Evaluating the immunogenicity of gold nanoparticles conjugated RBD with Freund's adjuvant as a potential vaccine against SARS-CoV-2

Abstract

Background: and Introduction: SARS-CoV-2 is currently considered as the most challenging issue in the field of health and medicine by causing a global pandemic. Vaccines are counted as a promising candidate to terminate this deadly pandemic. Various structural proteins in SARS-CoV-2 have recently drawn attention to be utilized as candidate vaccines to stimulate immune responses against COVID-19.

Materials and methods: In current study, the RBD protein was cloned and expressed in E. coli host. Then, the expressed RBD protein was purified and its characterizations were evaluated through various methods. Gold nanoparticles, which were utilized as a carrier for candidate Nano-vaccine, were synthesized via oxidation-reduction reaction. While Gold NPs-conjugated RBD was injected into the second treatment group, in the first candidate vaccine, RBD was injected into the first treatment group solely. Complete and Incomplete Freud's Adjuvant were also utilized for both treatment groups to enhance the immune responses against RBD antigen. Immunizations were repeated 2 times in 14-day intervals to boost the immune system of BALB/c mice. The humoral and cell-mediated immune responses were examined through immune and cytokine assays.

Results: Our outcomes demonstrate that strong short-term humoral immunity (IgM) was induced in both the first and second treatment group, while long-term humoral responses (IgG) were only observed in the second treatment group. While stronger short- and long-term humoral (IgM and IgG, respectively) were observed in the second treatment group, particular cytokines production (TNF-ɑ and IFN-γ) as a marker of cell-mediated responses were significantly higher in the first treatment group.

Discussion and conclusion: Our study results show the high potentiality of RBD protein as an appropriate stimulating antigen in vaccine synthesis and testifies RBD-based candidate vaccines to control the COVID-19 pandemic. Our outcomes also recommend that Nano-vaccines can be more suitable candidates when stronger long-term immune responses matter.

Keywords: COVID-19 vaccine; Freund's adjuvant; Gold nanoparticles; Immunogenic proteins; Nano-vaccines; RBD protein; SARS-CoV-2.

Fig. 1

The experiment process which was conducted in the current study: (A) A timeline of in vivo assay and immunization along with the route of administration, evaluated an immune response and ultimate purpose of collecting blood samples. (B) A schematic and straightforward figure about gold nanoparticles and RBD protein synthesis attachment reaction.

Fig. 2

Results of electrophoresis colony-PCR product (A) and evaluation of protein expression RBD in 12.5% SDS-PAGE gel (B). Electrophoresis: well M: Molecular Grower Marker (Thermo-Scientific Company, Iran); well C-: Negative control (no pattern DNA); C+ well: positive control (plasmid DNA pattern pET28a-RBD); 1 to 5 wells: Different transformed and cultured colonies (A). SDS-PAGE: well M: protein weight marker, Lane E1: Elution 1 of RBD protein after washing the affinity chromatography column, Lane E2: Elution 2 of RBD protein after washing the affinity chromatography column, Lane E3: Elution 3 of RBD protein after washing the affinity chromatography column. (B).

Fig. 3

Characterization of Gold nanoparticles. Gold NPs were synthesized following the procedure described. (A) dynamic light scattering (DLS); Polydispersity index (PDI) was 30.19 nm/59.92 nm (B) ultraviolet–visible spectroscopy (UV–Vis) and (C) scanning electron microscope (SEM); the scale bar of SEM was set to 300 nm.

Fig. 4

The effects of RBD immunization on levels of serum IgG isotypes (IgG1 as Th2-dependent isotype, IgG2a as Th1-biased IgG subclasses antibodies) and IgM. The RBD plus Freund's adjuvant and Gold NPs was a potent inducer for IgM (A), IgG1 (B), and IgG2a (C) in comparison to the mice inoculated with PBS. All data are presented as mean ± standard error of the mean *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S., not significant. (FA; Freund's Adjuvant, GNPs; Gold NPs).

Fig. 5

Analysis of total IgG antibody response to vaccine candidate. The experimental groups of BALB/c mice (n = 10) were vaccinated i.p. With three doses of RBD- Freund's Adjuvant, RBD-Freund's adjuvant-Gold NPs, and PBS (control) on days 0, 14, and 28. (A) Immunogenicity analysis of the protein RBD-Freund's Adjuvant. (B) Immunogenicity analysis of the protein RBD-Freund's adjuvant-Gold NPs. Antibody titers were measured by the ELISA method. All data are presented as mean ± standard error of the mean *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N·S., not significant.

Fig. 6

Cytokine release of mouse splenocytes stimulated by different components. Level of IL-4 (A), IFN-γ (B), and TNF-α (C) secretion was evaluated in mouse splenocytes stimulated by RBD protein along with Freund's adjuvant and Gold NPs. The level of responses was evaluated ten days after the final immunization. Data represented the mean ± standard deviation of ten mice per group (n = 10). Statistical analysis was performed using the T-test (GraphPad Prism8) and statistical significance was set at *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. n.s. Non-significant. (FA; Freund's Adjuvant, GNPs; Gold NPs).