Deep mutational scanning of whole SARS-CoV-2 spike in an inverted infection system (preprint)

In order to investigate SARS-CoV-2 mutations and their impact on immune evasion and infectivity, we developed a Deep Mutational Scanning (DMS) platform utilizing an inverted infection assay to measure spike expression, ACE2 affinity, and viral infectivity in human cells. Surprisingly, our analysis reveals that spike protein expression, rather than ACE2 affinity, is the primary factor affecting viral infectivity and correlated with SARS-CoV-2 evolution. Notably, within the N-terminal domain (NTD), spike expression and infectivity-enhancing mutations are concentrated in flexible loops. We also observed that Omicron variants BA.1 and BA.2 exhibit immune evasion through receptor binding domain (RBD) mutations, although these mutations reduce structural stability. Interestingly, the NTD has evolved to increase stability, compensating for the RBD instability and resulting in heightened overall infectivity. Our findings, available in SpikeScanDB, emphasize the importance of spike expression levels and compensatory mutations in both the NTD and RBD domains for shaping Omicron variant infectivity.

An engineered ACE2 decoy broadly neutralizes Omicron subvariants and shows therapeutic effect in SARS-CoV-2-infected cynomolgus macaques (preprint)

The Omicron variant continuously evolves under the humoral immune pressure obtained by vaccination and SARS-CoV-2 infection and the resultant Omicron subvariants exhibit further immune evasion and antibody escape. Engineered ACE2 decoy composed of high-affinity ACE2 and IgG1 Fc domain is an alternative modality to neutralize SARS-CoV-2 and we previously reported its broad spectrum and therapeutic potential in rodent models. Here, we show that engineered ACE2 decoy retains the neutralization activity against Omicron subvariants including the currently emerging XBB and BQ.1 which completely evade antibodies in clinical use. The culture of SARS-CoV-2 under suboptimal concentration of neutralizing drugs generated SARS-CoV-2 mutants escaping wild-type ACE2 decoy and monoclonal antibodies, whereas no escape mutant emerged against engineered ACE2 decoy. As the efficient drug delivery to respiratory tract infection of SARS-CoV-2, inhalation of aerosolized decoy treated mice infected with SARS-CoV-2 at a 20-fold lower dose than the intravenous administration. Finally, engineered ACE2 decoy exhibited the therapeutic efficacy for COVID-19 in cynomolgus macaques. Collectively, these results indicate that engineered ACE2 decoy is the promising therapeutic strategy to overcome immune-evading SARS-CoV-2 variants and that liquid aerosol inhalation can be considered as a non-invasive approach to enhance efficacy in the treatment of COVID-19.

Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 Spike protein (preprint)

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging showed that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Strikingly, cellular cryo-electron tomography revealed dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and found that S protein recognizes integrin v{beta}3. Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.

Engineered ACE2 counteracts vaccine-evading SARS-CoV-2 Omicron variant (preprint)

The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera of 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. However, some Delta patients have relatively preserved neutralization efficacy, comparable to 3-month double BNT162b2 vaccination. Domain-wise analysis using chimeric spike revealed that this efficient evasion was, at least in part, caused by multiple mutations in the N-terminal domain. Omicron escapes the therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective against Omicron. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from mutational escape. Deep mutational analysis demonstrated that, indeed, the engineered ACE2 overcomes every single-residue mutation in the receptor-binding domain, similar to immunized sera. Like previous SARS-CoV-2 variants, Omicron and some other sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.

Acquisition of resistance to wild-type spike-immune sera by emerging SARS-CoV-2 variants (preprint)

Breakthrough infection is often observed for the SARS-CoV-2 Delta variant, and neutralizing antibody levels are associated with vaccine efficiency 1 . Recent studies revealed that not only anti-receptor binding domain (RBD) antibodies 2 but also antibodies against the N-terminal domain (NTD) play important roles in positively 3,4 or negatively 4-8 controlling SARS-CoV-2 infectivity. Here, we found that the Delta variant completely escaped from anti-NTD neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Cryo-EM analysis of the Delta spike revealed that epitopes for anti-NTD neutralizing antibodies are structurally divergent, whereas epitopes for enhancing antibodies are well conserved with wild-type spike protein. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the original Delta variant, when major anti-RBD neutralizing antibody epitopes remaining in the Delta variant were disrupted, some BNT162b2-immune sera not only lost neutralizing activity but became infection-enhanced. The enhanced infectivity disappeared when the Delta NTD was substituted with the wild-type NTD. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta variant lacking anti-RBD antibody epitopes without enhancing infectivity. Importantly, SARS-CoV-2 variants with similar mutations in the RBD have already emerged according to the GISAID database and their pseudoviruses were resistant to some BNT162b2-immune sera. These findings demonstrate that mutations in the NTD, as well as the RBD, play an important role in antibody escape by SARS-CoV-2. Development of effective vaccines against emerging variants will be necessary, not only to protect against infection, but also to prevent further mutation of SARS-CoV-2.

High affinity modified ACE2 receptors prevent SARS-CoV-2 infection (preprint)

The SARS-CoV-2 spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor via receptor binding domain (RBD) to enter into the cell. Inhibiting this interaction is a main approach to block SARS-CoV-2 infection and it is required to have high affinity to RBD independently of viral mutation for effective protection. To this end, we engineered ACE2 to enhance the affinity with directed evolution in human cells. Three cycles of random mutation and cell sorting achieved more than 100-fold higher affinity to RBD than wild-type ACE2. The extracellular domain of modified ACE2 fused to the Fc region of the human immunoglobulin IgG1 had stable structure and neutralized SARS-CoV-2 pseudotyped lentivirus and authentic virus with more than 100-fold lower concentration than wild-type. Engineering ACE2 decoy receptors with directed evolution is a promising approach to develop a SARS-CoV-2 neutralizing drug that has affinity comparable to monoclonal antibodies yet displaying resistance to escape mutations of virus.