Spike protein of SARS-CoV-2 defines the tropism of the virus, facilitates its spread, and modulates host immune function. It is the viral entry point of the host cell by which it binds to host angiotensin-converting enzyme 2 (ACE2) receptor. It is composed of 1273 amino acids and harbours two subunits that are bridged by a furin cleavage site. Subunit 1 contains two important domains: N terminal domain (NTD) and receptor binding domain (RBD) (Figure 1). It is also the main target of vaccination efforts as the majority of serum neutralising activity is directed on its RBD (17, 18).

A primary infection with SARS-CoV-2 provokes immune responses that generate potent anti-SARS-CoV-2 neutralising antibodies and CD4⁺ and CD8⁺ T cell responses which clear the infection and culminate with an immune response memory to fight future infection. As the pandemic continues to sweep across the globe, it has gradually become apparent that a primary infection with SARS-CoV-2 does not provide a full protection. Although rare, cases of re-infection were documented as early as August 2020 and many are expected to be underreported as it can be difficult to distinguish between re-infection and prolonged viral shedding (19–22). Additionally, a comprehensive whole genome sequencing is necessary to identify two separate episodes of infection. A natural exposure to SARS-CoV-2 generate anti-SARS-CoV-2 IgG antibodies that are largely protective for at least 6 months post-infection (23, 24). Repeat infection can be caused by a low potency and quality of immune memory, or a decline in anti-SARS-CoV-2 antibody titre over time. However, a report of a re-infection case indicates that a secondary infection with SARS-CoV-2 occurred in the presence of intact anti-SARS-CoV-2 antibody titre, suggesting that a seropositivity does not necessarily guarantee immunity and raising concern about immune escape variants that can overcome previous immunity (25).

Reports on vaccine breakthrough infections in vaccinated individuals are also accumulating, raising concern of developing escape mutants in the face of immune selection (26–30). A breakthrough infection is defined as a detection of SARS-CoV-2 antigen or RNA more than 14 days after receiving a COVID-19 vaccine. To date, over 10, 000 breakthrough infections have been reported (31). The durability of vaccine-induced antibodies is yet to be determined, but vaccines against SARS-CoV-2 elicit more robust antibody responses than a natural infection which may suggest a more stable protection (32). Nevertheless, both natural- and vaccine-induced anti-SARS-CoV-2 antibodies demonstrate comparable neutralising power (33).

As of June 14^th, a total of 6,238 nucleotide substitutions, 56 insertions and 278 deletions in the spike protein have been recorded in clinical isolates (34, 35). The most profound mark of SARS-CoV-2 evolution is perhaps the D614G mutation that emerged as early as 6 months into the outbreak. D614G mutation is located outside of the RBD (Figure 1). D614G variant bypassed natural bottlenecks due to its selective advantages that lead to improved viral infectivity and transmissibility and reduced viral sensitivity to neutralising convalescent sera (36–38). It immediately outcompeted other sequence groups and became ubiquitous worldwide. All VOCs that are driving the current pandemic waves harbour this dominant mutation.

The main target of serum neutralising antibodies is the RBD of the virus spike protein, which upon recognition prevents the virus from gaining entry to the host cell. A growing list of mutations in the RBD and NTD of the spike protein has been reported to resist neutralisation by therapeutic monoclonal antibodies, convalescent sera and vaccinee sera (Figure 1). Among them, a few merit concern as they have also recently been identified in re-infection and breakthrough infections with VOCs, likely for their location in the immune epitope region of the RBD that may impact antigenicity, hinder immune neutralising antibody binding and promote pathogenesis (Table 1) (17, 18).

E484K mutation first emerged in late 2020 and has now been gaining prevalence among the circulating strains. It has been acquired by at least 9 strains including VOCs Alpha, Beta and Gamma, and other variants under monitoring Zeta (P.2), Eta (B.1.525), Iota (B.1.526), Kappa (B.1.617.1), B.1.620 and Mu (B.1.621) (39). It is noteworthy to mention that five reinfection cases of E484K-carrying variants (Gamma and Zeta), which primary infections were of non-E484K variants, have been reported in Brazil as early as December 2020. Two of the cases reported the presence of anti-SARS-CoV-2 IgG antibodies during re-infection suggesting the possibility of E484K mutation in aiding antibody evasion (59). Furthermore, breakthrough infections in vaccinated individuals are also growing in number. A cohort study identified a slight reduction of protection by vaccines against subsequent infection with E484K-carrying Alpha and Beta variants of concern (64). Another report also documented an infection of a vaccinated individual with E484K-carrying variant (65). In another study, a full vaccination elicited a high level of neutralising antibodies that were capable of inhibiting an E484K variant in vitro but failed to pre-empt a high viral replication (66).

In vitro studies showed that E484K mutation can significantly reduce binding and resist neutralisation by convalescent and vaccine-induced sera and monoclonal antibodies (33, 40–42, 46, 47, 49–51). Additionally, in silico and cryo-EM studies have found that E484K mutation enhances the virus affinity for host ACE2 receptor binding which may account for the increased infectivity of these mutant variants (53, 54). Its impact on infectivity in vitro has been revealed in an infection assay of murine ACE2-expressing HEK29T cells with E484K-carrying pseudotyped viruses whereby 3-fold more infection was observed (68). The prevalence of E484K-carrying variants has been increasingly reported in viral isolates, presenting at low frequency in the circulating strain populations (42), likely due to the positive selection that provides for an immune escape and a greater transmissibility. This is consistent with in vitro evolutionary studies that showed that E484K mutation is readily introduced in the viral genome when cultured in the presence of anti-SARS-CoV-2 neutralising antibodies or ACE2 receptor (42, 47, 54). Furthermore, four E484 mutational changes at this residue position demonstrated an immune escape phenotype in presence of vaccinee sera, highlighting the importance of this residue as part of the dominant neutralising epitope (40).

Also gaining in frequency among circulating variant sequences is N501Y substitution that has been acquired by three VOCs Alpha, Beta and Gamma. A report has demonstrated that N501Y mutation reduces the virus sensitivity to neutralising monoclonal antibodies and vaccine-induced polyclonal antibodies (33, 51). In vitro infection assay of murine ACE2-expressing HEK293T cells with N501Y-carrying pseudotyped virus also showed that this mutation caused a 5-fold increase in viral infectivity (68). Additionally, it was shown that it is positively selected in vitro when the virus is grown in the presence of ACE2 to which it developed a higher binding affinity, and in vivo in a chronically infected immunocompromised patient (54, 69, 70). The increase in ACE2 tropism consequently promotes viral replication, transmission and shedding (69). N501Y-carrying variants (VOCs Alpha and Gamma) have been reported in four re-infection cases in which primary episode of infection agents were of non-N501Y-carrying variant (25, 59). Three of these cases described the presence of anti-SARS-CoV-2 IgG antibodies at the timepoint of secondary infection, raising speculation of antibody escape that may be mediated by this mutation. Furthermore, several independent reports of breakthrough infections with N501Y-caryying variant had been reported in vaccinated individuals suggesting an evasion of humoral immunity (65, 67).

While E484K only mildly increases the affinity of the RBD for ACE2 receptor, N501Y appears to substantially enhance it by allowing it to engage the receptor for longer (71–73). This is in agreement with an observation of pseudotyped viruses carrying N501Y mutation demonstrating a higher infectivity in vitro than those carrying E484K mutation (68). In contrast, the E484 residue in RBD engages more with antibodies than with ACE2 receptor, which may explain the relevance of E484K mutation in mediating antibody escape (72). The co-existence of these two mutations can produce a synergistic effect whereby they dramatically improve spike binding affinity to ACE2 receptor and reinforce the virus ability to evade immunity (68, 72, 73). The importance of these mutations for viral fitness is highlighted by their convergence in certain variants, including Beta, Gamma, and Mu (Table 1).

Another mutation that has gained attention is L452R substitution which can resist neutralisation by monoclonal antibodies and vaccinee and convalescent sera (36, 40, 43, 46, 48, 52). It provides a greater affinity of binding to ACE2 receptor and thus promotes viral replication and infectivity (48, 55, 56). A report of breakthrough infections in fully or partially vaccinated healthcare workers and a secondary infection with L452R-carrying variants have been described, highlighting the relevance of this mutation in mediating viral immune escape (60, 67). L425R mutation has been identified in the circulating VOC Delta and other variants including Epsilon (B.1.427/B.1.429), Iota and Kappa. Of important note, Delta variant is currently posing the most concern and threat to public health, as it has displaced other variants owing to its pronounced ability to transmit and escape antibodies (monoclonal antibodies and convalescent and vaccinee sera) (74–76). Increasing numbers of breakthrough infection with Delta variant have been reported (57, 77). Other mutations, in addition to L452R of the RBD, have been attributed to its explosive spread. Upstream NTD mutation G142D has been shown to resist binding by monoclonal antibodies which may be mediated by the change of the conformation of the spike protein that deters antibody binding (44, 45). Downstream P681R mutation in furin cleavage site promotes cleavage and processing of the spike protein and hence the entry efficiency and infectivity of the virus in vitro (57, 58).

Although current available vaccines are engineered based on the spike protein of the Wuhan reference strain, vaccinated individuals are expected to carry potent neutralising antibodies that are cross-reactive which can circumvent the issue of variants (78). Nevertheless, the growing evidence for immune evasion by emerging spike variants prompts a heavier consideration for a more adequate and effective vaccine design guided by genomic surveillance that can stimulate a more durable protection from SARS-CoV-2 infection.