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Cited by 193SARS-CoV-2 and COVID-19 ResearchAnimal Virus Infections StudiesImmunotherapy and Immune Responses

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A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice

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de Alwis, Ruklanthi, Gan, Esther S., Chen, Shiwei, Leong, Yan Shan, Tan, Hwee Cheng, Zhang, Summer L., Yau, Clement, Low, Jenny G.H., Kalimuddin, Shirin, Matsuda, Daiki, Allen, Elizabeth C., Hartman, Paula, Park, Kyoung-Joo Jenny, Alayyoubi, Maher, Bhaskaran, Hari, Dukanovic, Adrian, Bao, Yanjie, Clemente, Brenda, Vega, Jerel, Roberts, Scott, Gonzalez, Jose A., Sablad, Marciano, Yelin, Rodrigo, Taylor, Wendy, Tachikawa, Kiyoshi, Parker, Suezanne, Karmali, Priya, Davis, Jared, Sullivan, Brian M., Sullivan, Sean M., Hughes, Steve G., Chivukula, Pad, Ooi, Eng Eong·2021·Molecular Therapy
DOI 10.1016/j.ymthe.2021.04.001PMC PMC8019652

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Abstract

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8 + T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4 + T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 μg and 10 μg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.

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Graphical abstract
Graphical abstract CC BY-NC-ND · PubMed Central
Figure 1 Design and expression of a SARS-COV-2 vaccine with conventional mRNA and self-transcribing and replicating RNA (STARR) platforms (A) Schematic diagram of the SARS-CoV-2 self-replicating STARR RNA (LUNAR-COV19) and conventional mRNA vaccine constructs. The STARR construct encodes for the four non-structural proteins, ns1–ns4, from Venezuelan equine encephalitis virus (VEEV) and the unmodified full-length pre-fusion spike protein of SARS-CoV-2. The mRNA construct also codes for the same SARS-CoV-2 full-length spike protein. (B) Physical characteristics and RNA trapping efficiency of the LNPs encapsulating conventional mRNA and LUNAR-COV19 vaccines. (C) Western blot detection of SARS-CoV-2 Spike protein following transfection of Hep3b cells with LUNAR-COV19 and conventional mRNA. (D) In vivo comparison of protein expression following i.m. administration of LNPs containing luciferase-expressing STARR RNA or conventional mRNA. BALB/c mice (n = 3/group) were injected i.m. with 0.2 μg, 2.0 μg, or 10.0 μg of STARR RNA or conventional mRNA formulated with the same LNPs. Luciferase expression was measured by in vivo bioluminescence on days 1, 3, and 7 post-i.m. administration. Results are shown as mean with standard deviation error bars. S1, S domain 1; S2, S domain 2; TM, transmembrane domain; CP, cytoplasmic domain; aka, also known as.
Figure 1 Design and expression of a SARS-COV-2 vaccine with conventional mRNA and self-transcribing and replicating RNA (STARR) platforms (A) Schematic diagram of the SARS-CoV-2 self-replicating STARR RNA (LUNAR-COV19) and conventional mRNA vaccine constructs. The STARR construct encodes for the four non-structural proteins, ns1–ns4, from Venezuelan equine encephalitis virus (VEEV) and the unmodified full-length pre-fusion spike protein of SARS-CoV-2. The mRNA construct also codes for the same SARS-CoV-2 full-length spike protein. (B) Physical characteristics and RNA trapping efficiency of the LNPs encapsulating conventional mRNA and LUNAR-COV19 vaccines. (C) Western blot detection of SARS-CoV-2 Spike protein following transfection of Hep3b cells with LUNAR-COV19 and conventional mRNA. (D) In vivo comparison of protein expression following i.m. administration of LNPs containing luciferase-expressing STARR RNA or conventional mRNA. BALB/c mice (n = 3/group) were injected i.m. with 0.2 μg, 2.0 μg, or 10.0 μg of STARR RNA or conventional mRNA formulated with the same LNPs. Luciferase expression was measured by in vivo bioluminescence on days 1, 3, and 7 post-i.m. administration. Results are shown as mean with standard deviation error bars. S1, S domain 1; S2, S domain 2; TM, transmembrane domain; CP, cytoplasmic domain; aka, also known as.CC BY-NC-ND · PubMed Central
Figure 2 Clinical scores, mouse weights, and transcriptomic analysis of immune genes after vaccination with LUNAR-COV19 (A) C57BL/6 mice (n = 5/group) were immunized with either PBS, mRNA, or LUNAR-COV19 (doses 0.2 μg, 2 μg, or 10 μg), had weight and clinical scores assessed every day, were bled at day 1 post-immunization, and were sacrificed at 7 days post-vaccination, and lymph nodes were harvested. Gene expression of inflammatory genes and immune genes were measured in whole blood (at day 1) and lymph nodes (at day 7), respectively. (B) Expression of IFN and inflammatory response genes in whole blood presented as heatmap of Z scores. (C) Lymph node weights at 7 days post-vaccination. (D–F) Principal-component analysis (PCA) of immune gene expression following vaccination with LUNAR-COV19 or conventional mRNA control at doses 0.2 μg (D), 2 μg (E), and 10 μg (F). (G–I) Volcano plots of fold change of LUNAR-COV19 versus conventional mRNA control (x axis) and log10 p value of LUNAR-COV19 versus conventional mRNA control (y axis) for doses 0.2 μg (G), 2 μg (H), and 10 μg (I). Study design schematic diagram created with BioRender.com. Weights of lymph nodes were compared between groups with a two-tailed Mann-Whitney U test; ∗0.05 < p < 0.01.
Figure 2 Clinical scores, mouse weights, and transcriptomic analysis of immune genes after vaccination with LUNAR-COV19 (A) C57BL/6 mice (n = 5/group) were immunized with either PBS, mRNA, or LUNAR-COV19 (doses 0.2 μg, 2 μg, or 10 μg), had weight and clinical scores assessed every day, were bled at day 1 post-immunization, and were sacrificed at 7 days post-vaccination, and lymph nodes were harvested. Gene expression of inflammatory genes and immune genes were measured in whole blood (at day 1) and lymph nodes (at day 7), respectively. (B) Expression of IFN and inflammatory response genes in whole blood presented as heatmap of Z scores. (C) Lymph node weights at 7 days post-vaccination. (D–F) Principal-component analysis (PCA) of immune gene expression following vaccination with LUNAR-COV19 or conventional mRNA control at doses 0.2 μg (D), 2 μg (E), and 10 μg (F). (G–I) Volcano plots of fold change of LUNAR-COV19 versus conventional mRNA control (x axis) and log10 p value of LUNAR-COV19 versus conventional mRNA control (y axis) for doses 0.2 μg (G), 2 μg (H), and 10 μg (I). Study design schematic diagram created with BioRender.com. Weights of lymph nodes were compared between groups with a two-tailed Mann-Whitney U test; ∗0.05 < p < 0.01.CC BY-NC-ND · PubMed Central
Figure 3 Cellular immune responses following vaccination with LUNAR-COV19 C57BL/6 mice (n = 5 per group) were immunized i.m. with 0.2 μg, 2.0 μg, or 10.0 μg of LUNAR-COV19 or conventional mRNA control and sacrificed at day 7 post-vaccination, and spleens were analyzed for cellular T cell responses by flow cytometry and ELISpot. (A–C) CD8+ (A and B) and CD4+ (C) T effector cells were assessed in vaccinated animals with surface staining for T cell markers and flow cytometry. (D–F) IFNγ+ CD8+ T cells (D and E) and ratio of IFNγ+ IL-4+ CD4+ T cells (F) in spleens of immunized mice were assessed after ex vivo stimulation with PMA/IO and intracellular staining. (G and H) SARS-CoV-2 Spike protein-specific responses to pooled Spike protein peptides were assessed with IFNγ ELISpot assays after vaccination with conventional mRNA controls or LUNAR-COV19. Percentage of CD8+ cells, CD4+ cells, and IFNγ- and IL-4-producing T cells were compared between groups with two-tailed Mann-Whitney U test; ∗0.05 < p < 0.01; ∗∗0.01 < p < 0.001.
Figure 3 Cellular immune responses following vaccination with LUNAR-COV19 C57BL/6 mice (n = 5 per group) were immunized i.m. with 0.2 μg, 2.0 μg, or 10.0 μg of LUNAR-COV19 or conventional mRNA control and sacrificed at day 7 post-vaccination, and spleens were analyzed for cellular T cell responses by flow cytometry and ELISpot. (A–C) CD8+ (A and B) and CD4+ (C) T effector cells were assessed in vaccinated animals with surface staining for T cell markers and flow cytometry. (D–F) IFNγ+ CD8+ T cells (D and E) and ratio of IFNγ+ IL-4+ CD4+ T cells (F) in spleens of immunized mice were assessed after ex vivo stimulation with PMA/IO and intracellular staining. (G and H) SARS-CoV-2 Spike protein-specific responses to pooled Spike protein peptides were assessed with IFNγ ELISpot assays after vaccination with conventional mRNA controls or LUNAR-COV19. Percentage of CD8+ cells, CD4+ cells, and IFNγ- and IL-4-producing T cells were compared between groups with two-tailed Mann-Whitney U test; ∗0.05 < p < 0.01; ∗∗0.01 < p < 0.001.CC BY-NC-ND · PubMed Central
Figure 4 Antibody response following LUNAR-COV19 vaccination (A) BALB/c and C57BL/6J mice were i.m. immunized with 0.2 μg, 2 μg, or 10 μg of LUNAR-COV19 or conventional mRNA control (n = 5/group). Blood sampling was conducted at baseline and days 10, 19, 30, 40, 50, and 60 post-vaccination for BALB/c and days 10, 20, and 30 for C57BL/6J. (B–E) IgM (B and C) and IgG (D and E) against the SARS-CoV-2 Spike protein over time, assessed with insect cell-derived whole Spike protein in a Luminex immuno-assay (measured as MFI). (F and G) IgG endpoint titers to mammalian-derived whole Spike protein and S1, S2, and RBD proteins to mammalian-derived whole Spike protein at day 30 post-vaccination were assessed in BALB/c (F) and C57BL/6J (G). (H) Avidity of SARS-CoV-2 Spike protein-specific IgG at day 30 post-immunization was measured with 8 M urea washes. (I) Neutralizing antibody (PRNT50 titers) at day 30 post-vaccination against a clinically isolated live SARS-CoV-2 virus measured in both BALB/c and C57BL/6J. Gray dashed lines depict the serum dilution range (i.e., from 1:20 to 1:320) tested by PRNT. (J and K) PRNT50 (J) and PRNT70 (K) of SARS-CoV-2 neutralization at day 30 and day 60 post-vaccination in BALB/c and convalescent sera from COVID-19 patients. (L) Correlation analysis of Spike-specific IgG endpoint titers against SARS-CoV-2 neutralization (PRNT50). Antibody data were compared between groups with a two-tailed Mann-Whitney U test; ∗0.05 < p < 0.01; ∗∗0.01 < p < 0.001.
Figure 4 Antibody response following LUNAR-COV19 vaccination (A) BALB/c and C57BL/6J mice were i.m. immunized with 0.2 μg, 2 μg, or 10 μg of LUNAR-COV19 or conventional mRNA control (n = 5/group). Blood sampling was conducted at baseline and days 10, 19, 30, 40, 50, and 60 post-vaccination for BALB/c and days 10, 20, and 30 for C57BL/6J. (B–E) IgM (B and C) and IgG (D and E) against the SARS-CoV-2 Spike protein over time, assessed with insect cell-derived whole Spike protein in a Luminex immuno-assay (measured as MFI). (F and G) IgG endpoint titers to mammalian-derived whole Spike protein and S1, S2, and RBD proteins to mammalian-derived whole Spike protein at day 30 post-vaccination were assessed in BALB/c (F) and C57BL/6J (G). (H) Avidity of SARS-CoV-2 Spike protein-specific IgG at day 30 post-immunization was measured with 8 M urea washes. (I) Neutralizing antibody (PRNT50 titers) at day 30 post-vaccination against a clinically isolated live SARS-CoV-2 virus measured in both BALB/c and C57BL/6J. Gray dashed lines depict the serum dilution range (i.e., from 1:20 to 1:320) tested by PRNT. (J and K) PRNT50 (J) and PRNT70 (K) of SARS-CoV-2 neutralization at day 30 and day 60 post-vaccination in BALB/c and convalescent sera from COVID-19 patients. (L) Correlation analysis of Spike-specific IgG endpoint titers against SARS-CoV-2 neutralization (PRNT50). Antibody data were compared between groups with a two-tailed Mann-Whitney U test; ∗0.05 < p < 0.01; ∗∗0.01 < p < 0.001.CC BY-NC-ND · PubMed Central
Figure 5 LUNAR-COV19 elicits Th1-biased immune responses (A and B) SARS-CoV-2 spike-specific IgG subclasses and the ratio of IgG2a/c/IgG1 at 30 days post-vaccination with LUNAR-COV19 compared with conventional mRNA control in BALB/c (A) and C57BL/6J (B) mice (n = 5/group). (C and D) Th2 cytokine and Th1/Th2 skew in CD4 T cells at day 7 post-vaccination in C57BL/6J mice measured by ICS as percentage of IL-4+ CD4 T cells (C) and ratio of IFNγ+/IL-4+ CD4+ T cells (D). Antibody titers and T cell data were compared between groups with a two-tailed Mann-Whitney U test; ∗0.05 < p < 0.01; ∗∗0.01 < p < 0.001.
Figure 5 LUNAR-COV19 elicits Th1-biased immune responses (A and B) SARS-CoV-2 spike-specific IgG subclasses and the ratio of IgG2a/c/IgG1 at 30 days post-vaccination with LUNAR-COV19 compared with conventional mRNA control in BALB/c (A) and C57BL/6J (B) mice (n = 5/group). (C and D) Th2 cytokine and Th1/Th2 skew in CD4 T cells at day 7 post-vaccination in C57BL/6J mice measured by ICS as percentage of IL-4+ CD4 T cells (C) and ratio of IFNγ+/IL-4+ CD4+ T cells (D). Antibody titers and T cell data were compared between groups with a two-tailed Mann-Whitney U test; ∗0.05 < p < 0.01; ∗∗0.01 < p < 0.001.CC BY-NC-ND · PubMed Central
Figure 6 Single dose of LUNAR-COV19 protects hACE2 mice against a lethal challenge of SARS-CoV-2 virus (A) hACE2 transgenic mice were immunized with a single dose of either PBS or 2 μg or 10 μg of LUNAR-COV19 (n = 5 per group), then challenged with live SARS-CoV-2 at 30 days post-vaccination, and either assessed for survival (with daily weights and clinical scores) or sacrificed at day 5 post-challenge for measurement of lung and brain tissue viral loads. (B) Live SARS-CoV-2 neutralizing antibody titers (PRNT70) measured at 28 days post-vaccination. (C–E) Weight (C), clinical score (D), and survival (E) were estimated after challenge with a lethal dose (5 × 105 TCID50) of live SARS-CoV-2 virus. (F and G) Viral RNA (F) and infectious virus (G) in the lungs and brain of challenged mice were measured with qRT-PCR and plaque assay, respectively. (H) The role of B cell and T cell depletion in LUNAR-COV19-vaccinated mice was studied after challenge with SARS-CoV-2 virus. (I) At 5 dpi, viral RNA was assessed in both mouse lungs and brain. Study design schematic diagrams were created with BioRender.com. PRNT70 and viral titers (RNA and plaque titers) were compared across groups with the non-parametric Mann-Whitney U test. Weights and clinical scores at different time points were compared between PBS and 10 μg LUNAR-COV19-immunized mice with multiple t tests. ∗0.05 < p < 0.01; ∗∗0.01 < p < 0.001; ∗∗∗0.001 < p < 0.0001; ∗∗∗∗p < 0.00001.
Figure 6 Single dose of LUNAR-COV19 protects hACE2 mice against a lethal challenge of SARS-CoV-2 virus (A) hACE2 transgenic mice were immunized with a single dose of either PBS or 2 μg or 10 μg of LUNAR-COV19 (n = 5 per group), then challenged with live SARS-CoV-2 at 30 days post-vaccination, and either assessed for survival (with daily weights and clinical scores) or sacrificed at day 5 post-challenge for measurement of lung and brain tissue viral loads. (B) Live SARS-CoV-2 neutralizing antibody titers (PRNT70) measured at 28 days post-vaccination. (C–E) Weight (C), clinical score (D), and survival (E) were estimated after challenge with a lethal dose (5 × 105 TCID50) of live SARS-CoV-2 virus. (F and G) Viral RNA (F) and infectious virus (G) in the lungs and brain of challenged mice were measured with qRT-PCR and plaque assay, respectively. (H) The role of B cell and T cell depletion in LUNAR-COV19-vaccinated mice was studied after challenge with SARS-CoV-2 virus. (I) At 5 dpi, viral RNA was assessed in both mouse lungs and brain. Study design schematic diagrams were created with BioRender.com. PRNT70 and viral titers (RNA and plaque titers) were compared across groups with the non-parametric Mann-Whitney U test. Weights and clinical scores at different time points were compared between PBS and 10 μg LUNAR-COV19-immunized mice with multiple t tests. ∗0.05 < p < 0.01; ∗∗0.01 < p < 0.001; ∗∗∗0.001 < p < 0.0001; ∗∗∗∗p < 0.00001.CC BY-NC-ND · PubMed Central

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  1. Walsh, 2020, Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates, N. Engl. J. Med., 383, 2439, 10.1056/NEJMoa2027906 · DOI · Paper page

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Metadata

Authors
de Alwis, R., Gan, E. S., Chen, S., Leong, Y. S., Tan, H. C., Zhang, S. L., Yau, C., Low, J. G. H., Kalimuddin, S., Matsuda, D., Allen, E. C., Hartman, P., Park, K.-J. J., Alayyoubi, M., Bhaskaran, H., Dukanovic, A., Bao, Y., Clemente, B., Vega, J., Roberts, S., Gonzalez, J. A., Sablad, M., Yelin, R., Taylor, W., Tachikawa, K., Parker, S., Karmali, P., Davis, J., Sullivan, B. M., Sullivan, S. M., Hughes, S. G., Chivukula, P., & Ooi, E. E.
Affiliations
  • Duke-NUS Medical School (SG)
  • SingHealth (SG)
  • National University of Singapore (SG)
  • Arcturus Therapeutics (United States) (US)
Corresponding author
Sullivan, Sean M.
ORCID
Venue
Molecular Therapy
Publisher
Elsevier BV
Published
2021-06
Volume
29
Issue
6
Pages
1970-1983
ISSN
1525-0016 (ISSN-L)
DOI
10.1016/j.ymthe.2021.04.001
PMC
PMC8019652
OpenAlex
W3139674533
Cited by
191
References
53
Citations by year
2026: 15 · 2025: 24 · 2024: 34 · 2023: 35 · 2022: 36 · 2021: 44
FWCI
11.80
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • National Medical Research Council
  • Economic Development Board - Singapore
Topics
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
COVID-19 vaccines, alphavirus replicon, spike protein, single-dose vaccination, neutralizing antibodies, CD8+ T cell responses, Th1 response, vaccine protection
MeSH terms
Angiotensin-Converting Enzyme 2, COVID-19, SARS-CoV-2, COVID-19 Vaccines, mRNA Vaccines, Alphavirus, Animals, Antibodies, Viral, Female, Humans, Immunity, Cellular, Interferon-gamma, Mice, Transgenic, Replicon, Vaccination, Vaccines, Synthetic, Interleukin-4, Gene Expression, Treatment Outcome, CD8-Positive T-Lymphocytes, Th1 Cells, Transgenes, Mice, Immunity, Humoral, Antibodies, Neutralizing, Spike Glycoprotein, Coronavirus

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