Development of a SARS-CoV-2 Vaccine Candidate Using Plant-Based Manufacturing and a Tobacco Mosaic Virus-like Nano-Particle

Stable, effective, easy-to-manufacture vaccines are critical to stopping the COVID-19 pandemic resulting from the coronavirus SARS-CoV-2. We constructed a vaccine candidate CoV-RBD121-NP, which is comprised of the SARS-CoV-2 receptor-binding domain (RBD) of the spike glycoprotein (S) fused to a human IgG1 Fc domain (CoV-RBD121) and conjugated to a modified tobacco mosaic virus (TMV) nanoparticle. In vitro, CoV-RBD121 bound to the host virus receptor ACE2 and to the monoclonal antibody CR3022, a neutralizing antibody that blocks S binding to ACE2. The CoV-RBD121-NP vaccine candidate retained key SARS-CoV-2 spike protein epitopes, had consistent manufacturing release properties of safety, identity, and strength, and displayed stable potency when stored for 12 months at 2–8 °C or 22–28 °C. Immunogenicity studies revealed strong antibody responses in C57BL/6 mice with non-adjuvanted or adjuvanted (7909 CpG) formulations. The non-adjuvanted vaccine induced a balanced Th1/Th2 response and antibodies that recognized both the S1 domain and full S protein from SARS2-CoV-2, whereas the adjuvanted vaccine induced a Th1-biased response. Both adjuvanted and non-adjuvanted vaccines induced virus neutralizing titers as measured by three different assays. Collectively, these data showed the production of a stable candidate vaccine for COVID-19 through the association of the SARS-CoV-2 RBD with the TMV-like nanoparticle.

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CoV-RBD121-NP Vaccine Candidate Protects against Symptomatic Disease Following SARS-CoV-2 Challenge in K18-hACE2 Mice and Induces Protective Responses That Prevent COVID-19-Associated Immunopathology

Vaccines ◽

10.3390/vaccines9111346 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1346

Author(s):

Jennifer K. DeMarco ◽

Joshua M. Royal ◽

William E. Severson ◽

Jon D. Gabbard ◽

Steve Hume ◽

...

Keyword(s):

Antibody Response ◽

Neutralizing Antibodies ◽

Potential Role ◽

Vaccine Candidate ◽

Severe Disease ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Receptor Binding Domain ◽

Symptomatic Disease ◽

Human Igg1

We developed a SARS-CoV-2 vaccine candidate (CoV-RBD121-NP) comprised of a tobacco mosaic virus-like nanoparticle conjugated to the receptor-binding domain of the spike glycoprotein of SARS-CoV-2 fused to a human IgG1 Fc domain. CoV-RBD121-NP elicits strong antibody responses in C57BL/6 mice and is stable for up to 12 months at 2–8 or 22–28 °C. Here, we showed that this vaccine induces a strong neutralizing antibody response in K18-hACE2 mice. Furthermore, we demonstrated that immunization protects mice from virus-associated mortality and symptomatic disease. Our data indicated that a sufficient pre-existing pool of neutralizing antibodies is required to restrict SARS-CoV-2 replication upon exposure and prevent induction of inflammatory mediators associated with severe disease. Finally, we identified a potential role for CXCL5 as a protective cytokine in SARS-CoV-2 infection. Our results suggested that disruption of the CXCL5 and CXCL1/2 axis may be important early components of the inflammatory dysregulation that is characteristic of severe cases of COVID-19.

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Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

Journal of Virology ◽

10.1128/jvi.74.24.11671-11680.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11671-11680 ◽

Cited By ~ 49

Author(s):

T. A. M. Osman ◽

C. L. Hemenway ◽

K. W. Buck

Keyword(s):

Rna Polymerase ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Untranslated Region ◽

Rna Synthesis ◽

Central Core ◽

Minus Strand ◽

Stem Loop ◽

Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

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An analysis of tobacco mosaic virus replicative structures synthesized in vitro

Plant Molecular Biology ◽

10.1007/bf00027137 ◽

1986 ◽

Vol 6 (6) ◽

pp. 455-465 ◽

Cited By ~ 15

Author(s):

Nevin Dale Young ◽

Milton Zaitlin

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus

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Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

Journal of Virology ◽

10.1128/jvi.00540-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8329-8344 ◽

Cited By ~ 67

Author(s):

Jamie Ashby ◽

Emmanuel Boutant ◽

Mark Seemanpillai ◽

Adrian Sambade ◽

Christophe Ritzenthaler ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Movement Protein ◽

Cell Extract ◽

Transport Processes ◽

In Vitro Assays ◽

Protein Functions ◽

Intercellular Spread

ABSTRACT The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

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In vitro and in vivo aggregation of the defective PM2 tobacco mosaic virus protein

Journal of Molecular Biology ◽

10.1016/s0022-2836(66)80056-6 ◽

1966 ◽

Vol 19 (1) ◽

pp. 140-IN8 ◽

Cited By ~ 21

Author(s):

Albert Siegel ◽

G.J. Hills ◽

Roy Markham

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Virus Protein

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On in vitro and in vivo binding of tobacco mosaic virus to cytoplasmic membranes

Biochemie und Physiologie der Pflanzen ◽

10.1016/s0015-3796(84)80090-6 ◽

1984 ◽

Vol 179 (6) ◽

pp. 507-516 ◽

Cited By ~ 4

Author(s):

Barbara Pustowoit ◽

Wladimir Pustowoit ◽

Gottfried Schuster

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

In Vivo Binding ◽

Cytoplasmic Membranes

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Visualization by atomic force microscopy of tobacco mosaic virus movement protein–RNA complexes formed in vitro

Journal of General Virology ◽

10.1099/0022-1317-82-6-1503 ◽

2001 ◽

Vol 82 (6) ◽

pp. 1503-1508 ◽

Cited By ~ 45

Author(s):

O. I. Kiselyova ◽

I. V. Yaminsky ◽

E. M. Karger ◽

O. Yu. Frolova ◽

Y. L. Dorokhov ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Movement Protein ◽

Structural Conversion ◽

Force Microscopy ◽

Rna Transcripts ◽

Atomic Force ◽

Rna Complexes

The structure of complexes formed in vitro by tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short (890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on a mica surface. MP molecules were found to be distributed along the chain of RNA and the structure of MP–RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60–100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive ‘beads-on-a-string’ into a ‘thick string’ form that was partly resistant to RNase. The ‘thick string’-type RNase-resistant complexes were also produced by short synthetic RNA transcripts at different MP:RNA ratios. The ‘thick string’ complexes are suggested to represent clusters of MP molecules cooperatively bound to discrete regions of TMV RNA and separated by protein-free RNA segments.

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