Nanoparticular CpG-adjuvanted SARS-CoV-2 S1 protein elicits broadly neutralizing and Th1-biased immunoreactivity in mice

Abstract

The spike (S) protein is a leading vaccine candidate against SARS-CoV-2 infection. The S1 domain of S protein, which contains a critical receptor-binding domain (RBD) antigen, potentially induces protective immunoreactivities against SARS-CoV-2. In this study, we presented preclinical evaluations of a novel insect cell-derived SARS-CoV-2 recombinant S1 (rS1) protein as a potent COVID-19 vaccine candidate. The native antigenicity of rS1 was characterized by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody targeting the RBD antigen. To improve its immunogenicity, rS1-adjuvanted with fucoidan/trimethylchitosan nanoparticles (FUC-TMC NPs) and cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODNs) were investigated using a mouse model. The S1-specific immunoglobulin G (IgG) titers, FluoroSpot assay, pseudovirus- and prototype SARS-CoV-2-based neutralization assays were assessed. The results showed that the rS1/CpG/ FUC-TMC NPs (rS1/CpG/NPs) formulation induced a broad-spectrum IgG response with potent, long-lasting, and cross-protective neutralizing activity against the emerging SARS-CoV-2 variant of concern, along with a Th1-biased cellular response. Thus, the rS1/CpG/NPs formulation presents a promising vaccination approach against COVID-19.

Keywords: CpG adjuvant; Fucoidan-trimethylchitosan nanoparticles; Immunogenicity enhancement; Neutralizing antibody; Recombinant S1 protein; SARS-CoV-2 infection; Th1-biased cellular immune responses.

Fig. 1

Purification and identification of rS1. (A) Western blot analysis to detect rS1 glycoprotein. Protein sizes (kDa) of markers are shown on the left. rS1 is indicated by the arrow. (B) Coomassie gel analysis of a representative purification run of rS1. Proteins were separated on SDS-PAGE and stained with SimplyBlue SafeStain. Original: starting material; TFF flow through (ft): TFF flow-through; TFF 10× con.: 10-fold TFF concentration; Co²⁺ ft.: flow-through from Co²⁺ column; Washing: 10, 20, and 40 mM imidazole wash; Elution: elution from HisTrap with 150 and 250 mM imidazole treatments; The respective molecular weights (kDa) of the proteins are indicated on the left. rS1 is indicated by the arrow. (C) Purified rS1 analyzed via mass spectrometry. The amino acid sequences of SARS-CoV-2 S glycoprotein are shown along with the matching peptides obtained from LC–MS/MS analysis (shown in green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Representative TEM images of (A) FUC-TMC NPs and (B–D) rS1/CpG/NPs of different magnification.

Fig. 3

rS1 possess a native RBD structure in rS1/CpG/NPs formulation. rS1 alone or rS1/CpG/NPs was coated onto a 96-well plate and incubated with a neutralizing monoclonal antibody (NT mAb) or control antibody (anti-Flavivirus mAb) at a range of antibody concentrations (0.1 to 100 ng). The absorbance value was measured at 450 nm.

Fig. 4

Immunization schedule and S-specific IgG responses. (A) Schedule presenting mice immunizations, bleeds, and FluoroSpot assay. (B) SARS-CoV-2 S-specific IgG responses after the second and third injections. (C) Stratified ratios of IgG2a (Th1) vs. IgG1 (Th2) subclasses.

Fig. 5

Neutralizing antibody responses. (A) NT with pseudotype virus. WT SARS-CoV-2 S protein pseudotyped virus applied to determine the individual ID50 titers of neutralization antibodies for comparison with ID50 titers of individual serum of SARS-CoV-2 patients. The statistical significance between the groups was analyzed using Student's t-test (*p < 0.05; **p < 0.01). (B) PRNT50 with WT SARS-CoV-2 virus. Pooled sera of mouse group and individual serum of COVID-19 patients were used to determine the PRNT50 titers with the prototype SARS-CoV-2 virus. (C) Cross reactivity of NT. Pooled sera of mouse group were subjected to NT assay with variant SARS-CoV-2 S proteins pseudotyped viruses. (D) Duration of NT induced by rS1/CpG/NPs. Sera of three mice immunized with rS1/CpG/NPs were harvested at 0.5 and 5 months after the third injection and subjected to NT assay with WT SARS-CoV-2 S protein pseudotyped virus.

Fig. 6

Cellular responses. Splenocytes from immunized mice (1 mouse/group) were collected and stimulated either without (none), with pooled peptides of S (S peptides) or with Con A followed by detection of IFN-γ or IL-4 cytokines in triplicate using FluoroSpot assay. (A) FluoroSpot wells show the IFN-γ or IL-4 released/5 × 10⁵ cells. (B) Spot-forming cells (SFC) from (A) were quantitated and calculated. Empty and slash columns indicate none and S peptides stimulations, respectively. The data are presented as mean ± SEM, and the statistical significance between the groups under peptide stimulation was analyzed using Student's t-test (*p < 0.05). (C) Stratified SFC ratios of IFN-γ (Th1) vs. IL-4 (Th2).