Conjugation with 8-arm PEG and CRM197 enhances the immunogenicity of SARS-CoV-2 ORF8 protein

Abstract

Safe and effective vaccines are urgently needed to combat the COVID-19 pandemic. However, the SARS-CoV-2 variants raise concerns about the effectiveness of vaccines. As a SARS-CoV-2 antigen target, ORF8 strongly inhibits the IFN-β and NF-κB-responsive promoter, and can be potentially used for the development of SARS-CoV-2 vaccine. However, it is necessary to improve the immunogenicity of ORF8 by adjuvants or delivery systems. CRM₁₉₇ was a carrier protein with the ability to activate T helper cells for antigens. Eight-arm PEG could conjugate multiple antigen molecules in one entity with inherent adjuvant effect. In the present study, ORF8 was conjugated with CRM₁₉₇ and 8-arm PEG, respectively. The cellular and humoral immune responses to the conjugates (ORF8-CRM and ORF8-PEG) were evaluated in the BALB/c mice. As compared with ORF8-CRM and ORF8 administrated with aluminum adjuvant (ORF8/AL), ORF8-PEG induced a higher ORF8-specific IgG titer (2.6 × 10⁴), higher levels of cytokines (IFN-γ, TNF-α, IFN-β, and IL-5), stronger splenocyte proliferation. Thus, conjugation with 8-arm PEG was an effective method to improve the immune response to ORF8. Moreover, ORF8-PEG did not lead to apparent toxicity to the cardiac, liver and renal functions. ORF8-PEG was expected to act as an effective vaccine to provide the immune protection against SARS-CoV-2.

Keywords: CRM(197); ORF8; SARS-CoV-2; Vaccine; eight-arm PEG.

Fig. 1

Preparation scheme of ORF8-CRM and ORF8-PEG.

Fig. 2

Purification and characterization of ORF8. The cell cultures were analyzed by SDS-PAGE (a). Lane 1, the cell culture without IPTG induction; Lane 2, the cell culture with IPTG induction; Lane 3, the inclusion body; Lane 4, the supernatant. ORF8 was purified from the cell culture by a Ni Sepharose HP column (b). The two conjugates were analyzed by an analytical Superdex 200 column (c) and SDS-PAGE (d). Lane 1, marker; Lane 2, ORF8; Lane 3, CRM₁₉₇; Lane 4, ORF8-CRM; Lane 5, ORF8-PEG.

Fig. 3

Antibody response after immunization of the samples. The measurements of ORF8-specific IgG (a), IgG1 (b), IgG2a (c) and IgM (d) were carried out using ELISA. Blood samples after immunization on days 7, 14 and 21 were obtained for antibody measurement. Values represent mean ± S.D. from six mice per group.

Fig. 4

Mice lymphocyte proliferation and antibody avidity analysis of ORF8-specific IgG. The lymphocyte proliferation of mice spleen suspensions was restimulated by ORF8 (a). The mouse sera on day 21 were used for antibody avidity analysis (b). Bar represents mean ± S.D. from 6 mice per group.

Fig. 5

Determination of the cytokine secretions in the immunized BALB/c mice. IFN-γ (a), IFN-β (b), TNF-α (c) and IL-5 (d) in the culture supernatant were analyzed using the ELISA kits. Values represented the mean value ± S.D. from 6 mice per group.