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An Alphavirus-derived replicon RNA vaccine induces SARS-CoV-2 neutralizing antibody and T cell responses in mice and nonhuman primates

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Science Translational Medicine  05 Aug 2020:
Vol. 12, Issue 555, eabc9396
DOI: 10.1126/scitranslmed.abc9396
  • Fig. 1 repRNA-CoV2S vaccine design and formulation.

    (A) Shown is the codon-optimized full-length spike (S) protein open reading frame, including the S1, S2, transmembrane (TM), and cytoplasmic domains (CD), corresponding to positions 21,536 to 25,384 in the S protein of SARS-CoV-2 isolate Wuhan-Hu-1 (GenBank: MN908947.3). This construct was fused to a C-terminal v5 epitope tag and then was cloned into an alphavirus replicon encoding the four nonstructural protein (nsP1 to nsP4) genes of Venezuelan equine encephalitis virus, strain TC-83. After RNA transcription and capping, repRNA-CoV2S was transfected into BHK cells. Twenty-four hours later, the transfected BHK cells were analyzed by (B) anti-v5 immunofluorescence and (C) Western blot using either convalescent human serum or anti-v5 serum for immunodetection. Recombinant SARS-CoV2 S protein (rCoV2-Spike) and repRNA-GFP were used as positive and negative controls, respectively. (D) Shown is a graphical representation of LION and formation of the vaccine complex after mixing with repRNA. (E) Shown is the evolution of LION particle size over 15 weeks measured by dynamic light scattering during storage at 4°, 25°, or 42°C. (F) After mixing LION particles and repRNA, complex formation was confirmed by a shift in size distribution. (G) Gel electrophoresis analysis of triplicate preparations of repRNA extracted from LION particles after a concentrated RNase challenge showed substantial protection relative to a triplicate preparation of a dose-matched naked RNA after RNAse challenge. The formulated vaccine was stable for at least 1 week after mixing and storage at 4° or 25°C as determined by (H) gel electrophoresis of repRNA extracted by phenol-chloroform treatment and (I) particle size of the complex. Data in (B) and (C) are representative of two independent experiments. Data in (E), (H), and (I) are from a single experiment, whereas data in (F) and (G) are representative of three independent experiments. Data in (E), (G), and (I) are shown as means ± SD of three technical replicates. Scale bar, 100 μm (B).

  • Fig. 2 The LION/repRNA-CoV2S vaccine induces TH1-biased and -neutralizing antibodies in C57BL/6 mice.

    Six- to 8-week-old C57BL/6 mice (n = 5 per group) received 10, 1, or 0.1 μg of LION/repRNA-CoV2S via the intramuscular route on days 0 and 28. (A) Anti-S IgG antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA) on days 14, 28, and 40. For day 14 samples, (B) 50% inhibitory concentrations (IC50) were determined by pseudovirus (SARS-CoV-2 Wuhan-Hu-1 pseudotype) neutralization assays. For day 14 samples, (C) anti-S IgG1 and IgG2c antibody end point titers and (D) ratios were determined by ELISA. On day 40, 12 days after a booster immunization, (E) spleens and (F) lungs were harvested, and IFN-γ responses were measured by enzyme-linked immune absorbent spot (ELISpot) assay after an 18-hour stimulation with 10 peptide pools encompassing the S protein and consisting of 15-nucleotide oligomer overlapping by 11 amino acids (see fig. S1). Data in (A), (C), and (D) are representative of three independent experiments; data in (B), (E), and (F) are from a single experiment. Dotted lines in (A), (B), (E), and (F) represent the lower limit of detection. All data are represented as individual values and means ± SD. *P < 0.05 as determined by one-way ANOVA with Tukey’s multiple comparison test.

  • Fig. 3 LION/repRNA-CoV2S induces TH1-biased antibodies in aged BALB/c mice.

    Two-, eight-, or seventeen-month-old BALB/c mice (n = 5 per group) received 10 or 1 μg of LION/repRNA-CoV2S via the intramuscular route on days 0 and 28. On day 14 after prime and day 12 after boost, (A) anti-S IgG was measured by ELISA. On day 40, 12 days after the boost, spleens were harvested and (B) IFN-γ responses were measured by ELISpot assay after an 18-hour stimulation with 10 peptide pools encompassing the S protein and consisting of 15-nucleotide oligomer overlapping by 11 amino acids (see fig. S1). (C) Anti-S IgG1 and IgG2a antibody end point titers and (D) ratios were determined by ELISA 14 days after the prime immunization. Data in 17-, 8-, and 2-month-old BALB/c mice are from a single experiment; data for the 2-month-old BALB/c mice were replicated in a second experiment. All data are represented as individual values and means ± SD. *P < 0.05 as determined by one-way ANOVA with Tukey’s multiple comparison test between the 17-month-old animals and either the 8- or 2-month-old animals.

  • Fig. 4 LION/repRNA-CoV2S induces neutralizing antibody responses in pigtailed macaques.

    (A) Pigtail macaques were vaccinated with 250 μg (n = 3) or with 50 μg (n = 2) of LION/repRNA-CoV2S via the intramuscular route, and serum was collected on days 10, 14, 28, 42, 56, and 70. The 50-μg-dose group received a boost vaccination on day 28, and blood was collected 14, 28, and 42 days later. (B) Using preimmunization blood draws to establish a baseline, serum anti-S IgG. ELISAs were performed on the post-immunization serum samples. (C) Pseudovirus (SARS-CoV-2 Wuhan-Hu-1 pseudotype) neutralization assays were performed on serum samples collected on days 14, 28, and 42 to determine mean IC50 of each sample. In addition, (D) 80% plaque reduction neutralizing antibody titers (PRNT80) against the SARS-CoV2/WA/2020 isolate were measured at days 28 and 42 alongside seven human convalescent serum samples collected from confirmed patients with COVID-19 (see table S1). The experiment was performed once. Each line in (B) and (C) represents each individual animal. Data in (D) are reported as individual values and means ± SD. *P < 0.05 as determined by Student’s t test comparing 250-μg-dose groups at days 14 and 28. There was no significant difference (ns) between mean PRNT80 titers in all five animals at day 42 and titers in sera from seven convalescent humans, as measured by Mann-Whitney U test. ns, not significant.

  • Fig. 5 LION/repRNA-CoV2S induces T cell responses in pigtail macaques.

    Pigtail macaques were vaccinated with 250 μg (n = 3) or with 50 μg (n = 2) of LION/repRNA-CoV2S via the intramuscular route. PBMCs were isolated from blood at baseline and on days 10, 14, 28, and 42 after prime immunization for T cell analysis. Shown are (A) magnitude and (B) breadth of IFN-γ responses measured in PBMCs by ELISpot assay after 24-hour stimulation with 11 peptide pools encompassing the S protein including the full-length S protein and S1, S2, and RBDs. (B) Data are presented as percent of total full-length S protein response. (C and D) The frequency of S-specific CD4+ or CD8+ T cells producing any cytokine (IFN-γ, IL-2, IL-17A, TNF, and/or MIP-1β, granzyme B/CD107a) or IFN-γ alone was determined using cryopreserved PBMCs stimulated overnight with S protein peptides. Shown are the frequencies of S-specific CD4+ or CD8+ T cells after subtraction of background (DMSO vehicle). Data are from a single experiment. In (A), (C), and (D), each symbol/line is an individual animal. Data in (B) are representative of each individual animal. (A) Friedman test with multiple comparisons. *P < 0.05. (C and D) Wilcoxon matched-pairs signed rank test; P values are shown.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/555/eabc9396/DC1

    Fig. S1. Breadth of T cell responses in C57BL/6 and BALB/c mice.

    Fig. S2. Vaccination did not induce adverse reactions in pigtail macaques.

    Fig. S3. Raw ELISA absorbance values for pigtail macaques.

    Fig. S4. Neutralization curves for pigtail macaque and human samples against SARS-CoV-2/WA/2020 or pseudotyped virus.

    Fig. S5. Gating strategies used for evaluation of peptide-specific intracellular cytokine staining.

    Fig. S6. LION/repRNA-CoV2S induces memory T cell responses in pigtail macaques.

    Table S1. Convalescent sera from individuals with COVID-19.

    Data file S1. Source data for all figures.

  • The PDF file includes:

    • Fig. S1. Breadth of T cell responses in C57BL/6 and BALB/c mice.
    • Fig. S2. Vaccination did not induce adverse reactions in pigtail macaques.
    • Fig. S3. Raw ELISA absorbance values for pigtail macaques.
    • Fig. S4. Neutralization curves for pigtail macaque and human samples against SARS-CoV-2/WA/2020 or pseudotyped virus.
    • Fig. S5. Gating strategies used for evaluation of peptide-specific intracellular cytokine staining.
    • Fig. S6. LION/repRNA-CoV2S induces memory T-cell responses in pigtail macaques.
    • Table S1. Convalescent sera from individuals with COVID-19.

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    Other Supplementary Material for this manuscript includes the following:

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