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Researchers developed a trimeric immunogen derived from the SARS-CoV-2 receptor binding domain and found it to be stable up to 100 ° C with high levels of antibodies in mice and guinea pigs.
Significant efforts have been made around the world to combat COVID-19, the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). An important strategy has been to develop vaccines against the virus. Many candidate vaccines are in development, and a handful have now been approved for use by several countries.
However, most vaccines must be frozen or stored in the refrigerator, requiring cold chain storage and transport facilities, increasing logistical challenges. Thus, there is a need for a vaccine which can be stored at higher temperatures while retaining its efficacy.
The receptor binding domain (RBD) on the SARS-CoV-2 virus spike protein is responsible for binding and subsequent fusion with host cells. Most neutralizing antibodies target RBD. There are several methods of increasing the amounts of neutralizing antibodies, but they may cause unwanted side effects in some people or may not be as effective with repeated immunizations.
Elevated levels of antibodies seen in animals
To get around this, a team of researchers reports connecting the N-terminus of a heat-tolerant RBD they had previously engineered to a disulfide-linked trimerization domain derived from the human cartilage matrix protein (hCMP ), which when added with adjuvants elicited guinea pig and mouse antibodies and did not need a cold room. The team reported on their results in the bioRxiv * pre-print server.
Their previously designed RBD comprising residues 332-532 with an additional glycosylation site at N532 (mRBD) induces neutralizing antibodies in guinea pigs. Since binding of slightly 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 by intermolecular disulfide bonds, and it has been hypothesized that it will not dissociate.
The hCMP-mRBD trimeric protein retained stability for up to four weeks at 37 ° C and can tolerate higher temperatures, up to 100 ° C for shorter periods. This property will make it suitable for the deployment of vaccines in areas without cold rooms.
The hCMP trimerization sequence is 81% and 91% similar to the corresponding orthologs for mice and guinea pigs, respectively, resulting in lower hCMP titers in guinea pigs. The match is 100% for human proteins, so the titers of hCMP will be relatively low in humans.
The team studied the immune response of hCMP-mRBD in guinea pigs and mice with different adjuvants. Animals were vaccinated with three doses, the last doses being given 21 and 42 days after the first dose.
Two weeks after the first dose, with AddaVaxTM Adjuvantly, mice showed 16-fold higher spike protein binding antibody levels compared to guinea pigs and 14-fold higher spike protein antibody levels in guinea pigs compared to monomeric RBD. Thus, mice seemed 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 (ACE2) assay, mice showed approximately 3.5 times more titers than guinea pigs.
When the adjuvant used was Alhydrogel with CpG, mice and guinea pigs showed a similar response to mRBD, while a significantly higher response, approximately 28 times, to pic-2P proteins in mice. Mice also showed higher antibody levels against hCMP-mRBD compared to guinea pigs. In the competition test with ACE2 receptors, mice showed 11 times higher competition than guinea pigs.
High temperature tolerant vaccine candidate
Antibodies to the pseudovirus were also higher in mice than in guinea pigs, and levels in both animals were higher than those in sera from recovering COVID-19 patients. In mice, the neutralizing antibodies produced are more than 110 times higher than in convalescent sera.
The team also investigated the expression of hCMP-mRBD from permanent cell lines using Chinese hamster ovary (CHO) and HEK293 cells for further clinical development. They obtained protein yields of 80-100 mg / L with disulfide bound trimers. Mice immunized with these proteins showed high levels of antibodies directed against the spike protein and competing antibodies to ACE2.
Thus, according to the authors, this one-component, trimeric RBD may be easier to manufacture compared to other multi-component nanoparticle systems. It is also expected to induce higher levels of anti-RBD antibodies due to the host-derived trimerization sequence. The availability of permanent cell lines for expression of the immunogen will allow further clinical development of this high temperature stable protein as a vaccine candidate.
*Important Notice
bioRxiv publishes preliminary scientific reports which are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behaviors, or treated as established information.
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