Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination

Abstract

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on modified vaccinia virus Ankara (MVA) against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust expression of S protein as antigen, make it a suitable candidate vaccine for industrial-scale production. Vaccinated mice produced S-specific CD8⁺ T cells and serum antibodies binding to S protein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19.

Keywords: nonclinical testing; poxvirus; vaccine vector; vaccinia virus.

Fig. 1.

Construction and virological characterization of MVA-SARS-2-S. (A) Schematic diagram of the MVA genome with the major deletion sites I to VI. The site of deletion III served for insertion of the SARS-CoV-2 S gene sequence (SARS-2-S). SARS-2-S was controlled by the virus-specific promoter PmH5 and inserted via homologous recombination between MVA DNA sequences (flank-1 and flank-2) adjacent to deletion site III in the MVA genome and copies cloned in the MVA vector plasmid pIIIH5red-SARS-2-S. Expression of the red fluorescent marker protein mCherry was used during plaque purification. Repetition of short flank-1 derived DNA sequences (del) served to remove the marker gene by intragenomic homologous recombination (marker gene deletion). (B) Genetic integrity of MVA-SARS-2-S (MVA-S). PCR analysis of viral DNA with deletion III site-specific oligonucleotide primers confirmed insertion of the SARS-2-S sequence and intragenomic deletion of the marker gene mCherry. PCR amplified a characteristic 4.8-kb DNA product from MVA-S genomic DNA compared to vector plasmid DNA (pIII-S). The expected 0.762-kb DNA fragment was obtained from nonrecombinant MVA DNA. (C) Multiple-step growth analysis of recombinant MVA-SARS-2-S (MVA-S) and nonrecombinant MVA (MVA). Differences in virus growth were determined by area under curve (AUC) prior to analysis by one-way ANOVA test. Error bars indicate the interquartile range (IQR) from the median. Asterisks represent statistically significant differences between groups: ns, nonsignificant; ****P < 0.0001.

Fig. 2.

Synthesis of full-length S glycoprotein in MVA-SARS-2-S (MVA-S)–infected cells. (A) Permeabilized or nonpermeabilized infected cells were probed with monoclonal antibodies directed against the HA-tag or the S protein of SARS-CoV (SARS-1-S). Polyclonal goat anti-mouse antibody served for S-specific fluorescent staining (red). Cell nuclei were counterstained with DAPI (blue). (B) Chicken embryonic fibroblasts (CEFs) and Vero cells were infected with a multiplicity of infection (MOI) of 10 and collected 24 h postinfection (hpi). (C and D) Vero cells were infected with MVA-SARS-2-S (MVA-S) at a MOI of 10 and collected at indicated time points. PNGase F was used for deglycosylation (MVA-Sd). Polypeptides in cell lysates were separated by SDS-PAGE and analyzed with a monoclonal antibody against the HA-tag (1:8,000) (B and C) or with human serum (1:200) (D). Lysates from noninfected (Mock) or nonrecombinant MVA-infected (MVA) cells were used as controls.

Fig. 3.

Antigen-specific humoral immunity induced by MVA-SARS-2-S (MVA-S). BALB/c mice were i.m. vaccinated in a prime-boost regime (21-d interval) with 10⁷ or 10⁸ PFU of MVA-S. Mice inoculated with saline (PBS) served as controls. Sera were collected 18 d after the first immunization (prime n = 7–8) and 14 d after the second immunization (prime-boost n = 6–8). (A and B) Sera were analyzed for S-specific IgG by ELISA and (C–E) SARS-CoV-2 neutralizing antibodies by plaque reduction assay (PRNT₅₀), virus neutralization (VNT₁₀₀), or surrogate virus neutralization test (sVNT).

Fig. 4.

Activation of S-specific CD8⁺ T cells after prime-boost immunization with MVA-SARS-2-S. Groups of BALB/c mice were i.m. immunized twice with 10⁷ or 10⁸ PFU MVA-SARS-2-S (MVA-S). Mock-immunized mice (PBS) were negative controls. (A–C) Splenocytes (n = 6) were collected and prepared on day 8 after prime, or (D–F) boost immunization on day 21 (n = 4). Splenocytes were stimulated with the H2-K^d–restricted peptide S_268–276 (S1; GYLQPRTFL) and tested by IFN-γ ELISPOT assay and IFN-γ/TNF-α ICS plus FACS analysis. (A and D) IFN-γ SFCs measured by ELISPOT assay. (B and E) IFN-γ–producing CD8⁺ T cells measured by FACS analysis. Graphs show the frequency and absolute number of IFN-γ⁺ CD8⁺ T cells. (C and F) IFN-γ– and TNF-α–producing CD8⁺ T cells measured by FACS analysis. Graphs show the frequency and absolute number of IFN-γ⁺ TNF-α⁺ CD8⁺ T cells. Differences between groups were analyzed by one-way ANOVA and Tukey post hoc test. Asterisks represent statistically significant differences between two groups: *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.

Protective capacity of MVA-SARS-2-S immunization against SARS-CoV-2 infection in human ACE2-transduced (hACE2) BALB/c mice. Groups of BALB/c mice (n = 4–6) were i.m. immunized twice with 10⁷ or 10⁸ PFU of MVA-SARS-2-S (MVA-S) over a 21-d interval. Mock-immunized mice (PBS) served as controls. About 2 wk after the last immunization, mice were sensitized with an adenovirus expressing hACE2 and mCherry and infected with SARS-CoV-2 3 d after transduction. Four days post challenge, the animals were killed and samples were taken for further analysis. (A) Lung tissues were harvested to determine SARS-CoV-2 gRNA copies, (B) the amounts of infectious SARS-CoV-2 by TCID₅₀/mL, and (D) lung histopathology. (C) Sera were tested for SARS-CoV-2 neutralizing antibodies by virus neutralization (VNT₁₀₀). (D) Fixed tissue was stained with hematoxylin and eosin (HE) or with in situ probes. Images show medium- (10×) and high-power (40×) magnification; images are representative of n = 4–6 per group. Statistical evaluation was performed with GraphPad Prism for Windows. Statistical significance of differences between groups is indicated as follows: ***P < 0.001.