SARS-CoV-2 virus lacking the envelope and membrane open-reading frames as a vaccine platform

Abstract

To address the need for broadly protective SARS-CoV-2 vaccines, we developed an attenuated a SARS-CoV-2 vaccine virus that lacks the open reading frames of two viral structural proteins: the envelope (E) and membrane (M) proteins. This vaccine virus (ΔEM) replicates in a cell line stably expressing E and M but not in wild-type cells. Vaccination with ΔEM elicits a CD8 T-cell response against the viral spike and nucleocapsid proteins. Two vaccinations with ΔEM provide better protection of the lower respiratory tissues than a single dose against the Delta and Omicron XBB variants in hamsters. Moreover, ΔEM is effective as a booster in hamsters previously vaccinated with an mRNA-based vaccine, providing higher levels of protection in both respiratory tissues compared to the mRNA vaccine booster. Collectively, our data demonstrate the feasibility of a SARS-CoV-2 ΔEM vaccine candidate virus as a vaccine platform.

Fig. 1. Generation of the SARS-CoV-2 ΔEM vaccine candidate virus and its attenuation in rodent models.

a Schematic diagram of the viral genomes of the parental virus (top) and the ΔEM vaccine candidate virus (bottom) with the dashed lines around ORF E and ORF indicating the removal of the ORFs from the genome. b Infection of Vero TMPRSS2/EM cells (left panel) or Vero TMPRSS2 cells (right panel) with the ΔEM vaccine candidate virus. The SARS-CoV-2 N protein was visualized by DAB staining using an anti-N antibody. c Growth kinetics of the ΔEM vaccine candidate virus in Vero TMPRSS2/EM cells (MOI of 0.1 or 0.01) or Vero TMPRSS2 cells (MOI of 0.1). Virus titers in the cell supernatants were determined on Vero TMPRSS2/EM cells. The Day 0 samples were collected from media added immediately after washing unbound virus from the cells. Data represent the mean of experiments repeated three times (Vero TMPRSS2/EM cells) or twice (Vero TMPRSS2 cells), respectively. Error bars represent standard deviation (SD). ND; not detected. The limit of detection of infectious virus was 20 pfu/ml. d Body weight changes in hACE2 mice infected with the recombinant parental virus (Wuhan-Hu-1) or the ΔEM virus (mean with SD, n = 6 females/group). Statistical significance was determined by use of the unpaired two-tailed Student’s t-test (*P  <  0.05, **P  <  0.01, ****P  <  0.0001). Exact Pvalues: day 4 (P =  0.013253), day 5 (P =  0.000006), day 6 (P < 0.000001), day 7 (P = 0.000001), day 8 (P = 0.000051), day 9 (P =  0.000042), day 10 (P =  0.000119), day 11 (P =  0.002217). e Survival of hACE2 mice infected with the recombinant parental virus (Wuhan-Hu-1) or the ΔEM virus (mean with SD, n = 6 females/group). Statistical significance was determined by use of the Log-rank Mantel-Cox test (**P  = 0.004). f, g Virus titers of the recombinant parental virus (Wuhan-Hu-1) or the ΔEM vaccine candidate virus in lung, nasal turbinate (NT), and brain tissues of infected hACE2 mice (f) and hACE2 hamsters (g) (mean with SD, n = 4 females/virus). Tissues were collected 3 days after infection. Each dot in the bar graph indicates an individual animal in each group. ND; not detected. The dotted lines indicate the lower limit of detection (1.3 log₁₀ pfu/g). Source data are provided as a Source Data file.

Fig. 2. Protective efficacy of the ΔEM vaccine candidate virus in K18-hACE2 mice.

a, b Body weight changes (a) and survival rate (b) (n = 12 females in each vaccinated group, n = 8 females in the control group) of hACE2 mice vaccinated once or twice with the ΔEM virus and then challenged with an ancestral SARS-CoV-2 isolate. Data represent the mean, and error bars indicate SD (a). Statistical significance was determined by use of the two-tailed Dunnett’s multiple comparisons test (a; *P  <  0.05, **P  <  0.01, ****P  <  0.0001) and the Log-rank Mantel-Cox test (b; *P  = 0.033). Exact P values: (a) control vs. ΔEM x2: day 4 (P =  0.0360), day 5 (P =  0.0012), day 6 (P < 0.0001), day 7 (P < 0.0001), day 8 (P =  0.0022), day 9 (P =  0.0348); control vs. ΔEM x1: day 5 (P =  0.0020), day 6 (P < 0.0001), day 7 (P = 0.0001), day 8 (P =  0.0064). c Efficacy of one or two vaccinations of the ΔEM vaccine candidate virus. Virus titers in the lung and nasal turbinate (NT) tissues of K18-hACE2 mice (mean with SD, n = 8 females/group) at 3 days after challenge with an ancestral SARS-CoV-2 isolate. The dotted line indicates the limit of detection (1.3 log₁₀ pfu/g). Each dot in the bar graph indicates an individual mouse in each group. Statistical significance was determined by use of a one-way ANOVA with Dunnett’s multiple comparisons test (****P  <  0.0001). Source data are provided as a Source Data file.

Fig. 3. Respiratory tract immune response in ΔEM virus-vaccinated K18-hACE2 mice.

a Induction of spike-specific IgA in nasal wash and bronchoalveolar lavage fluid (BALF) of ΔEM virus-vaccinated mice (n = 4 females for the mock infection group; n = 6 females for the infection groups except that nasal wash samples on day 5 post-infection was collected from 7 females), mRNA-vaccinated mice (n = 6 females for both the mock and infection groups), or mock-vaccinated mice (n = 4 females) as measured by an ELISA. Samples were collected on days 2 or 5 following infection with an ancestral SARS-CoV-2 isolate. b, c Induction of an antigen-specific T cell population producing IFN-γ in the lungs of ΔEM virus- or mRNA-vaccinated mice (n = 6 females/group) as measured by use of an ELISpot assay. Representative ELISpot wells of cells isolated from lung tissue stimulated with S peptide pool (b; 2 x 10⁵ cells/well) or N peptide pool (c; 4 x 10⁵ cells/well) are shown. d, e The frequency of IFN-γ- and TNF-α-positive CD4⁺ and CD8⁺ T cells in the lungs of ΔEM virus- or mRNA-vaccinated mice as measured by flow cytometry. Cells collected from lung tissue were stimulated with S peptide pool (d, n = 6 females) or N peptide pool (e, n = 6 females). Box plots show the median center line and 10/90 percentiles. Whiskers show min and max values. Statistical significance was determined by use of a one-way ANOVA with Tukey’s multiple comparisons test (a, d, e) and a one-way ANOVA with Dunnett’s multiple comparisons test (b, c) (*P  <  0.05, **P  <  0.01, ****P  <  0.0001; ns, not significant). Exact P values: (a) nasal wash samples on day 5 post-infection: ΔEM x1 vs. mock vaccination control (P =  0.0051), ΔEM x1 vs. mRNA x1 (P =  0.0944); BALF samples on day 5 post-infection: ΔEM x1 vs. mock vaccination control (P =  0.0492), ΔEM x1 vs. mRNA x1 (P =  0.0262). (b) ΔEM vs. mock (P =  0.0284), ΔEM vs. mRNA 0.1 μg (P =  0.0452), ΔEM vs. mRNA 1.0 μg (P =  0.7588). (c) ΔEM vs. mock (P <  0.0001), ΔEM vs. mRNA 0.1 μg (P <  0.0001), ΔEM vs. mRNA 1.0 μg (P <  0.0001). (d) S-reactive CD4⁺ T cells frequency: ΔEM vs. mock (P =  0.0109), ΔEM vs. mRNA 0.1 μg (P =  0.0494), ΔEM vs. mRNA 1.0 μg (P =  0.9897), mock vs. mRNA 1.0 μg (P =  0.0213); S-reactive CD8⁺ T cells frequency: ΔEM vs. mock (P =  0.0015), ΔEM vs. mRNA 0.1 μg (P =  0.0310), ΔEM vs. mRNA 1.0 μg (P =  0.9140), mock vs. mRNA 1.0 μg (P =  0.0066). (e) N-reactive CD4⁺ T cells frequency: ΔEM vs. mock (P =  0.9932), ΔEM vs. mRNA 0.1 μg (P =  0.9847), ΔEM vs. mRNA 1.0 μg (P =  0.5050), mock vs. mRNA 1.0 μg (P =  0.3592); N-reactive CD8⁺ T cells frequency: ΔEM vs. mock (P =  0.0076) ΔEM vs. mRNA 0.1 μg (P =  0.0076), ΔEM vs. mRNA 1.0 μg (P =  0.0144), mock vs. mRNA 1.0 μg (P =  0.9915). Source data are provided as a Source Data file.

Fig. 4. Protective efficacy of ΔEM vaccination against SARS-CoV-2 variants in hamsters.

Efficacy of two vaccinations with the ΔEM vaccine candidate virus in hamsters. a Virus titers in the lung and nasal turbinate (NT) tissues on day 3 after challenge with a Delta variant (n = 7 female and n = 4 male control animals; n = 8 female and n = 4 male vaccinated animals) or an Omicron XBB variant (n = 6 female and n = 4 male control animals; n = 8 female and n = 4 male vaccinated animals). b Virus titers on day 6 after challenge with a Delta variant (n = 6 female and n = 4 male control animals; n = 6 female and n = 6 male vaccinated animals) or an Omicron XBB variant (n = 6 female and n = 6 male control animals; n = 6 female and n = 6 male vaccinated animals). Data represent the mean, and each dot in the bar graph indicates an individual hamster. Error bars represent SD. The dotted line indicates the limit of detection (1.3 log₁₀ pfu/g). Statistical significance was determined by use of a two-way ANOVA with Šídák’s multiple comparisons test (*P  <  0.05, **P  <  0.01, ***P  <  0.001, ****P  <  0.0001). Exact P values: (a) Delta-challenged female lung (P  <  0.0001), female NT (P  <  0.0001), male lung (P  <  0.0001), male NT (P  <  0.0001); XBB-challenged female lung (P  <  0.0001), female NT (P  <  0.0001), male lung (P  <  0.0001), male NT (P  <  0.0001). (b) Delta-challenged female lung (P  =  0.0036), female NT (P  = 0.0171), male lung (P  <  0.0001), male NT (P  =  0.0128); XBB-challenged female lung (P  =  0.0165), female NT (P  =  0.0004), male lung (P  =  0.0209), male NT (P =  0.0012). Source data are provided as a Source Data file.

Fig. 5. Pathological features in the lungs of ΔEM virus-vaccinated hamsters.

Semi-quantitative pathological scores of individual hamsters analyzed using the indicated four parameters (a). A 5-point scoring system (0-within normal limits, 1-mild, 2-moderate, 3-marked, 4-severe) was used. A total pathology score was calculated for each hamster by adding the individual histopathological feature scores (b). A maximum total pathology score of 16 is possible for an individual hamster (n = 4 females, n = 4 males for non-vaccinated controls, n = 6 females, n = 6 males for vaccinated hamsters). Statistical significance was determined by use of a two-way ANOVA with Šídák’s multiple comparisons test (****P  <  0.0001). Data represent the mean, and error bars indicate SD (a and b). Each symbol indicates an individual hamster. Source data are provided as a Source Data file.

Fig. 6. Potential of the ΔEM vaccine candidate virus to serve as a booster vaccine in hamsters.

a Total IgG antibody endpoint titers against the spike proteins of an ancestral isolate (spike D614G) or Omicron XBB using hamster sera after one or two vaccinations with the ΔEM virus. Each dot in the bar graph indicates an individual hamster in each group (mean with SD, n = 4 females/group). Statistical significance was determined by using a two-way ANOVA with Šídák’s multiple comparisons test (*P  <  0.05, **P  <  0.01, ***P  <  0.001). b Efficacy of the ΔEM vaccine virus as a booster vaccine. Virus titers of the Omicron XBB variant in lung and nasal turbinate (NT) tissues at day 3 after challenge. Each dot in the bar graph indicates an individual hamster in each group (mean with SD, n = 4 females/group). Statistical significance was determined by using the unpaired two-tailed Student’s t-test. (*P  <  0.05, ***P  <  0.001, ****P  <  0.0001). c Induction of a spike-specific T cell population producing IFN-γ in the lungs of ΔEM virus- or mRNA-boosted hamsters that had previously received a prime mRNA vaccination as measured by use of an ELISpot assay (n = 6 females/group). Box plots show the median center line and 10/90 percentiles. Whiskers show min and max values. Statistical significance was determined by using a one-way ANOVA with Tukey’s multiple comparison test (*P  <  0.05, **P  <  0.01; ns, not significant). Exact P values: (a) Ancestral D614G spike: mRNA x2 (P  = 0.0001), mRNA + ΔEM (P  = 0.0018), ΔEM x2 (P  = 0.0004); XBB spike: mRNA x2 (P  = 0.0011), mRNA + ΔEM (P  = 0.0003), ΔEM x2 (P  = 0.0348). (b) lung: control vs. mRNA x2 (P  = 0.0002), mRNA x2 vs. mRNA + ΔEM (P  < 0.0001); NT: control vs. mRNA x2 (P  = 0.0122), mRNA x2 vs. mRNA + ΔEM (P  = 0.0129). (c) control vs. mRNA x2 (P  = 0.2058), control vs. mRNA + ΔEM (P  = 0.0011), mRNA x2 vs. mRNA + ΔEM (P  = 0.0381). Source data are provided as a Source Data file.