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.