SARS-CoV2 variant-specific replicating RNA vaccines protect from disease and pathology and reduce viral shedding following challenge with heterologous SARS-CoV2 variants of concern

Abstract

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late-2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoC) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.

Figure 1:

repRNA constructs evaluated in this study. Schematic representations of the SARS-CoV2 spike are shown with the signal peptide (sp), S1 and S2 domains, transmembrane domain (TM), and cytoplasmic domain (CD) indicated. The repRNAs are identified as 600 expressing the A.1 spike, 675 (B.1 spike), 604 (A.1. pre-fusion stabilized spike), 676 (B.1. pre-fusion stabilized spike), 673 (B.1.1.7 pre-fusion stabilized spike) and 671 (B.1.351 pre-fusion stabilized spike) with the mutations relative to the A.1 strain shown.

Figure 2:

Relative neutralizing antibody responses induced by each vaccine candidate as measured against 4 variants of concern. C57BL/6 mice (n=8/group) received a 1ug intramuscular injection on days 0 and 28 of LION/repRNA encoding either the native conformation of spike derived from A.1, or B.1 viruses, or the prefusion-stabilized conformation of spike derived from B.1, B.1.1.7, or B.1.351 viruses. Mice were bled 14 days after the boost immunization and neutralizing antibody responses measured by 80% plaque reduction neutralization tests (PRNT₈₀) against A.1 (A), B.1.1.7 (B), or B.1.351 (C) viruses. Data in A-C are presented as geometric mean and geometric standard deviation with each individual sample data point overlaid and were analyzed by one-way ANOVA. P < 0.05 were considered statistically significant.

Figure 3:

Post-boost neutralizing antibody responses in Syrian golden hamsters. Sera from animals immunized with LION complexed with repRNA vaccine variants A.1, B.1.1.7, or B.1.351 were incubated with live virus of variant A.1 (black), B.1.1.7 (pink), B.1.351 (green), or B.1.617.2 (purple) as indicated. Plaque-reduction neutralizing titers are indicated by individual symbols with geometric mean titers represented by the height of the bars. Indicated statistical comparisons performed using a two-way ANOVA with Tukey’s multiple comparisons test. ns P > 0.05, * P < 0.05, *** P < 0.001, **** P < 0.0001.

Figure 4:

repRNA vaccination significantly reduces viral shedding. Mock or repRNA vaccinated hamsters were challenged with 1000 TCID50 of the indicated SARS-CoV2 strains via the IN route. At day 2 or 4 PI, oral swabs were collected. SARS-CoV2 in the swabs was quantified by qRT-PCR specific for the SgE RNA (A, C, E) or infectious virus by TCID50 assay (B, D, F). N = 5 (mock-vaccinated, B.1.351 challenge) or 6 (all other groups). A two-way ANOVA with Dunnett’s multiple comparison test against mock-vaccinated hamsters was performed. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Comparisons without indicated P-values were considered non-significant (P > 0.05).

Figure 5:

repRNA vaccination significantly reduces viral burden in the lungs. Mock or repRNA vaccinated hamsters were challenged with 1000 TCID50 of the indicated SARS-CoV2 strains via the IN route. At day 4 PI, hamsters were euthanized, and lung tissue collected. SARS-CoV2 burden in the lung was quantified by qRT-PCR specific for the SgE RNA (A, C, E). Infectious virus in the lungs was quantified by TCID50 assay (B, D, F). Indicated statistical comparisons performed using a One-way ANOVA with Dunnett’s multiple comparison test against mock-vaccinated hamsters. * P < 0.05, **** P < 0.0001. Comparisons without indicated P-values were non-significant (P > 0.05)

Figure 6:

repRNA vaccination reduces lung pathology and SARS-CoV2 antigen burden in lung tissue. Mock or repRNA vaccinated hamsters were challenged with 1000 TCID50 of the indicated SARS-CoV2 strains via the IN route. At day 4 PI, hamsters were euthanized and lungs formalin-fixed and paraffin-embedded. Sections were stained with H&E (top-row of each challenge) or for SARS-CoV2 viral antigen (bottom-row of each challenge). Representative images of each group are shown.

Figure 7:

repRNA vaccination significantly protects against lung pathology in hamsters. Mock or repRNA vaccinated hamsters were challenged with 1000 TCID50 of the indicated SARS-CoV2 strains via the IN route. At day 4 PI, hamsters were euthanized, lung weighed and lungs formalin-fixed and paraffin-embedded. Lung weights as percentage of body weight are reported (A – C). Lung sections were stained with H&E (D – F) or for SARS-CoV2 viral antigen (G – I). Sections were scored by a pathologist blind to study groups and assigned a score for percent area affected by SARS-CoV2 lesions and cumulative score presented (A – C) or presence of viral antigen (D – F). Indicated statistical comparisons performed using a one-way ANOVA with Dunnett’s multiple comparison test against mock-vaccinated hamsters. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Comparisons without indicated P-values were considered non-significant (P > 0.05).