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Link to original content: http://www.ncbi.nlm.nih.gov/pubmed/34063562
Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein - PubMed Skip to main page content
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. 2021 May 3;9(5):449.
doi: 10.3390/vaccines9050449.

Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein

Asha Ann Philip  1 , John Thomas Patton  1
Affiliations

Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein

Asha Ann Philip et al. Vaccines (Basel). .

Abstract

Among vaccines administered to children are those targeting rotavirus, a segmented double-stranded RNA virus that represents a major cause of severe gastroenteritis. To explore the feasibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding cassettes for NSP3, a translational 2A stop-restart signal, and a FLAG-tagged portion of the SARS-CoV-2 spike (S) protein: S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended RBD (ExRBD), or S2 core (CR) domain. Generation of rSA11 containing the S1 coding sequence required a sequence insertion of 2.2 kbp, the largest such insertion yet introduced into the rotavirus genome. Immunoblotting showed that rSA11 viruses containing the smaller NTD, RBD, ExRBD, and CR coding sequences expressed S-protein products of expected size, with ExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, the rSA11 virus containing the full-length S coding sequence (rSA11/NSP3-fS1) failed to express its expected 80 kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of immunogenic portions of the SARS-CoV-2 S protein, including NTD, RBD, ExRBD, and CR, that have sizes smaller than the S1 fragment.

Keywords: COVID-19 vaccine; Reoviridae; SARS-CoV-2; expression vector; reverse genetics; rotavirus; rotavirus vaccine; spike protein.

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Conflict of interest statement

J.T.P. is an inventor on a patent application related to the content of this paper.

Figures

Figure 1
Figure 1
Domains of the SARS-CoV-2 S protein expressed by rSA11. (A) S protein trimers are cleaved at the S1/S2 junction by furin proconvertase and at the S2′ site by the TMPRSS2 serine protease. The S1 fragment contains a signal sequence (SS), N-terminal domain (NTD), receptor-binding domain (RBD), receptor-binding motif (RBM), coiled-coil (CC), and two heptad repeats (HR1, HR2). The S2 fragment contains a trimeric core region, transmembrane anchor (TM), and fusion domain. (B) Portions of the S protein expressed by recombinant rotaviruses are indicated. (C) Ribbon representations of the closed conformation of the trimeric S protein (PDB 6VXX) showing locations of the RBD (magenta), extended RBD (ExRBD, cyan), NTD (blue), core (CR, gold) domains and the S1 cleavage product (green).
Figure 2
Figure 2
Plasmids with modified segment 7 (NSP3) cDNAs used to generate rSA11 viruses expressing regions of the SARS-CoV-2 S protein. Illustration indicates nucleotide positions of the coding sequences for NSP3, porcine teschovirus 2A element, 3xFLAG (FL), and the complete S1 or portions of the S1 (NTD, ExRBD, and RBD) and S2 (CR) proteins. The red arrow notes the position of the 2A translational stop-restart site, and the asterisk (*) notes the end of the ORF. Sizes (aa) of encoded NSP3 and S products are in parenthesis. T7 (T7 RNA polymerase promoter sequence), Rz (Hepatitis D virus ribozyme), and UTR (untranslated region).
Figure 3
Figure 3
Properties of rSA11/NSP3-CoV2/S viruses expressing regions of the SARS-CoV-2 S protein. (A,B) dsRNA was recovered from MA104 cells infected with plaque-purified rSA11 isolates, resolved by gel electrophoresis, and detected by ethidium-bromide staining. RNA segments of rSA11/wt are labeled 1 to 11. Sizes (kbp) of segment 7 RNAs (black arrows) of rSA11 isolates are indicated. Double-stranded RNA of rSA11/NSP3-fS1 serially passaged twice (P1 and P2) in MA104 cells is shown in (A). (C) Plaque assays were performed using MA104 cells and detected by crystal-violet staining. (D) Titers reached by rSA11 isolates were determined by plaque assay. Bars indicate standard deviations calculated from three separate determinations.
Figure 4
Figure 4
Expression of SARS-CoV-2 S products by rSA11 viruses. (A,B) Whole cell lysates (WCL) were prepared from cells infected with rSA11 viruses and examined by immunoblot assay using (A) FLAG antibody to detect S products (NTD, ExRBD, RBD, CR, S1, and 2A read-through products) and antibodies specific for rotavirus NSP3 and VP6 and proliferating cell nuclear antigen (PCNA). Red asterisks (*) identify 2A read-through products and blue asterisks (*) identify 2A cleavage products. Cleaved fS1 product failed to be detected in five separate immunoblot assays analyzing two independently generated samples of MA104 cells infected with rSA11/NSP3-fS1 virus. (B) Lysates prepared from MA104 cells infected with rSA11wt, rSA11/NSP3-fRBD and rSA11/NSP3-fExRBD were examined by immunoblot assay using antibodies specific for RBD (ProSci 9087), rotavirus VP6, and PCNA. (C) Lysates prepared from MA104 cells infected with rSA11/wt, rSA11/NSP3-fRBD and rSA11/NSP3-fExRBD viruses were examined by immunoprecipitation assay using a SARS-CoV-2 S1 specific monoclonal antibody (GeneTex CR3022). Lysates were also analyzed with a NSP2-specific polyclonal antibody. Antigen-antibody complexes were recovered using IgA/G beads, resolved by gel electrophoresis, blotted onto nitrocellulose membranes, and probed with FLAG (fRBD and fExRBD) and NSP2 antibody. Molecular weight markers are indicated (kDa). Red arrows indicate fRBD and fExRBD. fRBD comigrates near the Ig light chain (Ig/L). Ig heavy chain, Ig/H).
Figure 5
Figure 5
Production of RBD and ExRBD by rSA11 viruses during infection. MA104 cells were mock infected or infected with rSA11/wt, rSA11/NSP3-fRBD, or rSA11/NSP3-fExRBD (MOI of 5). Lysates were prepared from the cells at 0, 4, 8, or 12 h p.i. and analyzed by immunoblot assay using antibodies specific for FLAG, NSP3, VP6, and PCNA. Red asterisks (*) identify 2A read-through products. Positions of molecular weight markers are indicated (kDa).
Figure 6
Figure 6
Impact of genome size on rotavirus particle density. MA104 cells were infected with rSA11/wt, rSA11/NSP3-fExRBD, or rSA11/NSP3-fS1 viruses at an MOI of 5. At 12 h p.i., the cells were recovered, lysed by treatment with non-ionic detergent, and treated with EDTA to convert rotavirus virions into DLPs. (A,B) DLPs were banded by centrifugation in CsCl gradients and densities (g/cm3) were determined using a refractometer. (C) Lysates from rSA11/wt and rSA11/NSP3-fS1 infected cells were combined and their DLP components banded by centrifugation in a CsCl gradient. (D,E) Electrophoretic profile of the dsRNA genomes of DLPs recovered from CsCl gradients. Panel D RNAs derive from DLPs in panel A and panel E RNAs derive from DLPs in panel B and C. RNA segments of rSA11/wt are labeled 1 to 11. Positions of segment 7 RNAs are indicated with red arrows.
Figure 7
Figure 7
Genetic stability of rSA11 strains expressing SARS-CoV-2 S domains. rSA11 strains were serially passaged five to six times (P1 to P5 or P6) in MA104 cells. (A) Genomic RNAs were recovered from infected cell lysates and analyzed by gel electrophoresis. Positions of viral genome segments are labeled. Position of modified segment 7 (NSP3) dsRNAs introduced into rSA11 strains are denoted with black arrows. Genetic instability of the modified segment 7 (NSP3) dsRNA of rSA11/NSP3-fS1 yielded R1-R4 RNAs during serial passage. (B) Genomic RNAs prepared from large (L1–L4) and small (S1–S4) plaque isolates of P6 rSA11/NSP3-fS1. Segment 7 RNAs are identified as R1–R3, as in (A). (C) Organization of R1–R3 sequences determined by sequencing of segment 7 RNAs of L1, S1, and S3 plaque isolates. Sequence deletions are indicated with dashed lines. Regions of the S1 ORF that are no longer encoded by the R1–R3 segment 7 RNAs are indicated by slashed green-white boxes.
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