The minimal mutation of SARS-CoV-2 since December 2019 suggests a global vaccine is feasible

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Genetic analysis of sequences from more than 27,000 individuals infected with the coronavirus that causes COVID-19 reveals that the virus has mutated minimally since December 2019, suggesting one vaccine would be sufficient to combat global infections.

The study was conducted by a team of scientists from the Walter Reed Army Institute of Research led by Morgane Rolland, chief of viral genetics and systems serology for the WRAIR Military HIV Research Program and Dr. Kayvon Modjarrad, director of the institute’s Emerging Infectious Diseases Program.

A manuscript detailing the findings was published in Proceedings of the National Academy of Sciences.

To characterize SARS-CoV-2 coronavirus diversification since the beginning of the pandemic they aligned 18,514 independent virus genome sequences sampled from individuals in 84 countries and scanned them for variations.

Analyses reveal low estimates of genetic differentiation following the initial outbreak, and indicate that, so far, the SARS-CoV-2 genome has evolved through a mostly random process rather than through adaptation to the human hosts it encounters.

“Like other reports, we noticed that the D614G mutation in the Spike has rapidly increased in frequency since the beginning of the epidemic, but we could not link this mutation to specific adaptive forces,” said Rolland.

“When viruses replicate and spread in the population, we expect to see some mutations and some can become fixed very rapidly in an epidemic just by random chance.”

Rolland noted that linking genotypes to phenotypes is complicated and more research is needed to fully understand the functional consequences of the D614G mutation in SARS-CoV-2.

Given the low level of genetic variation, a promising vaccine candidate would likely be equally efficacious against all currently circulating strains of the COVID-19 coronavirus.

“Viral diversity has challenged vaccine development efforts for other viruses such as HIV, influenza and dengue, but global samples show SARS-CoV-2 to be less diverse than these viruses,” said Rolland.

“We can therefore be cautiously optimistic that viral diversity should not be an obstacle for the development of a broadly protective vaccine against COVID-19 infection.”

Modjarrad co-leads the institute’s COVID-19 response efforts, including the development of a vaccine against COVID-19.

WRAIR’s leading vaccine candidate is built on a Spike Ferritin Nanoparticle platform and is expected to enter human testing before 2021.

The vaccine is paired with a proprietary adjuvant that was also developed at WRAIR, the Army Liposome Formulation, to further boost the immune response.

“Scientists are working hard to accelerate the development of a COVID-19 vaccine that is safe and effective for the entire world, now and in the years to come.

These data are critical to informing the field’s collective efforts in getting a vaccine that is rapidly scalable and universally applicable to all populations,” said Modjarrad.

He added, “Based upon WRAIR’s long experience developing vaccines for other viruses and recent work on coronaviruses, we have been able to move quickly to accelerate research efforts to combat this pandemic that has threatened global health and military readiness.”

WRAIR was established 127 years ago to combat these types of health threats and has played a role in the development of nearly half of the vaccines in public use today.

Rolland, whose research usually focuses on HIV viral genetics, has shifted her attention to COVID-19 during the current global health emergency. “It’s critical that people in various fields come together as we focus on learning everything we can about this virus,” she said.

“Teamwork will be vitally important to stem the tide of this pandemic.” Rolland is employed by the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and has conducted research at WRAIR since 2010.


WRAIR’s Emerging Infectious Diseases Branch (EIDB) is leading efforts to develop a safe and effective vaccine to prevent infection with COVID-19. WRAIR initially developed more than two dozen prototypes, administered to nearly one thousand mice, to study the most promising binding and neutralizing antibody response in preclinical studies.

In June, researchers identified the most promising vaccine prototype, along with two backups, for future testing in human clinical trials later this year.  WRAIR’s vaccine, called the Severe Acute Respiratory Syndrome Coronavirus-2 Spike Ferritin Nanoparticle (SpFN) is one of many currently in development.

Researchers hope WRAIR’s ferritin vaccine platform could also pave the way for a universal vaccine to protect against not only the current virus, but also other currently known coronaviruses and unknown species that could arise in the future. 

EIDB scientists are also working closely with other institutions through Operation Warp Speed to advance other vaccine candidates. 

“Based upon WRAIR’s long experience developing vaccines for other viruses and recent work on coronaviruses, we’ve been able to move quickly in advancing a vaccine candidate,” said Dr. Kayvon Modjarrad, Director of EIDB. Modjarrad recently published the results of the first-in-human trial of a Middle East respiratory syndrome (MERS) vaccine candidate. MERS, another coronavirus in the same family as the coronavirus that causes COVID-19, is a deployment and global health concern due to its high fatality rate of nearly 40%. 

The Spike Protein Target

SARS-CoV-2 enters the host cell by binding via its spike protein to the angiotensin-converting enzyme 2 (ACE), the host cell receptor. The spike protein exists as a trimer, with two subunits, S1 and S2, that play a role in viral attachment and fusion, respectively.

The S1 subunit has a receptor-binding domain (RBD) that undergoes functional folding when separated from the rest of the subunit.

Recovered COVID-19 patients show high titers of neutralizing antibodies directed against this protein, indicating that it could be used to produce a protective vaccine. Indeed, the spike glycoprotein has been the focus of intensive vaccine development efforts.

Construct design for SARS-CoV-2 spike-functionalized ferritin nanoparticles. All constructs are based on the Wuhan-Hu-1 amino acid sequence (GenBank MN9089473) of SARS-CoV-2 spike. Spike-functionalized ferritin constructs were made by fusing spike ectodomain (residues 1-1213) or spikeΔC (residues 1-1143) to the H. pylori ferritin subunit separated by an SGG linker. A structural representation based on the spike trimer cryo-EM structure (PDB 6VXX) and the H. pylori ferritin crystal structure (PDB 3BVE) depicts the 24-subunit particle displaying spike or spikeΔC on the surface. The estimated size of the spike-functionalized ferritin particles based on structural data is ~ 300 Å. The S-GCN4 and SΔC-GCN4 trimer constructs were made by fusing either the full-length spike residues (1-1213) or spikeΔC (1-1137) to a modified GCN4 trimerization domain followed by a hexahistidine tag. A structural representation of the spike trimers based on the cryo-EM structure (PDB 6VXX) is shown with an estimate length of ~ 100 Å. The RBD spans residues 319-541 of the spike protein and is preceded by the native signal peptide (not shown) and followed by a hexahistidine tag.

Construct design for SARS-CoV-2 spike-functionalized ferritin nanoparticles. All constructs are based on the Wuhan-Hu-1 amino acid sequence (GenBank MN9089473) of SARS-CoV-2 spike. Spike-functionalized ferritin constructs were made by fusing spike ectodomain (residues 1-1213) or spikeΔC (residues 1-1143) to the H. pylori ferritin subunit separated by an SGG linker. A structural representation based on the spike trimer cryo-EM structure (PDB 6VXX) and the H. pylori ferritin crystal structure (PDB 3BVE) depicts the 24-subunit particle displaying spike or spikeΔC on the surface. The estimated size of the spike-functionalized ferritin particles based on structural data is ~ 300 Å. The S-GCN4 and SΔC-GCN4 trimer constructs were made by fusing either the full-length spike residues (1-1213) or spikeΔC (1-1137) to a modified GCN4 trimerization domain followed by a hexahistidine tag. A structural representation of the spike trimers based on the cryo-EM structure (PDB 6VXX) is shown with an estimate length of ~ 100 Å. The RBD spans residues 319-541 of the spike protein and is preceded by the native signal peptide (not shown) and followed by a hexahistidine tag.

Advantages of a Subunit Vaccine

The current study focuses on the production of a subunit vaccine, rather than a virus-based vaccine. The reasons for this choice include improved storage stability, which includes a greater range of distribution, ease of manufacture, safety, and greater consistency in the vaccine quality. However, the limiting factor is the weaker immune response associated with them.

Ferritin-Based Vaccine

To overcome this, adjuvants are typically added to enhance their immunogenicity. However, presenting the antigen in a multivalent format is also an effective way to achieve this, and the use of nanoparticles is one such approach. The current study uses Helicobacter pylori ferritin, which has been employed to display antigens from the influenza virus, HIV, and Epstein-Barr virus.

The H. pylori ferritin is a protein that displays the property of self-assembly, forming particles containing 24 subunits, with eight 3-fold symmetries. This enables it to fuse with one spike protomer per subunit, allowing the display of eight trimeric spike antigen subunits at the surface of the assembled particle. Moreover, earlier research shows that antigens mounted on ferritin induce greater immunity compared to immunization with the antigen alone.

Two vaccines using influenza virus proteins on a ferritin platform have been demonstrated to be clinically viable in clinical trials. The facilities for large-scale ferritin vaccine manufacture are also ready.

Different Spike-Based Vaccine Designs

In the current study, the researchers used two designs to construct ferritin vaccines. One, they used the full-length spike ectodomain, while in the second case, they used it after deleting the 70 residues at the C-terminal end of the ectodomain. The reason for this deletion was the possible conformational flexibility in this region and the presence of a linear epitope, which elicited a robust immune response.

These were attached to ferritin to form S-Fer and SΔC-Fer, respectively. Both were stabilized in the prefusion conformation, which leads to better expression and enhanced immunogenicity. As controls, they also used three other antigens, namely, a spike trimer with a trimerization domain in full-length or SΔC form, and a monomeric RBD.

Functionalized Ferritin Shows No Disadvantages

They found that spike-ferritin nanoparticles can be produced within mammalian cells in culture and purified to obtain a homogeneous result. The fusion of the ferritin to the spike protein did not adversely impact protein expression; indeed, SΔC-Fer actually showed improved expression. The functionalization of the H. pylori ferritin with the spike did not disrupt the nanoparticle self-assembly, either.

After checking the ferritin constructs for stability and homogeneity, they examined the folding using various tests for biophysical, structural, and binding properties. They employed size-exclusion chromatography multi-angle light scattering (SEC-MALS), cryo-electron microscopy (cryo-EM), and bio-layer interferometry (BLI) to evaluate these characteristics.

They found that the functionalized nanoparticles displayed stable folding, and the epitopes of interest were demonstrated properly. They also found that the spike-functionalized particles were bound by both the virus and ACE2 to the same extent as trimeric spike proteins and the RBD.

Effective Immune Response After Single Dose

The researchers then immunized mice with a single dose of these vaccines to study the immune response in vivo. Using pseudotyped viruses to avoid biosafety issues, they confirmed that SΔC-Fer elicited a markedly higher neutralizing antibody response compared to all the non-ferritin vaccines. Both S-Fer and SΔC-Fer vaccines induced neutralizing antibodies at double the titer following natural infection.

The researchers comment, “These results demonstrate that spike-functionalized ferritin nanoparticles elicit an enhanced antibody response compared to the spike trimers or RBD alone.”

Second Dose Improves Efficacy for Other Vaccine Candidates

A second dose of the five antigens on day 21 showed that the immune response was boosted for all groups to an almost complete blocking of ACE2 at a serum dilution of 1:50. Another observation was that ACE2 blocking is not always correlated to the trend of neutralizing activity. 

This could be because other neutralizing epitopes are present on the spike glycoprotein. However, SΔC-Fer-immunized mice continued to show the highest neutralizing titers, showing that a multivalent spike presentation is necessary for a better and more reliable immune response.

Implications

The conclusion is that these spike ferritin nanoparticles are superior candidates for vaccine development to either spike trimer or RBD by themselves, and that, in their words, “SΔC-Fer is the best-performing antigen out of those we tested here.”

The high efficacy of a vaccine candidate in achieving neutralizing antibody response after a single dose could be the tipping point in achieving worldwide coverage and stopping the pandemic. Thus, this study gains more importance in demonstrating the favorable profile of this construct in developing an effective and widely deployable vaccine against COVID-19.


More information: Bethany Dearlove et al, A SARS-CoV-2 vaccine candidate would likely match all currently circulating variants, Proceedings of the National Academy of Sciences (2020). DOI: 10.1073/pnas.2008281117

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