiently than existing vaccines in human use. In our prior work, we characterised an attenuated SARS-CoV-2 variant, designated d16, which harbours a D130A mutation in the NSP16 protein, inactivating its 2'-O-methyltransferase function. The d16 variant has demonstrated an ability to induce both mucosal and sterilising immunity in animal models. However, further investigation is required to identify any additional modifications to d16 that could mitigate concerns regarding potential virulence reversion and the suboptimal regulation of the proinflammatory response. Mutations were introduced into molecular clone of SARS-CoV-2 and live attenuated virus was recovered from cultured cells. Virological, biochemical and immunological assays were performed in vitro and in two animal models to access the protective efficacies of the candidate vaccine strain. Here we describe evaluation of a derivative of d16. We further modified the d16 variant by inverting the open reading frame of the ORF3a accessory protein, resulting in the d16i3a strain. This modification is anticipated to enhance safety and reduce pathogenicity. d16i3a appeared to be further attenuated in hamsters and transgenic mice compared to d16. Intranasal vaccination with d16i3a stimulated humoural, cell-mediated and mucosal immune responses, conferring sterilising protection against SARS-CoV-2 Delta and Omicron variants in animals. A version of d16i3a expressing the XBB.1.16 spike protein further expanded the vaccine's protection spectrum against circulating variants. Notably, this version has demonstrated efficacy as a booster in hamsters, providing protection against Omicron subvariants and achieving inhibition of viral transmission. Our work established a platform for generating safe and effective live attenuated vaccines by dual inactivation of NSP16 and ORF3a of SARS-CoV-2. This work was supported by National Key Research and Development Program of China (2021YFC0866100, 2023YFC3041600, and 2023YFE0203400), Hong Kong Health and Medical Research Fund (COVID190114, CID-HKU1-9, and 23220712), Hong Kong Research Grants Council (C7142-20GF and T11-709/21-N), Hong Kong Innovation and Technology Commission grant (MHP/128/22), Guangzhou Laboratory (EKPG22-01) and Health@InnoHK (CVVT). Funding sources had no role in the writing of the manuscript or the decision to submit it for publication.