tetano
Editor, Senior Moderator
J Biol Chem
. 2020 Nov 5;jbc.RA120.016284.
doi: 10.1074/jbc.RA120.016284. Online ahead of print.
Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment
Sameer Kumar Malladi[SUP] 1 [/SUP], Randhir Singh[SUP] 2 [/SUP], Suman Pandey[SUP] 2 [/SUP], Savitha Gayathri[SUP] 3 [/SUP], Kawkab Kanjo[SUP] 3 [/SUP], Shahbaz Ahmed[SUP] 3 [/SUP], Mohammad Suhail Khan[SUP] 3 [/SUP], Parismita Kalita[SUP] 3 [/SUP], Nidhi Girish[SUP] 2 [/SUP], Aditya Upadhyaya[SUP] 2 [/SUP], Poorvi Reddy[SUP] 2 [/SUP], Ishika Pramanick[SUP] 3 [/SUP], Munmun Bhasin[SUP] 3 [/SUP], Shailendra Mani[SUP] 4 [/SUP], Sankar Bhattacharyya[SUP] 4 [/SUP], Jeswin Joseph[SUP] 5 [/SUP], Karthika Thankamani[SUP] 5 [/SUP], V Stalin Raj[SUP] 5 [/SUP], Somnath Dutta[SUP] 3 [/SUP], Ramandeep Singh[SUP] 6 [/SUP], Gautham Nadig[SUP] 2 [/SUP], Raghavan Varadarajan[SUP] 1 [/SUP]
Affiliations
Abstract
Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214 mg/L in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 ?C when lyophilized, up to 70 ?C in solution and stable for over four weeks at 37 ?C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax™, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.
Keywords: ACE2; Pichia; coronavirus; glycoprotein structure; glycosylation; infectious disease; microbial; protein engineering; protein stability; thermostable; vaccine; viral protein.
. 2020 Nov 5;jbc.RA120.016284.
doi: 10.1074/jbc.RA120.016284. Online ahead of print.
Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment
Sameer Kumar Malladi[SUP] 1 [/SUP], Randhir Singh[SUP] 2 [/SUP], Suman Pandey[SUP] 2 [/SUP], Savitha Gayathri[SUP] 3 [/SUP], Kawkab Kanjo[SUP] 3 [/SUP], Shahbaz Ahmed[SUP] 3 [/SUP], Mohammad Suhail Khan[SUP] 3 [/SUP], Parismita Kalita[SUP] 3 [/SUP], Nidhi Girish[SUP] 2 [/SUP], Aditya Upadhyaya[SUP] 2 [/SUP], Poorvi Reddy[SUP] 2 [/SUP], Ishika Pramanick[SUP] 3 [/SUP], Munmun Bhasin[SUP] 3 [/SUP], Shailendra Mani[SUP] 4 [/SUP], Sankar Bhattacharyya[SUP] 4 [/SUP], Jeswin Joseph[SUP] 5 [/SUP], Karthika Thankamani[SUP] 5 [/SUP], V Stalin Raj[SUP] 5 [/SUP], Somnath Dutta[SUP] 3 [/SUP], Ramandeep Singh[SUP] 6 [/SUP], Gautham Nadig[SUP] 2 [/SUP], Raghavan Varadarajan[SUP] 1 [/SUP]
Affiliations
- PMID: 33154165
- DOI: 10.1074/jbc.RA120.016284
Abstract
Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield of 214 mg/L in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 ?C when lyophilized, up to 70 ?C in solution and stable for over four weeks at 37 ?C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax™, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.
Keywords: ACE2; Pichia; coronavirus; glycoprotein structure; glycosylation; infectious disease; microbial; protein engineering; protein stability; thermostable; vaccine; viral protein.