This page has only limited features, please log in for full access.
SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1–6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7–17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (K D = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 μg mL−1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.
Tingting Li; Hongmin Cai; Hebang Yao; Bingjie Zhou; Ning Zhang; Martje Fentener van Vlissingen; Thijs Kuiken; Wenyu Han; Corine H. GeurtsvanKessel; Yuhuan Gong; Yapei Zhao; Quan Shen; Wenming Qin; Xiao-Xu Tian; Chao Peng; Yanling Lai; Yanxing Wang; Cedric A. J. Hutter; Shu-Ming Kuo; Juan Bao; Caixuan Liu; Yifan Wang; Audrey S. Richard; Hervé Raoul; Jiaming Lan; Markus A. Seeger; Yao Cong; Barry Rockx; Gary Wong; Yuhai Bi; Dimitri Lavillette; Dianfan Li. A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection. Nature Communications 2021, 12, 1 -13.
AMA StyleTingting Li, Hongmin Cai, Hebang Yao, Bingjie Zhou, Ning Zhang, Martje Fentener van Vlissingen, Thijs Kuiken, Wenyu Han, Corine H. GeurtsvanKessel, Yuhuan Gong, Yapei Zhao, Quan Shen, Wenming Qin, Xiao-Xu Tian, Chao Peng, Yanling Lai, Yanxing Wang, Cedric A. J. Hutter, Shu-Ming Kuo, Juan Bao, Caixuan Liu, Yifan Wang, Audrey S. Richard, Hervé Raoul, Jiaming Lan, Markus A. Seeger, Yao Cong, Barry Rockx, Gary Wong, Yuhai Bi, Dimitri Lavillette, Dianfan Li. A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection. Nature Communications. 2021; 12 (1):1-13.
Chicago/Turabian StyleTingting Li; Hongmin Cai; Hebang Yao; Bingjie Zhou; Ning Zhang; Martje Fentener van Vlissingen; Thijs Kuiken; Wenyu Han; Corine H. GeurtsvanKessel; Yuhuan Gong; Yapei Zhao; Quan Shen; Wenming Qin; Xiao-Xu Tian; Chao Peng; Yanling Lai; Yanxing Wang; Cedric A. J. Hutter; Shu-Ming Kuo; Juan Bao; Caixuan Liu; Yifan Wang; Audrey S. Richard; Hervé Raoul; Jiaming Lan; Markus A. Seeger; Yao Cong; Barry Rockx; Gary Wong; Yuhai Bi; Dimitri Lavillette; Dianfan Li. 2021. "A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection." Nature Communications 12, no. 1: 1-13.
The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most of the currently approved SARS-CoV-2 vaccines use the prototype strain-derived spike (S) protein or its receptor-binding domain (RBD) as the vaccine antigen. The emergence of several novel SARS-CoV-2 variants has raised concerns about potential immune escape. In this study, we performed an immunogenicity comparison of prototype strain-derived RBD, S1, and S ectodomain trimer (S-trimer) antigens and evaluated their induction of neutralizing antibodies against three circulating SARS-CoV-2 variants, including B.1.1.7, B.1.351, and B.1.617.1. We found that, at the same antigen dose, the RBD and S-trimer vaccines were more potent than the S1 vaccine in eliciting long-lasting, high-titer broadly neutralizing antibodies in mice. The RBD immune sera remained highly effective against the B.1.1.7, B.1.351, and B.1.617.1 variants despite the corresponding neutralizing titers decreasing by 1.2-, 2.8-, and 3.5-fold relative to that against the wild-type strain. Significantly, the S-trimer immune sera exhibited comparable neutralization potency (less than twofold variation in neutralizing GMTs) towards the prototype strain and all three variants tested. These findings provide valuable information for further development of recombinant protein-based SARS-CoV-2 vaccines and support the continued use of currently approved SARS-CoV-2 vaccines in the regions/countries where variant viruses circulate.
Yong Yang; Jinkai Zang; Shiqi Xu; Xueyang Zhang; Sule Yuan; Haikun Wang; Dimitri Lavillette; Chao Zhang; Zhong Huang. Elicitation of Broadly Neutralizing Antibodies against B.1.1.7, B.1.351, and B.1.617.1 SARS-CoV-2 Variants by Three Prototype Strain-Derived Recombinant Protein Vaccines. Viruses 2021, 13, 1421 .
AMA StyleYong Yang, Jinkai Zang, Shiqi Xu, Xueyang Zhang, Sule Yuan, Haikun Wang, Dimitri Lavillette, Chao Zhang, Zhong Huang. Elicitation of Broadly Neutralizing Antibodies against B.1.1.7, B.1.351, and B.1.617.1 SARS-CoV-2 Variants by Three Prototype Strain-Derived Recombinant Protein Vaccines. Viruses. 2021; 13 (8):1421.
Chicago/Turabian StyleYong Yang; Jinkai Zang; Shiqi Xu; Xueyang Zhang; Sule Yuan; Haikun Wang; Dimitri Lavillette; Chao Zhang; Zhong Huang. 2021. "Elicitation of Broadly Neutralizing Antibodies against B.1.1.7, B.1.351, and B.1.617.1 SARS-CoV-2 Variants by Three Prototype Strain-Derived Recombinant Protein Vaccines." Viruses 13, no. 8: 1421.
A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.
Hebang Yao; Hongmin Cai; Tingting Li; Bingjie Zhou; Wenming Qin; Dimitri Lavillette; Dianfan Li. A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2. PLOS Pathogens 2021, 17, e1009328 .
AMA StyleHebang Yao, Hongmin Cai, Tingting Li, Bingjie Zhou, Wenming Qin, Dimitri Lavillette, Dianfan Li. A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2. PLOS Pathogens. 2021; 17 (3):e1009328.
Chicago/Turabian StyleHebang Yao; Hongmin Cai; Tingting Li; Bingjie Zhou; Wenming Qin; Dimitri Lavillette; Dianfan Li. 2021. "A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2." PLOS Pathogens 17, no. 3: e1009328.
The ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Neutralizing antibodies against SARS-CoV-2 are an option for drug development for treating COVID-19. Here, we report the identification and characterization of two groups of mouse neutralizing monoclonal antibodies (MAbs) targeting the receptor-binding domain (RBD) on the SARS-CoV-2 spike (S) protein. MAbs 2H2 and 3C1, representing the two antibody groups, respectively, bind distinct epitopes and are compatible in formulating a noncompeting antibody cocktail. A humanized version of the 2H2/3C1 cocktail is found to potently neutralize authentic SARS-CoV-2 infection in vitro with half inhibitory concentration (IC50) of 12 ng/mL and effectively treat SARS-CoV-2-infected mice even when administered at as late as 24 h post-infection. We determine an ensemble of cryo-EM structures of 2H2 or 3C1 Fab in complex with the S trimer up to 3.8 Å resolution, revealing the conformational space of the antigen–antibody complexes and MAb-triggered stepwise allosteric rearrangements of the S trimer, delineating a previously uncharacterized dynamic process of coordinated binding of neutralizing antibodies to the trimeric S protein. Our findings provide important information for the development of MAb-based drugs for preventing and treating SARS-CoV-2 infections.
Chao Zhang; Yifan Wang; Yuanfei Zhu; Caixuan Liu; Chenjian Gu; Shiqi Xu; Yalei Wang; Yu Zhou; Yanxing Wang; Wenyu Han; Xiaoyu Hong; Yong Yang; Xueyang Zhang; Tingfeng Wang; Cong Xu; Qin Hong; Shutian Wang; Qiaoyu Zhao; Weihua Qiao; Jinkai Zang; Liangliang Kong; Fangfang Wang; Haikun Wang; Di Qu; Dimitri Lavillette; Hong Tang; Qiang Deng; Youhua Xie; Yao Cong; Zhong Huang. Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections. Nature Communications 2021, 12, 1 -16.
AMA StyleChao Zhang, Yifan Wang, Yuanfei Zhu, Caixuan Liu, Chenjian Gu, Shiqi Xu, Yalei Wang, Yu Zhou, Yanxing Wang, Wenyu Han, Xiaoyu Hong, Yong Yang, Xueyang Zhang, Tingfeng Wang, Cong Xu, Qin Hong, Shutian Wang, Qiaoyu Zhao, Weihua Qiao, Jinkai Zang, Liangliang Kong, Fangfang Wang, Haikun Wang, Di Qu, Dimitri Lavillette, Hong Tang, Qiang Deng, Youhua Xie, Yao Cong, Zhong Huang. Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections. Nature Communications. 2021; 12 (1):1-16.
Chicago/Turabian StyleChao Zhang; Yifan Wang; Yuanfei Zhu; Caixuan Liu; Chenjian Gu; Shiqi Xu; Yalei Wang; Yu Zhou; Yanxing Wang; Wenyu Han; Xiaoyu Hong; Yong Yang; Xueyang Zhang; Tingfeng Wang; Cong Xu; Qin Hong; Shutian Wang; Qiaoyu Zhao; Weihua Qiao; Jinkai Zang; Liangliang Kong; Fangfang Wang; Haikun Wang; Di Qu; Dimitri Lavillette; Hong Tang; Qiang Deng; Youhua Xie; Yao Cong; Zhong Huang. 2021. "Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections." Nature Communications 12, no. 1: 1-16.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: Spike (S), Envelope (E), Membrane (M) and Nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of β-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected vs. transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is re-localized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon co-expression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M and N are required for optimal production of virus- like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.
Bertrand Boson; Vincent Legros; Bingjie Zhou; Eglantine Siret; Cyrille Mathieu; François-Loïc Cosset; Dimitri Lavillette; Solène Denolly. The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles. Journal of Biological Chemistry 2021, 296, 100111 -100111.
AMA StyleBertrand Boson, Vincent Legros, Bingjie Zhou, Eglantine Siret, Cyrille Mathieu, François-Loïc Cosset, Dimitri Lavillette, Solène Denolly. The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles. Journal of Biological Chemistry. 2021; 296 ():100111-100111.
Chicago/Turabian StyleBertrand Boson; Vincent Legros; Bingjie Zhou; Eglantine Siret; Cyrille Mathieu; François-Loïc Cosset; Dimitri Lavillette; Solène Denolly. 2021. "The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles." Journal of Biological Chemistry 296, no. : 100111-100111.
A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research have been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.
Hebang Yao; Hongmin Cai; Tingting Li; Bingjie Zhou; Wenming Qin; Dimitri Lavillette; Dianfan Li. A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2. 2020, 1 .
AMA StyleHebang Yao, Hongmin Cai, Tingting Li, Bingjie Zhou, Wenming Qin, Dimitri Lavillette, Dianfan Li. A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2. . 2020; ():1.
Chicago/Turabian StyleHebang Yao; Hongmin Cai; Tingting Li; Bingjie Zhou; Wenming Qin; Dimitri Lavillette; Dianfan Li. 2020. "A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2." , no. : 1.
SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.
Dianfan Li; Tingting Li; Hongmin Cai; Hebang Yao; Bingjie Zhou; Ning Zhang; Yuhuan Gong; Yapei Zhao; Quan Shen; Wenming Qin; Cedric Hutter; Yanling Lai; Shu-Ming Kuo; Juan Bao; Jiaming Lan; Markus Seeger; Gary Wong; Yuhai Bi; Dimitri Lavillette. A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection. 2020, 1 .
AMA StyleDianfan Li, Tingting Li, Hongmin Cai, Hebang Yao, Bingjie Zhou, Ning Zhang, Yuhuan Gong, Yapei Zhao, Quan Shen, Wenming Qin, Cedric Hutter, Yanling Lai, Shu-Ming Kuo, Juan Bao, Jiaming Lan, Markus Seeger, Gary Wong, Yuhai Bi, Dimitri Lavillette. A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection. . 2020; ():1.
Chicago/Turabian StyleDianfan Li; Tingting Li; Hongmin Cai; Hebang Yao; Bingjie Zhou; Ning Zhang; Yuhuan Gong; Yapei Zhao; Quan Shen; Wenming Qin; Cedric Hutter; Yanling Lai; Shu-Ming Kuo; Juan Bao; Jiaming Lan; Markus Seeger; Gary Wong; Yuhai Bi; Dimitri Lavillette. 2020. "A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection." , no. : 1.
Jinkai Zang; Chenjian Gu; Bingjie Zhou; Chao Zhang; Yong Yang; Shiqi Xu; Lulu Bai; Rong Zhang; Qiang Deng; Zhenghong Yuan; Hong Tang; Di Qu; Dimitri Lavillette; Youhua Xie; Zhong Huang. Immunization with the receptor-binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement. Cell Discovery 2020, 6, 1 -4.
AMA StyleJinkai Zang, Chenjian Gu, Bingjie Zhou, Chao Zhang, Yong Yang, Shiqi Xu, Lulu Bai, Rong Zhang, Qiang Deng, Zhenghong Yuan, Hong Tang, Di Qu, Dimitri Lavillette, Youhua Xie, Zhong Huang. Immunization with the receptor-binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement. Cell Discovery. 2020; 6 (1):1-4.
Chicago/Turabian StyleJinkai Zang; Chenjian Gu; Bingjie Zhou; Chao Zhang; Yong Yang; Shiqi Xu; Lulu Bai; Rong Zhang; Qiang Deng; Zhenghong Yuan; Hong Tang; Di Qu; Dimitri Lavillette; Youhua Xie; Zhong Huang. 2020. "Immunization with the receptor-binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement." Cell Discovery 6, no. 1: 1-4.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins. The involvement of each of these proteins and their interplays during the assembly process of this new virus are poorly-defined and are likely β-coronavirus-type different. Therefore, we sought to investigate how SARS-CoV-2 behaves for its assembly by expression assays of S, in combination with E, M and/or N. By combining biochemical and imaging assays, we showed that E and M regulate intracellular trafficking of S and hence its furin-mediated processing. Indeed, our imaging data revealed that S remains at ERGIC or Golgi compartments upon expression of E or M, like for SARS-CoV-2 infected cells. By studying a mutant of S, we showed that its cytoplasmic tail, and more specifically, its C-terminal retrieval motif, is required for the M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlighted that E and M induce a specific maturation of S N-glycosylation, which is observed on particles and lysates from infected cells independently of its mechanisms of intracellular retention. Finally, we showed that both M, E and N are required for optimal production of virus-like-proteins. Altogether, our results indicated that E and M proteins influence the properties of S proteins to promote assembly of viral particles. Our results therefore highlight both similarities and dissimilarities in these events, as compared to other β-coronaviruses. Author Summary The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Its viral particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins, though their involvement in the virion assembly remain unknown for this particular coronavirus. Here we showed that presence of E and M influence the localization and maturation of S protein, in term of cleavage and N-glycosylation maturation. Indeed, E protein is able to slow down the cell secretory pathway whereas M-induced retention of S requires the retrieval motif in S C-terminus. We also highlighted that E and M might regulate the N glycosylation maturation of S independently of its intracellular retention mechanism. Finally, we showed that the four structural proteins are required for optimal formation of virus-like particles, highlighting the involvement of N, E and M in assembly of infectious particles. Altogether, our results highlight both similarities and dissimilarities in these events, as compared to other β-coronaviruses.
Bertrand Boson; Vincent Legros; Bingjie Zhou; Cyrille Mathieu; François-Loïc Cosset; Dimitri Lavillette; Solène Denolly. The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein. 2020, 1 .
AMA StyleBertrand Boson, Vincent Legros, Bingjie Zhou, Cyrille Mathieu, François-Loïc Cosset, Dimitri Lavillette, Solène Denolly. The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein. . 2020; ():1.
Chicago/Turabian StyleBertrand Boson; Vincent Legros; Bingjie Zhou; Cyrille Mathieu; François-Loïc Cosset; Dimitri Lavillette; Solène Denolly. 2020. "The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein." , no. : 1.
SARS-CoV-2, the Covid-19 causative virus, adheres to human cells through binding of its envelope Spike protein to the receptor ACE2. The Spike receptor-binding domain (S-RBD) mediates this key event and thus is a primary target for therapeutic neutralizing antibodies to mask the ACE2-interacting interface. Here, we generated 99 synthetic nanobodies (sybodies) using ribosome and phage display. The best sybody MR3 binds the RBD with KD of 1.0 nM and neutralizes SARS-CoV-2 pseudovirus with IC50 of 0.40 μg mL-1. Crystal structures of two sybody-RBD complexes reveal a common neutralizing mechanism through which the RBD-ACE2 interaction is competitively inhibited by sybodies. The structures allowed the rational design of a mutant with higher affinity and improved neutralization efficiency by ∼24-folds, lowering the IC50 from 12.32 to 0.50 μg mL-1. Further, the structures explain the selectivity of sybodies between SARS-CoV strains. Our work presents an alternative approach to generate neutralizers against newly emerged viruses.One sentence summaryStructural and biochemical studies revealed the molecular basis for the neutralization mechanism of in vitro-selected and rationally designed nanobody neutralizers for SARS-CoV-2 pseudovirus.
Tingting Li; Hongmin Cai; Hebang Yao; Bingjie Zhou; Yapei Zhao; Wenming Qin; Cedric A.J. Hutter; Yanling Lai; Juan Bao; Jiaming Lan; Gary Wong; Markus Seeger; Dimitri Lavillette; Dianfan Li. Potent synthetic nanobodies against SARS-CoV-2 and molecular basis for neutralization. bioRxiv 2020, 1 .
AMA StyleTingting Li, Hongmin Cai, Hebang Yao, Bingjie Zhou, Yapei Zhao, Wenming Qin, Cedric A.J. Hutter, Yanling Lai, Juan Bao, Jiaming Lan, Gary Wong, Markus Seeger, Dimitri Lavillette, Dianfan Li. Potent synthetic nanobodies against SARS-CoV-2 and molecular basis for neutralization. bioRxiv. 2020; ():1.
Chicago/Turabian StyleTingting Li; Hongmin Cai; Hebang Yao; Bingjie Zhou; Yapei Zhao; Wenming Qin; Cedric A.J. Hutter; Yanling Lai; Juan Bao; Jiaming Lan; Gary Wong; Markus Seeger; Dimitri Lavillette; Dianfan Li. 2020. "Potent synthetic nanobodies against SARS-CoV-2 and molecular basis for neutralization." bioRxiv , no. : 1.
Zika virus (ZIKV) infection is a serious public health concern due to its ability to induce neurological defects and its potential for rapid transmission at a global scale. However, no vaccine is currently available to prevent ZIKV infection. Here, we report the development of a yeast-derived subunit protein vaccine for ZIKV. The envelope protein domain III (EDIII) of ZIKV was produced as a secretory protein in the yeast Pichia pastoris. The yeast-derived EDIII could inhibit ZIKV infection in vitro in a dose-dependent manner, suggesting that it had acquired an appropriate conformation to bind to cellular receptors of ZIKV. Immunization with recombinant EDIII protein effectively induced antigen-specific binding antibodies and cellular immune responses. The resulting anti-EDIII sera could efficiently neutralize ZIKV representative strains from both Asian and African lineages. Passive transfer with the anti-EDIII neutralizing sera could confer protection against lethal ZIKV challenge in mice. Importantly, we found that purified anti-EDIII antibodies did not cross-react with closely related dengue virus (DENV) and therefore did not enhance DENV infection. Collectively, our results demonstrate that yeast-produced EDIII is a safe and effective ZIKV vaccine candidate.
Wei Zhang; Panke Qu; Dapeng Li; Chao Zhang; Qingwei Liu; Gang Zou; Myrielle Dupont-Rouzeyrol; Dimitri Lavillette; Xia Jin; Feifei Yin; Zhong Huang. Yeast-produced subunit protein vaccine elicits broadly neutralizing antibodies that protect mice against Zika virus lethal infection. Antiviral Research 2019, 170, 104578 .
AMA StyleWei Zhang, Panke Qu, Dapeng Li, Chao Zhang, Qingwei Liu, Gang Zou, Myrielle Dupont-Rouzeyrol, Dimitri Lavillette, Xia Jin, Feifei Yin, Zhong Huang. Yeast-produced subunit protein vaccine elicits broadly neutralizing antibodies that protect mice against Zika virus lethal infection. Antiviral Research. 2019; 170 ():104578.
Chicago/Turabian StyleWei Zhang; Panke Qu; Dapeng Li; Chao Zhang; Qingwei Liu; Gang Zou; Myrielle Dupont-Rouzeyrol; Dimitri Lavillette; Xia Jin; Feifei Yin; Zhong Huang. 2019. "Yeast-produced subunit protein vaccine elicits broadly neutralizing antibodies that protect mice against Zika virus lethal infection." Antiviral Research 170, no. : 104578.
The incidence of chikungunya virus (CHIKV) infection has increased dramatically in recent decades. Effective diagnostic methods must be available to optimize patient management. IgM-capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) is routinely used for the detection of specific CHIKV IgM. This method requires inactivated CHIKV viral lysate (VL). The use of viral bioparticles such as Virus-Like Particles (VLPs) and Pseudotyped-Particles (PPs) could represent an alternative to VL. Bioparticles performances were established by MAC-ELISA; physico-chemical characterizations were performed by field-flow fractionation (HF5) and confirmed by electron microscopy. Non-purified PPs give a detection signal higher than for VL. Results suggested that the signal difference observed in MAC-ELISA was probably due to the intrinsic antigenic properties of particles. The use of CHIKV bioparticles such as VLPs and PPs represents an attractive alternative to VL. Compared to VL and VLPs, non-purified PPs have proven to be more powerful antigens for specific IgM capture.
Gérald Theillet; Jérôme Martinez; Christophe Steinbrugger; Dimitri Lavillette; Bruno Coutard; Nicolas Papageorgiou; Pascal Dalbon; Isabelle Leparc-Goffart; Frédéric Bedin. Comparative study of chikungunya Virus-Like Particles and Pseudotyped-Particles used for serological detection of specific immunoglobulin M. Virology 2019, 529, 195 -204.
AMA StyleGérald Theillet, Jérôme Martinez, Christophe Steinbrugger, Dimitri Lavillette, Bruno Coutard, Nicolas Papageorgiou, Pascal Dalbon, Isabelle Leparc-Goffart, Frédéric Bedin. Comparative study of chikungunya Virus-Like Particles and Pseudotyped-Particles used for serological detection of specific immunoglobulin M. Virology. 2019; 529 ():195-204.
Chicago/Turabian StyleGérald Theillet; Jérôme Martinez; Christophe Steinbrugger; Dimitri Lavillette; Bruno Coutard; Nicolas Papageorgiou; Pascal Dalbon; Isabelle Leparc-Goffart; Frédéric Bedin. 2019. "Comparative study of chikungunya Virus-Like Particles and Pseudotyped-Particles used for serological detection of specific immunoglobulin M." Virology 529, no. : 195-204.
ZIKV has emerged as a significant human pathogene for the severe neurological complications, including Guillain-Barré(GBS) syndrome in adults and a variety of fetal abnormalities such as microcephaly. A stable and efficient infectious clone of Brazilian ZIKV isolate is required to study pathogenesis of epidemic ZIKV and virus evolution impact on it. Here we successfully constructed infectious cDNA clone on an early Brazilian isolate by eliminating the activity of predicted bacterial promoter in 1–3000 nt of ZIKV genome, leading to a stable infectious cDNA clone (pZL1). pZL1 derived virus could infect different cell lines and cause lethal effect to AG6 mice. We further investigated the role of a recent emerged substitution in NS5 (M2634V). We found that a reverse mutation (V2634M) caused negligible effect on the ZIKV viral genome replication and infectious progeny production in multiple cell culture systems. Additionally, this mutation did not alter the pathogenesis feature and virulence of ZIKV in AG6 mice. In summary, our results present another robust infectious ZIKV clone from Brazilian isolate and provide evidences to support that M2634V single mutation did not alter virus life cycle in cell culture and pathogenesis in AG6 mouse model.
Fanfan Zhao; Yongfen Xu; Dimitri Lavillette; Jin Zhong; Gang Zou; Gang Long. Negligible contribution of M2634V substitution to ZIKV pathogenesis in AG6 mice revealed by a bacterial promoter activity reduced infectious clone. Scientific Reports 2018, 8, 1 -12.
AMA StyleFanfan Zhao, Yongfen Xu, Dimitri Lavillette, Jin Zhong, Gang Zou, Gang Long. Negligible contribution of M2634V substitution to ZIKV pathogenesis in AG6 mice revealed by a bacterial promoter activity reduced infectious clone. Scientific Reports. 2018; 8 (1):1-12.
Chicago/Turabian StyleFanfan Zhao; Yongfen Xu; Dimitri Lavillette; Jin Zhong; Gang Zou; Gang Long. 2018. "Negligible contribution of M2634V substitution to ZIKV pathogenesis in AG6 mice revealed by a bacterial promoter activity reduced infectious clone." Scientific Reports 8, no. 1: 1-12.
Hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer to constitute viral envelope proteins, which play an essential role in virus entry. E1 does not directly interact with host receptors, and its functions in viral entry are exerted mostly through its interaction with E2 that directly binds the receptors. HCV enters the host cell via receptor-mediated endocytosis during which the fusion of viral and host endosomal membranes occurs to release viral genome to cytoplasm. A putative fusion peptide in E1 has been proposed to participate in membrane fusion, but its exact role and underlying molecular mechanisms remain to be deciphered. Recently solved crystal structures of the E2 ectodomains and N-terminal of E1 fail to reveal a classical fusion-like structure in HCV envelope glycoproteins. In addition, accumulating evidence suggests that E1 also plays an important role in virus assembly. In this mini-review, we summarize current knowledge on HCV E1 including its structure and biological functions in virus entry, fusion, and assembly, which may provide clues for developing HCV vaccines and more effective antivirals.
Yimin Tong; Dimitri Lavillette; Qingchao Li; Jin Zhong. Role of Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly. Frontiers in Immunology 2018, 9, 1411 .
AMA StyleYimin Tong, Dimitri Lavillette, Qingchao Li, Jin Zhong. Role of Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly. Frontiers in Immunology. 2018; 9 ():1411.
Chicago/Turabian StyleYimin Tong; Dimitri Lavillette; Qingchao Li; Jin Zhong. 2018. "Role of Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly." Frontiers in Immunology 9, no. : 1411.
Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses. Several virus-mediated molecular processes remain poorly described, which dampen the development of potent anti-viral therapies. Hence, new experimental strategies need to be undertaken to improve and accelerate our understanding of these processes. Here, as a proof of concept, we employ amino-acid coevolution as a tool to gain insights into the structural rearrangements of Hepatitis C Virus (HCV) envelope glycoproteins E1 and E2 during virus fusion with the cell membrane, and provide a basis for the inhibition of this process. Our coevolution analysis predicted that a specific domain of E2, the Back Layer (BL) is involved into significant conformational changes with E1 during the fusion of the HCV membrane with the cellular membrane. Consistently, a recombinant, soluble form of the BL was able to inhibit E1E2 fusogenic rearrangements and HCV infection. Moreover, predicted coevolution networks involving E1 and BL residues, as well as E1 and BL-adjacent residues, were found to modulate virus fusion. Our data shows that coevolution analysis is a powerful and underused approach that can provide significant insights into the functions and structural rearrangements of viral proteins. Importantly, this approach can also provide structural and molecular basis for the design of effective anti-viral drugs, and opens new perspectives to rapidly identify effective antiviral strategies against emerging and re-emerging viral pathogens.
Florian Douam; Floriane Fusil; Margot Enguehard; Linda Dib; Francesca Nadalin; Loïc Schwaller; Gabriela Hrebikova; Jimmy Mancip; Laurent Mailly; Roland Montserret; Qiang Ding; Carine Maisse; Emilie Carlot; Ke Xu; Els Verhoeyen; Thomas F. Baumert; Alexander Ploss; Alessandra Carbone; François-Loïc Cosset; Dimitri Lavillette. A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism. PLOS Pathogens 2018, 14, e1006908 .
AMA StyleFlorian Douam, Floriane Fusil, Margot Enguehard, Linda Dib, Francesca Nadalin, Loïc Schwaller, Gabriela Hrebikova, Jimmy Mancip, Laurent Mailly, Roland Montserret, Qiang Ding, Carine Maisse, Emilie Carlot, Ke Xu, Els Verhoeyen, Thomas F. Baumert, Alexander Ploss, Alessandra Carbone, François-Loïc Cosset, Dimitri Lavillette. A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism. PLOS Pathogens. 2018; 14 (3):e1006908.
Chicago/Turabian StyleFlorian Douam; Floriane Fusil; Margot Enguehard; Linda Dib; Francesca Nadalin; Loïc Schwaller; Gabriela Hrebikova; Jimmy Mancip; Laurent Mailly; Roland Montserret; Qiang Ding; Carine Maisse; Emilie Carlot; Ke Xu; Els Verhoeyen; Thomas F. Baumert; Alexander Ploss; Alessandra Carbone; François-Loïc Cosset; Dimitri Lavillette. 2018. "A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism." PLOS Pathogens 14, no. 3: e1006908.
The histone demethylase LSD1 has been known as a key transcriptional coactivator for DNA viruses such as herpes virus. Inhibition of LSD1 was found to block viral genome transcription and lytic replication of DNA viruses. However, RNA virus genomes do not rely on chromatin structure and histone association, and the role of demethylase activity of LSD1 in RNA virus infections is not anticipated. Here, we identify that, contrary to its role in enhancing DNA virus replication, LSD1 limits RNA virus replication by demethylating and activating IFITM3 which is a host restriction factor for many RNA viruses. We have found that LSD1 is recruited to demethylate IFITM3 at position K88 under IFNα treatment. However, infection by either Vesicular Stomatitis Virus (VSV) or Influenza A Virus (IAV) triggers methylation of IFITM3 by promoting its disassociation from LSD1. Accordingly, inhibition of the enzymatic activity of LSD1 by Trans-2-phenylcyclopropylamine hydrochloride (TCP) increases IFITM3 monomethylation which leads to more severe disease outcomes in IAV-infected mice. In summary, our findings highlight the opposite role of LSD1 in fighting RNA viruses comparing to DNA viruses infection. Our data suggest that the demethylation of IFITM3 by LSD1 is beneficial for the host to fight against RNA virus infection. The viral genomes of DNA viruses but not RNA viruses form chromatin structure during infection. Thus, epigenetic modulators are not expected to have crucial roles in RNA viral infection. However, here, we identify for the first time, that, opposite to its role in enhancing DNA virus replication, LSD1, a histone demethylase, limits RNA virus replication. We show that, under IFNα treatment, LSD1 is involved in the demethylation of IFITM3, a well-known host restriction factor for many RNA viruses. To counteract IFITM3 activation by demethylation, several RNA viruses, such as VSV and IAV, but not Zika virus, have developed strategy to inactive IFITM3 by promoting its dissociation from LSD1. In agreement with our findings, the inhibition of the enzymatic activity of LSD1 by small molecule leads to more severe disease outcomes in IAV-infected mice. Our data suggest that although LSD1 inhibitor is beneficial for treating DNA virus infection, it could be harmful to the host suffering from RNA virus infection. On the contrary, developing strategies to stimulate LSD1 activity to demethylate of IFITM3 is essential to fight RNA viruses.
Jiaoyu Shan; Binbin Zhao; Zhao Shan; Jia Nie; Rong Deng; Rui Xiong; Andy Tsun; Weiqi Pan; Hanzhi Zhao; Ling Chen; Ying Jin; Zhikang Qian; KaWing Lui; Rui Liang; Dan Li; Bing Sun; Dimitri Lavillette; Ke Xu; Bin Li. Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation. PLOS Pathogens 2017, 13, e1006773 .
AMA StyleJiaoyu Shan, Binbin Zhao, Zhao Shan, Jia Nie, Rong Deng, Rui Xiong, Andy Tsun, Weiqi Pan, Hanzhi Zhao, Ling Chen, Ying Jin, Zhikang Qian, KaWing Lui, Rui Liang, Dan Li, Bing Sun, Dimitri Lavillette, Ke Xu, Bin Li. Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation. PLOS Pathogens. 2017; 13 (12):e1006773.
Chicago/Turabian StyleJiaoyu Shan; Binbin Zhao; Zhao Shan; Jia Nie; Rong Deng; Rui Xiong; Andy Tsun; Weiqi Pan; Hanzhi Zhao; Ling Chen; Ying Jin; Zhikang Qian; KaWing Lui; Rui Liang; Dan Li; Bing Sun; Dimitri Lavillette; Ke Xu; Bin Li. 2017. "Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation." PLOS Pathogens 13, no. 12: e1006773.
Arthropod-borne virus (arbovirus) infections cause several emerging and resurgent infectious diseases in humans and animals. Chikungunya-affected areas often overlap with dengue-endemic areas. Concurrent dengue virus (DENV) and chikungunya virus (CHIKV) infections have been detected in travelers returning from regions of endemicity. CHIKV and DENV co-infected Aedes albopictus have also been collected in the vicinity of co-infected human cases, emphasizing the need to study co-infections in mosquitoes. We thus aimed to study the pathogen-pathogen interaction involved in these co-infections in DENV/CHIKV co-infected Aedes aegypti mosquitoes. In mono-infections, we detected CHIKV antigens as early as 4 days post-virus exposure in both the midgut (MG) and salivary gland (SG), whereas we detected DENV serotype 2 (DENV-2) antigens from day 5 post-virus exposure in MG and day 10 post-virus exposure in SG. Identical infection rates were observed for singly and co-infected mosquitoes, and facilitation of the replication of both viruses at various times post-viral exposure. We observed a higher replication for DENV-2 in SG of co-infected mosquitoes. We showed that mixed CHIKV and DENV infection facilitated viral replication in Ae. aegypti. The outcome of these mixed infections must be further studied to increase our understanding of pathogen-pathogen interactions in host cells.
Alain Le Coupanec; Stéphane Tchankouo-Nguetcheu; Pascal Roux; Huot Khun; Michel Huerre; Ronald Morales-Vargas; Margot Enguehard; Dimitri Lavillette; Dorothée Missé; Valérie Choumet. Co-Infection of Mosquitoes with Chikungunya and Dengue Viruses Reveals Modulation of the Replication of Both Viruses in Midguts and Salivary Glands of Aedes aegypti Mosquitoes. International Journal of Molecular Sciences 2017, 18, 1708 .
AMA StyleAlain Le Coupanec, Stéphane Tchankouo-Nguetcheu, Pascal Roux, Huot Khun, Michel Huerre, Ronald Morales-Vargas, Margot Enguehard, Dimitri Lavillette, Dorothée Missé, Valérie Choumet. Co-Infection of Mosquitoes with Chikungunya and Dengue Viruses Reveals Modulation of the Replication of Both Viruses in Midguts and Salivary Glands of Aedes aegypti Mosquitoes. International Journal of Molecular Sciences. 2017; 18 (8):1708.
Chicago/Turabian StyleAlain Le Coupanec; Stéphane Tchankouo-Nguetcheu; Pascal Roux; Huot Khun; Michel Huerre; Ronald Morales-Vargas; Margot Enguehard; Dimitri Lavillette; Dorothée Missé; Valérie Choumet. 2017. "Co-Infection of Mosquitoes with Chikungunya and Dengue Viruses Reveals Modulation of the Replication of Both Viruses in Midguts and Salivary Glands of Aedes aegypti Mosquitoes." International Journal of Molecular Sciences 18, no. 8: 1708.
Hepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCV JFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders. IMPORTANCE Hepatitis C virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytes in vitro and in vivo , highlighting the existence of lymphotropism determinants within HCV viral proteins. We isolated HCV envelope glycoproteins from patient B lymphocytes that conferred to a nonlymphotropic HCV the ability to enter B cells, thus providing a platform for characterization of HCV entry into lymphocytes. This unusual tropism was accompanied by a loss of entry function into hepatocytes, suggesting that HCV lymphotropic variants likely constitute a distinct but parallel source for viral persistence and immune escape within chronically infected patients. Moreover, the level of genetic divergence of B-cell-derived envelopes correlated with their degree of lymphotropism, underlining a long-term specialization of some viral populations for B-lymphocytes. Consequently, the clearance of both hepatotropic and nonhepatotropic HCV populations may be important for effective treatment of chronically infected patients.
Florian Douam; Louis-Marie Bobay; Guillemette Maurin; Judith Fresquet; Noémie Calland; Carine Maisse; Tony Durand; François-Loïc Cosset; Cyrille Féray; Dimitri Lavillette. Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients. Journal of Virology 2016, 90, 992 -1008.
AMA StyleFlorian Douam, Louis-Marie Bobay, Guillemette Maurin, Judith Fresquet, Noémie Calland, Carine Maisse, Tony Durand, François-Loïc Cosset, Cyrille Féray, Dimitri Lavillette. Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients. Journal of Virology. 2016; 90 (2):992-1008.
Chicago/Turabian StyleFlorian Douam; Louis-Marie Bobay; Guillemette Maurin; Judith Fresquet; Noémie Calland; Carine Maisse; Tony Durand; François-Loïc Cosset; Cyrille Féray; Dimitri Lavillette. 2016. "Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients." Journal of Virology 90, no. 2: 992-1008.
Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCE Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts.
Caroline Marnata; Aure Saulnier; Dimitri Mompelat; Thomas Krey; Lisette Cohen; Célia Boukadida; Lucile Warter; Judith Fresquet; Ieva Vasiliauskaite; Nicolas Escriou; François-Loïc Cosset; Felix A. Rey; Robert E. Lanford; Peter Karayiannis; Nicola J. Rose; Dimitri Lavillette; Annette Martin. Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction. Journal of Virology 2015, 89, 12131 -12144.
AMA StyleCaroline Marnata, Aure Saulnier, Dimitri Mompelat, Thomas Krey, Lisette Cohen, Célia Boukadida, Lucile Warter, Judith Fresquet, Ieva Vasiliauskaite, Nicolas Escriou, François-Loïc Cosset, Felix A. Rey, Robert E. Lanford, Peter Karayiannis, Nicola J. Rose, Dimitri Lavillette, Annette Martin. Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction. Journal of Virology. 2015; 89 (23):12131-12144.
Chicago/Turabian StyleCaroline Marnata; Aure Saulnier; Dimitri Mompelat; Thomas Krey; Lisette Cohen; Célia Boukadida; Lucile Warter; Judith Fresquet; Ieva Vasiliauskaite; Nicolas Escriou; François-Loïc Cosset; Felix A. Rey; Robert E. Lanford; Peter Karayiannis; Nicola J. Rose; Dimitri Lavillette; Annette Martin. 2015. "Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction." Journal of Virology 89, no. 23: 12131-12144.
The development of lentiviral vectors (LVs) for expression of a specific antibody can be achieved through the transduction of mature B-cells. This approach would provide a versatile tool for active immunotherapy strategies for infectious diseases or cancer, as well as for protein engineering. Here, we created a lentiviral expression system mimicking the natural production of these two distinct immunoglobulin isoforms. We designed a LV (FAM2-LV) expressing an anti-HCV-E2 surface glycoprotein antibody (AR3A) as a membrane-anchored Ig form or a soluble Ig form, depending on the B-cell maturation status. FAM2-LV induced high-level and functional membrane expression of the transgenic antibody in a nonsecretory B-cell line. In contrast, a plasma cell (PC) line transduced with FAM2-LV preferentially produced the secreted transgenic antibody. Similar results were obtained with primary B-cells transduced ex vivo. Most importantly, FAM2-LV transduced primary B-cells efficiently differentiated into PCs, which secreted the neutralizing anti-HCV E2 antibody upon adoptive transfer into immunodeficient NSG (NOD/SCIDγc(-/-)) recipient mice. Altogether, these results demonstrate that the conditional FAM2-LV allows preferential expression of the membrane-anchored form of an antiviral neutralizing antibody in B-cells and permits secretion of a soluble antibody following B-cell maturation into PCs in vivo.
Floriane Fusil; Sara Calattini; Fouzia Amirache; Jimmy Mancip; Caroline Costa; Justin B Robbins; Florian Douam; Dimitri Lavillette; Mansun Law; Thierry Defrance; Els Verhoeyen; François-Loïc Cosset. A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status. Molecular Therapy 2015, 23, 1734 -1747.
AMA StyleFloriane Fusil, Sara Calattini, Fouzia Amirache, Jimmy Mancip, Caroline Costa, Justin B Robbins, Florian Douam, Dimitri Lavillette, Mansun Law, Thierry Defrance, Els Verhoeyen, François-Loïc Cosset. A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status. Molecular Therapy. 2015; 23 (11):1734-1747.
Chicago/Turabian StyleFloriane Fusil; Sara Calattini; Fouzia Amirache; Jimmy Mancip; Caroline Costa; Justin B Robbins; Florian Douam; Dimitri Lavillette; Mansun Law; Thierry Defrance; Els Verhoeyen; François-Loïc Cosset. 2015. "A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status." Molecular Therapy 23, no. 11: 1734-1747.