Elucidation of the mechanism of severe viral infections
| Project/Area Number |
22KJ2159
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| Project/Area Number (Other) |
22J13220 (2022)
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Multi-year Fund (2023)
Single-year Grants (2022) |
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| Section | 国内 |
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| Review Section |
Basic Section 49060:Virology-related
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| Research Institution | Osaka University |
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Principal Investigator |
LIU YAFEI 大阪大学, 医学系研究科, 特別研究員(DC2)
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| Project Period (FY) |
2023-03-08 – 2024-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2023: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2022: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | SARS-CoV2 / enhancing antibody / NTD-antibody / RBD-antibody |
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| Outline of Research at the Start |
Specific antibodies for SARS-CoV2 enhance the binding of ACE2 to the Wild-type spike. The omicron variant has a large number of mutations. This study followed up on our previous findings by investigating whether Omicron spikes induce specific enhancing antibodies against Omicron variants.
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| Outline of Annual Research Achievements |
Many variants of Omicron have emerged in the last year. In our previous study, we first showed that SARS-CoV2-specific N-terminal structural domain (NTD) antibodies enhance ACE2 binding to spike proteins by changing the conformation of spike proteins. (Liu Y., et al., 2021 Cell) Thus, it is possible that the enhancing antibody changes the conformation of Omicron spikes and then enhances the binding of the receptor binding domain (RBD) to their receptors.
Last year, I found that most of the neutralizing antibodies against NTD or RBD lost their neutralizing effect due to a large number of mutations on the Omicron spikes. On the other hand, NTD-enhancing antibodies against Wild-type spike did not bind to the Omicron spike and lost their enhancing function in the Omicron pseudovirus infection assay. To investigate whether enhancing antibodies against Omicron are produced during Omicron infection or vaccination, first, I immunized mice with protein of Omicron BA.1/BA.5 spike and tested the neutralization effect with sera from these immunized mice. These mice sera were more effective in neutralizing Omicron BA.1/BA.5 than Wild-Type, Delta or XBB. Furthermore, by using these immunized mice, I performed BCR single-cell sequencing in which BA.1/BA.5 NTD-positive memory B cells were sorted. Currently, I have identified and prepared a batch of monoclonal antibodies specific for the Omicron BA.1/BA.5 NTD. It is important for vaccine development to investigate whether there are enhancing antibodies against BA.5/XBB and whether BA.5 spike induces the production of such antibodies.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Based on the previous plan, enhancing antibody against other variants need to be screened from Covid-19 convalescent who infected with wild-type SARS-CoV2. However, NTD-enhancing antibodies against Wild-type spike did not bind to the Omicron spike. To investigate whether enhancing antibodies against Omicron are produced during Omicron infection and to isolate these antibodies, I performed BCR single-cell sequencing in which BA.5 NTD-positive memory B cells were sorted from immunized mice. Although progress is similar to that planned, for example, a number of NTD monoclonal antibodies have been cloned against BA.5, the preliminary functional analysis showed that none of these antibodies enhance BA.5/XBB infection. This result is not as expected. Additional anti-NTD antibodies still need to be identified, and analyze whether they can enhance the infection of the Omicron variant.
On the other hand, I found differences between monkey and human ACE2 in the neutralizing assay. The enhancing antibodies enhance virus infection significantly on ACE2 transfected cells, but not on VERO cells. The function of the enhancing antibody may also be related to the expression of TMPRSS2. these result goes beyond the previous plan, and need to be investigate in future. In addition, I unexpectedly found that some RBD antibodies enhances the infection of the Delta variant on high expression ACE2 transfected cells. The mechanism by which the RBD antibody enhances the infection of the Delta variant on high expression ACE2 transfected cells is currently unknown and requires further investigation.
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| Strategy for Future Research Activity |
In order to find some enhancing antibodies against Omicron BA.5 or XBB, I will complete the functional analysis of the anti-NTD antibodies obtained by BCR single cell sequencing last year by receptor binding assay and pseudovirus infection assay. If I can find some enhancing antibodies against Omicron, I will identify the epitopes of these enhancing antibodies and check the titer of enhancing antibodies in the vaccine sera. Furthermore, in order to check whether the enhanced epitope reduced the efficiency of the vaccine, we prepared Omicron spikes containing the enhancing epitope and Omicron spikes without the enhancing epitope and used them to immunize mice. The presence of enhancing antibodies in mouse sera will be tested, and the neutralization of SARS-CoV2 pseudovirus by different groups of mouse sera will be compared by bead assay. If not, I will try to re-isolate additional anti-NTD antibodies by altering condition such as using NTD protein of omicron or changing adjuvant to induce more positive B cells.
On the other hand, based on my previous findings, I will investigate the mechanism by which the RBD antibody enhances the infection of Delta variants on high expression ACE2 transfected cells. Moreover, I will perform western blotting to confirm whether TMPRSS2 and cleavage of spike is associated with the function of the enhancing antibody by using TMPRSS2 inhibitors. Furthermore, the function of enhancing antibody will be elucidated in physiological condition by using TMPRSS2 inhibitor in mouse.
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Report
(1 results)
Research Products
(5 results)
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[Journal Article] SARS-CoV-2 S2?targeted vaccination elicits broadly neutralizing antibodies2022
Author(s)
Ng Kevin W.、Faulkner Nikhil、Finsterbusch Katja、Wu Mary、Harvey Ruth、Hussain Saira、Greco Maria、Liu Yafei、Kjaer Svend、Swanton Charles、Gandhi Sonia、Beale Rupert、Gamblin Steve J.、Cherepanov Peter、McCauley John、Daniels Rodney、Howell Michael、Arase Hisashi、Wack Andreas、Bauer David L.V.、Kassiotis George
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Journal Title
Science Translational Medicine
Volume: 14
Issue: 655
DOI
Related Report
Peer Reviewed / Int'l Joint Research
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