SARS-CoV-2 Variants: An Emerging Landscape of Therapeutic Targets

As SARS-CoV-2 variants have emerged the CDC has systematically classified them as variants of interest, variants of concern (VOC), and variants of high concern.1 Three independent SARS-CoV-2 VOCs have emerged in the UK (B.1.1.7), South Africa (B.1.351), and Brazil (P.1) (Table 1). The rapid dominance of these variants within their originating countries, and in other countries to which they have spread, has raised concerns about their potential impact on current COVID-19 countermeasures, including vaccines, therapeutics, and diagnostics, thus highlighting the need for continual sequence surveillance.

Table 1
Variants of Concern
B.1.351 B.1.1.7 P.1
First Case UK, December 2020 South Africa, December 2020 Brazil, January 2021
Transmission increased increased unchanged
Disease severity potentially increased unchanged unchanged
mAb efficacy unaffected significantly reduced for many potentially
Neutralizing Ab efficacy minimal effects reduced; vaccine efficacy significantly reduced potentially
Spike Protein Mutations
in RBD E484K*, S494P*, N501Y, A507D K417N, E484K, N501Y K417N, E484K, N501Y
in NTD 𝝙69/70, 𝝙144 D80A, D215G, 𝝙241/242/243 L18F, T20N, P26S, D138Y, R190S
Other D614G, P681H, T716I, S982A, D1118H, K1191N* D614G, A701V D614G, H655Y, T1027I

*= detected in some sequences but not all.
RBD = receptor binding domain; NTD = N-terminal domain; mAB = monoclonal antibody.

First generation vaccines and therapeutics have been developed based on the wild-type (WT) Wuhan Spike protein sequence which was released in January 2020 (Genbank accession: QHD43416).2 The Spike protein’s receptor binding domain (RBD) binds cellular ACE2 to effect cell entry. The independent emergence of common Spike mutations in B.1.1.7, B.1.351, and P.1 strains indicates that these changes may confer an evolutionary advantage. Understanding the effects of these mutations on vaccine efficacy, disease progression, therapeutic response, and transmissibility is important for mitigating or limiting their effects.

Spike RBD variants are likely to affect measurable parameters because these residues have been targeted for vaccine and therapeutic purposes. Only a small subset of antibodies produced against a virus are considered to be neutralizing. Neutralizing antibodies bind the virus in a manner that prevents infection, for example, by blocking interaction with its receptor. The Spike RBD-ACE2 binding interface is a short 25 amino acid sequence essential for infection,3 which makes it a target for natural and vaccine-induced neutralizing antibodies. Furthermore, its small size means that sequence alterations can lead to immune escape. B.1.1.7, B.1.351, and P.1 VOCs have mutations in this region and the rapid worldwide spread of B.1.1.7 and B.1.351 is cause for concern. In the USA, the percentage of new COVID-19 cases caused by the B.1.1.7 and B1.351 variants between late January and early April 2021 rose from 1.2% to 59.6% and 0.1% to 1.0%, respectively.4

The transmissibility of the B.1.351 variant appears unaltered, but this strain is more difficult to neutralize than its parental strains. Of 20 monoclonal antibodies tested against B.1.351, 14 have compromised or abrogated effects and, in South Africa, vaccine efficacy against this strain is as low as 57%.3

First identified in B.1.351, the E484K “escape” mutation has since been observed in P.1 and in some B.1.1.7 strains. E484K helps the virus evade the immune response, and increased antibodies are required to prevent infection with B.1.1.7 variants with E484K5 Structurally, the E484K mutation results in a Spike protein side chain charge change, preventing salt bridge formation and allowing escape from the effects of some monoclonal antibodies.3

The N501Y RBD mutation, initially identified in B.1.1.7 and subsequently appearing in B.1.351 and P.1 strains, increases the RBD binding affinity for ACE2. This leads to enhanced viral transmission but not increased virulence.6,7. This mutation can decrease the efficacy of some monoclonal antibodies, supporting the therapeutic use of antibody cocktails.3

D614G is common to B.1.1.7, B.1.351, and P.1 VOCs, and is surrounded by conserved amino acids. D614G has led to increased transmissibility and is correlated with high viral loads. G614 stabilizes the Spike protein and prevents premature dissociation from the target cell, resulting in increased infectivity.8,9 Receptor binding is not altered by this mutation and vaccines generated against D614 (WT) are effective against G614.9

Mutations at K417 increase the affinity of the RBD to ACE2, increase chances of immune escape, and reduce monoclonal antibody response and convalescent plasma-mediated neutralization.10 K417N and K417T RBD mutations have been identified in B.1.351 and P.1, respectively. In combination with their common E484K and N501Y mutations, K417 alterations induce a relatively large conformational change that may increase the potential for immune escape.11

The N-terminal domain of the Spike protein subunit 1 (S1) is increasingly recognized as an important site for neutralizing antibodies. The B.1.1.7 N-terminal domain deletion, HV69-70 del, was thought to affect antibody neutralizing capacity, but recent conflicting results indicate that this requires additional investigation.12

The emergence and spread of multiple SARS-CoV-2 variants have led to a rush to understand the consequences of the changes. This has also caused a shift toward the development of second and third generation vaccines less reliant on disrupting the RBD-ACE2 interaction. Recent reports indicate that that previous COVID-19 infection can increase the efficacy of current vaccines against novel variants, including the B.1.351 ‘escape’ variant.5 However, as additional SARS-CoV-2 variants emerge, it will become increasingly important to evaluate their effects alone and in combination with existing variants. In support of these efforts, RayBiotech has developed a suite of tools to assist detection of neutralizing activity against Spike mutants – including from antibodies, sera, small molecules, or other agents with the potential to block the Spike-ACE2 interaction. Efficient surveillance of protective immune responses against new variants will require laboratories to adopt high throughput screening methods. RayBiotech’s range of Spike variant binding assays enable the researcher to screen dozens of Spike-ACE2 inhibitors in parallel. The Spike variant-specific kits include B.1.1.7 (including the N501Y and D614G mutations), B.1.351 (including K417N, E484K, and N501Y mutations), and P.1 (including K417T, E484K, and N501Y mutations) variants. Binding assays for the individual Spike protein mutations N501Y (useful for B.1.1.7, B.1.351, and P.1), E484K alone (in P.1 and B.1.351 but only in some B.1.1.7 strains), and D614G (useful for B.1.1.7, B.1.351, and P.1), are also available. More information about Spike-targeted binding assays can be found here.

References
  1. https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html (accessed 05/05/2021).
  2. Wu, F., Zhao, S., Yu, B. et al. A new coronavirus associated with human respiratory disease in China. Nature 579, 265–269 (2020). https://doi.org/10.1038/s41586-020-2008-3
  3. Zhou et al., 2021, Cell 189, 2348–2361. April 29, 2021 https://doi.org/10.1016/j.cell.2021.02.037
  4. https://covid.cdc.gov/covid-data-tracker/#variant-proportions (accessed 05/05/2021).
  5. BMJ 2021; 372: n359)
  6. Starr, T. N. et al. Cell 182, 1295–1310 (2020).
  7. Liu Y, Liu J, Plante KS, Plante JA, Xie X, Zhang X, Ku Z, An Z, Scharton D, Schindewolf C, Menachery VD, Shi PY, Weaver SC. The N501Y spike substitution enhances SARS-CoV-2 transmission. bioRxiv [Preprint]. 2021 Mar 9:2021.03.08.434499. doi: 10.1101/2021.03.08.434499. PMID: 33758836; PMCID: PMC7986995.
  8. Wang, R., Chen, J., Gao, K. et al. Analysis of SARS-CoV-2 mutations in the United States suggests presence of four substrains and novel variants. Commun Biol 4, 228 (2021). https://doi.org/10.1038/s42003-021-01754-6
  9. Zhang et al. Science 30 Apr 2021: Vol. 372, pp. 525-530 DOI: 10.1126/science.abf2303).
  10. Kai Wuet al. bioRxiv 2021.01.25.427948; doi: https://doi.org/10.1101/2021.01.25.427948
  11. Gard Nelson et al. bioRxiv 2021.01.13.426558; doi: https://doi.org/10.1101/2021.01.13.426558
  12. Rees-Spear, Chloe et al. Cell reports, Vol 134, issue 12, 23 March 2021.

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