A study involving more than 5,000 COVID-19 patients in Houston finds that the virus that causes the disease is accumulating genetic mutations, one of which may have made it more contagious.
According to the paper published in the peer-reviewed journal mBIO, that mutation, called D614G, is located in the spike protein that pries open our cells for viral entry. It’s the largest peer-reviewed study of SARS-CoV-2 genome sequences in one metropolitan region of the U.S. to date.
The paper shows “the virus is mutating due to a combination of neutral drift – which just means random genetic changes that don’t help or hurt the virus – and pressure from our immune systems,” said Ilya Finkelstein, associate professor of molecular biosciences at The University of Texas at Austin and co-author of the study. The study was carried out by scientists at Houston Methodist Hospital, UT Austin and elsewhere.
During the initial wave of the pandemic, 71% of the novel coronaviruses identified in patients in Houston had this mutation. When the second wave of the outbreak hit Houston during the summer, this variant had leaped to 99.9% prevalence.
This mirrors a trend observed around the world. A study published in July based on more than 28,000 genome sequences found that variants carrying the D614G mutation became the globally dominant form of SARS-CoV-2 in about a month. SARS-CoV-2 is the coronavirus that causes COVID-19.
So why did strains containing this mutation outcompete those that didn’t have it?
Perhaps they’re more contagious. A study of more than 25,000 genome sequences in the U.K. found that viruses with the mutation tended to transmit slightly faster than those without it and caused larger clusters of infections.
Natural selection would favor strains of the virus that transmit more easily. But not all scientists are convinced. Some have suggested another explanation, called “founder’s effects.”
In that scenario, the D614G mutation might have been more common in the first viruses to arrive in Europe and North America, essentially giving them a head start on other strains.
The spike protein is also continuing to accumulate additional mutations of unknown significance.
The Houston Methodist-UT Austin team also showed in lab experiments that at least one such mutation allows spike to evade a neutralizing antibody that humans naturally produce to fight SARS-CoV-2 infections.
This may allow that variant of the virus to more easily slip past our immune systems. Although it is not clear yet whether that translates into it also being more easily transmitted between individuals.
The good news is that this mutation is rare and does not appear to make the disease more severe for infected patients. According to Finkelstein, the group did not see viruses that have learned to evade first-generation vaccines and therapeutic antibody formulations.
“The virus continues to mutate as it rips through the world,” Finkelstein said. “Real-time surveillance efforts like our study will ensure that global vaccines and therapeutics are always one step ahead.”
The scientists noted a total of 285 mutations across thousands of infections, although most don’t appear to have a significant effect on how severe the disease is. Ongoing studies are continuing to surveil the third wave of COVID-19 patients and to characterize how the virus is adapting to neutralizing antibodies that are produced by our immune systems. Each new infection is a roll of the dice, an additional chance to develop more dangerous mutations.
“We have given this virus a lot of chances,” lead author James Musser of Houston Methodist told The Washington Post. “There is a huge population size out there right now.”
Several other UT Austin authors contributed to the work: visiting scholar Jimmy Gollihar, associate professor of molecular biosciences Jason S. McLellan and graduate students Chia-Wei Chou, Kamyab Javanmardi and Hung-Che Kuo.
The UT Austin team tested different genetic variants of the virus’s spike protein, the part that allows it to infect host cells, to measure the protein’s stability and to see how well it binds to a receptor on host cells and to neutralizing antibodies. Earlier in the year, McLellan and his team at UT Austin, in collaboration with researchers at the National Institutes of Health, developed the first 3-D map of the coronavirus spike protein for an innovation that now factors into several leading vaccine candidates’ designs.
The researchers found that SARS-CoV-2 was introduced to the Houston area many times, independently, from diverse geographic regions, with virus strains from Europe, Asia, South America and elsewhere in the United States. There was widespread community dissemination soon after COVID-19 cases were reported in Houston.
An earlier version of the paper was posted last month to the preprint server medRxiv.
COVID-19 vaccine candidates primarily target the trimeric ‘spike’ (S) glycoprotein, as this factor enables binding to the ‘angiotensin-converting enzyme 2’ (ACE2) host surface receptors and facilitates virus entry into the cells1.
Over the last few months, an Aspartate-to-Glycine amino acid change has arisen at position 614 of the S protein (resulting from a single A-to-G nucleotide change at position 23,403 in the Wuhan-Hu-1 reference genome), with G614 variants accounting for 75% of published genome sequences worldwide as of 1 July 2020.
This mutation has resulted in a number of articles and preprints postulating that isolates containing this ‘D614G’ mutation have a structural advantage2, including as a better substrate to the S1 furin cleavage domain3, and are associated with an increase in:
- (a) transmissibility and viral loads4;
- (b) transduction of human cells2,5;
- (c) pathogenicity and case fatality6.
These have led to speculation that the efficacy of vaccines and countermeasures which target the S protein could be adversely affected, necessitating frequent vaccine matching.
Results and discussion
We tested this hypothesis with sera from ferrets immunised with a COVID-19 vaccine candidate that targets the S protein (D614 variant), and used biomolecular modelling to interpret our results. We used Australian isolates which either possess or lack the D614G mutation but are otherwise comparable in S protein sequence and also devoid of significant mutations of consequence within viral proteins responsible for cell binding and entry (as discerned with Geneious Prime 2020.1 software; c.f. Supplementary Table 1).
Isolated at the Victorian Infectious Diseases Reference Laboratory7, the Australian isolates ‘VIC01’ and ‘SA01’ (which are D614) and ‘VIC31’ (which is G614), were used in standard virus neutralisation assays preformed at the Australian Centre for Disease Preparedness, as described under Methods.
Previous studies in rodents with INO-4800 have demonstrated the induction of humoural and cellular immune responses targeting SARS-CoV-2 spike protein8. In this study, ferrets were shown to have developed SARS-CoV-2 neutralising antibody responses following vaccination with INO-4800, demonstrating that ferrets are an appropriate animal to model COVID-19 vaccine immunogenicity, and that this DNA vaccine stimulates an effective B cell response.
The overall median log2 neutralisation titre against the three virus isolates combined was 6.32 (range 4.32 to 8.32). Comparison of the titres by virus isolate (SA01, VIC01, and VIC31) revealed that the D614G mutation had little effect on neutralisation efficiency following vaccination (Fig. 1). Indeed, the overall log transformed mean neutralisation titres for the VIC31 variant (the G614 variant) were not significantly different than those for the SA01 and VIC01 isolates possessing the D614 (p > 0.05).
We used molecular models of the spike protein (both our molecular dynamics simulations and another spike model9) to examine the structural context of the D614G mutation to address possible concerns of adverse vaccine efficacy and the plausibility of recently-proposed selection advantages2–5.
The S protein of coronavirus comprises of two sections, S1 and S2, and forms a heavily glycosylated transmembrane trimer facilitating both host attachment (via the receptor binding domain ‘RBD’ in S1), and cell fusion/entry (via a trimeric membrane fusion S2 stalk) after proteolytic cleavage of the S1/S2 junction10 (Fig. (Fig.2a).2a).
The SARS-CoV-2 S protein is different to related coronaviruses as it contains a proprotein convertase (PPC) motif at the S1/S2 boundary, which has been shown to be pre-cleaved in pseudovirus cellular entry assays by the proprotein convertase furin10,11.
The 614 position in S is upstream of the S1/S2 furin cleavage site (R685/S686)9 and is recessed at a buried interface position of the adjacent monomer while additionally shielded by an N-linked glycan at position N61612 (Fig. 2b). Given its internal position, the G614 variant is unlikely to be a component of neutralising epitopes on the S protein and thus a lack of adverse effects on neutralisation efficiency of antibodies generated following vaccination with D614-derived vaccines is not unexpected.
Indeed, the location of residue 614 in S is distinct from both of the linear neutralising epitopes13.
Whilst we agree with studies suggesting that the D614G mutation introduces an elastase cleavage site14, our molecular dynamics simulations do not support their inference of increased replication efficiency by promoting elastase-mediated S1/S2 cleavage.
Modelling of S in the presence of both furin and elastase proteases suggest that, while furin can access the S1/S2 cleavage site (Fig. S1), elastase is unable to access the putative G614-introduced elastase cleavage site due to its interface positioning and glycan blocking (Fig. S2a) unless the S1 trimer cap and S2 separate (Fig. S2b).
Recent findings2 suggested the superior infectivity of G614 is through the D614G mutation stabilising the interaction between the S1 and S2 domains. However, our examination of D614 (located in S1) reveals interactions with S2, not only through hydrogen bond interactions with an adjacent Thr859 as previously noted4, but also a salt bridge with residue Lys854 (Fig. (Fig.2c).
The aspartate-to-glycine change at 614 removes the inter-chain D614-K854 and might actually destabilise the interaction between S1 and S2 domains.
Our in silico approach has shed additional insights into a couple of mechanisms that warrant further investigation, including experiments. Firstly, although our simulation considered interactions between human ACE2 and the RBD in the traditional ‘up’ orientation, over the course of a short 100-ns simulation we observed the ACE2 tilt and contact the adjacent RBD via V445 making close contact with P321 and F555 on ACE2.
This suggests that the adjacent RBD in the ‘down’ conformation could contribute to ACE2 binding and specificity (Fig. S3), a hypothesis supported by previous work15 highlighting structural flexibility with three distinct conformations of SARS-CoV S protein in complex with ACE2.
Secondly, a parallel C-to-U substitution at position 14,408 in the genome, resulting in a P323L (P314L in orf1b) mutation in the RNA-dependent polymerase (RdRp/nsp12), has been associated with D614G16 (including in VIC31) and should therefore be considered for potential contribution to infectivity. The P323 position is in a hydrophobic cleft approximately 30 Å from the catalytic site of RdRp, but close to the nsp8 interface (Fig. S4).
It is not immediately clear what effect the P323L mutation has on virulence. The Pro323 to Leu mutation may relieve some backbone constraints and contribute local conformational stability. We noted in our model based on pdb structure 6YYT17 that the cleft in nsp12 is predominately lined with arginine residues (R173, R249, R349 & R457) which may contribute to nucleotide binding due to the strong association with arginine and phosphate groups18 (Fig. S4). The P323L mutation appears to partially obstruct R349 in the cleft. We are yet to establish the significance of this mutation.
Given the rapidity with which this virus has emerged, correlates of protection have yet to be established for immune responses. Ideally, passive-protection assays would also be performed in future studies to determine in vivo effects. The experimental and biomolecular modelling approaches described above are available at many organisations around the world such as ours.
Therefore, it would be desirable to analyse the impact of identified mutations, in collaboration with such organisations, before speculating on potential adverse effects on vaccines. As infectious clones are established19–21, it will be easier to study accumulated mutations, which are inevitable for this RNA virus, even with an exoribonuclease ‘proof-reading’ capacity.
Therefore, we urge caution when these mutations are described in preprint articles2–5 because premature inferences on their effects without supporting experimental evidence could result in a media frenzy and potentially undermine public confidence in vaccines.
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More information: Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area, mBIO, DOI: 10.1128/mBio.02707-20 , mbio.asm.org/content/11/6/e02707-20