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REVIEW

SARS-CoV-2 E484K Mutation Narrative Review: Epidemiology, Immune Escape, Clinical Implications, and Future Considerations

, , , , & ORCID Icon
Pages 373-385 | Published online: 03 Feb 2022

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread rapidly over the world and claimed million lives. The virus evolves constantly, and a swarm of mutants is a now major concern globally. Distinct variants could have independently converged on same mutation, despite being detected in different geographic regions, which suggested it could confer an evolutionary advantage. E484K has rapidly emerged and has frequently been detected in several SARS-CoV-2 variants of concern. In this study, we review the epidemiology and impact of E484K, its effects on neutralizing effect of several monoclonal antibodies, convalescent plasma, and post-vaccine sera.

Introduction

The emergence of the SARS‐CoV‐2 strain of the human coronavirus has led to a new pandemic. According to data from the World Health Organization (WHO), as of 31 July 2021, there have been around 194 million confirmed cases of Covid-19, including more than 4 million deaths globally.Citation1

SARS-CoV-2 is an enveloped virus, belonging to the genus Coronavirus, with a non-segmented, positive-sense single-strand RNA genome, measuring 29,903 kilobytes.Citation2 Entry of SARS-CoV-2 into host cells is mediated by the transmembrane spike (S) glycoprotein. The S glycoprotein comprises two functional subunits. S1 subunit amino acids 318–510 (S318–510) contains the receptor-binding domains (RBD), which bind to the host cell receptor, angiotensin-converting enzyme 2, whereas the S2 subunit is responsible for fusing of cellular membranes.Citation3,Citation4 Mutations on the spike protein are common features of many SARS-CoV-2 variants.

Receptor binding domain (RBD) mutations enhance the structural stability and increase the human ACE2 receptor-binding capacity of the spike protein, which could be an essential characteristic that would explain the high virus spread and the severe infectivity.Citation5 E484K, a mutation of one of the RBD residues, has a glutamate (E) to lysine (K) substitution at position 484.

The mutation in the spike protein has an impact on the convalescent sera, monoclonal antibody treatment, and vaccine efficacy. E484K has been identified as an escape mutation that emerges during convalescent plasma.Citation6 E484K reduces the convalescent sera antibody binding affinity by more than order of magnitude. Similar experimental results reported by Andreano et al, Greaney et al, Liu et al and Weisblum et al show that mutations at the site E484 reduce the neutralization potency of some human plasmas by >10-fold.Citation7–10

In the research of Liu et al, 0.03% of sequenced isolates exhibited variation at E484, as of October 2020, and this increased to 0.09% of substitutions at this position by January 2021.Citation10 The variation at E484 emerged more frequently with viral replication. In an escape mutation study, which using 19 monoclonal antibodies, substitutions were found at E484 with higher incidence than at any other residue, and four variants at this position (E484A, E484D, E484G, and E484K) exhibited resistance to each of the human convalescent sera tested.Citation10

There is a gap in our current knowledge, with few available data on E484K mutations. In this review, E484K mutations are discussed particularly in the context of observed frequencies in global sequence datasets. The binding-strengthening mutations of E484K can play a vital role in the development of infectivity, transmissibility, and/or antigenicity, and therefore it is important to gain insights into the underlying mechanisms involved. Herein, we analyze the percentage of E484K present in the variants and the global prevalence based on geographic location in order to surveil the unfolding pandemic, comprehend the roles of epidemiological variables, identify SARS-CoV-2 variants and their genetic sequences, and recommend public health policy strategies.

Significance of E484K in SARS-CoV-2 Variant

The E484K mutation is present in several variants and popped up in rapid succession in different geographical regions of five continents, especially in Southern Africa and South America.

The E484K mutation was first identified in the Beta variant (B1.351) in South Africa and was also detected in the Alpha variant (B.1.1.7) in the United Kingdom, as well as in the Gamma variant (P.1) in Brazil, all of which are classified as SARS-CoV-2 variants of concern (VOC). These variants were found to have higher transmissibility, increased fatality rates, and according to the WHO, the effectiveness of vaccines, therapies, and other health measures used against these variants were significantly lower.Citation11 According to the data from Outbreak Information, the presence of the E484K mutation in the Gamma variant is 91%, compared with 86% in the Beta variant, and less than 0.5% in the Alpha variant. illustrates the prevalence rates of E484K in the other variants.Citation12

Table 1 E484K Prevalence in SARS-CoV-2 Variant

The Eta variant (B.1.525), Lota variant (B.1.526), Kappa variant (B.1.617.1), and Lambda variant (C.37) are all considered Variants of Interest (VOI). The prevalence of the E484K mutation in the Eta variant is 97%, compared with 43% in Iota, 1% in Lambda, and less than 0.5% in Kappa. According to the WHO, a VOI is a SARS CoV-2 variant with a genetic capability that affects characteristics of the virus, such as disease severity, immune escape, transmissibility, and diagnostic escape. The WHO further confirmed that VOIs are responsible for a consequential volume of community transmission. A global increase in VOI cases poses a public health risk to large portions of populations worldwide.Citation11,Citation12

The E484K mutation is present in Zeta variant (P.2) with a prevalence rate of 96%, compared to 85% in P.3. Evidence of the phenotypic or epidemiological impact of the Zeta variant and P.3 variant is currently unclear, and thus enhanced monitoring and repeat assessment is required.Citation12

E484K Global Presence Rate

Geographically, E484K mutations are highly prevalent in countries in Southern Africa and neighboring islands. Prevalence of positive E484K mutation over 50% in all confirmed cases with genetic sequence is listed in descending order as follows: Mozambique 66%, Mauritius 65%, Reunion 63%, Malawi 61%, South Africa 54%, and Mayotte 51%. E484K mutation prevalence rates over 50% in Southern America, according to data from Outbreak Information, were reported to be 62% in Suriname, 70% in French Guiana, 72% in Brazil, and 78% in both Trinidad and Tobago.Citation12 summarizes the E484K global presence rate, and the world map is created by the Microsoft Excel™ on the Bing platform and reprinted under a CC BY license.

Figure 1 E484K Global Presence Rate.

Notes: No current data for areas in gray color. This map was developed on the Bing platform by Microsoft Excel™, Microsoft product screen shot(s) reprinted with permission from Microsoft Corporation under a CC BY license.
Figure 1 E484K Global Presence Rate.

E484K Reduced Neutralization by Convalescence Sera

Deep mutational scanning (DMS) is a novel method that analyzes how a mutation changes the structure and function of a protein.Citation13 DMS creates a library of mutated gene variants, cloned into the appropriate vector, and mostly utilizes yeast-display. Greaney et al recognized that E484 plays a crucial role in neutralization by convalescent sera by mutated spike receptor-binding domain yeast library.Citation7 Assays such as authentic viruses and pseudoviruses with mutated spike protein were applied to evaluate the impacts of mutations on convalescent sera. Inhibitory concentration 50% (IC50) of mutated virus and wild-type virus were compared.Citation14 Most pseudoviruses assays assessing the neutralization impact on SARS-CoV-2 use lentiviral or vesicular stomatitis virus (VSV) vector-based SARS-CoV-2 pseudoviruses. Convalescence sera obtained from different countries and periods have consistently demonstrated that E484K or triple mutation (K417N/E484K/N501Y) decreased neutralization in pseudo-viral assays.

Jangra et al compared the neutralization of the USA-WA1/2020 virus and a recombinant (r)SARS-CoV-2 virus with the E484K mutation, and found a 2·4-fold to 4·2-fold reduction in neutralization when E484K was present.Citation6 Greaney et al used lentiviral vector-based SARS-CoV-2 pseudoviruses and reported amino acid changes in E484 to K, P, or Q reduced binding of polyclonal convalescent plasma, and in some individuals, mutations at E484K could decrease neutralization by >100-fold.Citation7 Gaebler et al and Wang et al obtained convalescent plasma at 1.3 months after infection, and the neutralization against the K417N/E484K/N501Y mutant was reduced 0.5- to 29-fold (P = 0.001).Citation15,Citation16 Chen et al used Vero-hACE2-TMPRSS2 cells, and found neutralization titer of convalescent plasma decreased fivefold against E484K/N501Y compared with the WA1/2020 D614G virus.Citation17 Wibmer et al obtained convalescent sera from blood donors in South Africa and discovered 27% of the samples lost neutralization against the pseudovirus containing K417N, E484K, and N501Y.Citation18 Collier et al found a similar result showing that the Alpha variant combined with E484K reduced the neutralization 11.4-fold in comparison with the wild-type spike protein.Citation19 summarizes studies on the impact of E484K on the neutralization of convalescent sera.

Table 2 Studies Demonstrating E484K Reduced Neutralization of Convalescent Sera

Monoclonal antibodies derived from convalescent patients were further investigated. Cryo-electron microscopy is an electron microscopy technique capable of capturing images of flash-frozen molecules in liquid nitrogen with a special camera.Citation20 Cryo-electron microscopy can be used to analyze the structure of RBD-binding neutralizing antibodies and the interaction between RBD mutants and human neutralizing antibodies. Antibodies are categorized into four classes. Class 1 antibodies bind to an epitope only in the RBD “up” conformation, and are the most abundant. Class 2 antibodies bind to the RBD both in “up” and “down” conformations. Class 3 and class 4 antibodies both bind outside the ACE2 binding site. Class 3 antibodies bind the RBD in both the open and closed conformation, while class 4 antibodies bind only in the open conformation. Class 2 antibodies resist E484K mutations.Citation15,Citation21

Using Cryo-EM, Barnes et al identified three class 2 antibodies, and all of them commonly interact with E484.Citation21 They further detected molecular interactions by surface plasmon resonance (SPR). In the SPR assay, class 2 antibodies lose binding affinity with E484K mutations.Citation21 Other studies using pseudovirus assay similarly proved class 2 antibodies conferred resistance to E484K mutation.Citation9,Citation10

E484K Reduced Neutralization by Post-Vaccination Sera

Among the various modalities that have been considered in the fight to bring an end to the COVID-19 pandemic, vaccination offers the most promise. To date, over 100 vaccines have been evaluated in human trials, and over 180 vaccines have been tested in preclinical trials.Citation22 Several vaccines using different platforms have been approved and are proven to be safe and effective. Platforms of currently available vaccines include inactivated virus vaccines, live attenuated virus vaccines, recombinant protein vaccines, vector vaccines, and RNA and DNA vaccines.Citation23

Several methods assess the impacts of mutations on vaccine efficacy both in vitro and in vivo. With respect to in vitro studies, as previously mentioned, authentic viruses and pseudoviruses holding spike mutations were utilized and the neutralization activity has been assessed. In vivo clinical trials of vaccine efficacy have also been conducted.

Studies have evaluated the post-vaccine sera from individuals who received mRNA vaccine, adenovirus vaccine, and protein subunit vaccine, including studies using authentic viruses and pseudovirus assays. Not only human post-vaccine sera were evaluated but also post-vaccine sera from macaque monkeys.Citation24

In one study, sera from individuals who received BNT162b2 vaccine were obtained, and the analysis found a 3.4-fold reduction in serum neutralization efficiency when E484K was present.Citation6 Pseudotyped viruses carrying the E484K mutation reduced the effectiveness by at least tenfold against the 17 most potent antibodies selected from eight vaccinated individuals who received the mRNA-1273 or BNT162b2 vaccine.Citation15 Another study obtained mRNA-1273 post-vaccine sera and compared the neutralizing activity against VSV-pseudovirus of the Alpha variant and the Alpha+E484K variant, and a significant reduction in neutralizing titers was evident when the E484K mutation was present.Citation24 BNT162b2 post-vaccine sera also showed a similar result. The Alpha variant alone reduced neutralization 1.9-fold; when adding E484K to the Alpha variant, the neutralization was reduced 6.7-fold relative to the wild-type virus.Citation19 In addition, a similar trend was found in mRNA-1273 post-vaccine sera from macaque monkeys when assessed in VSV and lentiviral pseudovirus neutralization assays.Citation24 The above studies verified that E484K decreased the neutralization in pseudovirus assays of post-mRNA-vaccine sera from human and macaque monkeys.

E484K together with other mutations, also decreased the neutralization effect of post-vaccine sera. Chen et al evaluated post-BNT162b vaccine sera using Vero-hACE2-TMPRSS2 cells and found E484K/N501Y mutation decreased neutralization fourfold compared to the WA1/2020 D614G variant.Citation17 Post-vaccine sera of mRNA-1273 against recombinant SARS-CoV-2 pseudovirus containing RBD mutations (K417N-E484K-N501Y- D614G) in the Beta variant decreased neutralization 2.7-fold.Citation24 Sera obtained from macaque monkeys showed a similar trend.Citation24 Wang et al obtained mRNA-1273 or BNT162b2 post-vaccine sera from 20 volunteers.Citation15 The neutralization decreased one- to three-fold when human immunodeficiency virus-1 (HIV-1) pseudoviruses possessed the E484K-, N501Y-, or K417N/E484K/N501-mutant. Adenoviral vector vaccine demonstrated similar results. Madhi et al investigated antibodies induced by ChAdOx1 nCoV-19 vaccine in a pseudovirus assay, and found that the geometric mean titers of the RBD triple mutant (K417N, E484K, and N501Y) decreased 3.49-fold relative to the original virus, and the live-virus assay showed lower neutralization than the pseudo-viral assay.Citation25

Variants containing the E484K mutation, such as the Beta and Gamma variants, reduced neutralization of post-vaccine sera, whereas there was a minimal effect on the Alpha variant. Garcia-Beltran et al utilized a lentiviral vector-based SARS-CoV-2 pseudovirus neutralization assay and compared the neutralizing capacity of BNT162b2 vaccine (n = 30) and mRNA-1273 vaccine (n = 35) post-vaccination sera against eight SARS-CoV-2 variants of interest or concern.Citation26 Pseudoviruses with Alpha variant mutants revealed non-statistically significant reductions in neutralization compared to wild-type viruses. The Beta variants showed significant decreases in neutralization activity; for BNT162b2 post-vaccination sera, the neutralizing responses decreased 34-fold to 42-fold; and for mRNA-1273 post-vaccination sera, the titer decreased 19.2-fold to 27.7-fold. The Gamma variant showed significant decreases in neutralizing antibody responses, with a 6.7-fold reduction for BNT162b2 and a 4.5-fold reduction for mRNA-1273. Both the Beta and Gamma variants contain E484K, and Alpha variants do not. Therefore, these data collectively indicate that the primary determinant responsible for decreased neutralization is related to E484K.Citation26 Wu et al demonstrated neutralization of mRNA-1273 postvaccine sera against pseudoviruses containing the complete set of S mutations in the Beta variant decreased 6.4-fold; conversely, there was a minimal effect on neutralization of the Alpha variant.Citation24 Liu et al obtained BNT162b2 post-vaccine sera from 15 participants.Citation27 They utilized recombinant isogenic SARS-CoV-2s possessing spike mutations, and implemented a conventional 50% plaque reduction neutralization test (PRNT50) to measure virus suppression. Geometric mean neutralizing titers against wild type, Alpha-variant-spike, Gamma-variant-spike, Beta-variant-spike were 532, 663, 437, and 194, respectively. Thus, they concluded that the neutralizing of Alpha-spike and Gamma-spike viruses were not significantly different from the wild type, and the neutralizing of Beta-spike virus decreased significantly.Citation27 Studies on the neutralization of post-mRNA vaccine sera consistently demonstrated that variants containing E484K, such as the Beta and Gamma variants, decreased the neutralizing effects. Although neutralization levels were lower against the Beta variant, the post-vaccine sera with mRNA-1273 retained neutralizing activity against the Beta variant.Citation24,Citation28 Antibodies induced by adenovirus vector vaccine, protein subunit vaccines, and inactivated vaccine also showed the neutralization effect was decreased against the Beta variant. For antibodies induced by the Ad26.COV2.S vaccine, the neutralization against the Beta variant decreased 5-fold when compared with the original virus in a pseudovirus neutralizing assay.Citation29 Antibodies induced by NVX-CoV2373, a recombinant protein nanoparticle vaccine, reduced neutralization 14.5-fold against the Beta variant relative to D614G in a pseudovirus assay.Citation28 Studies on BBIBP-CorV, an inactivated SARS-CoV-2 vaccine, also showed a similar trend. Huang et al demonstrated that the neutralizing effect of BBIBP-CorV against the Beta variant was 1.6-fold lower than against the original SARS-CoV-2.Citation30 However, Zhang et al measured the neutralizing activity of post-vaccine sera of individuals who received the BBIBP-CorV vaccine. Neutralizing activity against the Alpha lineage declined 2.2-fold compared to the wild-type strains, the Gamma variant declined 1.9-fold, the Lota variant declined 3.8-fold, and the Beta variant declined 4.6-fold.Citation31

In conclusion, studies on post-vaccine sera showed that E484K alone, or combined with other mutations or variants containing E484K, reduced the neutralization titer, regardless of the vaccine platform used. Studies evaluating the impact of E484K, as well as the mutations and variants of concern on post-vaccine sera are summarized in .

Table 3 Summary of Studies Evaluating the Impact of E484K, Mutations and Variants on Post-Vaccine Sera

The Impact of E484K on Vaccine Efficacy and Effectiveness

Changes in the in vitro neutralization of strains by vaccine-induced antibodies do not necessarily indicate reduced effectiveness; nevertheless, striking differences may correlate with clinical effectiveness.Citation32 We assessed the impact of E484K based on vaccine efficacy and effectiveness against variants containing the E484K mutation using real-world data.

A nationwide study evaluated the effectiveness of BNT162b2 in Qatar and the results demonstrated that the vaccine effectiveness was 90% (95% CI 86–92) for Alpha infection, compared with 75% (95% CI 71–79) for Beta infection.Citation33 Vaccine efficacy of adenovirus-based vaccines was also evaluated in South Africa where the Beta variant predominated. In a randomized, double-blind controlled trial of the ChAdOx1 nCoV-19 vaccine in South Africa, the vaccine efficacy for preventing mild-to-moderate Covid-19 was 21.9% (95% confidence interval [CI], −49.9 to 59.8), and against B.1.351 variant was 10.4%. Nevertheless, the trial was small, the confidence interval of vaccine efficacy was wide, and the trial concluded that two doses of the ChAdOx1 nCoV-19 vaccine were ineffective in preventing mild-to-moderate symptoms of the Beta variant.Citation25 In the international Phase 3 ENSEMBLE trial of Ad26.COV2.S vaccine, the overall vaccine efficacy varied by country: 74% in the United States, 66% in Brazil, and 52% in South Africa. In this trial, 95% of the Covid-19 cases in South Africa were caused by the Beta variant, whereas 69% of the cases in Brazil were caused by the Zeta variant (P.2 lineage), and both carry the E484K mutation.Citation34 NVX-CoV2373, in a Phase 2 trial in South Africa, where the Beta variant caused most COVID-19 cases, the vaccine efficacy was estimated at 49.4% (95% CI 6.1–72.8), which was lower than the vaccine efficacy 89.7% (95% CI 802–94.6) in the UK.Citation35,Citation36 Although the above trials showed most vaccines retained efficacy/effectiveness against the Beta and Zeta variants, a trend toward a lower level of vaccine efficacy was observed.

The available evidence suggests the vaccine efficacy of Ad26.COV2.S, NVX-CoV2373, and ChAdOx1 nCoV-19/AZD1222 were lowered but retained in Southern Africa, where the Beta variant is dominant.Citation25,Citation34–36 The effectiveness of BNT162b2 was also slightly lower with the Beta variant than with the Alpha variant in the observational study.Citation33 summarizes studies showing the impact of variants containing E484K on vaccine efficacy and effectiveness.

Table 4 Vaccine Efficacy/Effectiveness Against Variants Possessing E484K

E484K and Vaccine Breakthrough Infection

Several factors contribute to breakthrough infection, such as inadequate post-vaccination immune response, the immune response fading over time, and the constant change of the virus over time.Citation37 Several studies have analyzed how SARS-CoV-2 variants and mutations impact vaccine breakthrough infection.

Hacisuleyman et al investigated fully vaccinated patients who received mRNA-1273 and developed a breakthrough infection. Whole viral genome sequencing found E484K along with other mutations.Citation38 An institute in Washington state reported 20 vaccine breakthrough infections. All of the breakthrough infections were caused by variants of concern. Sixty percent of vaccine breakthrough cases were caused by variants reported to reduce the neutralization of post-vaccination sera, such as the Gamma, Beta, and Epsilon variants; while these variants only accounted for 36.7% of non-vaccinated cases, and a 1.63-fold change (p=0.037) was found.Citation39 Kustin et al conducted a matched cohort study in Israel to evaluate the distribution of variants of concern in individuals who had a BNT162b2 mRNA vaccine breakthrough infection. An increased proportion of the Beta variant was found in vaccinated cases with breakthrough infection when compared with unvaccinated cases.Citation40 Other reports also demonstrated that some variants of concern were more prevalent in breakthrough infections.Citation38,Citation40,Citation41

While some studies reported a significantly higher proportion of variants of concern among vaccine breakthrough infection cases relative to the general population, other reports have suggested the rate of variants of concern was no different from that of the general population. A report on fully vaccinated health care workers in Israel found 85% were caused by the Alpha variant, which was similar to the prevalence in non-vaccinated people.Citation42 The United States Centers for Disease Control and Prevention (CDC) reported that among 101 million fully vaccinated individuals, 10,262 breakthrough infections occurred, with a breakthrough rate of 0.01%, from January 1 to April 30, 2021. Sixty-four percent of the viral sequences obtained were variants of concern, including the Alpha variant (56%), the Epsilon variant (33%), the Gamma variant (8%), and the Beta variant (4%). Nevertheless, the proportions caused by variants of concern were no different from the circulating rates of those variants, suggesting that the proportions of SARS-CoV-2 variants were not significantly greater in breakthrough cases.Citation14 Other studies also demonstrated that the proportions of variants of concern in vaccinated and non-vaccinated individuals were similar.Citation43–45 lists reports on vaccine breakthrough infection and proportions of variants in vaccinated and non-vaccinated individuals.

Table 5 Reports of Vaccine Breakthrough Infection and Proportions of Variants in Vaccinated and Non-Vaccinated Individuals

Despite inconsistent data, some studies demonstrated that in comparison to non-vaccinated individuals, a higher proportion of variants of concern was observed in vaccinated individuals who had a breakthrough infection. A few studies revealed an increased proportion of variants containing E484K (such as Beta and Gamma variants) in cases of breakthrough. Whether variants possessing E484K play a role in breakthrough infection is not clear currently. The correlation between mutation and vaccine breakthrough infection warrants further investigation.

The Effects of E484K on Therapeutic Monoclonal Antibodies

Many therapeutic monoclonal antibodies are currently under development. Monoclonal antibodies for Covid-19 have been granted emergency use authorization (EUA) from the US Food and Drug Administration (FDA) to treat outpatients with mild-to-moderate disease, and include casirivimab–imdevimab, sotrovimab, bamlanivimab–etesevimab.Citation46,Citation47

Bamlanivimab (also known as LY-CoV555 and LY3819253) and Etesevimab (LY-CoV016 and LY3832479) are neutralizing monoclonal antibodies that bind to the RBD of the SARS-CoV-2 S protein. In phase 3 of BLAZE-1, treatment with bamlanivimab and etesevimab reduced Covid-19-related hospitalizations and deaths by 70% relative to placebo treatment.Citation48 Based on these data, EUAs for bamlanivimab and etesevimab were issued, but later revoked due to the growing number of SARS-CoV-2 variants resistant to bamlanivimab.Citation49 In a pseudovirus harboring the E484K substitution, bamlanivimab and etesevimab reduced susceptibility by 17-fold.Citation49 Moreover, it was reported that in patients with a high risk of severe Covid-19 treated with bamlanivimab, viral rebound was found in five of six severely immunodeficient patients, and E484K was found in the five patients. The study revealed that the E484K escape mutation can develop during treatment with monoclonal antibodies.Citation50

E484K reduced susceptibility to casirivimab (25-fold). In contrast, imdevimab and casirivimab combined with imdevimab retained their activity against VSV pseudovirus with E484K.Citation49,Citation51,Citation52 REGN-COV2 is an antibody cocktail composed of Casirivimab and imdevimab. In the preliminary results of a phase 3 randomized controlled trial, casirivimab-imdevimab reduced hospitalization and death compared with placebo.Citation49 It has been granted an EUA for treatment of mild-to-moderate Covid-19.

Sotrovimab, a recombinant human IgG1 monoclonal antibody, interacts with a highly conserved epitope on the RBD.Citation53 The epitope is different from the sites of mutations among SARS-CoV-2 variants of concern and interest. In an unpublished randomized trial for patients with one or more risk factors for severe disease, who have mild-to-moderate Covid-19, sotrovimab reduced the combined rates of hospitalization and death. In both pseudovirus and authentic virus assays, sotrovimab retains activity against the Alpha, Beta, Gamma, Epsilon and lota variants.Citation53

AZD7442 is a combination of two human monoclonal antibodies, AZD8895 and AZD1061.Citation54 In vitro, AZD8895, AZD1061, and AZD8895+ AZD1061 reduced the inhibitory capacity slightly (2- to fivefold increases in IC50 values) when tested in recombinant isogenic SARS-CoV-2s possessing the E484K mutation.Citation55 Other studies also proved that AZD7442 is effective against the Beta variant.Citation15,Citation55,Citation56

Regdanvimab, a monoclonal antibody, is also known as CT-P59. A pseudovirus with the E484K mutation resulted in a 2-fold reduction individually. The neutralization against the Beta variant strain was reduced 19-fold compared to the wild type.Citation57

In vitro assays have demonstrated that sotrovimab, AZD7442, regdanvimab, and REGN-CoV2 retained their activity against SARS-CoV-2 variants containing E484K, while bamlanivimab and etesevimab reduced neutralization. summarizes monoclonal antibodies under Phase III clinical trials and the impact of E484K on neutralization of these monoclonal antibodies.

Table 6 The Impact of E484K on Neutralization of Monoclonal Antibodies Under Phase III Clinical Trial

Conclusion

E484K has emerged frequently in several SARS-CoV-2 variants of concern/interest. E484K escapes the neutralizing effect of several monoclonal antibodies, convalescent plasma, and post-vaccine sera.

Several studies have revealed that the E484K mutation reduces the neutralization of convalescent sera, as proved by a pseudoviral assay. Monoclonal antibodies derived from convalescent patients were further studied, and class 2 antibodies, which bind to RBD both in “up” and “down” conformations of the spike, were proved to be resistant to the E484K mutation.

E484K mutation also reduced neutralization of post-vaccination sera. An in vitro analysis of post-vaccination sera from different vaccines simultaneously showed significantly decreased activity against the variants of concern containing E484K. Clinical trials of Ad26.COV2.S, NVX-CoV2373, and ChAdOx1 nCoV-19 showed reduced efficacy in Southern Africa, where the Beta variant is dominant.Citation25,Citation34–36 Despite the inconsistency of reported data, some studies have revealed an increased proportion of variants of concern, especially the Beta variant, in vaccine breakthrough infections.Citation39,Citation40

Therapeutic monoclonal antibodies are being developed rapidly. The impact of E484K on the susceptibility to monoclonal antibodies were assessed. E484K reduced the susceptibility to casirivimab (25-fold), but retained susceptibility when casirivimab was combined with imdevimab. Other monoclonal antibodies, such as sotrovimab, AZD7442, regdanvimab, and REGN-COV2, also retained their activity against E484K in vitro. However, bamlanivimab and etesevimab exhibited significantly reduced neutralization. Due to the reduced susceptibility of variants to bamlanivimab and etesevimab, the FDA has revoked the EUA for this combination therapy.Citation49,Citation51,Citation53,Citation56,Citation57

E484K can be selected when a culture recombinant virus expressing SARS-CoV-2 S is treated with antibody cocktails, and new variants with novel mutations are on the rise.Citation8–10,Citation15 Studies are being conducted with the aim of developing a new vaccine booster or a modified vaccine to provide increased protection against variants. In January 2021, Moderna developed a modified vaccine, a boosting dose of mRNA-1273.351, targeting the beta variant and three mutations (E484K, N501Y, and K417N). mRNA-1273.351 resulted in increased neutralizing antibody levels against the Beta and Gamma (P1) variants compared with levels elicited following the initial vaccine series.Citation58 As the virus is rapidly evolving and new mutations are emerging, it is necessary to continue to develop new vaccines and antibody therapies for Covid-19 over time.

Abbreviations

WHO, World Health Organization; RBD, receptor binding domain; E, glutamate; K, lysine; VOC, variants of concern; VOI, variants of interest; DMS, deep mutational scanning; IC50, Inhibitory concentration 50%; VSV, vesicular stomatitis virus; SPR, surface plasmon resonance; HIV-1, human immunodeficiency virus-1; PRNT50, conventional 50% plaque reduction neutralization testing; CDC, Centers for Disease Control and Prevention; EUA, emergency use authorization; FDA, Food and Drug Administration.

Disclosure

The authors report no conflicts of interest in this work.

References