4. DISCUSSION
ORF8 has the potential as hotspot of SARS-CoV-2 vulnerability. In order to improve the immunogenicity of ORF8, a SARS-CoV-2 vaccine was formulated by covalent conjugation of ORF8 with 8-arm PEG or CRM197 in the present study. ORF8/AL acted as the control. The immunological properties and preliminary safety of the two conjugates were determined in the present study.
ORF8 was expressed in the form of inclusion body by E.coli . After denaturation of the inclusion body and renaturation process, the resultant protein was loaded on a Ni Sepharose HP column. However, ORF8 could not be bound by the column, due to that the His tag of ORF8 was shielded after renaturation (data not shown). As an improved process, the inclusion body was denatured and then directly loaded on the column, which could ensure the full exposure of His tag of ORF8 to the column. ORF8 was then purified with high purity and renatured as the antigen.
Here, ORF8 was self-conjugated with 8-arm PEG and multiple ORF8 molecules were in one entity. ORF8-PEG has a high-loading capacity of ORF8 with the ability to deliver multiple ORF8 molecules, which could facilitate the antigen presenting and the bioavailability to the immune cells. Thus, ORF8-PEG per se showed the inherent adjuvant activity. CRM197 is an effective carrier protein possessing T-helper cell epitopes. Conjugation with CRM197 can activate T helper cells for ORF8 and strongly improved the immunogenicity of ORF8. Presumably, the T-helper cell epitopes of CRM197 could confer on the ORF8-specific immune cells the ability to activate T helper cells. Interestingly, ORF8-PEG showed higher immunogenicity than ORF8-CRM.
The splenocytes of the ORF8-PEG group stimulated with ORF8 could generate high secretion levels of IFN-γ (2675.1 pg/mL), TNF-α (3221.4 pg/mL), IFN-β (47.9 pg/mL) and IL-5 (17.0 pg/mL). IFN-γ, IFN-β and TNF-α were two Th1-type cytokines related to the innate immunity essential to control the viral infection. ORF8-PEG could strongly stimulate the splenocyte proliferation with a PI value of 3.2. Thus, ORF8-PEG elicited a potent cellular immune response. IL-5 was a Th2 -type cytokine related to the humoral immunity and antibody response. ORF8-PEG also elicited high ORF8-specific IgG titers (2.6×104). Thus, ORF8-PEG elicited a strong humoral immune response to ORF8. Moreover, the IgG2a and IgG1 levels increased after three immunizations of ORF8-PEG, indicating that booster immunizations led to memory B-cell activation.
In recent years, several vaccines based on spike protein or RBD have launched on the market. However, the emergence of the virus variants (e.g., Delta and Omicron strains) significantly lowered the effectiveness of the vaccines. As a different antiviral target, the vaccine based on ORF8 could be a supplement to the current vaccines, which could provide full immune protection.
In summary, ORF8-PEG induced high ORF8-specific IgG titer, high levels of IFN-γ, TNF-α and IL-5, and strong splenocyte proliferation. Moreover, ORF8-PEG showed higher immunogenicity than ORF8-CRM and ORF8/AL. Thus, conjugation with 8-arm PEG is an effective method to improve the humoral and cellular immune response to SARS-CoV-2 ORF8. Moreover, ORF8-PEG did not lead to apparent toxicity to the cardiac, liver and renal functions. ORF8-PEG was expected to act as a candidate vaccine against SARS-CoV-2.