SARS-CoV-2 Variants: Evading the Immune System

The COVID-19 pandemic, caused by the novel SARS-CoV-2 coronavirus, has ravaged the globe, infecting over 145 million people and causing over 3 million deaths as of the time of this article. Ending the pandemic will require a combination of effective interventions, including therapeutics, diagnostics, and vaccines.  Several monoclonal antibody therapeutics have received emergency use authorization, and a number of vaccines have been developed, at least two with ~95% protective efficacy.1 However, these interventions were directed toward the initial SARS-CoV-2 that emerged in 2019, Genbank #YP_009724390.1. The recent emergence of new SARS-CoV-2 variants is a severe concern, especially as many of these mutations occur within the Spike protein and appear to increase transmissibility and infectivity.

D614G Mutation

One particular mutation in the Spike protein, a change from Aspartate (D) to Glycine (G) at amino acid 614 (D614G) was identified very early in the pandemic. By April of 2020, this mutation had already become the dominant lineage of the virus in Europe, Australia, and North America, and by June was found in nearly 100% of new viral cases worldwide.2 Therefore, the D614G variant has become the pandemic (Figure 1).

This D614G mutation is particularly interesting because it lies outside the Receptor Binding Domain (RBD) of Spike, the region of that recognizes and binds to the ACE2 receptor on target cells in the lungs and airways.  The D614G mutation increases the flexibility of the Spike protein, which enhances the binding affinity for ACE2, and subsequently leads to increased efficiency for viral entry into the target cell (3,4). Research using D614G pseudovirus vs. D614 wildtype pseudovirus to infect ACE2- or TMPRSS2-expressing cells demonstrates 3-9x greater infectivity, based on eGFP or luciferase expression (Figure 2)(2,5).  Fortunately, despite evidence of higher viral loads in patients infected with the D614G variant, the mutation has not been directly linked to a phenotype of increased virulence or transmissibility (6). 

Part of the difficulty in determining the phenotype of D614G may be related to concurrent mutations in other viral proteins, such as mutations in the nucleocapsid protein or the P323L mutation in the RNA polymerase (RdRp). This RdRp P323L mutation usually occurs concurrently with Spike D614G, and the combination of mutations has been linked to disease severity (7). The P323L mutation appears to destabilize the RdRp:NSP8 interaction and decrease proof-reading ability, leading to an overall increased viral mutation rate. The RdRp complex is the target of key COVID-19 drugs such as remdesovir, so non-Spike mutations are also critical to the spread of viral variants (8).

B.1.1.7 Variant

While the D614G mutation does not appear to affect disease severity, that isn’t the case for some other variants. The B.1.1.7 variant was first detected in the U.K. in September 2020, and has since been identified in more than 100 other countries. The UK variant contains 23 mutations throughout the genome, including a critical N501Y mutation in the RBD region of the Spike protein. This N501Y mutation has been shown to enhance binding between the SARS-CoV-2 Spike protein and the human ACE2 receptor, which directly contributes to this variant’s higher contagiousness.9 The B.1.1.7 variant also has a P681H mutation near the S1/S2 furin cleavage site, which may affect Spike cleavage rates and viral fusion with the target cell membrane.

The B.1.1.7 variant is thought to be about 40%-70% more transmissible than previous dominant variants, and B.1.1.7 infections were associated with higher viral loads than non-B.1.1.7 infections. Even worse, B.1.1.7 “has the potential to cause substantial additional mortality compared with previously circulating variants” (10). The B.1.1.7 strain has been designated as a “variant of concern” and epidemiologists believe the B.1.1.7 variant is fueling the recent surge in infections. By late March, 2021, the B.1.1.7 variant had become the most common variant identified in the United States (11). The B.1.1.7 variant has a deletion at the N-terminus (Del 69.70) that can interfere with some of the currently used PCR diagnostic tests, complicating epidemiological analysis. The B.1.1.7 mutations may also affect the efficacy of some of the current vaccines. Thus, variant-specific antibodies that can differentiate the B.1.1.7 variant from the wild-type (Figure 3) are valuable tools for studying the effectiveness of vaccines to neutralize infection. 

P.1 Variant

Unfortunately, other variants have also emerged and taken hold globally. In late 2020, a variant of SARS-CoV-2 known as P.1 emerged in Brazil; this variant was detected in the USA by the end of January 2021. The P.1 variant has 17 unique mutations, including three (K417T, E484K, and again, N501Y) within the RBD of Spike. Evidence suggests that some of the mutations in the P.1 variant affect the ability of antibodies (from either natural infection or vaccination) to recognize and neutralize the virus.12 Of particular concern, E484K Spike mutation has been linked to confirmed cases of re-infection with SARS-CoV-2, making the study of these mutations even more critical for successful vaccination and antibody-based therapeutic strategies.13

Figure 4. Neutralization of binding of spike protein variants to ACE2.  The Spike RBD region (left) and full-length trimer (right) from the wildtype, B.1.1.7 (UK) and B.1.351 (SA) strains of SARS-Cov-2 was inhibited from binding to the ACE2 receptor by a neutralizing monoclonal antibody, clone C-A11 (#101024). Spike RBD and trimer binding to ACE2 was detected using variant-specific colorimetric assay kits (#79999, #78152, #78155, #78133, #78018). 

B.1.351 Variant

Another important variant, known as B.1.351 or 501Y.V2, arose in South Africa independently of the B.1.1.7 or P.1 mutants. However, it shares both the E484K and N501Y mutations. This strain is particularly worrisome, since it is refractory to neutralization by antibodies to the RBD region as well as to the N-terminal region of Spike. It is also about 10x more resistant to neutralization by convalescent plasma or vaccine sera.14 The availability of new research tools such as the C-A11 clone (#101024) that can neutralize the binding of the B.1.351 Spike protein (Figure 4) will be critical for research to develop therapeutic antibodies and vaccines.

B.1.427 & B.1.429 Variants

Additional variants continue to emerge. BPS will soon offer Spike proteins and pseudovirus for the new “California variants” (B.1.427 and B.1.429) and “New York” Variant (B.1.526). As of March 5, 2021, the New York variant had been identified in 7 countries, and the “California” variants in 21 or 23 countries, respectively, suggesting these variants will continue to spread rapidly.15 In Jan. 2021, the New York variant represented only 3% of samples analyzed by researchers, but had risen to 12.3% by mid-February. Many of the cases occurred in people who already had recovered from the coronavirus, intensifying the threat of reinfection. Perhaps not surprisingly, the New York variant includes the same E484K variation found in the Brazilian variant. Moreover, the E484K mutation has been shown to lower the effectiveness of monoclonal antibody treatments against the B.1.351 variant (16).

Interestingly, instead of the E484K mutation, the new B.1.429 California variant contains three spike protein mutations: S13l, W152C and L452R. The B.1.427 variant also contains this L452R mutation, which is thought to stabilize the interaction between the spike protein and the host cell and thereby increase infectivity (17). Resistance of this mutation to antibodies (obtained either natural infection or COVID-19 vaccines) is still unknown.

New variants are continuing to emerge at a rapid pace.  The most notable of the new variants is the B.1.617 variant, often called the “double mutant” variant, since this variant contains not only the L452R mutation found in the California variants, but also a variation of the E484 mutation (E484Q) found in the New York and Brazilian variants.  First identified in India, this mutant is thought to exhibit both higher infectivity and transmission rates, making it both more infectious and more deadly.  Researchers are concerned that the double mutant variant may promote “immune escape” and increased infection rates post-vaccination. A “triple mutant” (B.1.618), which also includes P681H, has also been recently identified.

Tracking mutations as new variants emerge is absolutely essential for effective treatments and vaccines. As a global leader in developing new tools for drug discovery, BPS Bioscience has remained at the leading edge of scientific research, offering a full array of mutant proteins, and variant-specific assay kits that allow researchers to screen for inhibitors of ACE2 binding to SARS-CoV-2 Spike variants. Currently BPS offers kits to identify antibodies to the wildtype, D614G, B.1.1.7, B.1.351, and P1 variants, and products for the California B.1.427/9 and New York variants are expected to be released shortly.

BPS has developed variant-specific kits that target the RBD region of Spike (a.a. 319-591) that is responsible for binding to the ACE2 receptor on target cells.  BPS has also developed variant-specific kits that include the full S1 subunit of the Spike protein (a.a. 14-685), since mutations outside of the RBD in the S1 subunit of spike are important for influencing the immunogenicity, conformation, and flexibility of the spike protein. Even more valuable, BPS offers a unique panel of variant-specific pseudoviruses. These pseudoviruses incorporate various wild-type or mutant Spike proteins into a lentiviral vector to allow the study of viral fusion and entry in a cellular context.  

Emerging variants present new challenges for mAb therapy and threaten the protective efficacy of current vaccines. Investigations on the effects of mutations on viral replication and pathogenesis will be critical for developing effective strategies for vaccines and antibody therapies to fight the devastating impact of COVID-19.  BPS Bioscience will continue to lead the way with innovative assays and tools to aid researchers in developing new attacks to neutralize and limit the spread of SARS-CoV-2. 

ORFs 2, 4, 5, and 9a encode the four major SARS-CoV-2 structural proteins: Spike (S), Envelope (E), Membrane (M), and Nucleocapsid, respectively [3]. Other ORFs encode for accessory proteins with no known role in viral replication, such as ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8a, and ORF8b, some of which may or may not be present in a particular coronavirus genome. 

References

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