BREAKING! COVID-19 Vaccine: New Discovery About SARS-CoV-2 Cold Sensitivity For Survival Adaptivity Could Impact Vaccine Studies And Developments
: American researchers from Duke University-Durham have made an important discovery about the spike protein SARS-CoV-2 that could have a broad impact on vaccine studies.
The S or spike protein is the structure that binds to host cell angiotensin-converting enzyme 2 (ACE2) to enable viral attachment and entry during the infection process. The spike protein is also the main target in vaccine development studies
The research team led Dr Robert Edwards from Duke Human Vaccine Institute demonstrated that storage temperature impacts the stability of the ectodomain on this spike protein, which significantly affects its ability to bind ACE2.
It was found that compared with storage at 22 °C or 37 °C, storage at 4 °C decreased the stability of the ACE-2 binding site and increased its exposure. This lower cold storage temperature significantly altered the antigenicity of the SARS-CoV-2 spike protein, while incubation at 37 °C quickly restored Spike integrity.
The study team warned that the findings could directly impact vaccine development studies that involve readouts of antigenic and binding measures.
Dr Edwards told Thailand Medical News, “This highly dynamic protein exhibits substantial conformational flexibility, transitioning between pre-fusion “down” and “up” states that alter the initial accessibility of the receptor-binding domain (RBD), and subsequently transitioning to a post-fusion conformation that mediates fusion of viral and cellular membranes.”
Scientists and researchers have developed stabilized spike ectodomain constructs with a native-like structure that resembles the ectodomain found on SARS-CoV-2. These ectodomain constructs, with their ability to bind ACE-2 and present surface epitopes for neutralizing antibodies, are widely used to study the spike protein and the mechanisms underlying viral entry and immune evasion.
Utilizing negative stain electron microscopy to track the behavior of the spike protein under different storage conditions, the researchers showed that the spike fraction was an average of 75% on freshly prepared spike samples. This decreased to 64% with one cycle of freezing and thawing and further decreased to 59% after storage at room temperature for one week. Storage at 37 °C for one week, on the other hand, increased the spike fraction to 83%.
Significantly the study team was surprised to find that storing the samples at 4°C dramatically decreased the fraction of intact spike to just 5%. This degraded spike could be restored through incubation at 37 °C for three hours.
As it was previously known that the spike ectodomain has been shown to undergo conformational changes according to pH, the team tested whether the degradation they observed was due to a temperature-dependent pH change that would be expected for the Tris buffer they used. Applying size exclusion chromatography, they purified one fraction of a split spike preparation into Tris buffer pH 8.0 and the other fraction into MOPS buffer pH 7.4.
After one-week of storage however with the Tris buffer at room temperature and the MOPS buffer at 4 °C, there was no significant difference in spik
e fraction reductions, compared with those previously observed.
Dr Edwards confirmed, “Thus, the primary cause of spike degradation here appears to be the temperature change and not the pH shift.”
Detailed analysis by differential scanning fluorimetry showed that the reduction in spike fraction following cold storage was accompanied by distinct profile shifts, indicating decreased stability of spike with storage at 4 °C.
To further check that the observed cold-sensitivity was not merely an artifact of the negative stain sample preparation, the researchers also measured stability using differential scanning calorimetry.
This further confirmed that storage at 4 °C destabilizes the spike protein, compared with storage at 22 °C or 37 °C, and that incubation at 37 °C for 3 hours substantially restored its stability.
The team next used ELISA to test the effects of this cold-induced instability on ACE2 binding.
It was observed that Spike stored at 4 °C exhibited higher binding to ACE-2 and an RBD-directed antibody that both require the protein to be in the “up” RBD conformation. Furthermore, an antibody isolated from a convalescent COVID-19 patient that was mapped to the ACE-2 binding site showed higher levels of binding to spike that had been stored at 4 °C.
Most significantly, a temperature-sensitive ectodomain has “direct implications for vaccine studies”
The researchers say the findings show that storage temperature has a significant impact on stability and, in turn, the antigenic and binding properties of the SARS-CoV-2 ectodomain.
The researchers warned, “The results presented here suggest a highly malleable internal architecture of the spike ectodomain consistent with its dynamic nature and our findings that point at a fragile and temperature-sensitive ectodomain have direct implications for vaccine studies.”
The research findings are published on a preprint server and are currently being peer-reviewed. https://www.biorxiv.org/content/10.1101/2020.07.12.199588v1
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