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.