Abstract
Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318–510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318–536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.
Highlights
Severe acute respiratory syndrome (SARS) was first diagnosed in Guangdong Province, China, in 2002, spreading to 29 countries where it caused 776 deaths and over 8000 respiratory infections.[1]
The high M.W. smear disappeared, and the size develop genetically engineered SARS-CoV S protein vaccines of RBD193-WT returned to the expected M.W. (23 kDa) were reviewed previously.[1]
This assay confirmed that the high M.W. smear recombinant protein adjuvanted with alum and a Venezuelan was from high glycosylation of the yeast-expressed RBDs, and equine encephalitis vector containing S-protein plasmid were not from aggregation
Summary
Severe acute respiratory syndrome (SARS) was first diagnosed in Guangdong Province, China, in 2002, spreading to 29 countries where it caused 776 deaths and over 8000 respiratory infections.[1]. National Institutes of Health (NIH) labelled it a Category C pathogen, along with other highly transmissible agents of potential biodefense importance.[4]. Because of the explosive nature of the 2002–03 SARS pandemic, an intensive effort has been underway to develop SARS countermeasures, including vaccines.[1] A stable and effective SARS-CoV vaccine could be stockpiled as part of national or global public health emergency preparedness efforts.[4] Initial efforts focused on developing whole virus vaccines that were often inactivated by chemical agents or radiation and adjuvanted on alum[1] in laboratory mice, it was observed that such vaccines elicited eosinophilic immunoenhancing pathology with evidence of TH2-linked alveolar damage.[5,6] Previously, immune enhancing pathology in vaccinated children derailed similar efforts to develop inactivated respiratory syncytial virus (RSV) vaccines.[7]
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