A dual-antigen self-amplifying RNA SARS-CoV-2 vaccine induces potent humoral and cellular immune responses and protects against SARS-CoV-2 variants through T cell-mediated immunity

Figure 1. Total IgG antibody titers and SARS-CoV-2 neutralizing titers in Swiss mice vaccinated with dual- or single-antigen saRNA vaccines Neutralizing titers of COVID-19 convalescent patients were used as reference. (A) Schematic overview of the i.m. vaccination of mice with LNP formulations containing 1 μg S-RBD-saRNA, N-saRNA, or both saRNA species (ZIP1642). ZIP1642 contains 0.5 μg of each saRNA. A prime-boost schedule with 21 days interval was used. Mice vaccinated with a saRNA encoding luciferase were used as control. In these mice, luciferase expression was measured via IVIS over a course of 22 days at indicated time points (orange star). Blood samples were collected just before the prime (day 0), 3 weeks post-prime (day 21), and 2 weeks post-boost (day 35) (red drops). Splenocytes were isolated at day 35 after euthanasia (brown spleen). (B) Serum SARS-CoV-2 S1-RBD and N-specific IgG endpoint titers post-prime (day 21) and post-boost (day 35) vaccination of 6 biologically independent animals. Data are presented as individual datapoints (overlay for luciferase saRNA control) with GMT ± geometric SD; limit of quantification (LOQ) is set at 512. Numerical data can be found in Table…

Figure 2. Cellular immune response in Swiss mice after vaccination with dual- or single-antigen saRNA vaccines (A) Flow cytometric quantification of the percentage of S- or N-specific IFN-γ+ cells in the CD3+CD4+ and CD3+CD8+ splenocyte subpopulations isolated from Swiss mice 2 weeks after booster vaccination with 1 μg S-RBD-saRNA, N-saRNA, or both saRNA vaccines (ZIP1642) (n = 6). Mice injected with a luciferase-encoding saRNA mock vaccine were used as negative controls. Data are presented as individual datapoints, with means ± SDs. Numerical data can be found in Table S3. (B–E) Cytokine release profile from antigen-stimulated splenocytes as measured by a multiplex mouse Th cytokine immunoassay (n = 6). All of the multiplex data are presented as minimum-to-maximum box-and-whiskers plots with median cytokine concentration and lower limit of detection (LLOD) < 0.1. Outliers were determined via the ROUT method (Q = 1%) and presented as small datapoints; outliers were not included in statistical analysis. The asterisks in all of the graphs indicate significance of ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, or ∗∗∗∗p < 0.0001, or ns, as determined by multiple Mann-Whitney tests.

Figure 3. Low doses of the dual-antigen saRNA vaccine ZIP1642 protects Syrian Golden hamsters against WA1/2020 SARS-CoV-2 infection (A) Schematic overview of i.m. vaccination and intranasal infection of a SARS-CoV-2 Syrian Golden hamster infection model. Female Syrian Golden hamsters (6- to 8-week-old) were vaccinated with 0.1, 1, or 5 μg LNP formulated dual-antigen saRNA vaccine (ZIP1642) following a prime-boost schedule with a 21-day interval. As controls we used hamsters treated with PBS (sham) or 5 μg luciferase saRNA (negative mock control). Blood was sampled at baseline before prime (day 0), before boost (day 21), and 2 weeks post-boost (day 35) (red drops). Two weeks after the booster vaccination, hamsters were intranasally challenged with 1 × 104 TCID50 of BetaCov/Belgium/GHB-03021/2020 (EPI ISL 109 407976|2020-02-03), a Wuhan strain-like SARS-CoV-2 virus, that was recovered from a nasopharyngeal swab taken from a qRT-PCR confirmed asymptomatic patient. Lungs were collected after sacrifice, at 4 days post-infection (dpi) (pink lungs). (B) SARS-CoV-2 S-specific IgG end titers at baseline (day 0), post-prime (day 21), and post-boost (day 35) vaccination as determined by IIFA in serum…

Figure 4. Protection of hamsters against WA1/2020 SARS-CoV-2 infection by ZIP1642 is associated with reduced virus-induced lung pathology (A) Lung tissue sections were analyzed after H&E staining and scored blindly for lung damage by an expert pathologist. Cumulative lung scores are presented as medians, and the average lung score of healthy hamsters is set at 1.25. The asterisks indicate the significance of ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, or ns, non-significant, as determined by a Kruskal-Wallis test. (B–F) H&E-stained representative lung tissue sections for each vaccination group (sham, luciferase saRNA mock control, and 0.1, 1, and 5 μg ZIP1642 saRNA). Hallmarks that resemble pulmonal histopathologic findings in COVID-19 patients are indicated, including broncho-pneumonia (green arrows) with apoptotic bodies in the bronchus wall, peribronchial inflammation (blue arrows), perivascular inflammation with perivascular cuffs (red arrows), and intra-alveolar edema (purple arrows). A representative lung tissue section from an untreated healthy hamster can be seen in Figure S7.

Figure 5. Immunization with saRNA vaccines encoding Wuhan SARS-CoV-2 nucleocapsid lowers viral load in hamster challenged with a SARS-CoV-2 Beta B.1.351 variant (A) Schematic overview of i.m. vaccination and intranasal infection of a SARS-CoV-2 Syrian Golden hamster infection model. Female Syrian Golden hamsters (6- to 8-week-old) were vaccinated with 2 μg of either S-RBD-saRNA, N-saRNA, or dual-antigen saRNA vaccine (ZIP1642). Hamsters vaccinated with 2 μg of a luciferase encoding saRNA served as negative mock controls. All saRNAs were formulated in LNPs and a prime-boost schedule with a 28 day interval was used. Blood samples were taken before prime (day 0), before boost (day 28), and 2 weeks post-boost (day 42) (red drops). Two weeks after the booster vaccination, hamsters were intranasally challenged with 1 × 104 TCID50 of South African variant B.1.351 virus (hCoV-19/Belgium/rega-1920/2021; EPI_ISL_896474, 2021-01-11). Lungs were collected after sacrifice, at 4 dpi (pink lungs). (B) SARS-CoV-2 S-specific IgG end titers at baseline (day 0), post-prime (day 28), and post-boost (day 42) vaccination as determined by IIFA in serum (n = 6 and n = 5 for ZIP1642 group). Data are presented as…