Heterologous mRNA/MVA delivering trimeric-RBD as effective vaccination regimen against SARS-CoV-2: COVARNA Consortium

Abstract

Despite the high efficiency of current SARS-CoV-2 mRNA vaccines in reducing COVID-19 morbidity and mortality, waning immunity and the emergence of resistant variants underscore the need for novel vaccination strategies. This study explores a heterologous mRNA/Modified Vaccinia virus Ankara (MVA) prime/boost regimen employing a trimeric form of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein compared to a homologous MVA/MVA regimen. In C57BL/6 mice, the RBD was delivered during priming via an mRNA vector encapsulated in nanoemulsions (NE) or lipid nanoparticles (LNP), followed by a booster with a replication-deficient MVA-based recombinant virus (MVA-RBD). This heterologous mRNA/MVA regimen elicited strong anti-RBD binding and neutralizing antibodies (BAbs and NAbs) against both the ancestral SARS-CoV-2 strain and different variants of concern (VoCs). Additionally, this protocol induced robust and polyfunctional RBD-specific CD4 and CD8 T cell responses, particularly in animals primed with mLNP-RBD. In K18-hACE2 transgenic mice, the LNP-RBD/MVA combination provided complete protection from morbidity and mortality following a live SARS-CoV-2 challenge compared with the partial protection observed with mNE-RBD/MVA or MVA/MVA regimens. Although the mNE-RBD/MVA regimen only protects half of the animals, it was able to induce antibodies with Fc-mediated effector functions besides NAbs. Moreover, viral replication and viral load in the respiratory tract were markedly reduced and decreased pro-inflammatory cytokine levels were observed. These results support the efficacy of heterologous mRNA/MVA vaccine combinations over homologous MVA/MVA regimen, using alternative nanocarriers that circumvent intellectual property restrictions of current mRNA vaccine formulations.

Keywords: SARS-CoV-2 vaccine; mRNA/MVA regimen; nanocarriers; trimeric-RBD; vaccine protection.

Graphical abstract

Figure 1.

MVA-RBD recombinant virus efficiently expresses the trimeric-RBD of SARS-CoV-2. (A) Schematic representation of MVA-RBD virus genome generated by the insertion of RBD gene into the viral HA locus. (B) Time-course expression of RBD protein in HeLa cells infected with MVA-RBD. The expression of RBD protein was analyzed by WB in cellular pellets (pellet) and supernatants (SN) under reducing (R) and non-reducing (NR) conditions using an anti-RBD antibody. (C, D) SEC of RBD. Supernatants of cells infected with MVA-RBD or MVA-RBDmono were processed and analyzed by SEC as described in M&M. WB of the collected fractions is show in C, and the SEC profile resulting from the quantified amount of protein in each fraction is shown in D. Size exclusion markers in the SEC are indicated by dashed lines: Alcohol dehydrogenase-150 kDa, bovine albumin-66 kDa, and a monomeric RBD protein-27 kDa. (E) Subcellular localization of RBD protein by confocal microscopy.

Figure 2.

mRNA/MVA-RBD vaccination regimen enhances RBD-specific IgG BAbs and NAbs and elicits polyfunctional T cell immune responses in C57BL/6 mice. (A) Immunization schedule (as described in M&M). (B) Analysis of RBD-specific IgGs in individual serum samples at d20, d42 and d54. Data are shown as colored forms for each animal with geometric mean and SD. The red dotted line represents the lower limit of detection (LLD) of the assay. (C) Analysis of NAbs in individual serum samples at d20, d42 and d54. Upper black dotted line represents the levels obtained with the NIBSC 20/136 international standard plasma (pooled from 11 individuals recovered from SARS-CoV-2 infection). Bottom red dotted line represents the LLD of the assay. (D) NAbs against SARS-CoV-2 VoCs at d54. (E) Magnitude of SARS-CoV-2 RBD-specific CD4 or CD8 T cells at d11 post-MVA-RBD boost. The overall response represents the sum of the percentages of RBD-specific CD4 or CD8 T cells expressing CD107a and/or secreting IFN-γ and/or IL-2 and/or TNF-α. Data are background-subtracted. (F) Polyfunctional profile of SARS-CoV-2 RBD-specific CD4 (left) or CD8 (right) T cells. The positive combinations of the responses are indicated on the x axis, while the percentages of the functionally different cell populations within the total CD4 or CD8 T cells are represented on the y axis. Specific responses are grouped and color-coded based on the number of functions. C: CD107a; I: IFN-γ; 2: IL-2; T: TNF-α.

Figure 3.

mRNA-LNP/MVA vaccination regimen elicits the highest levels of RBD-specific IgGs and NAbs and fully protects K18-hACE2 transgenic mice. (A) Immunization schedule (as described in M&M). (B) Analysis of RBD-specific IgGs in individual serum samples obtained at d20 and d42. The red dotted line represents the LLD of the assay. (C) Analysis of NAbs in individual serum samples at d20 and d42. Upper black dotted line represents the NIBSC 20/136 international standard plasma. Bottom red dotted line represented the LLD of the assay. (D) NAbs against VoCs at d42. (E) ADCC activity against SARS-CoV-2 trimeric-RBD protein in sera collected from vaccinated mice. Black dotted curve represents the levels obtained with the BNT162b2 mRNA vaccine. Red dotted line indicates the EC₅₀. (F) Monitoring of mortality during 14 days after the SARS-CoV-2 challenge. Mice that lost more than 25% of the initial body weight were euthanized. (G) Left panel: Viral copy number of genomic (RdRp) and subgenomic (N) SARS-CoV-2 RNAs detected by one step RT-qPCR in lungs from individual mice at d7 p.c. (G5 and 50% of G1 and G4) or d14 p.c. (G2, G3 and 50% of G1 and G4). Mean RNA levels (in copies/µL) and SEM from duplicates of each lung sample are represented. Relative values were referred to PBS-challenged mice (G5). The filled shapes of G1 and G4 correspond to non-protected mice. Right panel: SARS-CoV-2 infectious virus in lungs and nasal turbinates. Mean PFU (PFUs/g of lung tissue or PFUs/mL of nasal turbinates) and SEM from triplicates of each sample are represented. The filled shapes of G1 and G4 correspond to non-protected mice.

Figure 4.

Cytokine and chemokine profile in lungs from vaccinated K18-hACE2 transgenic mice after SARS-CoV-2 challenge. Detection of RNA levels of pro-inflammatory cytokines and chemokines RNA by one step RT-qPCR at d7 p.c. for non-protected mice (G5 and 50% of G1 and G4, filled shapes) or at d14 p.c. for protected mice (G2, G3 and 50% of G1 and G4, empty shapes). Values were normalized to cellular 28S. Mean RNA levels (in A.U.) and SD from duplicates for each animal were related to control animal mock.