SARS-CoV-2 ORF8 is a viral cytokine regulating immune responses (preprint)

Many patients with severe COVID-19 suffer from pneumonia, and thus elucidation of the mechanisms underlying the development of such severe pneumonia is important. The ORF8 protein is a secreted protein of SARS-CoV-2, whose in vivo function is not well understood. Here, we analyzed the function of ORF8 protein by generating ORF8-knockout SARS-CoV-2. We found that the lung inflammation observed in wild-type SARS-CoV-2-infected hamsters was decreased in ORF8-knockout SARS-CoV-2-infected hamsters. Administration of recombinant ORF8 protein to hamsters also induced lymphocyte infiltration into the lungs. Similar pro-inflammatory cytokine production was observed in primary human monocytes treated with recombinant ORF8 protein. Furthermore, we demonstrate that the serum ORF8 protein levels are correlated well with clinical markers of inflammation. These results demonstrated that the ORF8 protein is a viral cytokine of SARS-CoV-2 involved in the in the immune dysregulation observed in COVID-19 patients, and that the ORF8 protein could be a novel therapeutic target in severe COVID-19 patients.

Broad human and animal coronavirus neutralisation by SARS-CoV-2 S2-targeted vaccination (preprint)

Several common-cold coronaviruses (HCoVs) are endemic in humans and several variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged during the current Coronavirus disease 2019 (COVID-19) pandemic. Whilst antibody cross-reactivity with the Spike glycoproteins (S) of diverse coronaviruses has been documented, it remains unclear whether such antibody responses, typically targeting the conserved S2 subunit, contribute to or mediate protection, when induced naturally or through vaccination. Using a mouse model, we show that prior HCoV-OC43 S immunity primes neutralising antibody responses to otherwise subimmunogenic SARS-CoV-2 S exposure and promotes S2-targeting antibody responses. Moreover, mouse vaccination with SARS-CoV-2 S2 elicits antibodies that neutralise diverse animal and human alphacoronaviruses and betacoronaviruses in vitro, and protects against SARS-CoV-2 challenge in vivo. Lastly, in mice with a history of SARS-CoV-2 Wuhan-based S vaccination, further S2 vaccination induces stronger and broader neutralising antibody response than booster Wuhan S vaccination, suggesting it may prevent repertoire focusing caused by repeated homologous vaccination. The data presented here establish the protective value of an S2-targeting vaccine and support the notion that S2 vaccination may better prepare the immune system to respond to the changing nature of the S1 subunit in SARS-CoV-2 variants of concern (VOCs), as well as to unpredictable, yet inevitable future coronavirus zoonoses.

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.

The SARS-CoV-2 Delta variant is poised to acquire complete resistance to wild-type spike vaccines (preprint)

mRNA-based vaccines provide effective protection against most common SARS-CoV-2 variants. However, identifying likely breakthrough variants is critical for future vaccine development. Here, we found that the Delta variant completely escaped from anti-N-terminal domain (NTD) neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the Delta variant, when four common mutations were introduced into the receptor binding domain (RBD) of the Delta variant (Delta 4+), some BNT162b2-immune sera lost neutralizing activity and enhanced the infectivity. Unique mutations in the Delta NTD were involved in the enhanced infectivity by the BNT162b2-immune sera. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta 4+ variant without enhancing infectivity. Given the fact that a Delta variant with three similar RBD mutations has already emerged according to the GISAID database, it is necessary to develop vaccines that protect against such complete breakthrough variants.

SARS-CoV-2 Lambda variant exhibits higher infectivity and immune resistance (preprint)

SARS-CoV-2 Lambda, a new variant of interest, is now spreading in some South American countries; however, its virological features and evolutionary trait remain unknown. Here we reveal that the spike protein of the Lambda variant is more infectious and it is attributed to the T76I and L452Q mutations. The RSYLTPGD246-253N mutation, a unique 7-amino-acid deletion mutation in the N-terminal domain of the Lambda spike protein, is responsible for evasion from neutralizing antibodies. Since the Lambda variant has dominantly spread according to the increasing frequency of the isolates harboring the RSYLTPGD246-253N mutation, our data suggest that the insertion of the RSYLTPGD246-253N mutation is closely associated with the massive infection spread of the Lambda variant in South America.

An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies (preprint)

SARS-CoV-2 infection causes severe symptoms in a subset of patients, suggesting the presence of certain unknown risk factors. Although antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike have been shown prevent SARS-CoV-2 infection, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from COVID-19 patients, and found that some of antibodies against the N-terminal domain (NTD) dramatically enhanced the binding capacity of the spike protein to ACE2, and thus increased SARS-CoV2 infectivity. Surprisingly, mutational analysis revealed that all the infectivity-enhancing antibodies recognized a specific site on the surface of the NTD. The antibodies against this infectivity-enhancing site were detected in all samples of hospitalized COVID-19 patients in the study. However, the ratio of infectivity-enhancing antibodies to neutralizing antibodies differed among patients. Furthermore, the antibodies against the infectivity-enhancing site were detected in 3 out of 48 uninfected donors, albeit at low levels. These findings suggest that the production of antibodies against SARS-CoV-2 infectivity-enhancing site could be considered as a possible exacerbating factors for COVID-19 and that a spike protein lacking such antibody epitopes may be required for safe vaccine development, especially for individuals with pre-existing enhancing antibodies.

The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN (preprint)

The widespread occurrence of SARS-CoV-2 has had a profound effect on society and a vaccine is currently being developed. Angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor that interacts with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Although pneumonia is the main symptom in severe cases of SARS-CoV-2 infection, the expression levels of ACE2 in the lung is low, suggesting the presence of another receptor for the spike protein. In order to identify the additional receptors for the spike protein, we screened a receptor for the SARS-CoV-2 spike protein from the lung cDNA library. We cloned L-SIGN as a specific receptor for the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. The RBD of the spike protein did not bind to L-SIGN. In addition, not only L-SIGN but also DC-SIGN, a closely related C-type lectin receptor to L-SIGN, bound to the NTD of the SARS-CoV-2 spike protein. Importantly, cells expressing L-SIGN and DC-SIGN were both infected by SARS-CoV-2. Furthermore, L-SIGN and DC-SIGN induced membrane fusion by associating with the SARS-CoV-2 spike protein. Serum antibodies from infected patients and a patient-derived monoclonal antibody against NTD inhibited SARS-CoV-2 infection of L-SIGN or DC-SIGN expressing cells. Our results highlight the important role of NTD in SARS-CoV-2 dissemination through L-SIGN and DC-SIGN and the significance of having anti-NTD neutralizing antibodies in antibody-based therapeutics.