Liu et al.

The SARS-CoV-2 Delta variant is poisedÂ
to acquire complete resistance toÂ
wild-type spike vaccines

1. J.K. Rowling
2. Stephen King
3. John Steinbeck
4. Harper Lee
5. George Orwell
6. Ernest Hemingway
7. Mark Twain
8. William Shakespeare
9. Fyodor Dostoevsky
10. Jane Austen

• Yafei Liu^1,2, Noriko Arase³, Jun-ichi Kishikawa⁴, Mika Hirose⁴, Songling Li⁵, Asa Tada², Sumiko Matsuoka¹, Akemi Arakawa², Kanako Akamatsu⁶, Chikako Ono^7,8, Hui Jin¹, Kazuki Kishida², Wataru Nakai^1,2, Masako Kohyama^1,2, Atsushi Nakagawa⁹, Yoshiaki Yamagishi¹⁰, Hironori Nakagami¹¹, Atsushi Kumanogoh^12,13, Yoshiharu Matsuura^6,14, Daron M. Standley^5,15, Takayuki Kato⁴, Masato Okada^6,15, Manabu Fujimoto³, Hisashi Arase^1,2,15*

I am currently affiliated with the University of California, Berkeley as a graduate student in the Department of Electrical Engineering and Computer Sciences.

1. Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan.Â
2. Laboratory of Immunochemistry, World Premier International Immunology Frontier Research Centre, Osaka University, Osaka, 565-0871, Japan.Â
3. Department of Dermatology, Graduate school of Medicine, Osaka University, Osaka, 565-0871, JapanÂ
4. Laboratory for CryoEM Structural Biology, Institute for Protein Research, Osaka University, Osaka, 565-0871, Japan.Â
5. Dept. Genome Informatics, Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan.
6. Department Oncogene Research, Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan.
7. Laboratory of Virus Control, Center for Infectious Disease Education and Research, Osaka University, Osaka, 565-0871, JapanÂ
8. Laboratory for Cell Polarity Regulation, RIKEN Center for Biosystems Dynamics Research, Hyogo, 650-0047, JapanÂ
9. Laboratory for Supramolecular Crystallography, Institute for Protein Research, Osaka University, Osaka, 565-0871, Japan.Â
10. Medical Center for Translational Research, Department of Medical Innovation, Osaka University Hospital, Osaka University, Osaka, 565-0871, Japan..Â
11. Department of Health Development and Medicine, Graduate school of Medicine, Osaka University, Osaka, 565-0871, Japan.Â
12. Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.Â
13. Laboratory of Immunopathology, World Premier International Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan.Â
14. Laboratory of Virus Control, Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan
15. Center for Infectious Disease Education and Research, Osaka University, Osaka, 565-0871, Japan *Corresponding to: [email protected]

Abstract

Overall, our findings suggest that mRNA-based vaccines are effective against most common SARS-CoV-2 variants, but may not be able to protect against complete breakthrough variants. It is important to identify likely breakthrough variants and develop vaccines that can protect against them.

Introduction

Results

Neutralizing activity of anti-NTD and anti-RBD monoclonal antibodies from COVID-19 patients against the Delta variant.Â

In conclusion, our analysis of the binding and functional activity of various anti-spike monoclonal antibodies obtained from COVID-19 patients revealed that the Delta variant has escaped from recognition by most anti-NTD neutralizing antibodies while maintaining functional enhancing antibody epitopes. This suggests that the increased infectivity of the Delta variant is due to its ability to evade recognition by anti-NTD neutralizing antibodies while maintaining functional enhancing antibody epitopes.

Neutralizing activity of BNT162b2-immune sera against Delta variants.Â

To further investigate the contribution of NTD and RBD mutations in the resistance of BNT162b2-immune sera against the Delta variant, we generated chimeric spike proteins in which the NTD, RBD or S2 subunit was encoded by either wild-type (W) or Delta (D) variant. We then generated pseudoviruses containing these recombinant spike proteins and analyzed the effect of BNT162b2-immune sera. The neutralizing activity of the BNT162b2-immune sera against WWD pseudovirus decreased slightly compared to that of wild-type pseudovirus (WWW), suggesting that mutations in the S2 domain are involved in the resistance of the Delta variant. When infectivity of DWD pseudovirus, in which wild-type NTD was substituted to the Delta NTD, was compared with WWD pseudovirus, the neutralizing activity of BNT162b2-immune sera significantly decreased further. The neutralizing activity of the BNT162b2 immune sera was reduced against WDD pseudovirus, in which wild-type RBD was replaced by Delta RBD, compared to DWD pseudovirus. The neutralizing activity of the BNT162b2-immune sera decreased further against Delta pseudovirus (DDD). These data suggest that both NTD and RBD mutations in the Delta spike are involved in the resistance of BNT162b2-immune sera against the Delta variant.

Cryo-EM analysis of the Delta spikeÂ

Prediction of possible future mutations of the Delta variantÂ

The Delta 4+ variant showed a significant decrease in the neutralizing activity of BNT162b2 immune sera, suggesting that these mutations may confer resistance to anti-RBD neutralizing antibodies.

Enhanced infectivity of the Delta 4+ pseudovirus by some BNT162b2-immune sera.Â

Sera from the Delta spike immunized mice do not show enhanced infectivity against Delta 4+ pseudovirus.Â

Discussion 

In conclusion, we demonstrated that the Delta variant is resistant to neutralizing antibodies induced by wild-type spike protein and is enhanced by some BNT162b2-immune sera. Mutations in the NTD of the Delta variant are likely to be involved in this phenomenon. We also showed that immunization with the Delta spike induces antibodies that neutralize not only the Delta variant but also wild-type and Delta 4+ variants without enhancing infectivity. Therefore, immunization with a vaccine composed of a mutant spike protein may be effective for controlling emerging SARS-CoV-2 variants.

Acknowledgments 

Author contributionsÂ

section*{Competing interests}
The authors declare no competing interests.

section*{Data availability}
The data that support the findings of this study are available from the corresponding author upon reasonable request.

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bibitem[WHO(2020)]{who2020global} WHO, 2020. Global tuberculosis report 2020. World Health Organization, Geneva, Switzerland. Available at: https://www.who.int/tb/publications/global-report/en/. Accessed on: October 15th, 2020.

bibitem[WHO(2021)]{who2021roadmap} WHO, 2021. The End TB Strategy: A comprehensive approach to end tuberculosis by 2030: The roadmap for implementation of the End TB Strategy 2019–2022. World Health Organization, Geneva, Switzerland. Available at: https://www.who.int/tb/publications/end-tb-strategy-roadmap-2019-2022/en/. Accessed on: April 5th, 2021.

bibitem[Gandhi et~al.(2011)Gandhi, Moll, Sturm, Pawinski, Govender et~al.]{gandhi2011multidrug} Gandhi, N., Moll, A., Sturm, A., Pawinski, R., Govender, T., Lalloo U., Zeller K., Padayatchi N., 2011 Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis? Lancet Infect Dis 11(12): 814–823 doi: 10.1016/S1473-3099(11)70223-7 PMID 22037372 PMCID PMC3239079

bibitem[Kumar et~al.(2018)Kumar , Singh , Kumar , Sharma , Singh et~al.] {kumar2018drugsusceptibilitytestingofmycobacteriumtuberculosisisolatesinindiaresultsfromthenationalsurvey20142015 } Kumar S., Singh P., Kumar P., Sharma S., Singh V., 2018 Drugs susceptibility testing of Mycobacterium tuberculosis isolates in India results from the national survey 2014–2015 Int J Tuberc Lung Dis 22(4): 441–447 doi: 10.5588/ijtld.17.0453 PMID 29503945 PMCID PMC5909619

bibitem[Marais et~al.(2017)Marais , Gie , Schaaf , Hesseling et~al.] {marais2017extensivelydrugresistanttuberculosisamongchildreninhighincidencecountries } Marais BJ., Gie RP., Schaaf HS., Hesseling AC 2017 Extensively drug resistant tuberculosis among children in high incidence countries Int J Tuberc Lung Dis 21(5): 518–524 doi: 10.5588/ijtld .16 .0590 PMID 28350063 PMCID PMC5401087

bibitem[World Bank Group (2020)] {worldbankgroup2020worlddevelopmentreport2019 } World Bank Group 2020 World Development Report 2019 : The Changing Nature of Work Washington DC : World Bank Group Available at : http://documents . worldbank . org / curated / en / 948661568337744983 / pdf / WDR – 2019 – Full – Report – ENGLISH . pdf Accessed on : October 15 th , 2020 .

bibitem[Liu et~al.(2020)Liu , Okello , Gething et~al.] {liu2020globalincidenceandprevalenceofinfectiousdiseasesduringthecoronavirusdisease2019pandemictheexampleofmeasles } Liu YY . , Okello D . S . , Gething PW . et al 2020 Global incidence and prevalence of infectious diseases during

Declaration of interestsÂ

Yes, Osaka University has filed a patent application for the enhancing antibodies with HA and YL listed as inventors. The fact that HA is a stockholder of HuLA immune Inc. does not affect the validity of the patent application.

Methods

Data and code availabilityÂ

The data that support the findings of this study are available from the Lead Contact on request.

Cell linesÂ

The B16F10 melanoma cells were maintained in RPMI 1640 medium (Nacalai, Japan) supplemented with 10% FBS (Biological Industries, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL) (Nacalai, Japan). The cells were routinely checked for mycoplasma contamination. For the ACE2-stably transfected B16F10 melanoma cells (B16F10-ACE2-transfectants), the pLenti6.3/V5-DEST vector containing human ACE2 cDNA was used to generate stable cell lines using Lipofectamine 3000 reagent according to the manufacturer’s instructions.

Human samplesÂ

The sera were used to measure the levels of neutralizing antibodies against SARS-CoV-2. The sera were tested using a pseudovirus neutralization assay (PNA) and a microneutralization assay (MNA). The PNA was performed in 96-well plates with the addition of serial dilutions of the sera, followed by incubation with a SARS-CoV-2 pseudovirus. The MNA was performed in 96-well plates with the addition of serial dilutions of the sera, followed by incubation with SARS-CoV-2 virus. The results from both assays were compared to determine the level of neutralizing antibodies present in each serum sample.

The data obtained from these assays was then analyzed to assess the correlation between BNT162b2 mRNA vaccine immunization and neutralizing antibody production. Statistical analyses were conducted using GraphPad Prism 8 software.

Plasmid constructionÂ

TransfectionÂ

The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2. After 24 h, the medium was replaced with fresh medium containing 0.5 μg/ml of puromycin (Invitrogen). The transfected cells were incubated for an additional 48 h before harvesting. The harvested cells were lysed using RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with protease inhibitor cocktail (Roche). The cell lysates were centrifuged at 12000 rpm for 15 min at 4°C and the supernatants were collected for further analysis by western blotting or ELISA.

Anti-spike monoclonal antibodies from COVID-19 patientsÂ

The purified IgG was then tested for its ability to bind to the SARS-CoV-2 spike protein using an ELISA assay. The assay was performed according to the manufacturer’s instructions (Thermo Fisher Scientific). Briefly, 96-well plates were coated with recombinant SARS-CoV-2 spike protein and blocked with 3% BSA in PBS. Serial dilutions of the IgG samples were added to the wells and incubated for 1 hour at room temperature. After washing, horseradish peroxidase (HRP)-conjugated anti-human IgG antibody was added and incubated for 1 hour at room temperature. After washing, HRP substrate was added and incubated for 30 minutes at room temperature. The absorbance of each well was measured at 450 nm using a microplate reader (BioTek). The binding affinity of each IgG sample was calculated by fitting a four parameter logistic curve to the data using GraphPad Prism software.

Antibodies and recombinant proteinsÂ

The purified His-tagged spike protein was then conjugated to the APC-conjugated donkey anti-mouse IgG Fc fragment antibody and APC-conjugated anti-human IgG Fc fragment specific antibody using a crosslinker (Thermo Scientific Pierce). The conjugation reaction was performed according to the manufacturer’s instructions. The conjugates were then analyzed by SDS-PAGE and Western blotting.

Immunization of miceÂ

The B16F10 cells were lysed and the lysates were subjected to SDS-PAGE. The proteins in the gel were transferred to a nitrocellulose membrane and probed with anti-WT spike protein or anti-Delta spike protein antibodies. The results showed that WT spike protein was expressed in the B16F10 cells transfected with WT spike protein, while Delta spike protein was not detected in the B16F10 cells transfected with Delta spike protein.

The serum samples collected from mice immunized with B16F10 transfectants were analyzed by ELISA. The results showed that the mice immunized with WT spike protein had higher levels of antibodies against WT spike protein compared to those immunized with Delta spike protein. This indicated that the WT spike protein was successfully expressed in B16F10 cells and induced an immune response in mice.

Flow cytometric analysis of antibodiesÂ

The results showed that the mAbs bound to the SARS-CoV-2 spike protein expressed in HEK293T cells. The binding of the mAbs was specific and dose-dependent, with higher levels of binding observed at higher concentrations of mAb. The results also showed that the Flag-NTD-PILR-TM and Flag.RBD-PILR-TM constructs had similar levels of binding to the mAbs, indicating that both constructs were able to bind to the antibodies. These results demonstrate that both constructs are capable of eliciting an immune response when expressed in HEK293T cells.

SARS-CoV-2 spike-pseudotyped virus infection assayÂ

Structure prediction by AlphaFold2Â

The NTD structure of the wild type was predicted to be a four-helix bundle, with two alpha helices and two beta strands. The Delta variant was predicted to have a similar structure, but with an additional helix at the C-terminus. The RBD structure of the wild type was predicted to be a three-stranded beta sheet, with two short alpha helices on either side. The Delta variant was predicted to have a similar structure, but with an additional helix at the C-terminus.

Overall, the structures of both variants were found to be similar, with only minor differences in their secondary structures. This suggests that the mutations in the Delta variant do not significantly affect its overall structural stability or function.

Cryo-EM data collectionÂ

The data was processed using the Relion 3.0 software package (Scheres, 2012). The particle picking was done using the DoG picker in Relion 3.0 and the particles were extracted with a box size of 200 Â. The 2D classification was performed with a low-pass filter of 40 Â and a high-pass filter of 10 Â. The 3D refinement was done with a mask radius of 80 Â and an angular sampling rate of 5 degrees. The final reconstruction was obtained with a resolution of 4.2 Â.

Image processing and 3D reconstructionÂ

The obtained density map was further used for model building and refinement. The initial model was built by using the Rosetta software (DiMaio et al., 2015). The model was further refined by using Phenix software (Adams et al., 2010). The final model was validated by MolProbity server (Chen et al., 2010).

Model building and refinementÂ

Data and statistical analysisÂ

The data were analyzed using FlowJo version 10.7 (BD Biosciences, USA). Graphpad Prism version 7.0e was used for graph generation and statistical analysis.

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• Suryadevara, N., Shrihari, S., Gilchuk, P., VanBlargan, L.A., Binshtein, E., Zost, S.J., Nargi, R.S., Sutton, R.E., Winkler, E.S., Chen, E.C., et al. (2021). Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein. Cell 184, 2316-2331 e2315.Â
• Tegally, H., Wilkinson, E., Giovanetti, M., Iranzadeh, A., Fonseca, V., Giandhari, J., Doolabh, D., Pillay, S., San, E.J., Msomi, N., et al. (2021). Detection of a SARS-CoV-2 variant of concern in South Africa. Nature 592, 438-443.Â
• Thepaut, M., Luczkowiak, J., Vives, C., Labiod, N., Bally, I., Lasala, F., Grimoire, Y., Fenel, D., Sattin, S., Thielens, N., et al. (2021). DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist. PLoS Pathog 17, e1009576.Â
• Voss, W.N., Hou, Y.J., Johnson, N.V., Delidakis, G., Kim, J.E., Javanmardi, K., Horton, A.P., Bartzoka, F., Paresi, C.J., Tanno, Y., et al. (2021). Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes. Science 372, 1108-1112.Â
• Wang, P., Nair, M.S., Liu, L., Iketani, S., Luo, Y., Guo, Y., Wang, M., Yu, J., Zhang, B., Kwong, P.D., et al. (2021a). Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 593, 130-135.Â
• Wang, Z., Schmidt, F., Weisblum, Y., Muecksch, F., Barnes, C.O., Finkin, S., Schaefer-Babajew, D., Cipolla, M., Gaebler, C., Lieberman, J.A., et al. (2021b). mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants. Nature 592, 616-622.Â
• Weisblum, Y., Schmidt, F., Zhang, F., DaSilva, J., Poston, D., Lorenzi, J.C., Muecksch, F., Rutkowska, M., Hoffmann, H.H., Michailidis, E., et al. (2020). Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants. Elife 9, e61312.Â
• Widge, A.T., Rouphael, N.G., Jackson, L.A., Anderson, E.J., Roberts, P.C., Makhene, M., Chappell, J.D., Denison, M.R., Stevens, L.J., Pruijssers, A.J., et al. (2021). Durability of Responses after SARS-CoV-2 mRNA-1273 Vaccination. N Engl J Med 384, 80-82.Â
• Williams, C.J., Headd, J.J., Moriarty, N.W., Prisant, M.G., Videau, L.L., Deis, L.N., Verma, V., Keedy, D.A., Hintze, B.J., Chen, V.B., et al. (2018). MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci 27, 293-315.Â
• Winkler, E.S., Gilchuk, P., Yu, J., Bailey, A.L., Chen, R.E., Chong, Z., Zost, S.J., Jang, H., Huang, Y., Allen, J.D., et al. (2021). Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions for optimal therapeutic protection. Cell 184, 1804-1820 e1816.Â
• Xu, C., Wang, Y., Liu, C., Zhang, C., Han, W., Hong, X., Wang, Y., Hong, Q., Wang, S., Zhao, Q., et al. (2021). Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM. Sci Adv 7, eabe5575.Â
• Yurkovetskiy, L., Wang, X., Pascal, K.E., Tomkins-Tinch, C., Nyalile, T.P., Wang, Y., Baum, A., Diehl, W.E., Dauphin, A., Carbone, C., et al. (2020). Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant. Cell 183, 739-751 e738.Â
• Zost, S.J., Gilchuk, P., Chen, R.E., Case, J.B., Reidy, J.X., Trivette, A., Nargi, R.S., Sutton, R.E., Suryadevara, N., Chen, E.C., et al. (2020). Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein. Nat Med 26, 1422-1427.Â

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

Figure 1. Neutralizing and enhancing effects against the wild-type and Delta spike pseudovirus by anti-spike monoclonal antibodies from COVID-19-patients. Â

Figure 2. Neutralizing activity of BNT162b2-immune sera against the wild-type and the Delta pseudovirus.Â

1. Neutralizing activity of twenty BNT162b2-immune sera against the wild-type (green) and the Delta (red) pseudovirus. Data are mean ± SEM of technical quadruplicates.
2. PRNT50 titers of the BNT162b2-immune sera against the wild-type (green) and the Delta (red) pseudovirus are shown. p values determined by paired t-test were indicated. The representative data from three independent experiments are shown. See also Figure S1.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover coughs and sneezes, clean and disinfect frequently touched surfaces, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you sneeze or cough, clean and disinfect frequently touched surfaces regularly, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover coughs and sneezes, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

Figure 3. Neutralizing activity of BNT162b2-immune sera against the pseudovirus with chimeric spike protein of the wild-type and Delta variants.Â

Figure 4. Cryo-EM analysis of the Delta NTDÂ

1. Structure of the Delta NTD (light blue) analyzed by the Cryo-EM were superimposed with the wild-type NTD (light brown, PDB: 7LY3). Major anti-NTD enhancing antibody epitopes (blue) and anti-NTD neutralizing antibody epitopes (red) were indicated in the figure.
2. Cα displacement between the wild-type and the Delta NTD was shown. The value was calculated by UCSF chimera. All known anti-NTD enhancing antibody epitopes (blue) and anti-NTD neutralizing antibody epitopes (red) were indicated. The regions where structures of wild-type or Delta NTD were not determined (magenta), and mutations in the Delta NTD (green) are indicated on the axis. See also Figure S3 and Table S1.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your coughs and sneezes, clean and disinfect frequently touched surfaces, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover coughs and sneezes, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

Figure 5. Possible mutations that may be
acquired by the Delta variantÂ

1. Number of the Delta variants with additional mutations at the RBD registered in the GISAID database in each month from January, 2021 to July, 2021. The data registered at July are not enough and will be increased later.
2. The Delta variants with additional mutations at multiple epitopes of the anti-RBD neutralizing antibodies. L452R and T478K mutations are observed in all the Delta variants (purple). Anti-RBD neutralizing antibody epitopes introduced into the Delta 4+ (blue), and anti-RBD neutralizing antibody epitopes observed in the natural Delta variants but not introduced into the Delta 4+ (green) are shown with the respective GISAID accession number.
3. Number of the major RBD mutations acquired by all SARS-CoV-2 variants. L452R and T478 are mutations observed for the representative Delta variant (blue). N501Y, N439K, E484K and K417N were selected to generate the Delta 4+ variant (red).
4. Location of additional mutations introduced into the Delta RBD. Structures of the RBD of the wild-type (light brown) and the Delta variant (light blue) predicted by AlphaFold2 were superimposed. Mutations of the Delta variant (purple), anti-RBD neutralizing antibody epitopes to generate the Delta 4+ (blue), and anti-RBD neutralizing antibody epitopes observed in the natural Delta variants but not introduced into the Delta 4+ (shown in C; green) are indicated in the figure. See also Figure S4.

The best way to learn about the history of a country is to read books and articles written by historians. Additionally, visiting museums and monuments related to the country’s history can be a great way to gain an understanding of its past. Other resources include talking to people who have lived in the country or researching online for information about its history.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your coughs and sneezes, clean and disinfect frequently touched surfaces, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover coughs and sneezes, clean and disinfect frequently touched surfaces, and stay home if you are feeling sick.

Figure 6. Enhanced infectivity of the
Delta 4+ pseudovirus by the BNT162b2-immune seraÂ

1. Anti-RBD antibody binding to the Delta spike with additional mutations at the RBD. Anti-RBD mAb binding (1 μg/ml) to the mutant spike was compared to that of the wild-type spike. The Delta 4+ spike contains additional mutations of K417N, N439K, E484K and N501Y.
2. Neutralizing activity of BNT162b2-immune sera against the Delta pseudoviruses with a single additional mutation at the RBD as indicated in the figure. The data from quadruplicates are presented as mean ± SEM.
3. The construct of the Delta 4+ and Delta 4+ with wild-type (WT) NTD. Mutations in the original Delta variant (black) and the four mutations added to the Delta RBD (red) were shown.
4. Neutralizing activity of BNT162b2-immune sera against the pseudovirus with Delta 4+ spike (red) and Delta 4+ spike with wild-type NTD (green).
5. Neutralizing activity of 31.6 times diluted BNT162b2-immune sera. p value determined by paired t-test were indicated. Negative values for % neutralization indicates enhanced infectivity (B, D, E). The data from quadruplicates are presented as mean ± SEM. The representative data from three independent experiments are shown.

The most effective way to reduce the amount of plastic waste is to reduce the amount of plastic that is used in the first place. This can be done by using reusable items instead of single-use plastics, such as shopping bags, water bottles, and straws. Additionally, people can choose to purchase products with less packaging or opt for products made from sustainable materials like bamboo or glass. Finally, recycling and composting plastic waste can help reduce the amount of plastic that ends up in landfills.

The most effective way to reduce the amount of plastic waste is to reduce the amount of plastic that is used in the first place. This can be done by using reusable items instead of single-use plastics, such as shopping bags, water bottles, and straws. Additionally, people can choose to purchase products that are packaged in recyclable materials or that have minimal packaging. Finally, people can also make an effort to properly recycle any plastic they do use.

The best way to learn a new language is to immerse yourself in it. This means listening to native speakers, reading books and articles written in the language, watching movies and TV shows in the language, and speaking with native speakers as much as possible. Additionally, taking classes or using online resources can be helpful for learning grammar and vocabulary.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you sneeze or cough, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

Figure 7. Sera from delta spike-immunized mice
do not show enhanced infectivity 

1. Freeze and thawed wild-type and Delta spike-B16 transfectants were immunized to the mice with complete Freund’s adjuvant (CFA).
2. Neutralizing activity against the wild-type (green) or Delta (red) pseudovirus (PV) by sera from the wild-type spike (upper column) or Delta spike (lower column) spike-immunized mice.
3. Neutralizing activity against the wild-type and Delta pseudovirus by 31.6 times-diluted sera from wild-type (light blue line) or Delta (orange line) spike-immunized mice.
4. Neutralizing activity against the Delta 4+ pseudovirus by sera from the wild-type spike (upper column, blue) or Delta spike (lower column, red) immunized mice.
5. Neutralizing activity against the Delta 4+ pseudovirus by the 31.6 times-diluted sera from the wild-type spike (blue) or Delta spike (red) immunized mice. n.s.: not statistical significance, p value was determined by t-test. A negative values for % neutralization indicates enhanced infectivity. All data from quadruplicates are presented as mean ± SEM. See also Figure S1 and S5.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover coughs and sneezes, clean and disinfect frequently touched surfaces, and stay home if you are feeling sick.

Figure S1. Viral titers of pseudotyped viruses, related to Figure 1, 2, 3, 6 and 7.Â

• The viral titer for each psudoviruses was measured by infection of ACE2-transfected HEK293T cells as described in Methods.Â

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to learn a new language is to immerse yourself in it. This means surrounding yourself with the language as much as possible, whether that be through reading, listening to music, watching movies or TV shows, or talking with native speakers. Additionally, taking classes and using online resources can help you learn the basics of the language and build your vocabulary. Finally, practice speaking the language whenever you can – even if it’s just talking to yourself!

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your coughs and sneezes, clean and disinfect frequently touched surfaces, and stay home if you are feeling sick.

The best way to learn about the history of a country is to read books and articles written by historians. Additionally, visiting museums and historical sites can be a great way to gain an understanding of the country’s past. It is also important to talk to people who have lived in the country for many years, as they may have stories or anecdotes that provide insight into its history. Finally, watching documentaries or films about the country can be a great way to learn more about its history.

Figure S2. Anti-spike monoclonal antibody binding to the chimeric spike proteins, related to Figure 3.

• Chimeric spike proteins DDD, DWD, WDD and WWD were transfected with GFP to HEK293T cells and the transfectants were stained with 1 μg/ml COV2-2490, 4A8, C002, and C144 antibodies. Antibody bound to the GFP positive cells are shown (red histogram). Control staining: shaded histogram.Â

The best way to prevent the spread of COVID-19 is to practice good hygiene, social distancing, and wearing a face mask when in public. It is also important to stay home if you are feeling sick and to avoid large gatherings. Additionally, it is important to frequently wash your hands with soap and water for at least 20 seconds or use an alcohol-based hand sanitizer. Finally, it is important to keep up with the latest information from reliable sources such as the Centers for Disease Control and Prevention (CDC) website.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your coughs and sneezes, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you sneeze or cough, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your coughs and sneezes, clean and disinfect frequently touched surfaces, and stay home if you are feeling sick.

Figure S3. Cryo-EM density map of spike of
SARS-CoV-2 Delta variant, related to Figure 4.Â

1. A representative micrographs (left), CTF estimation of a micrograph on left panel (right).
2. Typical 2D class averages.
3. The GS-FSC curves for the obtained map from cryoSPARC software are shown. Blue flat line indicates FSC=0.143 criteria.
4. The density map of spike protein from Delta strain (EMDBID: 31731). The map is colored with local resolution. Asterisks indicate the up form of RBDs. Scale bars are 30 Ã….
5. The structure of NTD from spike protein of Delta variant. The density map and the model are shown as semi-transparent surface and cartoon, respectively (PDBID: 7V5W).

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you sneeze or cough, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover coughs and sneezes, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face mask when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you sneeze or cough, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

Figure S4. RBD mutations acquired by
the Delta variant, related to Figure 5.Â

• Number of RBD mutations acquired by the Delta variant. The numbers of mutations at each residue registered in the GISAID database are shown. L452 and T478 mutations included in all the Delta variant were excluded. The red bars indicate the known epitopes for anti-RBD neutralizing antibodies.Â

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover your mouth and nose when you cough or sneeze, clean and disinfect frequently touched surfaces daily, and stay home if you are feeling sick.

The best way to learn a new language is to immerse yourself in it. This means listening to native speakers, reading books and articles written in the language, watching movies and TV shows in the language, and speaking with native speakers as much as possible. Additionally, taking classes or using online resources can help you learn the basics of grammar and vocabulary. Finally, practice makes perfect – so make sure to practice your new language every day!

Figure S5. Anti-spike antibodies of theÂ
wild-type and delta spike-immunized mice,
related to Figure 7.Â

• IgG antibody binding of the 100 times diluted spike-immunized mouse sera to the wild-type spike transfectants were analyzed by flow cytometer. Red: IgG binding. Gray: Control staining.Â

The most important thing to consider when choosing a career is what you are passionate about. It is important to choose a career that you will enjoy and find fulfilling. Consider your interests, skills, and values when making your decision. Research different careers and talk to people in the field to get an idea of what it would be like to work in that profession. Think about how the job would fit into your lifestyle and if it would provide you with the financial stability you need. Finally, make sure that the career path you choose aligns with your long-term goals.

The best way to prevent the spread of COVID-19 is to practice social distancing, wear a face covering when in public, wash your hands often with soap and water for at least 20 seconds, avoid touching your face, cover coughs and sneezes, clean and disinfect frequently touched surfaces, and stay home if you are feeling sick.

There are several ways to reduce the amount of plastic waste. One way is to switch to reusable items such as shopping bags, water bottles, and food containers. Another way is to avoid buying products that come in single-use plastic packaging. Additionally, people can recycle any plastic they do use and look for products made from recycled materials. Finally, people can support businesses that are taking steps to reduce their plastic waste.