The perpetuation of the SARS-CoV-2 pandemic has permitted the continued evolution of mutations, many of which appear to promote infectivity, transmission, and immune evasion. Critically, several derivative lineages defined as variants of concern (VOCs) and variants of interest (VOIs) have emerged in the last year that possess a constellation of highly adaptive mutations that have resulted in unprecedented propagation. To better understand the significance of these mutations, we analyzed their molecular and immunological consequences against the immunogenetic profile of the United States population using immunoinformatics to analyze in silico data. Our findings indicate that several evolving mutations in the VOCs and VOIs appear to confer immune evasion properties leading to antigenic drift, specifically for Ab-mediated and Th cell–mediated immune recognition, whereas mutations leading to evasion from innate immune mechanisms are less common in the more successful VOC strains compared with the VOIs. Importantly, several of these mutations raise concerns for the effectiveness of anamnestic responses achieved through natural infection and vaccination as well as for the utility of Ab-based therapeutic interventions. The emergence of such adaptations underscores the need for vaccine enhancements as well as the continued need to for preventative hygiene measures to help minimize transmission.

Mutations are central to the evolutionary trajectory of viruses, allowing for sustained adaptation on the host. Indeed, since the emergence of SARS-CoV-2 in 2019 (1), its genome has undergone significant mutations marked by deletions and nonsynonymous substitutions. The potential impact of these mutations is a matter of intense investigation. Importantly, several SARS-CoV-2 variants, including those classified as variants of concern (VOCs) and variants of interest (VOIs), possess specific defining mutations that appear to confer challenges to the immunity conferred by natural infection, vaccination, and interventional therapies based on mAbs.

VOC is a designation used to denote variants that have evolved a constellation of specific point mutations that substantially enhance the ability of the virus to infect and rapidly transmit within the human population. More specifically, the Centers for Disease Control and Prevention defines VOC as “a variant for which there is evidence of an increase in transmissibility, more severe disease, significant reduction in neutralization by antibodies generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, or diagnostic detection failures” (2). Before receiving this designation, a strain must first receive designation as a VOI. VOIs are strains characterized with having VOC features and are accordingly under enhanced surveillance and investigation (2).

The Centers for Disease Control and Prevention currently lists four SARS-CoV-2 VOCs: the alpha variant (B.1.1.7), originating from the United Kingdom (2020-09), exhibits a 29% increased transmission rate and increased morbidity and mortality compared with the index strain (2, 3). The beta variant (B.1.351), first identified in South Africa (2020-09), similarly has an increased transmission rate (25%) and mortality (2, 3). The delta lineage (B.1.617.2), originating from India (2020-07), exhibits an increased transmission rate of 97% and a significant reduction in Ab neutralization (2, 3). Finally, the gamma lineage (P.1), originating from Brazil (2020-10), exhibits a significant reduction in Ab neutralization and an increased transmission rate of 38% (24). In addition, there are several VOIs that have prompted global concern, including the epsilon variant (B.1.427) from California (2020-04), kappa variant (B.1.617.1) from India (2020-03), the lambda variant (C.37) from Peru (2020-11), and the mu variant (B.1.621) from Colombia (2021-01) (Refs. 2, 5, 6; M.L. Acevedo, L.A. Palomares, A. Bustamante, A. Gaggero, F. Paredes, C.P. Cortés, F. Valiente-Echeverría, and R. Soto-Rifo, manuscript posted on medRxiv, DOI: 10.1101/2021.06.28.21259673). A timeline summarizing the emergence of these strains is depicted in (Fig. 1. Collectively, the mutations that define these variants appear to enhance infectivity, transmissibility, and contribute to their propagation and persistence. To better understand the significance of these lineage-defining mutations, their potential immune-evasion capacity was determined using immunoinformatics and assessed for resistance against current vaccines, mAb-based interventional strategies, and naturally acquired immunity within the U.S. population.

FIGURE 1.

Timeline for the emergence of VOC and VOI strains.

FIGURE 1.

Timeline for the emergence of VOC and VOI strains.

Close modal

The following representative mutant sequences from the GISDAID SARS-CoV-2 database were assessed: B.1.1.7 (alpha lineage, EPI_ISL_601443), P.1 (gamma lineage, formally B.1.1.28.1; EPI_ISL_833174), B.1.351 (beta lineage; EPI_ISL_736980), B.1.617.2 (delta lineage, EPI_ISL_2887781), B.1.617.1 (kappa lineage, EPI_ISL_1372093), B.1.427 (epsilon lineage, EPI_ISL_1675148), C.37 (lambda lineage, EPI_ISL_1111130), and B.1.621 (mu lineage, EPI_ISL_2179709). Specific variants were selected based on minimal sequence ambiguity and the earliest possible collection date. Comparisons were made against the reference strain sequence (NC_045512.2) and 44 randomly selected variants using the alignment tool EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle). The results were cataloged for each open reading frame and individual molecular assessments were manually performed.

The most frequent HLA class I and HLA class II alleles in the United States were identified from Maiers et al. 2007 (7) and from data curated at the Allele Frequency Net Database (www.allelefrequencies.net) and included HLA A*01:01, HLA A*02:01, HLA A*03:01, HLA B*07:02, HLA B*35:01, HLA B*44:03, HLA C*04:01, HLA C*07:01, HLA C*07:02, HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DQA01:02/DQB10301, HLA-DQA05:01:01/DQB10302, and HLA DQA05:03:01/DQB201.

The most wildly immunogenic T cell epitopes have been reported to occur in spike, matrix, and nucleocapsid (8). For T cell analysis, the index strain sequences for spike, matrix, and nucleocapsid were assessed using the immunoinformatic tools NetMHCpan (http://www.cbs.dtu.dk/services/NetMHCpan) and NetMHCIIpan (https://services.healthtech.dtu.dk/service.php?NetMHCIIpan-4.0) to determine the highest affinity binding epitope for each HLA allele defined above. Both tools predict binding of peptides to any MHC molecule of known sequence using artificial neural networks and exhibit significantly improved predictive power for MHC ligands and T cell epitopes relative to other available state-of-the-art algorithms (9). For mutant strains possessing amino acid mutations in any of these immunodominant epitopes, theoretical change in HLA-binding affinity (compared with reference strain) was calculated to predict the impact on CD4+ and CD8+ T cell recognition.

The most wildly immunogenic B cell epitopes have been reported to occur in structural proteins spike, matrix, and nucleocapsid (10, 11). Accordingly, epitope analysis was performed using S:552–562 LTESNKKFLPF, S:655–669 HVNNSYECDIPIGAG, S (spike):768–777 TGIAVEQDKN, S:796–805 DFGGFNFSQI, S:823–830 NKVTLAD, S:1157–1163 KNHTSPD, M (matrix):89–197 GDSGFAAYS, N:363–373 FPPTEPKKDKK, and N (nucleocapsid):153–160 NNAAIVLQ sequences from the reference strain. For mutant strains possessing amino acid mutations in these immunodominant epitopes, the theoretical change in binding affinity (compared with reference strain) was calculated to predict the relative impact on Ab binding using the enhanced VaxiJenv 2.0 Ag prediction server (12). We additionally assessed critical Ab-binding sites reported in the GISAID SARS-CoV-2 database as well as epitopes targeted by mAb-based anti–COVID-19 immunotherapies (13).

Summaries of each variant, its classification, defining spike mutations, earliest sampling date, current variant sequence count, and number of variant sublineages are reported in Table I. All four VOCs were identified within a 3-mo span in 2020 (July 15–October 1), with VOIs emerging before and after this period (Fig. 1, Table I). The event count for the VOCs ranges from 27,000 to 1,000,000, and the event count for the VOIs ranged from 4,000 to 18,000; the variant with the highest sequence count to date is alpha (B.1.1.7) at 1,020,185 reported events at GISAID (Table I).

Table I.

Lineage summary of VOCs and VOIs

Mutant StrainClassificationS Protein Lineage-Specific MutationsaEarliest Sampling Date/ OriginVariant Sequence CountVariant Sublineages
Alpha (B.1.1.7) VOC 69del, 70del, 144del, (E484K), (S494P), N501Y, A570D, D614G, P681H, T716I, S982A, D1118H (K1191N) December 9, 2020
(United Kingdom) 
1,020,185 
Beta (B.1.351) VOC D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, A701V December 9, 2020
(South Africa) 
27,706 
Gamma (P.1 or B.1.1.28.1) VOC L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I December 22, 2020
(Brazil) 
70,024 10 
Epsilon (B.1.617.2) VOC T19R, (V70F), T95I, (G142D), E156del, F157del, R158G, (A222V), (W258L), (K417N), L452R, T478K, D614G, P681R, D950N July 1, 2021
(India) 
375,934 34 
Delta (B.1.427) VOI L452R, D614G April 11, 2020
(California) 
17,816 
Kappa (B.1.617.1) VOI (T95I), G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H December 1, 2020
(India) 
5,886 
Lambda (C.37 or B.1.1.1.37) VOI G75V, T76I, R246del, S247del, Y248del, L249del, T250del, P251del, G252del, L452Q, F490S, D614G, T859N November 8, 2020
(Peru) 
6,630 
Mu (B.1.621) VOIb T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, D950N January 11, 2021
(Colombia) 
4,516 
44 Random variants (variable) Variable Variable May 2020– January 2021 Variable Variable 
Mutant StrainClassificationS Protein Lineage-Specific MutationsaEarliest Sampling Date/ OriginVariant Sequence CountVariant Sublineages
Alpha (B.1.1.7) VOC 69del, 70del, 144del, (E484K), (S494P), N501Y, A570D, D614G, P681H, T716I, S982A, D1118H (K1191N) December 9, 2020
(United Kingdom) 
1,020,185 
Beta (B.1.351) VOC D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, A701V December 9, 2020
(South Africa) 
27,706 
Gamma (P.1 or B.1.1.28.1) VOC L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I December 22, 2020
(Brazil) 
70,024 10 
Epsilon (B.1.617.2) VOC T19R, (V70F), T95I, (G142D), E156del, F157del, R158G, (A222V), (W258L), (K417N), L452R, T478K, D614G, P681R, D950N July 1, 2021
(India) 
375,934 34 
Delta (B.1.427) VOI L452R, D614G April 11, 2020
(California) 
17,816 
Kappa (B.1.617.1) VOI (T95I), G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H December 1, 2020
(India) 
5,886 
Lambda (C.37 or B.1.1.1.37) VOI G75V, T76I, R246del, S247del, Y248del, L249del, T250del, P251del, G252del, L452Q, F490S, D614G, T859N November 8, 2020
(Peru) 
6,630 
Mu (B.1.621) VOIb T95I, Y144S, Y145N, R346K, E484K, N501Y, D614G, P681H, D950N January 11, 2021
(Colombia) 
4,516 
44 Random variants (variable) Variable Variable May 2020– January 2021 Variable Variable 

aParentheses indicate that the mutation is present in many but not all mutants of this lineage.

bRedesignated as variant being monitored (VBM) as of September 21, 2021.

Genetic plasticity, as evidenced by the number of sublineages, ranged between 4 and 34 for the VOCs and 0 and 1 for the VOIs; the variant with the most genetic plasticity, as evidenced by number of sublineages, is delta (B.1.617.2), with 34 sublineages currently reported in Pango (Table I). Accordingly, VOCs exhibit increased propagation and subsequent genetic variation in relation to the VOIs.

Variability was additionally assessed using a representative variant for each VOC and VOI (see Materials and Methods). An unusually high number of nucleotide mutations were observed in the delta variant, at 76, compared with the predicted average of 6–11 nucleotide diseases (14). In comparison, the remaining VOCs/VOIs exhibited 21–45 mutations and the control group averaged 17 ± 6.8 mutations (Table II). To reconcile this, we additionally assessed NSP14, a proofreading enzyme that imparts high fidelity replication of the virus (15). NSP14 mutations were cataloged for both the delta variant as well as 13 of the 44 control variants (Table II). Specifically, the delta variant possessed the NSP14 A394V mutation (Table II), which occurs in ∼30% of total GISAID-archived variants. However, direct comparison of mutation frequency across distinct delta variant sequences did not support the conclusion that the A394V mutation conferred a reduction in enzyme function (data not shown). An additional consideration was NSP12, the RNA-dependent RNA polymerase that catalyzes viral replication and transcription (16). All VOCs and VOIs possessed the P323L mutation occurring in the non-catalytic domain of NSP12. Importantly, the GISAID database reports that the P323L mutation has 3,474,929 occurrences among all SARS-CoV-2 variants, indicating that it is a favorable mutation, not just in VOCs and VOIs.

Table II.

Mutation summary of variant sequences

StrainTop Mutated ORFNo. of NT MutationsaNS/S RatioC→T (U) Mutations (%)Amino Acid MutationNSP12 MutationNSP14 Mutation
Alpha (EPI_ISL_601443) Orf1abb>S>N 45  1.7 10 (22.2) 17 P323L No 
Beta (EPI_ISL_736980) S>Orf1ab>N 39  9.5 9 (23.1) 19 P323L No 
Gamma (EPI_ISL_833174) Orf1ab>S >N 40  2.1 10 (25) 21 P323L No 
Delta (EPI_ISL_2887781) Orf1ab>S>N 76  3.4 16 (21.05) 33 P323L A394V 
Epsilon (EPI_ISL_1675148) Orf1ab>S>N 21  3.2 8 (38) 16 P323L No 
Kappa (EPI_ISL_1372093) Orf1ab>S>N 28  3.5 10 (35.71) 21 P323L No 
Lambda (EPI_ISL_1111130) Orf1ab>S>N 27  6.75 13 (48.15) 27 P323L No 
Mu (EPI_ISL_2179709) S>Orf1ab>N 25  2.7 15 (44.18) 34 P323L No 
44 Random variants (variable) Orf1ab >N>S 17.6 ± 6.8  1.8 ± 0.8 8.0 (47.7) 10.5 ± 3 All: P323L
6/44: A423V
1/44: T803I, D303G 
13/44: P43L, D345Y, T16I, P24S, H26Y 
StrainTop Mutated ORFNo. of NT MutationsaNS/S RatioC→T (U) Mutations (%)Amino Acid MutationNSP12 MutationNSP14 Mutation
Alpha (EPI_ISL_601443) Orf1abb>S>N 45  1.7 10 (22.2) 17 P323L No 
Beta (EPI_ISL_736980) S>Orf1ab>N 39  9.5 9 (23.1) 19 P323L No 
Gamma (EPI_ISL_833174) Orf1ab>S >N 40  2.1 10 (25) 21 P323L No 
Delta (EPI_ISL_2887781) Orf1ab>S>N 76  3.4 16 (21.05) 33 P323L A394V 
Epsilon (EPI_ISL_1675148) Orf1ab>S>N 21  3.2 8 (38) 16 P323L No 
Kappa (EPI_ISL_1372093) Orf1ab>S>N 28  3.5 10 (35.71) 21 P323L No 
Lambda (EPI_ISL_1111130) Orf1ab>S>N 27  6.75 13 (48.15) 27 P323L No 
Mu (EPI_ISL_2179709) S>Orf1ab>N 25  2.7 15 (44.18) 34 P323L No 
44 Random variants (variable) Orf1ab >N>S 17.6 ± 6.8  1.8 ± 0.8 8.0 (47.7) 10.5 ± 3 All: P323L
6/44: A423V
1/44: T803I, D303G 
13/44: P43L, D345Y, T16I, P24S, H26Y 

Representative variant sequences were analyzed (see Materials and Methods).

aIndicates the number of nucleotide substitutions relative to the reference strain.

bORF1ab refers to the largest open reading frame in SARS-CoV-2, which encodes polyproteins PP1ab and PP1a, which are cleaved to generate 16 nonstructural proteins.

NT, nucleotide; ORF, open reading frame.

An accepted indicator applied to mutational and evolutionary analyses is the ratio of nonsynonymous to synonymous nucleotide mutations (NS/S). Critically, nonsynonymous mutations lead to amino acid substitutions that impart biological change and evolutionary consequences. For SARS-COV-2, the genome-wide NS/S ratio is 1.88 (14), a value that is orders of magnitude higher than the predicted values observed for other (+) sense mRNA viruses (17) and suggests significant accrual of adaptive mutations. Although all VOCs/VOIs exhibited more amino acid mutations, relative to the control variants (10.5 ± 3.3, mean ± SD), the delta variant and mu variant sequence possessed disproportionately more amino acid substitutions (compare 33 and 34 versus 16–21 mutations) (Table II). Relating these changes back to NS/S ratios, the beta, delta, epsilon, kappa, and lambda variant sequences observed NS/S ratios ranging from 3.2 to 9.5, indicating that they have undergone accelerated evolution (Table II). The reason for these unusually high ratio values cannot be rationalized based on mutation in NSP12 or NSP14, and thus it is likely that extrinsic factors related to chronic infection in the setting of immunodeficiency may have led to this pronounced evolution rate (18). In comparison, the alpha, gamma, and mu variants possessed NS/S ratios that approximated the predicted ratio, between 1.7 and 2.7, respectfully, indicating that they are evolving along a more predicted pattern, as likewise observed in the random control sequences (1.8 ± 0.8, mean ± SD) (Table II).

Mutations in SARS-CoV-2, as a whole, exhibit a strong bias for deamination of cytosine to uracil (C→U), comprising ∼35–50% of the total nucleotide mutations at the genomic level (14, 19). Of note, C→U mutations are postulated to be catalyzed by host RNA editing enzymes and appear to confer resistance to zinc finger antiviral protein (ZAP), an IFN-induced gene product that promotes degradation of viral RNA (20). Specifically, earlier studies assessing HIV have shown that C→U mutations reduce the occurrence of viral CpG motifs in ssRNA that in turn reduce the ability for recognition and subsequent elimination of viral RNA via a ZAP-dependent mechanism (21). All VOCs exhibited C→U mutation frequencies that were significantly lower than the predicted rate, ranging from 21 to 25%, whereas the VOI and control sequence values were within range, at 36–40% and 47.2 ± 10.85% (mean ± SD) (Fig. 2, Table II,). This dichotomy implies that distinct evolutionary pressures appear to be driving the mutation pattern in the highly successful VOCs. Indeed, excess C→U mutations likely confer amino acid substitutions that negatively affect protein function, thus reducing the overall fitness of the virus when occurring at exaggerated frequencies.

FIGURE 2.

Percent C→U mutations.

Values represent the percent of nucleotide mutations, relative to the reference strain, that represented C→U substitutions.

FIGURE 2.

Percent C→U mutations.

Values represent the percent of nucleotide mutations, relative to the reference strain, that represented C→U substitutions.

Close modal

Amino acid changes that influence Ab binding were assessed through three distinct approaches. First, high-frequency immunodominant epitopes described in the United States (see Materials and Methods) derived from reference strain spike, matrix, and nucleocapsid proteins were compared against the corresponding mutant sequences. Only gamma and mu possessed mutations in these previously defined immunodominant epitopes (Table III), specifically at spike H655Y, near the furin cleavage site associated with increased transmissibility, which modestly reduces the theoretical Ab-binding affinity to 68% (Table III). This mutation is observed in ∼3% of archived sequences and therefore does not appear to confer an overt selective advantage for these lineages. Correspondingly, the lambda variant EPI_ISL_1111130 possessed the nucleocapsid T366I mutation, although this is not lineage-defining, nor does it represent a high-frequency event.

Table III.

B cell immunodominant epitopes (spike, matrix, nucleocapsid): event summary in the United States

Mutant StrainAmino Acid Changes in S EpitopesAmino Acid Changes in N EpitopesGISAID Ab SitesAmino Acid Changes in mAb Tx Sites
Alpha (EPI_ISL_601443)   S: 144, 501, (484a(E484K) 
Beta (EPI_ISL_736980)   S: 417, 501, 484 E484K, K417N, N501Y 
Gamma (EPI_ISL_833174) H655Y:
68% affinity 
 S: 417, 484, 501 E484K, K417T, N501Y 
Delta (EPI_ISL_2887781)   S: 158, 452, 478 (K417N), L452R 
Epsilon (EPI_ISL_2304313)   S: 152, 452 L452R 
Kappa (EPI_ISL_1372093)   S: 452, 484 E484Q, L452R 
Lambda (EPI_ISL_1111130)  N: [T366I]
88% affinity 
S: 452, 471, 490 F490S 
Mu (EPI_ISL_21797090) H655Y:
68% affinity 
 S: Y144S, 145, 346, 484, 501 E484K 
44 Random variants (variable)   S: 144, 510 (co-occurring in same variant)  
Mutant StrainAmino Acid Changes in S EpitopesAmino Acid Changes in N EpitopesGISAID Ab SitesAmino Acid Changes in mAb Tx Sites
Alpha (EPI_ISL_601443)   S: 144, 501, (484a(E484K) 
Beta (EPI_ISL_736980)   S: 417, 501, 484 E484K, K417N, N501Y 
Gamma (EPI_ISL_833174) H655Y:
68% affinity 
 S: 417, 484, 501 E484K, K417T, N501Y 
Delta (EPI_ISL_2887781)   S: 158, 452, 478 (K417N), L452R 
Epsilon (EPI_ISL_2304313)   S: 152, 452 L452R 
Kappa (EPI_ISL_1372093)   S: 452, 484 E484Q, L452R 
Lambda (EPI_ISL_1111130)  N: [T366I]
88% affinity 
S: 452, 471, 490 F490S 
Mu (EPI_ISL_21797090) H655Y:
68% affinity 
 S: Y144S, 145, 346, 484, 501 E484K 
44 Random variants (variable)   S: 144, 510 (co-occurring in same variant)  

Representative variant sequences were analyzed (see Materials and Methods). Amino acid changes are shown that were documented to occur in S, M, and N, the most immunogenic proteins in SARS-CoV-2, relative to the reference strain. See Materials and Methods for details. GISAID Ab sites refer to mutations occurring at sites previously documented by GISAID to influence Ab binding. mAb Tx indicates mutations occurring at sites that are known to influence binding of therapeutic mAbs.

aParentheses indicate that the mutation is present in many but not all mutants of this lineage.

M, matrix; N, nucleocapsid; S, spike.

Second, key Ab-binding sites in spike protein previously cataloged at GISAID on the basis of their ability to impart antigenic shift were systematically assessed. All VOCs and VOIs possessed two or more amino acid substitutions at these defined sites (Table III). Importantly, the delta and mu variants possessed the most substitutions, with a total of four and five changes in Ab-binding sites, respectively, implying that both lineages pose significant challenges to humoral immunity, particularly to vaccine-induced immunity, which is singularly dependent on the spike protein. In comparison, only 1 of 44 (<3%) of the control sequences had mutations at these sites. Thus, mutations that confer evasion from Abs are selectively enriched in VOC and VOI lineages.

We additionally assessed lineage-specific mutations in the spike protein occurring at positions 417, 452, 484, 501, and 681, which likely represent adaptive traits based on their independent recurrence in distinct VOCs and VOIs (Table I). K417N and K417T mutations present in the beta, delta, and gamma variants appear to enhance affinity for the ACE2 receptor as well as reduce the in vitro neutralizing activity of vaccine and infection-acquired Abs (Ref. 22 and K. Wu, A.P. Werner, J.I. Moliva, M. Koch, A. Choi, G.B.E. Stewart-Jones, H. Bennett, S. Boyoglu-Barnum, W. Shi, B.S. Graham, manuscript posted on bioRxiv, DOI: 10.1101/2021.01.25.427948). The mutations E484K and E484Q occurring in the receptor-binding domain of the delta, epsilon, kappa, and mu variants have also been shown to reduce the binding of Abs derived from postvaccine and convalescent serum by 3- to 5-fold (6, 23). The N501Y mutation in alpha, beta, gamma, and mu variants is associated with increased viral replication in the nasal cavity as well as increased affinity for the ACE2 receptor (24). Furthermore, the N501Y mutation also appears to reduce reactivity with convalescent sera in vitro (25). The P681R mutation, located within the furin cleavage site, is thought to enhance delta variant infectivity through increasing S1/S2 cleavage (B. Lubinski, T. Tang, S. Daniel, J.A. Jaimes, and G.R. Whittaker, manuscript posted on bioRxiv, DOI: 10.1101/2021.01.25.427948). The L452R mutation, which presents in the delta, epsilon, and kappa variants, has been shown to increase viral infectivity and impairment of neutralizing Ab binding (26). Accordingly, mutations in spike protein amino acid residues 417, 452, 484, and 501 are associated with humoral immune evasion.

Finally, we assessed therapeutically significant amino acid substitutions that influence mAb binding to spike protein, specifically bamlanivimab, etesevimab, sotrovimab, and the combined casirivimab/imdevimab mixture, which are influenced by changes to spike protein at positions 417, 452, and 484 (2, 27, 28). To date, there are no reported mutants with in vitro susceptibility to sotrovimab or the combined casirivimab/imdevimab mixture. The beta and gamma VOCs, with the combined K417T, E484K, and N501Y mutations, show reduced binding to bamlanivimab and etesevimab. The L452R mutation present in delta and epsilon modestly reduces the neutralizing ability of these therapeutics as well. The N501Y mutation alone does not appear to influence any of the mAb-based therapies. The clinical significance of these in vitro findings remains unclear but they imply reduced reactivity in vivo. The propagation of these mutations in response to mAb-based interventions is unlikely given the low application rate of these drugs.

In comparison, to assess T cell–mediated recognition, we identified the most immunodominant epitopes of the spike, matrix, and nucleocapsid proteins from the reference strain and ran the sequences against the top three high alleles for HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ alleles seen in the U.S. population (see Materials and Methods). The mutant sequences were then compared against the reference strain epitopes to assess fold change in binding affinity. No lineage-defining mutations were observed for HLA class I binding immunodominant epitopes derived from spike, nucleocapsid, or matrix protein (Table IV). This finding implies that in the U.S. population, recognition of VOCs and VOIs by CD8+ T cells remains largely unaffected. Correspondingly, each VOC and VOI possessed at least one mutation in nucleocapsid protein affecting HLA-DQA/DQB binding, whereas immunodominant epitopes of spike and matrix remained unchanged (Table IV). In some instances, double mutations occurred in the same immunodominant epitope, as seen for alpha, gamma, and lambda (Table IV). This double mutation event was only observed to occur once in the randomly selected control strains. Critically, these mutations clustered around the same region and were shared across different lineages: nucleocapsid R203K and G204R double mutations were present in alpha, gamma, and lambda lineages, resulting in 37% original affinity. The nucleocapsid R203M single mutation was observed in epsilon and kappa lineages, resulting in 50% binding affinity, and T205I was observed in beta, epsilon, and mu lineages reducing relative affinity relative to 84% that of the reference strain. The R203K/G204R mutations have previously been associated with increased infectivity and transmissibility (29). Although the T205I mutation has been shown to reduce detection capability by SARS Ag fluorescent immunoassay tests (30), the outcome of this mutation of HLA binding or T cell recognition has not yet been established.

Table IV.

T cell immunodominant epitopes (spike, matrix, nucleocapsid): events summary in the United States

Mutant StrainHLA Class I (HLA-A, HLA-B, HLA-C) N EpitopeHLA Class II (HLA-DR, HLA-DQ) N Epitope
Alpha (EPI_ISL_601443)  HLA-DQA01:02/DQB10301: N199–N213: PGSSKRTSPARMAGN; 37% original affinity 
Beta (EPI_ISL_736980)  HLA-DQA01:02/DQB10301: N199–N213 PGSSRGISPARMAGN; 84% original affinity 
Gamma (EPI_ISL_833174)  HLA-DQA01:02/DQB10301: N199–N213: PGSSKRTSPARMAGN; 37% original affinity 
Epsilon (EPI_ISL_167514)  HLA-DQA01:02/DQB10301: N199–N213: PGSSRGISPARMAGN; 84% original affinity 
Delta (EPI_ISL_288778)  HLA-DQ: 1) N199–N213 (HLA-DQA01:02/DQB10301): PGSSMGTSPARMAGN; 50% original affinity. 2) N49–N63 (HLA-DQA050301/DQB201): TASWFTALTQHGKEG;87% original affinity. 3) N49-N63 (HLA-DQA050101/DQB10302): TASWFTALTQHGKEG; 81% original affinity 
Kappa (EPI_ISL_137209)  HLA-DQA01:02/DQB10301: N199–N213: PGSSMGTSPARMAGN; 50% original affinity 
Lambda (EPI_ISL_111113) HLA-B3501: N322–N333: MEVXPSGTWLTY; unknown effect
HLA-B4403:N319-330: RIGMEVXPSGW; unknown effect 
HLA-DQA01:02/DQB10301: N199–N213: PGSSKRTSPARMAGN; 37% original affinity
HLADRB1-15:01:N354–368: NKHIDAYKTFPPIEP; 99% original affinity (non-lineage specific) 
Mu (EPI_ISL_21797090)  HLA-DQA0102/DQB10301:N199–N213:PGSSRGISPARMAGN; 84% original affinity 
44 Random variants (variable) HLA-C0701: N148–N159: TRNLANNAAIVL; 20% original affinity HLA-DQA0102/DQB10301, HLA-C0701: HLA-DQ: N199-N213: PGSSKRTSPARMAGN;37% original affinity 
Mutant StrainHLA Class I (HLA-A, HLA-B, HLA-C) N EpitopeHLA Class II (HLA-DR, HLA-DQ) N Epitope
Alpha (EPI_ISL_601443)  HLA-DQA01:02/DQB10301: N199–N213: PGSSKRTSPARMAGN; 37% original affinity 
Beta (EPI_ISL_736980)  HLA-DQA01:02/DQB10301: N199–N213 PGSSRGISPARMAGN; 84% original affinity 
Gamma (EPI_ISL_833174)  HLA-DQA01:02/DQB10301: N199–N213: PGSSKRTSPARMAGN; 37% original affinity 
Epsilon (EPI_ISL_167514)  HLA-DQA01:02/DQB10301: N199–N213: PGSSRGISPARMAGN; 84% original affinity 
Delta (EPI_ISL_288778)  HLA-DQ: 1) N199–N213 (HLA-DQA01:02/DQB10301): PGSSMGTSPARMAGN; 50% original affinity. 2) N49–N63 (HLA-DQA050301/DQB201): TASWFTALTQHGKEG;87% original affinity. 3) N49-N63 (HLA-DQA050101/DQB10302): TASWFTALTQHGKEG; 81% original affinity 
Kappa (EPI_ISL_137209)  HLA-DQA01:02/DQB10301: N199–N213: PGSSMGTSPARMAGN; 50% original affinity 
Lambda (EPI_ISL_111113) HLA-B3501: N322–N333: MEVXPSGTWLTY; unknown effect
HLA-B4403:N319-330: RIGMEVXPSGW; unknown effect 
HLA-DQA01:02/DQB10301: N199–N213: PGSSKRTSPARMAGN; 37% original affinity
HLADRB1-15:01:N354–368: NKHIDAYKTFPPIEP; 99% original affinity (non-lineage specific) 
Mu (EPI_ISL_21797090)  HLA-DQA0102/DQB10301:N199–N213:PGSSRGISPARMAGN; 84% original affinity 
44 Random variants (variable) HLA-C0701: N148–N159: TRNLANNAAIVL; 20% original affinity HLA-DQA0102/DQB10301, HLA-C0701: HLA-DQ: N199-N213: PGSSKRTSPARMAGN;37% original affinity 

Amino acid changes are reported for predicted immunodominant epitopes based on high-frequency HLAs in the United States. Boldface and underlined amino acid residues represent the mutant sequences (relative to the reference sequence).

S, spike; M, matrix; N, nucleocapsid.

Our in silico data support the conclusion that these VOC/VOI strains have evolved lineage-specific mutations in immunodominant epitopes for both B cells and T cells as well as mutations that influence innate immune recognition. Specifically, changes to immunodominant B cell (Ab) epitopes in spike protein appear to present certain challenges in immunity conferred by vaccination or through natural infection. The in vitro consequences of these mutations affecting humoral immunity are previously addressed in the literature (see Results). With respect to the T cell compartment, all of the VOCs/VOIs demonstrated significant and recurring alterations in class II HLA-binding epitopes derived from the nucleocapsid protein that are predicted to negatively impact the anamnestic Th cell response in patients having experienced natural infection. Modifications to HLA class II–restricted epitopes is especially problematic in that the Th cellular response governs the host’s control of viral infection. The potential immunological consequences of these T cell–related epitope mutations have not yet been validated in vitro or ex vivo. Finally, C→U mutations, which are thought to evoke evasion from the antiviral IFN-inducible ZAP, are not as prevalent in the VOCs compared with the VOIs or the control cohort, signifying that successful VOCs have likely refined the balanced higher fidelity protein function in exchange for enhanced evasion of the innate immune response.

Although these results are derived from in silico analyses and are therefore limited in the scope of their biological conclusions, the overriding trend observed in the VOI and VOC mutant strains is the evolution of mutations that impair adaptive immune functions, thereby likely contributing to their aggressive propagation. The potential role of vaccination in evolving new mutations will be a matter of future studies. Regardless, the observation that the virus is adapting against these elements serves as further evidence to implement stringent public health measures to minimize community spread and propagation and warrants consideration for vaccination enhancements to improve humoral immunity.

The authors have no financial conflicts of interest.

Abbreviations used in this article

NS/S

nonsynonymous to synonymous nucleotide mutations

VOC

variant of concern

VOI

variant of interest

ZAP

zinc finger antiviral protein

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