1. INTRODUCTION
SARS-CoV-2 is highly pathogenic in human, which leads to the COVID-19 pandemic and poses immeasurable public health challenges to the world.[1] The infection of SARS-CoV-2 exhibits a diverse clinical presentation, ranging from mild cases to a respiratory distress syndrome with high morbidity and mortality.[2] The importance of effective vaccines is highlighted to control the COVID-19 pandemic. The effectiveness of vaccines based on spike protein or receptor binding domain (RBD) has been established in clinical trials.[3] Recently, several vaccines have been authorized for emergent use against SARS-CoV-2, based on the inactivated viruses, recombinant viral vectors, mRNA, and recombinant proteins.[4-6] However, the emergence of SARS-CoV-2 variants such as Delta and Omicron strains significantly lowered the effectiveness of vaccines.[7]
ORF8 is a rapidly evolving accessory protein that has been proposed to interfere with immune responses.[8] ORF8 showed strong inhibition on IFN-β and NF-κB-responsive promoter.[9] ORF8 disrupts antigen presentation and reduces the recognition and the elimination of virus-infected cells by cytotoxic T lymphocytes (CTLs).[10] Thus, ORF8 is a potential antigen target for the vaccine development and a supplement to S protein, due to its lower mutation rate than S protein.[11-12]
However, ORF8 lacks the pathogen-associated molecular patterns and displays low immunogenicity, which necessitates the adjuvants or the delivery system to stimulate the immune system. Alum salts, MF59, AS03, AS04, AF03 and QS-21 have been approved as vaccine adjuvants for clinical use, which have been used for SARS-CoV-2 vaccines. However, these adjuvants may lead to the undesirable effects such as inflammation, toxicity, allergy, and immunosuppression.[13]
Antigen delivery system with the inherent adjuvant activity is highly desirable for development of SARS-CoV-2 vaccines. In particular, covalent conjugation of a carrier protein with antigens or self-conjugation of antigens is a potent antigen delivery strategy.[14-15] CRM197 is a carrier protein possessing T-helper cell epitopes and can activate T helper cells for antigens.[16] Recently, CRM197 has been covalently conjugated with bacterial capsular polysaccharide to enhance the activation of B and T cells.[17] Eight-arm polyethylene glycol (PEG) could covalently conjugate multiple antigen molecules in one entity and improve the immunogenicity of antigens.[18-19]
In the present study, SARS-CoV-2 ORF8 was expressed by E.coli , denatured and purified with high purity. ORF8 was covalently conjugated with CRM197 and self-conjugated with 8-arm PEG to improve the immunogenicity of ORF8, respectively. ORF8 administrated with aluminum adjuvant acted as the control. The cellular and humoral immune responses to the conjugates (ORF8-CRM and ORF8-PEG) were evaluated in the BALB/c mice. The potential toxicity of the conjugates to the cardiac, liver and renal functions was measured. ORF8-CRM and ORF8-PEG were expected to act as an effective vaccine to provide the immune protection against SARS-CoV-2.