Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative

Sameer Kumar Malladi, Unnatiben Rajeshbhai Patel, Raju S. Rajmani, Randhir Singh, Suman Pandey, Sahil Kumar, Sara Khaleeq, Petrus Jansen Van Vuren, Shane Riddell, Sarah Goldie, Savitha Gayathri, Debajyoti Chakraborty, Parismita Kalita, Ishika Pramanick, Nupur Agarwal, Poorvi Reddy, Nidhi Girish, Aditya Upadhyaya, Mohammad Suhail Khan, Kawkab KanjoMadhuraj Bhat, Shailendra Mani, Sankar Bhattacharyya, Samreen Siddiqui, Akansha Tyagi, Sujeet Jha, Rajesh Pandey, Shashank Tripathi, Somnath Dutta, Alexander J. McAuley, Nagendrakumar Balasubramanian Singanallur, Seshadri S. Vasan, Rajesh P. Ringe, Raghavan Varadarajan

Research output: Contribution to journalArticlepeer-review

26 Scopus citations


The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Original languageEnglish
Pages (from-to)2546-2564
Number of pages19
JournalACS Infectious Diseases
Issue number8
StatePublished - 13 Aug 2021
Externally publishedYes


  • glycosylation
  • oligomerization
  • thermostable
  • vaccine
  • variant of concern


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