Rapid evaluation of heterologous chimeric RBD-dimer mRNA vaccine for currently-epidemic Omicron sub-variants as booster shot after inactivated vaccine

Graphical With continuous mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the severe immune escape of Omicron sub-variants urges the development of next-generation broad-spectrum vaccines, especially as booster jabs after high-level vaccination coverage of inactivated vaccines in China and many other countries. Previously, we developed a coronavirus disease 2019 (COVID-19) protein subunit vaccine ZF2001® based on the tandem homo-prototype receptor-binding domain (RBD)-dimer of the SARS-CoV-2 spike protein. We upgraded the antigen into a hetero-chimeric prototype (PT)-Beta or Delta-BA.1 RBD-dimer to broaden the cross-protection efficacy and prove its efficiency with protein subunit and mRNA vaccine platforms. Herein, we further explored the hetero-chimeric RBD-dimer mRNA vaccines and evaluated their broad-spectrum activities as booster jabs following two doses of inactivated vaccine in mice. Our data demonstrated that the chimeric vaccines significantly boosted neutralizing antibody levels and specific T-cell responses against the variants, and PT-Beta was superior to Delta-BA.1 RBD as a booster in mice, shedding light on the antigen design for the next-generation COVID-19 vaccines.

Rapid evaluation of heterologous chimeric RBD-dimer mRNA vaccine for currently-epidemic Omicron sub-variants as booster shot after inactivated vaccine.

With continuous mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the severe immune escape of Omicron sub-variants urges the development of next-generation broad-spectrum vaccines, especially as booster jabs after high-level vaccination coverage of inactivated vaccines in China and many other countries. Previously, we developed a coronavirus disease 2019 (COVID-19) protein subunit vaccine ZF2001® based on the tandem homo-prototype receptor-binding domain (RBD)-dimer of the SARS-CoV-2 spike protein. We upgraded the antigen into a hetero-chimeric prototype (PT)-Beta or Delta-BA.1 RBD-dimer to broaden the cross-protection efficacy and prove its efficiency with protein subunit and mRNA vaccine platforms. Herein, we further explored the hetero-chimeric RBD-dimer mRNA vaccines and evaluated their broad-spectrum activities as booster jabs following two doses of inactivated vaccine (IV) in mice. Our data demonstrated that the chimeric vaccines significantly boosted neutralizing antibody levels and specific T-cell responses against the variants, and PT-Beta was superior to Delta-BA.1 RBD as a booster in mice, shedding light on the antigen design for the next-generation COVID-19 vaccines.

Two pan-SARS-CoV-2 nanobodies and their multivalent derivatives effectively prevent Omicron infections in mice.

With the widespread vaccinations against coronavirus disease 2019 (COVID-19), we are witnessing gradually waning neutralizing antibodies and increasing cases of breakthrough infections, necessitating the development of drugs aside from vaccines, particularly ones that can be administered outside of hospitals. Here, we present two cross-reactive nanobodies (R14 and S43) and their multivalent derivatives, including decameric ones (fused to the immunoglobulin M [IgM] Fc) that maintain potent neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after aerosolization and display not only pan-SARS-CoV-2 but also varied pan-sarbecovirus activities. Through respiratory administration to mice, monovalent and decameric R14 significantly reduce the lung viral RNAs at low dose and display potent pre- and post-exposure protection. Furthermore, structural studies reveal the neutralizing mechanisms of R14 and S43 and the multiple inhibition effects that the multivalent derivatives exert. Our work demonstrates promising convenient drug candidates via respiratory administration against SARS-CoV-2 infection, which can contribute to containing the COVID-19 pandemic.

Molecular Basis of Mink ACE2 Binding to SARS-CoV-2 and Its Mink-Derived Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted between humans and minks, and some mutations in the spike (S) protein, especially in the receptor-binding domain (RBD), have been identified in mink-derived viruses. Here, we examined binding of the mink angiotensin-converting enzyme 2 (ACE2) receptor to mink-derived and important human-originating variants, and we demonstrated that most of the RBD variants increased the binding affinities to mink ACE2 (mkACE2). Cryo-electron microscopy structures of the mkACE2-RBD Y453F (with a Y-to-F change at position 453) and mkACE2-RBD F486L complexes helped identify the key residues that facilitate changes in mkACE2 binding affinity. Additionally, the data indicated that the Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and human vaccinated sera efficiently prevented infection of human cells by pseudoviruses expressing Y453F, F486L, or N501T RBD. Our findings provide an important molecular mechanism for the rapid adaptation of SARS-CoV-2 in minks and highlight the potential influence of the main mink-originating variants for humans. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a broad range of hosts. Mink-derived SARS-CoV-2 can transmit back to humans. There is an urgent need to understand the binding mechanism of mink-derived SARS-CoV-2 variants to mink receptor. In this study, we identified all mutations in the receptor-binding domain (RBD) of spike (S) protein from mink-derived SARS-CoV-2, and we demonstrated the enhanced binding affinity of mink angiotensin-converting enzyme 2 (ACE2) to most of the mink-derived RBD variants as well as important human-originating RBD variants. Cryo-electron microscopy structures revealed that the Y453F and F486L mutations enhanced the binding forces in the interaction interface. In addition, Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and the SARS-CoV-2 pseudoviruses with Y453F, F486L, or N501T mutations were neutralized by human vaccinated sera. Therefore, our results provide valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

Atlas of currently available human neutralizing antibodies against SARS-CoV-2 and escape by Omicron sub-variants BA.1/BA.1.1/BA.2/BA.3.

SARS-CoV-2 Omicron variant has presented significant challenges to current antibodies and vaccines. Herein, we systematically compared the efficacy of 50 human monoclonal antibodies (mAbs), covering the seven identified epitope classes of the SARS-CoV-2 RBD, against Omicron sub-variants BA.1, BA.1.1, BA.2, and BA.3. Binding and pseudovirus-based neutralizing assays revealed that 37 of the 50 mAbs lost neutralizing activities, whereas the others displayed variably decreased activities against the four Omicron sub-variants. BA.2 was found to be more sensitive to RBD-5 antibodies than the other sub-variants. Furthermore, a quaternary complex structure of BA.1 RBD with three mAbs showing different neutralizing potencies against Omicron provided a basis for understanding the immune evasion of Omicron sub-variants and revealed the lack of G446S mutation accounting for the sensitivity of BA.2 to RBD-5 mAbs. Our results may guide the application of the available mAbs and facilitate the development of universal therapeutic antibodies and vaccines against COVID-19.

Molecular insights into receptor binding of recent emerging SARS-CoV-2 variants.

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.