Biology subjects

Tada, T.

Publications and source records attributed to Tada, T..

6 recordsLinked to original sources

bioRxiv2021

Decreased neutralization of SARS-CoV-2 global variants by therapeutic anti-spike protein monoclonal antibodies

Monoclonal antibodies against the SARS-CoV-2 spike protein, notably, those developed by Regeneron Pharmaceuticals and Eli Lilly and Company have proven to provide protection against severe COVID-19. The emergence of SARS-CoV-2 variants with heavily mutated spike proteins raises the concern that the therapy could become less effective if any of the mutations disrupt epitopes engaged by the antibodies. In this study, we tested monoclonal antibodies REGN10933 and REGN10987 that are used in combination, for their ability to neutralize SARS-CoV-2 variants B.1.1.7, B.1.351, mink cluster 5 and COH.20G/677H. We report that REGN10987 maintains most of its neutralization activity against viruses with B.1.1.7, B.1.351 and mink cluster 5 spike proteins but that REGN10933 has lost activity against B.1.351 and mink cluster 5. The failure of REGN10933 to neutralize B.1.351 is caused by the K417N and E484K mutations in the receptor binding domain; the failure to neutralize the mink cluster 5 spike protein is caused by the Y453F mutation. The REGN10933 and REGN10987 combination was 9.1-fold less potent on B.1.351 and 16.2-fold less potent on mink cluster 5, raising concerns of reduced efficacy in the treatment of patients infected with variant viruses. The results suggest that there is a need to develop additional monoclonal antibodies that are not affected by the current spike protein mutations.

immunology↗

bioRxiv2021

Neutralization of viruses with European, South African, and United States SARS-CoV-2 variant spike proteins by convalescent sera and BNT162b2 mRNA vaccine-elicited antibodies

The increasing prevalence of SARS-CoV-2 variants with mutations in the spike protein has raised concerns that recovered individuals may not be protected from reinfection and that current vaccines will become less effective. The B.1.1.7 isolate identified in the United Kingdom and B.1.351 isolate identified in the Republic of South Africa encode spike proteins with multiple mutations in the S1 and S2 subunits. In addition, variants have been identified in Columbus, Ohio (COH.20G/677H), Europe (20A.EU2) and in domesticated minks. Analysis by antibody neutralization of pseudotyped viruses showed that convalescent sera from patients infected prior to the emergence of the variant viruses neutralized viruses with the B.1.1.7, B.1.351, COH.20G/677H Columbus Ohio, 20A.EU2 Europe and mink cluster 5 spike proteins with only a minor decrease in titer compared to that of the earlier D614G spike protein. Serum specimens from individuals vaccinated with the BNT162b2 mRNA vaccine neutralized D614G virus with titers that were on average 7-fold greater than convalescent sera. Vaccine elicited antibodies neutralized virus with the B.1.1.7 spike protein with titers similar to D614G virus and neutralized virus with the B.1.351 spike with, on average, a 3-fold reduction in titer (1:500), a titer that was still higher than the average titer with which convalescent sera neutralized D614G (1:139). The reduction in titer was attributable to the E484K mutation in the RBD. The B.1.1.7 and B.1.351 viruses were not more infectious than D614G on ACE2.293T cells in vitro but N501Y, an ACE2 contacting residue present in the B.1.1.7, B.1.351 and COH.20G/677H spike proteins caused higher affinity binding to ACE2, likely contributing to their increased transmissibility. These findings suggest that antibodies elicited by primary infection and by the BNT162b2 mRNA vaccine are likely to maintain protective efficacy against B.1.1.7 and most other variants but that the partial resistance of virus with the B.1.351 spike protein could render some individuals less well protected, supporting a rationale for the development of modified vaccines containing E484K.

microbiology↗

bioRxiv2020

The ACE2-binding interface of SARS-CoV-2 Spike inherently deflects immune recognition

The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.

biochemistry↗

bioRxiv2020

A soluble ACE2 microbody protein fused to a single immunoglobulin Fc domain is a potent inhibitor of SARS-CoV-2 infection in cell culture

Soluble forms of ACE2 have recently been shown to inhibit SARS-CoV-2 infection. We report on an improved soluble ACE2, termed a "microbody" in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibited entry of lentiviral SARS-CoV-2 spike protein pseudotyped virus and live SARS-CoV-2 with a potency 10-fold higher than unmodified soluble ACE2 and was active after initial virus binding to the cell. The ACE2 microbody inhibited the entry of ACE2-specific {beta} coronaviruses and viruses with the high infectivity variant D614G spike. The ACE2 microbody may be a valuable therapeutic for COVID-19 that is active against SARS-CoV-2 variants and future coronaviruses that may arise.

microbiology↗

bioRxiv2020

High titers of multiple antibody isotypes against the SARS-CoV-2 spike receptor-binding domain and nucleoprotein associate with better neutralization.

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Here, we determine the ability of sera from 101 recovered healthcare workers to neutralize both authentic SARS-CoV-2 and SARS-CoV-2 pseudotyped virus and address their antibody titers against SARS-CoV-2 nucleoprotein and spike receptor-binding domain. Interestingly, the majority of individuals have low neutralization capacity and only 6% of the healthcare workers showed high neutralizing titers against both authentic SARS-CoV-2 virus and the pseudotyped virus. We found the antibody response to SARS-CoV-2 infection generates antigen-specific isotypes as well as a diverse combination of antibody isotypes, with high titers of IgG, IgM and IgA against both antigens correlating with neutralization capacity. Importantly, we found that neutralization correlated with antibody titers as quantified by ELISA. This suggests that an ELISA assay can be used to determine seroneutralization potential. Altogether, our work provides a snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides evidence that possessing multiple antibody isotypes may play an important role in SARS-CoV-2 neutralization.

microbiology↗

bioRxiv2020

MARCH8 inhibits viral infection by two different mechanisms

Membrane-associated RING-CH 8 (MARCH8) inhibits infection with both HIV-1 and vesicular stomatitis virus G-glycoprotein (VSV-G)-pseudotyped viruses by reducing virion incorporation of envelope glycoproteins. The molecular mechanisms by which MARCH8 targets envelope glycoproteins remain unknown. Here, we show two different mechanisms by which MARCH8 inhibits viral infection. Viruses pseudotyped with the VSV-G mutant, in which cytoplasmic lysine residues were mutated, were insensitive to the inhibitory effect of MARCH8, whereas those with a similar lysine mutant of HIV-1 Env remained sensitive to it. Indeed, the wild-type VSV-G, but not its lysine mutant, was ubiquitinated by MARCH8. Furthermore, the MARCH8 mutant, which had a disrupted cytoplasmic tyrosine motif that is critical for intracellular protein sorting, did not inhibit HIV-1 Env-mediated infection, while it still impaired infection by VSV-G-pseudotyped viruses. Overall, we conclude that MARCH8 reduces viral infectivity by downregulating envelope glycoproteins through two different mechanisms mediated by a ubiquitination-dependent or tyrosine motif-dependent pathway.

microbiology↗