Researchers have developed a trimeric immunogen derived from the SARS-CoV-2 receptor binding domain and found that it is stable up to 100 ° C with high levels of antibodies in mice and guinea pigs.
Significant efforts have been made worldwide to counteract COVID-19, the disease caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) syndrome. One obvious strategy has been the development of vaccines against the virus. A number of vaccine candidates are being developed, and a handful have now been approved for use by a number of countries.
However, most vaccines need to be frozen or stored in the refrigerator, which requires cold chain storage and transportation facilities, increasing supply challenges. Therefore, there is a need for a vaccine that can be stored at a higher temperature but still maintain efficacy.
The receptor binding domain (RBD) of the SARS-CoV-2 virus spike protein is responsible for binding and subsequent binding to the host cells. Most neutralization antibodies target the RBD. There are a number of ways to neutralize the amount of antibodies, but they can cause side effects in some people or they may not be as effective with a repeat vaccine.
High levels of antibody seen in animals
To get around this, a team of researchers report that they bind heat-tolerant RBD N-terminus previously designed to a disulfide-linked trimerization ground derived from human cartilage matrix proteins ( hCMP), added when added with admitted strong antibodies into guinea pigs and mice and does not require cold storage. The team reported their findings in the bioRxiv * preprint server.
Their previously designed RBD consisted of residues 332–532 with an additional glycosylation site at N532 (mRBD) stimulating neutral antibodies in guinea pigs. Since binding a few more of the same molecules together, or oligomerization, is believed to increase antibody titers, the authors combined their RBD with the trimerization motif. The trimer is stabilized with intermolecular disulfide bands, and it was thought that it would not separate.
The trimeric protein hCMP-mRBD maintained its stability for up to four weeks at 37 ° C and tolerates higher temperatures, up to 100 ° C for shorter periods. This premise makes it ideal for administering vaccines in areas where there are no cold storage facilities.
The hCMP trimerization sequence is similar to 81% and 91% of the corresponding orthologs for mice and guinea pigs, respectively, leading to lower hCMP titers in guinea pigs. The contact is 100% for human proteins, so the hCMP titers will be relatively low in humans.
The team studied the immune response of hCMP-mRBD in guinea pigs and mice with different confessions. The animals were vaccinated with three doses, the last doses given 21 and 42 days after the first dose.
Two weeks after the first dose, with the AddaVaxTM adjuvant, mice showed higher 16-fold spike-protein binding antibody levels compared to guinea pigs and 14-fold higher spike-protein antibody levels in guinea pigs compared to the monomeric RBD. Thus, mice appeared to show a better immune response compared to guinea pigs, possibly due to differences in the immune response of the animals. In a competitive angiotensin-converting enzyme 2 (ACE2) assay, about 3.5-fold mice showed more titers compared to guinea pigs.
When the adjuvant was used Alhydrogel with CpG, both mice and guinea pigs showed a mRBD-like response, while there was a significantly higher response, approximately 28-fold, to spike-2P proteins in mice. Mice also showed higher antibody levels to hCMP-mRBD compared to guinea pigs. In an ACE2 receptor competition assay, mice showed 11-fold higher competition compared to guinea pigs.
High temperature tolerant vaccine candidate
Antibodies to pseudovirus were also higher in mice than in guinea pigs, and levels in both animals were higher than those in sera from COVID-19 convalescent patients. In mice, the neutralization antibodies produced are more than 110-fold higher than in the convalescent sera.
The team also examined the expression of hCMP-mRBD from permanent cell lines using Chinese hamster ovary (CHO) and HEK293 cells for further clinical improvement. They obtained a protein yield of 80–100 mg / L with disulfide-linked trimers. Mice vaccinated with these proteins showed elevated levels of antibodies to spike proteins and ACE2 competing antibodies.
Thus, according to the authors, this single-component trimeric RBD may be easier to fabricate compared to other multi-component nanoparticle systems. It should also give higher levels of antibodies to RBD due to the host-derived trimerization sequence. The permanent cell lines for the expression of the immunogen allow further clinical development of this high-temperature stable protein as a vaccine candidate.
* Important message
bioRxiv publish preliminary scientific reports that are not peer-reviewed and, therefore, should not be seen as final, guiding health-related clinical / behavioral practice, or be treated as information established.