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1.
PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-333828

ABSTRACT

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC 50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. ONE-SENTENCE SUMMARY: We designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

2.
PubMed; 2020.
Preprint in English | PubMed | ID: ppcovidwho-331914

ABSTRACT

A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.

3.
Science ; 375(6583):864-+, 2022.
Article in English | Web of Science | ID: covidwho-1769817

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.

4.
Meng, B.; Ferreira, I. A. T. M.; Abdullahi, A.; Goonawardane, N.; Saito, A.; Kimura, I.; Yamasoba, D.; Gerba, P. P.; Fatihi, S.; Rathore, S.; Zepeda, S. K.; Papa, G.; Kemp, S. A.; Ikeda, T.; Toyoda, M.; Tan, T. S.; Kuramochi, J.; Mitsunaga, S.; Ueno, T.; Shirakawa, K.; Takaori-Kondo, A.; Brevini, T.; Mallery, D. L.; Charles, O. J.; Bowen, J. E.; Joshi, A.; Walls, A. C.; Jackson, L.; Cele, S.; Martin, D.; Smith, K. G. C.; Bradley, J.; Briggs, J. A. G.; Choi, J.; Madissoon, E.; Meyer, K.; Mlcochova, P.; Ceron-Gutierrez, L.; Doffinger, R.; Teichmann, S.; Pizzuto, M.; de Marco, A.; Corti, D.; Sigal, A.; James, L.; Veesler, D.; Hosmillo, M.; Lee, J. H.; Sampaziotis, F.; Goodfellow, I. G.; Matheson, N. J.; Thukral, L.; Sato, K.; Gupta, R. K.; Kawabata, R.; Morizako, N.; Sadamasu, K.; Asakura, H.; Nagashima, M.; Yoshimura, K.; Ito, J.; Kimura, I.; Uriu, K.; Kosugi, Y.; Suganami, M.; Oide, A.; Yokoyama, M.; Chiba, M.; Saito, A.; Butlertanaka, E. P.; Tanaka, Y. L.; Ikeda, T.; Motozono, C.; Nasser, H.; Shimizu, R.; Yuan, Y.; Kitazato, K.; Hasebe, H.; Nakagawa, S.; Wu, J.; Takahashi, M.; Fukuhara, T.; Shimizu, K.; Tsushima, K.; Kubo, H.; Kazuma, Y.; Nomura, R.; Horisawa, Y.; Nagata, K.; Kawai, Y.; Yanagida, Y.; Tashiro, Y.; Tokunaga, K.; Ozono, S.; Baker, S.; Dougan, G.; Hess, C.; Kingston, N.; Lehner, P. J.; Lyons, P. A.; Matheson, N. J.; Owehand, W. H.; Saunders, C.; Summers, C.; Thaventhiran, J. E. D.; Toshner, M.; Weekes, M. P.; Maxwell, P.; Shaw, A.; Bucke, A.; Calder, J.; Canna, L.; Domingo, J.; Elmer, A.; Fuller, S.; Harris, J.; Hewitt, S.; Kennet, J.; Jose, S.; Kourampa, J.; Meadows, A.; O’Brien, C.; Price, J.; Publico, C.; Rastall, R.; Ribeiro, C.; Rowlands, J.; Ruffolo, V.; Tordesillas, H.; Bullman, B.; Dunmore, B. J.; Fawke, S.; Gräf, S.; Hodgson, J.; Huang, C.; Hunter, K.; Jones, E.; Legchenko, E.; Matara, C.; Martin, J.; Mescia, F.; O’Donnell, C.; Pointon, L.; Pond, N.; Shih, J.; Sutcliffe, R.; Tilly, T.; Treacy, C.; Tong, Z.; Wood, J.; Wylot, M.; Bergamaschi, L.; Betancourt, A.; Bower, G.; Cossetti, C.; de Sa, A.; Epping, M.; Fawke, S.; Gleadall, N.; Grenfell, R.; Hinch, A.; Huhn, O.; Jackson, S.; Jarvis, I.; Krishna, B.; Lewis, D.; Marsden, J.; Nice, F.; Okecha, G.; Omarjee, O.; Perera, M.; Potts, M.; Richoz, N.; Romashova, V.; Yarkoni, N. S.; Sharma, R.; Stefanucci, L.; Stephens, J.; Strezlecki, M.; Turner, L.; de Bie, E. M. D. D.; Bunclark, K.; Josipovic, M.; Mackay, M.; Mescia, F.; Michael, A.; Rossi, S.; Selvan, M.; Spencer, S.; Yong, C.; Allison, J.; Butcher, H.; Caputo, D.; Clapham-Riley, D.; Dewhurst, E.; Furlong, A.; Graves, B.; Gray, J.; Ivers, T.; Kasanicki, M.; Le Gresley, E.; Linger, R.; Meloy, S.; Muldoon, F.; Ovington, N.; Papadia, S.; Phelan, I.; Stark, H.; Stirrups, K. E.; Townsend, P.; Walker, N.; Webster, J.; Scholtes, I.; Hein, S.; King, R.; Márquez, S.; Prado-Vivar, B.; Becerra-Wong, M.; Caravajal, M.; Trueba, G.; Rojas-Silva, P.; Grunauer, M.; Gutierrez, B.; Guadalupe, J. J.; Fernández-Cadena, J. C.; Andrade-Molina, D.; Baldeon, M.; Pinos, A..
Web of Science; 2021.
Preprint in English | Web of Science | ID: ppcovidwho-331154

ABSTRACT

The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and is characterised by multiple spike mutations across all spike domains. Here we show that Omicron BA.1 has higher affinity for ACE2 compared to Delta, and confers very significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesevir and molnupiravir retain efficacy against Omicron BA.1. We found that in human nasal epithelial 3D cultures replication was similar for both Omicron and Delta. However, in lower airway organoids, Calu-3 lung cells and gut adenocarcinoma cell lines live Omicron virus demonstrated significantly lower replication in comparison to Delta. We noted that despite presence of mutations predicted to favour spike S1/S2 cleavage, the spike protein is less efficiently cleaved in live Omicron virions compared to Delta virions. We mapped the replication differences between the variants to entry efficiency using spike pseudotyped virus (PV) entry assays. The defect for Omicron PV in specific cell types correlated with higher cellular RNA expression of TMPRSS2, and accordingly knock down of TMPRSS2 impacted Delta entry to a greater extent as compared to Omicron. Furthermore, drug inhibitors targeting specific entry pathways demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that mediates cell entry via plasma membrane fusion. Instead, we demonstrate that Omicron spike has greater dependency on cell entry via the endocytic pathway requiring the activity of endosomal cathepsins to cleave spike. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was dramatically impaired compared to the Delta spike. Overall, Omicron appears to have gained significant evasion from neutralising antibodies whilst maintaining sensitivity to antiviral drugs targeting the polymerase. Omicron has shifted cellular tropism away from TMPRSS2 expressing cells that are enriched in cells found in the lower respiratory and GI tracts, with implications for altered pathogenesis.

6.
Embase;
Preprint in English | EMBASE | ID: ppcovidwho-327034

ABSTRACT

The SARS-CoV-2 Omicron variant of concern evades antibody mediated immunity with an unprecedented magnitude due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and X-ray crystal structures of the spike and RBD bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a structural framework for understanding the marked reduction of binding of all other therapeutic mAbs leading to dampened neutralizing activity. We reveal electrostatic remodeling of the interactions within the spike and those formed between the Omicron RBD and human ACE2, likely explaining enhanced affinity for the host receptor relative to the prototypic virus.

7.
Embase;
Preprint in English | EMBASE | ID: ppcovidwho-326896

ABSTRACT

Numerous safe and effective COVID-19 vaccines have been developed that utilize various delivery technologies and engineering strategies. The influence of the SARS-CoV2 spike (S) glycoprotein conformation on antibody responses induced by vaccination or infection in humans remains unknown. To address this question, we compared plasma antibodies elicited by six globally-distributed vaccines or infection and observed markedly higher binding titers for vaccines encoding a prefusion-stabilized S relative to other groups. Prefusion S binding titers positively correlated with plasma neutralizing activity, indicating that physical stabilization of the prefusion conformation enhances protection against SARS-CoV-2. We show that almost all plasma neutralizing activity is directed to prefusion S, in particular the S1 subunit, and that variant cross-neutralization is mediated solely by RBD-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants and longer durability than current technologies.

8.
Embase;
Preprint in English | EMBASE | ID: ppcovidwho-326798

ABSTRACT

The recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb1, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab2, S2X2593and S2H974, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

9.
Embase;
Preprint in English | EMBASE | ID: ppcovidwho-326771

ABSTRACT

The SARS-CoV-2 Delta variant is currently responsible for most infections worldwide, including among fully vaccinated individuals. Although these latter infections are associated with milder COVID-19 disease relative to unvaccinated subjects, the specificity and durability of antibody responses elicited by Delta breakthrough cases remain unknown. Here, we demonstrate that breakthrough infections induce serum binding and neutralizing antibody responses that are markedly more potent, durable and resilient to spike mutations observed in variants of concern than those observed in subjects who were infected only or received only two doses of COVID-19 vaccine. However, wee show that Delta breakthrough cases, subjects who were vaccinated after SARS-CoV-2 infection and individuals vaccinated three times (without infection) have serum neutralizing activity of comparable magnitude and breadth indicate that multiple types of exposure or increased number of exposures to SARS-CoV-2 antigen(s) enhance spike-specific antibody responses. Neutralization of the genetically divergent SARS-CoV, however, was moderate with all four cohorts examined, except after four exposures to the SARS-CoV-2 spike, underscoring the importance of developing vaccines eliciting broad sarbecovirus immunity for pandemic preparedness.

10.
MEDLINE;
Preprint in English | MEDLINE | ID: ppcovidwho-326698

ABSTRACT

Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the "class 3" epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.

11.
MEDLINE;
Preprint in English | MEDLINE | ID: ppcovidwho-326665

ABSTRACT

With global vaccination efforts against SARS-CoV-2 underway, there is a need for rapid quantification methods for neutralizing antibodies elicited by vaccination and characterization of their strain dependence. Here, we describe a designed protein biosensor that enables sensitive and rapid detection of neutralizing antibodies against wild type and variant SARS-CoV-2 in serum samples. More generally, our thermodynamic coupling approach can better distinguish sample to sample differences in analyte binding affinity and abundance than traditional competition based assays.

12.
MEDLINE;
Preprint in English | MEDLINE | ID: ppcovidwho-326613

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 out of 31), but not in neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect newly emerging viruses in the UK led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b.

13.
PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-296521

ABSTRACT

The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor-binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion;AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines;AS37, a TLR-7 agonist adsorbed to Alum;CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum;or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.

14.
Cell ; 176(5):1026-1039.e15, 2019.
Article in English | EMBASE | ID: covidwho-1092987

ABSTRACT

Recent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism. Structural analysis of the SARS-CoV S and MERS-CoV S glycoproteins in complex with neutralizing antibodies from human survivors sheds light into the mechanisms of membrane fusion and neutralization

15.
Advances in virus research ; 105:93-116, 2019.
Article in English | MEDLINE | ID: covidwho-1044443

ABSTRACT

Coronaviruses (CoVs) have caused outbreaks of deadly pneumonia in humans since the beginning of the 21st century. The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 and was responsible for an epidemic that spread to five continents with a fatality rate of 10% before being contained in 2003 (with additional cases reported in 2004). The Middle-East respiratory syndrome coronavirus (MERS-CoV) emerged in the Arabian Peninsula in 2012 and has caused recurrent outbreaks in humans with a fatality rate of 35%. SARS-CoV and MERS-CoV are zoonotic viruses that crossed the species barrier using bats/palm civets and dromedary camels, respectively. No specific treatments or vaccines have been approved against any of the six human coronaviruses, highlighting the need to investigate the principles governing viral entry and cross-species transmission as well as to prepare for zoonotic outbreaks which are likely to occur due to the large reservoir of CoVs found in mammals and birds. Here, we review our understanding of the infection mechanism used by coronaviruses derived from recent structural and biochemical studies.

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