Study looks at the impact of Stabilising SARS-Cov-2 Spike through “PP” mutations and Cleavage site removal. Combination of PP and Cleavage site removal (as seen in J&J/Novavax vaccine) was beneficial, “PP” mutation alone (as seen in Pfizer/Moderna) was not

The spike protein of (SARS-CoV-2) the virus that causes COVID is the prime focus of vaccine development. The spike protein enables both binding to host cells and membrane fusion and is the only known viral target of neutralizing antibodies. 

The virus spike protein needs to be in the pre-fusion conformation in order to attach to our body cells. The Spike protein has to undergo a conformational change to enable membrane fusion, resulting in the post-fusion confirmation.  This involves the cleavage of the two subunits (S1 and S2) that make up the pre-fusion Spike.

Antibody is only effective at preventing the virus from infecting our cells if it can bind to the spike protein in its pre-fusion conformation state. In order to be successful in preventing infections antibody must have a complementary shape to the the pre-fusion spike protein.  An effective vaccine must expose individuals to the pre-fusion confirmation spike protein; this will result in the individual making large quantities of antibody that is capable of attaching to the spike protein in its prefusion shape. 

The Spike proteins can sometimes spontaneously switch from its pre-fusion to post-fusion confirmation. One solution used by vaccine manufacturers to avoid this problem is to alter the genetic information coding for the spike protein, to stabilise (lock) it in the pre-fusion conformation. The aim being to increase the effectiveness of the neutralising antibody response generated.

Moderna’s and Pfizer’s mRNA vaccines are based on a spike construct that includes a stabilising double Proline, “PP” mutation, which aims to maintain pre-fusion conformation. The J&J and Novavax vaccines include this stabilising mutation in their vaccines but make an additional change, deleting a site from the Spike protein that is used to cleave the pre-fusion spike into the two subunits. This deleted cleavage site is referred to as the ”DCP” in this paper.  

The ChAdOx-based vaccine candidate developed by Oxford AstraZeneca, as well as the CanSino- and Gamaleya (Sputnik)-vectored candidates, use a wild-type version of the spike protein (with a genetic sequence unchanged from the original virus variant). The same is of course true for the inactivated vaccines produced by Sinovac and Sinopharm . 

A study has just been released, March 2nd 2021 that looks at how Stabilisation changes to the Spike protein impact vaccine effectiveness. “Introduction of Two Prolines and Removal of the Polybasic Cleavage Site Lead to Higher Efficacy of a Recombinant Spike- Based SARS-CoV-2 Vaccine in the Mouse Model” The study compared the “Wild type” Spike (Oxford AstraZeneca), “PP” Spike (Pfizer/Moderna) and “DCS-PP” (J&J/Novavax) > https://mbio.asm.org/content/12/2/e02648-20/article-info

The study reported that vaccination with all of the Spikes induced neutralizing antibodies that led to control of virus replication in the lung. Despite this there were notable differences observed. 

All animals (except the negative control animals) made anti-RBD (Receptor binding domain) responses after the first vaccine dose

These response were higher in the “DCS” and “DCS-PP” groups than in the Wildtype “WT” or “PP” group

Removing the cleavage site “DCP” did increase the humoral immune response in ELISAs. However it was noted that the majority of the protein was not cleaved, even when the cleavage site was present. They speculate that removal of the cleavage site “DCP” might make the protein more stable in the body after vaccination. 

Lung titers on day 2 suggested low virus replication in the “WT”, “PP”, and “DCS” groups, with some animals having no detectable virus. There however was no presence of replication-competent virus in the “DCS-PP” animals.

Conclusion

The combination of deleting the cleavage site “DCP” plus introducing the PP mutations performed best. All animals (except the negative control animals) made anti-RBD responses after the priming, but they were higher in the “DCS” and “DCS-PP” groups than in the Wildtype (WT) (Oxford AstraZeneca) and “PP group” (Pfizer/Moderna). Interestingly the “PP” stabilising mutation on its own did not induce higher levels of RBD neutralising antibody over the Wildtype Spike.  

It is currently unclear if addition of these modifications (including both DCS and PP would enhance the immunogenicity of others the other vaccines (Oxford AZ, Pfizer and Moderna). However, it might be worth testing if these vaccine candidates can be improved by this strategy. A study in nonhuman primates with adenovirus 26-vectored vaccine candidates (from J&J) expressing different versions of the spike protein also showed that the “DCS-PP candidate” performed best. As a result it was incorporated into the J&J vaccine that has just been licensed for use in the USA. 

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