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Prefusion-Stabilized Lassa Virus Trimer Identifies Neutralizing Nanobodies and Reveals an Apex-Situated Site of Vulnerability

Jason Gorman, Crystal Sao-Fong Cheung, Zhijian Duan, Yaping Sun, Pengfei Wang, Jeffrey C. Boyington, Andrea Biju, Tatsiana Bylund, Cheng Cheng, Li Ou, Tyler Stephens, Yaroslav Tsybovsky, Raffaello Verardi, Shuishu Wang, Yongping Yang, Baoshan Zhang, ChengYan Zheng, Tongqing Zhou, John R. Mascola, David D. Ho, View ORCID ProfileMitchell Ho, Peter D. Kwong
doi: https://doi.org/10.1101/2022.04.21.488985
Jason Gorman
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Crystal Sao-Fong Cheung
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Zhijian Duan
bNCI Antibody Engineering Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Yaping Sun
bNCI Antibody Engineering Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Pengfei Wang
cAaron Diamond AIDS Research Center, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032
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Jeffrey C. Boyington
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Andrea Biju
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Tatsiana Bylund
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Cheng Cheng
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Li Ou
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Tyler Stephens
dElectron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
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Yaroslav Tsybovsky
dElectron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
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Raffaello Verardi
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Shuishu Wang
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Yongping Yang
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Baoshan Zhang
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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ChengYan Zheng
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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Tongqing Zhou
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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John R. Mascola
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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David D. Ho
cAaron Diamond AIDS Research Center, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032
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  • For correspondence: dh2994@cumc.columbia.edu homi@mail.nih.gov pdkwong@nih.gov
Mitchell Ho
bNCI Antibody Engineering Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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  • ORCID record for Mitchell Ho
  • For correspondence: dh2994@cumc.columbia.edu homi@mail.nih.gov pdkwong@nih.gov
Peter D. Kwong
aVaccine Research Center, National Institutes of Health, Bethesda, MD 20892, USA
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  • For correspondence: dh2994@cumc.columbia.edu homi@mail.nih.gov pdkwong@nih.gov
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ABSTRACT

Lassa virus (LASV) is responsible for 100,000-300,000 zoonotic infections annually and poses a threat to public health. Development of antibody-based therapeutics or vaccines has been challenging because neutralizing antibodies – even among Lassa hemorrhagic fever survivors – are generally of low titer, and the target of neutralizing antibodies, the trimeric glycoprotein complex (GPC), a type 1-fusion machine with GP1 and GP2 subunits, has been difficult to produce. Here, we use structure-based design to obtain a soluble LASV GPC by engineering an inter-protomer disulfide (R207GCGP1-L326CGP2) and appending the T4-fibritin trimerization domain. We verified the antigenicity of this prefusion-stabilized LASV GPC against a panel of human antibodies and used electron microscopy (EM) to confirm its trimeric association. We panned the prefusion-stabilized LASV GPC against single domain ‘nanobody’ libraries and identified one of camel origin, which we named D5, which bound GPC with 27 nM affinity and neutralized the Josiah strain of LASV with an IC50 of 12 µg/ml when formatted into a bivalent IgG2a context. The cryo-EM structure of a ternary complex of the D5 nanobody, the antigen-binding fragment of human antibody 8.11G, and LASV GPC revealed D5 to recognize a site-of-vulnerability at the trimer apex. The recognized site appeared to be specific to GPC lacking cleavage of between GP1 and GP2 subunits. Collectively, our findings suggest that GPC-cleavage intermediates may be targets for LASV neutralization and define an apex-situated site of vulnerability for vaccine development.

Significance Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a biothreat to the world. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into requirements for antibody-mediated neutralization of LASV, we developed a prefusion-stabilized LASV glycoprotein trimer (GPC), panned it against phage libraries comprised single-domain antibodies or nanobodies from shark and camel, and identified one, D5, which – when placed into bivalent IgG2a context – could neutralize LASV. Cryo-EM analysis revealed D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. We propose this apex-situated site to be an attractive target for LASV vaccine and therapeutic development.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
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Posted April 21, 2022.
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Prefusion-Stabilized Lassa Virus Trimer Identifies Neutralizing Nanobodies and Reveals an Apex-Situated Site of Vulnerability
Jason Gorman, Crystal Sao-Fong Cheung, Zhijian Duan, Yaping Sun, Pengfei Wang, Jeffrey C. Boyington, Andrea Biju, Tatsiana Bylund, Cheng Cheng, Li Ou, Tyler Stephens, Yaroslav Tsybovsky, Raffaello Verardi, Shuishu Wang, Yongping Yang, Baoshan Zhang, ChengYan Zheng, Tongqing Zhou, John R. Mascola, David D. Ho, Mitchell Ho, Peter D. Kwong
bioRxiv 2022.04.21.488985; doi: https://doi.org/10.1101/2022.04.21.488985
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Prefusion-Stabilized Lassa Virus Trimer Identifies Neutralizing Nanobodies and Reveals an Apex-Situated Site of Vulnerability
Jason Gorman, Crystal Sao-Fong Cheung, Zhijian Duan, Yaping Sun, Pengfei Wang, Jeffrey C. Boyington, Andrea Biju, Tatsiana Bylund, Cheng Cheng, Li Ou, Tyler Stephens, Yaroslav Tsybovsky, Raffaello Verardi, Shuishu Wang, Yongping Yang, Baoshan Zhang, ChengYan Zheng, Tongqing Zhou, John R. Mascola, David D. Ho, Mitchell Ho, Peter D. Kwong
bioRxiv 2022.04.21.488985; doi: https://doi.org/10.1101/2022.04.21.488985

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