Preclinical evaluation of a COVID-19 vaccine candidate based on a recombinant RBD fusion heterodimer of SARS-CoV-2

Abstract

Current COVID-19 vaccines have been associated with a decline in infection rates, prevention of severe disease, and a decrease in mortality rates. However, SARS-CoV-2 variants are continuously evolving, and development of new accessible COVID-19 vaccines is essential to mitigate the pandemic. Here, we present data on preclinical studies in mice of a receptor-binding domain (RBD)-based recombinant protein vaccine (PHH-1V) consisting of an RBD fusion heterodimer comprising the B.1.351 and B.1.1.7 SARS-CoV-2 variants formulated in SQBA adjuvant, an oil-in-water emulsion. A prime-boost immunisation with PHH-1V in BALB/c and K18-hACE2 mice induced a CD4⁺ and CD8⁺ T cell response and RBD-binding antibodies with neutralizing activity against several variants, and also showed a good tolerability profile. Significantly, RBD fusion heterodimer vaccination conferred 100% efficacy, preventing mortality in SARS-CoV-2 infected K18-hACE2 mice, but also reducing Beta, Delta and Omicron infection in lower respiratory airways. These findings demonstrate the feasibility of this recombinant vaccine strategy.

Keywords: Health sciences; Immune response; Immunology; Microbiology.

Graphical abstract

Figure 1

Structure and characterization of the B.1.351 (Beta) - B.1.1.7 (Alpha) receptor-binding domain (RBD) heterodimer, immunogen of PHH-1V (A) Structural representation of the RBD heterodimer. Top: sequence diagram. Bottom left: front view of the RBD heterodimer cartoon structure. Bottom right: top view of the antigen surface structure. Mutations are highlighted in green (K417N), cyan (E484K) and yellow (N501Y). (B) Computation modeling for PHH-1V vaccine. Top: AlphaFold2 results for the B.1.351-B.1.1.7 construct. This highlights the presence of two different construct conformations: (1) Stabilized N-/C-terminal conformation and (2) adopting protein-protein interactions. Bottom: hACE2 receptor-construct model derived from MD calculations of the B.1.351-B.1.1.7 construct. RBD residues 1 to 219 and 220 to 439 are shown in gray and orange, respectively, whereas ACE2 monomers are shown as a transparent surface and cartoon representation in violet and purple. (C) SDS-PAGE. The reduced and non-reduced purified antigens were loaded at three serial dilutions: 1/10, 1/20 and 1/40. M: molecular weight ladder. C: BSA control. (D) SEC-HPLC chromatogram of the purified antigen. (E) Surface plasmon resonance (SPR) for the quantitative evaluation of the affinity between the antigen and its natural ligand, the human ACE2 receptor. RU: resonance units.

Figure 2

Schematic representation of the experimental protocol in BALB/c and K18-hACE2 mice for the safety, immunogenicity, and efficacy assessment For safety and immunogenicity assays (in the top side), 48 five-week-old female BALB/c mice were allocated to 6 groups (n = 8) and were injected intramuscularly with two doses of 0.1 mL of the PHH1-1V vaccine on days 0 (prime) and 21 (boost). Then, animals were monitored daily for clinical signs and bodyweight was recorded weekly until D35/D37, when they were euthanized and both spleens and blood were collected. For safety, immunogenicity and efficacy assays (in the bottom side), K18-hACE2 mice were allocated to 4 groups (efficacy against SARS-CoV-2 D614G) or 3 groups (efficacy against different VoCs), and were injected intramuscularly with two doses of 0.1 mL of the PHH1-1V vaccine on days 0 (prime) and 21 (boost). On D35 animals were challenged with 10³ TCID₅₀ of the SARS-CoV-2 or different VoCs, blood samples were collected to analyze neutralizing activity, and they were monitored daily for clinical signs and mortality. Then, challenged animals were chronologically euthanized on D37, D39 and D42/D43; and several tissue samples were collected for several analyses. Schematic artwork used in this figure is provided by Servier Medical Art under a Creative Commons Attribution 3.0 Unported License.

Figure 3

Analysis of the antibody response on PHH-1V vaccination in mice (A) SARS-CoV-2 RBD-specific IgG responses in groups A to F on days D0, D21 and D35/D37. Endpoint antibody titers determined by ELISA in female BALB/c mice are shown. Log₁₀ IgG titers were analyzed by means of a linear mixed effects model. (B) Endpoint titer ratios of IgG2a to IgG1 in female mice vaccinated with PHH-1V vaccine (groups E and F). Analyses of IgG1 and IgG2 subclasses in groups E and F were performed by ELISA on serum samples taken on day D35/D37. Data was analyzed by means of a Mann-Whitney U-test. (C) Neutralizing antibody responses in groups A to F. SARS-CoV-2 neutralizing antibody titers in sera, against pseudoviruses that express the S protein with the Alpha sequence, were determined by PBNA 14/16 days after the second dose of each vaccine (D35/D37). Sera from female BALB/c mice collected on D35/D37 were assessed for pseudovirus-neutralizing activity. Log₁₀ IC₅₀ data was analyzed using a generalized least squares (GLS) model, employing one-sample tests against the null H₀: μ = 1.78 for comparison of estimated marginal mean against groups A and B. (D) Neutralizing antibody responses against multiple SARS-CoV-2 variants (Alpha, Beta, Delta, Omicron BA.1) by PBNA on 20-μg RBD fusion heterodimer/dose immunization. Sera mice from groups A and F collected on D35/D37 were assessed for pseudovirus-neutralizing activity as pool sera or individual sera, respectively. For the analysis of this data, one-sample t-tests against the null H₀: μ = 1.78 were employed. Each data point represents an individual mouse serum, with bars representing the mean titer per group ± SD. Statistically significant differences between groups are indicated with a line on top of each group: ^∗p<0.05; ^∗∗p<0.01; ⁺0.05<p<0.1.

Figure 4

PHH-1V-induced CD4⁺ and CD8⁺ T cell responses, and extracellular cytokine levels in vaccine-induced splenocytes from mice (A and B) Splenocytes from vaccinated female BALB/c mice were isolated 14/16 days after boost immunization (D35/D37), stimulated with RBD peptide pools, and analyzed by intracellular cytokine staining. The frequencies of cytokine expressing CD4⁺ T cells (A) and CD8⁺ T cells (B) are shown. The frequencies of CD4⁺ and CD8⁺ T cells expressing Th1 cytokines (sum of IFN-γ, TNF-α, IL-2) are also shown. The cytokine expression in splenocytes stimulated with the medium was considered the background value and this was subtracted from peptide-specific responses. Data were analyzed using a GLS model on the arcsine-square root-transformed percentage values. (C) Splenocytes from vaccinated BALB/c mice were isolated 14/16 days after boost immunization (D35/D37), stimulated with RBD peptide pools, and analyzed by IFN-γ and IL-4-specific ELISpot assays. Data were analyzed using a GLS model on the arcsine-square root-transformed percentage values. (D) Extracellular cytokines were measured by Luminex Multiplex in supernatants from BALB/c splenocytes stimulated with a pool of peptides from SARS-CoV-2 RBD. Cytokine levels in splenocytes stimulated with the medium were considered the background value and these were subtracted from the responses measured from the RBD peptide pool for each individual mouse. Data were analyzed using Kruskal-Wallis’ H test and Dunn’s post-hoc with Holm’s correction for multiple testing or Mann-Whitney’s U-test. Each data point represents an individual mouse value, with bars representing the mean ± SD Statistically significant differences between groups are indicated with a line on top of each group: ^∗p<0.05; ^∗∗p<0.01; ⁺0.05<p<0.1.

Figure 5

Protective efficacy of PHH-1V vaccine in K18-hACE2 mice on SARS-CoV-2 challenge Group A (n = 8, 4F + 4M), group B (n = 18, 9F + 9M), group C (n = 18, 9F + 9M), and group D (n = 18, 9F + 9M). (A) Survival curves for groups of immunized K18-hACE2 mice with PHH-1V vaccine and control groups. Survival analysis (Kaplan-Meier estimates and logrank test to compare groups) was performed to study differences in time to/before clinical signs and mortality. (B) Mean weight change after SARS-CoV-2 challenge calculated as a percentage of the pre-challenge weight in K18-hACE2 mice. A linear mixed effects model on the body weight change data was performed considering groups B, C and D. Points represent the average weight variation in each group and error bars depict a ± SD interval. (C) SARS-CoV-2 RT-qPCR (number of copies) in the lungs, nasal turbinate, oropharyngeal swabs and brain collected from challenged animals. (D) Viral titers were determined using a standard TCID₅₀ assay on positive samples of RT-qPCR (in some exceptional cases, RT-qPCR and viral isolation were performed in parallel for logistical reasons). RT-qPCR-negative samples are represented as 0 TCID₅₀/mL. The detection limit was set at 1.8 TCID₅₀/mL. (E) Histopathological analyses from the lungs and brain were determined for all animals. For each tissue sample, lesions were classified as follows: multifocal broncho-interstitial pneumonia; multifocal lymphoplasmacytic rhinitis; multifocal lymphoplasmacytic meningoencephalitis; and multifocal mononuclear inflammatory infiltrates within and around muscular fibers. Lesions were evaluated with the following score: 0 (no lesion); 1 (mild lesion); 2 (moderate lesion); and 3 (severe lesion). Samples of groups A, C and D correspond to 2 (D37), 4 (D39) and 7 dpi (D42 for males) or 8 dpi (D43 for females); samples of group B were taken 2 (D37), 4 (D39), and 5 dpi (D40; n = 3) or 6 dpi (D41; n = 3), when animals reached the endpoint criteria. GLS models or Kruskal-Wallis and Dunn’s post-hoc tests were employed for the analysis of the RT-qPCR, TCID₅₀ and histopathological data depending on verification of assumptions. Each data point represents an individual mouse value, with bars representing the mean ± SD. Statistically significant differences between groups are indicated with a line on top of each group: ^∗p<0.05; ^∗∗p<0.01; ⁺0.05<p<0.1. DPI: days post infection. See also Figures S1–S4.

Figure 6

Protective efficacy of PHH-1V vaccine in K18-hACE2 mice on challenge with SARS-CoV-2 Beta variant Group A (n = 8, 4F + 4M), group B (n = 18, 9F + 9M), and group C (n = 18, 9F + 9M). (A) Survival curves of animals from PHH-1V vaccinated groups and non-vaccinated groups. Survival analysis (Kaplan-Meier estimates and logrank test to compare groups) was performed to study differences in time to/before clinical signs and mortality. (B) Mean weight change after Beta variant challenge calculated as a percentage of the pre-challenge weight in K18-hACE2 mice. A linear mixed effects model on the body weight change data was performed considering groups B and C. Points represent the average weight variation in each group and error bars depict a ± SD interval. (C) SARS-CoV-2 RT-qPCR (number of copies) in the lungs and oropharyngeal swabs collected from challenged animals. (D) Viral titers were determined using a standard TCID₅₀ assay on positive samples of RT-qPCR. Negative samples are represented as 0 TCID₅₀/mL. The detection limit was set at 1.8 TCID₅₀/mL. (E) Histopathological analyses from the lungs were determined for all animals. For each tissue sample, lesions were classified as follows: multifocal broncho-interstitial pneumonia; multifocal lymphoplasmacytic rhinitis; multifocal lymphoplasmacytic meningoencephalitis; and multifocal mononuclear inflammatory infiltrates within and around muscular fibers. Lesions were evaluated with the following score: 0 (no lesion); 1 (mild lesion); 2 (moderate lesion); and 3 (severe lesion). All the samples correspond to 2 (D37), 4 (D39) and 7 dpi (D42 for males) or 8 days post infection (D43 for females); or at the time of euthanasia in animals reaching endpoint criteria before the scheduled euthanasia day. GLS models or Mann-Whitney tests were employed for the analysis of the RT-qPCR, TCID₅₀ and histopathological data depending on verification of assumptions. Each data point represents an individual mouse value, with bars representing the mean ± SD. Statistically significant differences between groups are indicated with a line on top of each group: ^∗p<0.05; ^∗∗p<0.01; ⁺0.05<p<0.1. DPI: days post infection. See also Figure S2.

Figure 7

Protective efficacy of PHH-1V vaccine in K18-hACE2 mice on challenge with SARS-CoV-2 Delta variant (A) Group A (n = 8, 4F + 4M), group B (n = 18, 9F + 9M), and group C (n = 18, 9F + 9M), (A) Survival curves of animals from PHH-1V vaccinated groups and non-vaccinated groups. Survival analysis (Kaplan-Meier estimates and logrank test to compare groups) was performed to study differences in time to/before clinical signs and mortality. (B) Mean weight change after Delta variant challenge calculated as a percentage of the pre-challenge weight in K18-hACE2 mice. A linear mixed effects model on the body weight change data was performed considering groups B and C. Points represent the average weight variation in each group and error bars depict a ± SD interval. (C) SARS-CoV-2 RT-qPCR (number of copies) in the lungs and oropharyngeal swabs collected from challenged animals. (D) Viral titers were determined using a standard TCID₅₀ assay on positive samples of RT-qPCR. Negative samples are represented as 0 TCID₅₀/mL. The detection limit was set at 1.8 TCID₅₀/mL. (E) Histopathological analyses from the lungs were determined for all animals. For each tissue sample, lesions were classified as previously assays. Lesions were evaluated with the following score: 0 (no lesion); 1 (mild lesion); 2 (moderate lesion); and 3 (severe lesion). All the samples correspond to 2 (D37), 4 (D39) and 7 dpi (D42 for males) or 8 days post infection (D43 for females); or at the time of euthanasia in animals reaching endpoint criteria before the scheduled euthanasia day. GLS models or Mann-Whitney tests were employed for the analysis of the RT-qPCR, TCID₅₀ and histopathological data depending on verification of assumptions. Each data point represents an individual mouse value, with bars representing the mean ± SD Statistically significant differences between groups are indicated with a line on top of each group: ^∗p<0.05; ^∗∗p<0.01; ⁺0.05<p<0.1. DPI: days post infection. See also Figure S2.

Figure 8

Protective efficacy of PHH-1V vaccine in K18-hACE2 mice on challenge with SARS-CoV-2 Omicron BA.1 variant (A) Group A (n = 8, 4F + 4M), group B (n = 18, 9F + 9M), and group C (n = 18, 9F + 9M), (A) Survival curves of animals from PHH-1V vaccinated groups and non-vaccinated groups. Survival analysis (Kaplan-Meier estimates and logrank test to compare groups) was performed to study differences in time to/before clinical signs and mortality. (B) Mean weight change after Omicron BA.1 variant challenge calculated as a percentage of the pre-challenge weight in K18-hACE2 mice. A linear mixed effects model on the body weight change data was performed considering groups B and C. Points represent the average weight variation in each group and error bars depict a ± SD interval. (C) SARS-CoV-2 RT-qPCR (number of copies) in the lungs and oropharyngeal swabs collected from challenged animals. (D) Viral titers were determined using a standard TCID₅₀ assay on positive samples of RT-qPCR. Negative samples are represented as 0 TCID₅₀/mL. The detection limit was set at 1.8 TCID₅₀/mL. (E) Histopathological analyses from the lungs were determined for all animals. For each tissue sample, lesions were classified as previously assays. Lesions were evaluated with the following score: 0 (no lesion); 1 (mild lesion); 2 (moderate lesion); and 3 (severe lesion). All the samples correspond to 2 (D37), 4 (D39) and 7 dpi (D42 for males) or 8 days post infection (D43 for females); or at the time of euthanasia in animals reaching endpoint criteria before the scheduled euthanasia day. GLS models or Mann-Whitney tests were employed for the analysis of the RT-qPCR, TCID₅₀ and histopathological data depending on verification of assumptions. Each data point represents an individual mouse value, with bars representing the mean ± SD. Statistically significant differences between groups are indicated with a line on top of each group: ^∗p<0.05; ^∗∗p<0.01; ⁺0.05<p<0.1. DPI: days post infection. See also Figure S2.