Self-Assembled Particles Combining SARS-CoV-2 RBD Protein and RBD DNA Vaccine Induce Synergistic Enhancement of the Humoral Response in Mice

Abstract

Despite the fact that a range of vaccines against COVID-19 have already been created and are used for mass vaccination, the development of effective, safe, technological, and affordable vaccines continues. We have designed a vaccine that combines the recombinant protein and DNA vaccine approaches in a self-assembled particle. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 was conjugated to polyglucin:spermidine and mixed with DNA vaccine (pVAXrbd), which led to the formation of particles of combined coronavirus vaccine (CCV-RBD) that contain the DNA vaccine inside and RBD protein on the surface. CCV-RBD particles were characterized with gel filtration, electron microscopy, and biolayer interferometry. To investigate the immunogenicity of the combined vaccine and its components, mice were immunized with the DNA vaccine pVAXrbd or RBD protein as well as CCV-RBD particles. The highest antigen-specific IgG and neutralizing activity were induced by CCV-RBD, and the level of antibodies induced by DNA or RBD alone was significantly lower. The cellular immune response was detected only in the case of DNA or CCV-RBD vaccination. These results demonstrate that a combination of DNA vaccine and RBD protein in one construct synergistically increases the humoral response to RBD protein in mice.

Keywords: DNA vaccine; RBD protein; SARS-CoV-2; cellular response; humoral response; self-assembled particles; virus-neutralizing activity.

Figure 1

Verification of the RBD expression in transfected cells. (A1) HEK293T cells were transfected with pVAXrbd or pVAX (negative control). The target gene expression was confirmed with corresponding mRNA detection using RT-PCR. Electrophoretic analysis of RT-PCR products in 1% agarose gel: lanes 1 and 3 are products obtained from total RNA of HEK293T cells transfected with pVAX and pVAXrbd, respectively; lane 2 is the product obtained by PCR of plasmid pVAXrbd. (A2) Analysis of the RBD protein production by Western blot in HEK293T cells: lanes 1 and 2 are lysates of HEK293T transfected with pVAXrbd and pVAX, respectively; lanes 3 and 4 are culture medium from HEK293T cells transfected with pVAX and pVAXrbd, respectively; lane 5 is purified recombinant RBD. (B) SDS-PAGE analysis of purified recombinant RBD produced in CHO-K1 cells transfected with pVEALrbd.

Figure 2

General scheme for DNA/protein complexation and characterization of their components. (A1) Schematic representation of the pVAXrbd-PGS particle (control) assembly. (A2) Electron micrograph of pVAXrbd-PGS particles. (B1) Schematic representation of the particle assembly of the combined CCV-RBD vaccine. (B2) Electron micrograph of CCV-RBD particles. (C) Confirmation of DNA encapsulation in the shell of PGS and PGS-RBD by electrophoresis in a 1% agarose gel: 1, CCV-RBD; 2, pVAXrbd-PGS; and 3, naked plasmid pVAXrbd. (D) UV spectra of the CCV-RBD and pVAXrbd-PGS structures and their components. 1, PGS; 2, RBD; 3, PGS-RBD; 4, pVAXrbd plasmid; 5 and 6, CCV-RBD and pVAXrbd-PGS particles, respectively. (E) Gel filtration on a Sepharose CL-6B column (chromatographic profile). Blue line, CCV-RBD; green line, pVAXrbd-PGS; red line, pVAXrbd plasmid. (F1) Octet binding of the RBD to iB14, SARS-CoV-2 nAb. (F2) Octet binding of the CCV-RBD to iB14, SARS-CoV-2 nAb. (G1) Schematic representation of RBD protein and antibody interaction. (G2) Schematic representation of CCV-RBD particle and antibody interaction.

Figure 3

Humoral immune response in BALB/c mice. (A) Mice were immunized intramuscularly with CCV-RBD, pVAXrbd-PGS, or RBD protein twice on days 0 and 21 and serum samples were collected 10 days after the second immunization (day 31). (B) Titers of specific IgG antibodies to SARS-CoV-2 RBD (1) and SARS-CoV-2 S (2) were determined by ELISA. (C) The virus-neutralizing activity of sera from mice immunized with CCV-RBD, pVAXrbd-PGS, and RBD protein was determined by using the SARS-CoV-2 nCoV/Victoria/1/2020 strain (100 TCID₅₀). In panels (B,C), data are presented as median reciprocals of titers. Significance was calculated using nonparametric Mann-Whitney method (* p < 0.01, ** p < 0. 05).

Figure 4

Cellular immune response in BALB/c mice. (A) The number of splenocytes releasing IFN-γ in response to specific stimulation with peptides from the RBD protein was counted in an ELISpot. (B) The percentage of SARS-CoV-2-specific IFN-γ-producing CD4+ (2) and CD8+ (3) T cells was analyzed by ISC and flow cytometry (1). Statistical analysis was performed by using GraphPad Prism 6.0 software. The significance of differences between samples was determined by using the nonparametric Mann–Whitney method (* p < 0.01).