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

In recent months, viral strains of SARS-CoV-2 have been detected that are unusual in that they have accumulated mutations in the virus' genetic code that diverges from the wild-type (WT) Wuhan SARS-CoV-2 strain. These viral strains
with accumulated genome mutations are known as SARS-CoV-2 variants. As SARS-CoV-2 variants continue to emerge, the U.S. Centers for Disease Control (CDC) has systematically classified them in three categories: variants of high consequence, variants of concern, and variants of interest.¹ Currently, no SARS-CoV-2 variant occupies the highest category, variants of high consequence. This category would include variants that fail to be detected by diagnostics, have a significant reduction in susceptibility to vaccines, and present with more severe clinical disease. The lowest category, variants of interest, contains variants with suspected implications on viral transmission and immune escape and often lead to demographic cluster infections. Current variants of interest include Zeta, Eta, Theta, Iota, Kappa, and Lambda. Four independent SARS-CoV-2 variants of concern (VOCs) have emerged in the UK (Alpha; B.1.1.7), South Africa (Beta; B.1.351), Brazil (Gamma; P.1), and, most recently, India (Delta; B.1.617.2) with mutations of high interest (Figure 1) (Table 1). These variants show reduced susceptibility to vaccines, decreased neutralization by antibodies, an increase in transmissibility, along with an increase in disease severity (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

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

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 gain cell entry. The independent emergence of common Spike mutations in alpha, beta, gamma, and delta variants 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 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. All four VOCs have mutations in this region and the rapid worldwide spread of alpha, beta, and delta variants is cause for concern. In the US, the percentage of new COVID-19 cases caused by the delta variant as of August 14th, 2021 is 86%. Furthermore, the variant causing the first cases of COVID-19 in the U.S. in January 2020 is no longer detected in the population.⁴

The transmissibility of the beta variant appears unaltered, but this strain is more difficult to neutralize than its parental strains. Of 20 monoclonal antibodies (mAbs) 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 the beta variant, the E484K "escape" mutation has since been observed in gamma and in some alpha variant strains. E484K helps the virus evade the immune response, and increased antibodies are required to prevent infection with multiple variants exhibiting the E484K mutation.⁵ Structurally, the E484K mutation results in a Spike protein side chain charge change, preventing salt bridge formation and allowing the virus to bind more tightly to human cells as well as escape from the effects of some mAbs.³

The N501Y RBD mutation, initially identified in the alpha variant and subsequently appearing in both beta and gamma variants, increases the RBD binding affinity for ACE2. This leads to enhanced viral transmission but not increased virulence.^6,7. This mutation has shown a decrease in the efficacy of some mAb treatments such as bamlanivimab and estesevimab, supporting the therapeutic use of antibody cocktails.³

D614G is common to all four 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 mAb response and convalescent plasma-mediated neutralization.¹⁰ K417N and K417T RBD mutations have been identified in beta and gamma variants, 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

E484Q and L452R are a unique combination of mutations found in the fast-emerging delta variant. These mutations together result in increased ACE2 binding and rate of S1-S2 cleavage leading to better transmissibility and a higher viral load in infected individuals.¹² Furthermore, the RBD carrying this pair of mutations exhibits decreased binding to selected mAbs and may affect their neutralization potential.¹² More experimental validation is warranted of the effectiveness of commonly elicited neutralizing mAbs against delta variant strains carrying these mutations.

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 an individual having previously had a COVID-19 infection can increase the efficacy of current vaccines against novel variants, including the beta "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 RBD-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 RBD-ACE2 inhibitors in parallel. The Spike variant-specific kits include the alpha variant (including the N501Y and D614G mutations), the beta variant (including K417N, E484K, and N501Y mutations), and the gamma variant (including K417T, E484K, and N501Y mutations). Binding assays for the individual Spike protein mutations N501Y (useful for alpha, beta, and gamma variant strains), E484K alone (in beta and gamma variants but only in some alpha variant strains), and D614G (useful for alpha, beta, gamma, and delta variant strains), are also available. More information about Spike-targeted binding assays can be found here.

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