Double-layered N-S1 protein nanoparticle immunization elicits robust cellular immune and broad antibody responses against SARS-CoV-2.

BACKGROUND: The COVID-19 pandemic is a persistent global threat to public health. As for the emerging variants of SARS-CoV-2, it is necessary to develop vaccines that can induce broader immune responses, particularly vaccines with weak cellular immunity. METHODS: In this study, we generated a double-layered N-S1 protein nanoparticle (N-S1 PNp) that was formed by desolvating N protein into a protein nanoparticle as the core and crosslinking S1 protein onto the core surface against SARS-CoV-2. RESULTS: Vaccination with N-S1 PNp elicited robust humoral and vigorous cellular immune responses specific to SARS-CoV-2 in mice. Compared to soluble protein groups, the N-S1 PNp induced a higher level of humoral response, as evidenced by the ability of S1-specific antibodies to block hACE2 receptor binding and neutralize pseudovirus. Critically, N-S1 PNp induced Th1-biased, long-lasting, and cross-neutralizing antibodies, which neutralized the variants of SARS-CoV-2 with minimal loss of activity. N-S1 PNp induced strong responses of CD4+ and CD8+ T cells, mDCs, Tfh cells, and GCs B cells in spleens. CONCLUSIONS: These results demonstrate that N-S1 PNp vaccination is a practical approach for promoting protection, which has the potential to counteract the waning immune responses against SARS-CoV-2 variants and confer broad efficacy against future new variants. This study provides a new idea for the design of next-generation SARS-CoV-2 vaccines based on the B and T cells response coordination.

Development and characterization of monoclonal antibody against the critical loop structure of african swine fever virus P72 protein.

African swine fever (ASF) is a highly infectious and lethal viral disease caused by the African swine fever virus (ASFV). The four prominent loop structures on the surface of the primary structural protein P72 are considered to be key protective epitopes. In this study, the four critical loops (ER1-4) of the ASFV p72 protein were individually fused to hepatitis B virus core particles (HBc) and self-assembled into nanoparticles to preserve the natural conformation of the loop structure and enhance its immunogenicity. Then, four recombinant proteins were obtained in E. coli expression system and monoclonal antibodies (mAbs) were developed and characterized. All 10 mAbs obtained were able to react with P72 protein and ASFV with potencies up to 1:204 800. Amino acids 250-274, 279-299 and 507-517 of the P72 protein were identified as linear epitopes and highly conserved. The mAb 4G8 showed the highest inhibition rate of 84% against ASFV positive sera. Importantly, neutralization experiments illustrated that mAb 4G8 has a 67% inhibition rate, indicating that its corresponding epitopes are potential candidates for ASFV vaccine. In conclusion, highly immunogenic nanoparticles of the ASFV P72 key loop were constructed to induce the production of highly effective mAbs and clarify their epitope information for the diagnosis and prevention of ASFV.

Spy&IAC enables specific capture of SpyTagged proteins for rapid assembly of plug-and-display nanoparticle vaccines.

From modular vaccine production to protein assembly on nanoparticles, the SpyCatcher/SpyTag system provides a convenient plug-and-display procedure. Here, we established a general-purpose immunoaffinity chromatography (IAC) method for SpyTagged proteins (Spy&IAC). SpyTags are displayed on the surface of nanoparticles to induce high-affinity monoclonal antibodies, allowing the specific capture of the target protein. Taking the key core antigenic regions of two coronaviruses that are currently more threatened in the field of human and animal diseases, the nucleocapsid (N) protein of SARS-CoV-2 and the COE protein of porcine epidemic diarrhea virus (PEDV) as model proteins, a purification model with SpyTag at the N-terminal or C-terminal expressed in E. coli or mammalian cells was constructed. After the efficient elution of Spy&IAC, the final yield of several proteins is about 3.5-15 mg/L culture, and the protein purity is above 90 %. Purification also preserves the assembly function and immunogenicity of the protein to support subsequent modular assembly and immunization programs. This strategy provides a general tool for the efficient purification of SpyTagged proteins from different expression sources and different tag positions, enabling the production of modular vaccines at lower cost and in a shorter time, which will prepare the public health field for potential pandemic threats.

Development and characterization of monoclonal antibodies against the extracellular domain of African swine fever virus structural protein, CD2v.

African swine fever virus (ASFV), a DNA double-stranded virus with high infectivity and mortality, causing a devastating blow to the pig industry and the world economy. The CD2v protein is an essential immunoprotective protein of ASFV. In this study, we expressed the extracellular region of the CD2v protein in the 293F expression system to achieve proper glycosylation. Monoclonal antibodies (mAbs) were prepared by immunizing mice with the recombinant CD2v protein. Eventually, four mAbs that target the extracellular region of the ASFV CD2v protein were obtained. All four mAbs responded well to the ASFV HLJ/18 strain and recognized the same linear epitope, 154SILE157. The specific shortest amino acid sequence of this epitope has been accurately identified for the first time. Meaningfully, the 154SILE157 epitope was highly conformed in the ASFV Chinese epidemic strain and Georgia2008/1 strains according to the analysis of the conservation and have a fair protective effect. These findings contribute to further understanding of the protein function of CD2v and provide potential support for the development of diagnostic tools and vaccines for ASFV.

Development and characterization of recombinant ASFV CD2v protein nanoparticle-induced monoclonal antibody.

African swine fever (ASF) caused by African swine fever virus (ASFV) is becoming a serious threat to the swine industry worldwide. CD2v is a key pathogenic factor of ASFV and the protective antigen with low immunogenicity, whereas viral protein-based nanoparticles have advantages of precise assembly and high immunogenicity. In this study, the CD2v protein fused with Norovirus (NoV) P particle assembled into nanoparticle for improved immunogenicity. Then, CD2v protein nanoparticle and monomer CD2v protein were expressed in HEK293F cells. The former induced higher levels of antibodies, and thus highly potent monoclonal antibodies (mAbs) were generated and characterized. The highest antibody titration of mAb 10A3 reached 1:2048000, and mAb 2E9 had the highest inhibition percent of 84% when competed with ASFV positive serum. Meanwhile, all mAbs reacted specifically with the denatured CD2v protein, and the linear epitope with the location of amino acids 28th to 51st of CD2v extracellular domain sequence was identified. In summary, this study produced a highly immunogenic CD2v protein and generated high-titer mAbs, the precise location of linear epitope on the CD2v was further determined. These findings may provide a powerful help for etiology and serological detection of ASFV.