Research progress in spike mutations of SARS‐CoV‐2 variants and vaccine development

The coronavirus disease 2019 (COVID‐19) pandemic can hardly end with the emergence of different variants over time. In the past 2 years, several variants of severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2), such as the Delta and Omicron variants, have emerged with higher transmissibility, immune evasion and drug resistance, leading to higher morbidity and mortality in the population. The prevalent variants of concern (VOCs) share several mutations on the spike that can affect virus characteristics, including transmissibility, antigenicity, and immune evasion. Increasing evidence has demonstrated that the neutralization capacity of sera from COVID‐19 convalescent or vaccinated individuals is decreased against SARS‐CoV‐2 variants. Moreover, the vaccine effectiveness of current COVID‐19 vaccines against SARS‐CoV‐2 VOCs is not as high as that against wild‐type SARS‐CoV‐2. Therefore, more attention might be paid to how the mutations impact vaccine effectiveness. In this review, we summarized the current studies on the mutations of the SARS‐CoV‐2 spike, particularly of the receptor binding domain, to elaborate on how the mutations impact the infectivity, transmissibility and immune evasion of the virus. The effects of mutations in the SARS‐CoV‐2 spike on the current therapeutics were highlighted, and potential strategies for future vaccine development were suggested.

the Delta and Omicron variants, have emerged with higher transmissibility, immune evasion and drug resistance, leading to higher morbidity and mortality in the population.
The prevalent variants of concern (VOCs) share several mutations on the spike that can affect virus characteristics, including transmissibility, antigenicity, and immune evasion.
Increasing evidence has demonstrated that the neutralization capacity of sera from COVID-19 convalescent or vaccinated individuals is decreased against SARS-CoV-2 variants. Moreover, the vaccine effectiveness of current COVID-19 vaccines against SARS-CoV-2 VOCs is not as high as that against wild-type SARS-CoV-2. Therefore, more attention might be paid to how the mutations impact vaccine effectiveness. In this review, we summarized the current studies on the mutations of the SARS-CoV-2 spike, particularly of the receptor binding domain, to elaborate on how the mutations impact the infectivity, transmissibility and immune evasion of the virus. The effects of mutations in the SARS-CoV-2 spike on the current therapeutics were a heavily glycosylated type I viral membrane fusion protein. It can form mushroom-like homotrimers on the surface of viral particles, which can bind to receptors to mediate viral entry and membrane fusion ( Figure 1). 10 During biogenesis or virus assembly, the spike is cleaved into S1 and S2 subunits by the receptor transmembrane protease serine 2 (TMPRSS2). 11 The S1 subunit is comprised of a signal peptide (SP), an N-terminal domain (NTD), a carboxy-terminal receptor-binding domain (RBD), and two additional domains (subdomains 1 and 2, SD1 and SD2). Previous studies have revealed that the S1 subunit is crucial for recognizing and binding to ACE2 through the RBD. A few studies have also reported that the NTD is involved in the entry of SARS-CoV-2 into host cells that highly express asialoglycoprotein receptor 1 (ASGR1) or Kremen 1. 12,13 The S2 subunit consists of a fusion peptide (FP), two heptad repeats (HR), a central helix (CH), and a connector domain (Figure 1). It plays a critical role in the fusion of the virus and host cell membranes. 14 In the spike homotrimer, three copies of the S1 subunit sit on the top of a stem of the S2 subunit. 14 The RBD structure in the S1 subunit exerts high flexibility, exhibiting "up" and "down" conformations. The spike trimer can bind ACE2 only when the RBD is in an "up" conformation. 15,16 In brief, the entry of the SARS-CoV-2 virus into human respiratory epithelial cells is divided into three steps. First, the spike is proteolytically cleaved at the S1/S2 cleavage site to make S1 able to attach the virus to the surface receptors of host cells. 14,17,18 After receptor engagement at the plasma membrane or virus endocytosis by human respiratory epithelial cells, a second cleavage, named S2′ site cleavage mediated by TMPRSS2 or by cathepsin L in the endosomal compartment following ACE2-regulated endocytosis, 19 is essential to release the fusion peptide from the Nterminus of S2, which may initiate the S2-mediated membrane fusion cascade. 20,21 Subsequently, the fusion peptide inserts into the host cell membrane to form the prehairpin intermediate state and rearranges conformation to facilitate membrane fusion. 12,21,22 The SASR-CoV-2 ancestor and previous variants mainly enter host cells via the plasma membrane pathway. In contrast, the Omicron variant prefers the TMPRSS2independent endosomal entry pathway rather than the plasma membrane entry route. 23,24 The successful F I G U R E 1 SARS-CoV-2 viral particle and spike composition. (A) SARS-CoV-2 viral particle and genome. SARS-CoV-2 is an enveloped RNA virus with a single-stranded positive-sense RNA and four major structural proteins: spike glycoprotein (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The spike was composed of the S1 subunit and S2 subunit.

| The outbreak of SARS-CoV-2 variants
The naming of the five SARS-CoV-2 VOCs follows a chronological order. The Alpha variant, also known as B.1. 1.7 or 501Y.V1, was first reported in September 2020 in southeast England and then spread rapidly to be the dominant variant in the United Kingdom (UK). [25][26][27] Several months later, the Alpha variant spread across more than 50 countries, indicating its higher infectivity than the ancestor. [28][29][30] The Alpha variant contains two deletions at amino acids 69/70 and 144/145 in the NTD, one substitution (N501Y) in the RBD, and three other substitutions (A570D, The life cycle of SARS-CoV-2. SARS-CoV-2 wild type or previous variants mainly infect host cells with high TMPRSS2 expression through the plasma membrane route. Omicron and its sublineages can enter cells with low TMPRSS2 expression via the endosomal entry pathway. Then, the virus shoots its RNA genome into the host cell, and related viral proteins are synthesized. The newly synthesized proteins are processed and packaged in the Golgi apparatus to assemble into a complete virus particle. Next, the spike is cut by furin to prepare for virus release through exocytosis. N: nucleocapsid protein, M: membrane protein, E: envelope protein, S: spike protein. SARS-CoV-2, severe acute respiratory syndrome coronavirus-2. HE ET AL. | 935 D614G, and P681H), which substantially increase the affinity of ACE2 for RBD by sevenfold. 31,32 The Beta variant, also known as B.1.351, 501Y.V2, or 20H, has nine mutations in the S1 subunit, including four substitutions and one deletion (Δ242-Δ244) in the NTD, three amino acid mutations at K417N, E484K, N501Y in RBD, and a substitution at D614G. 31,33 At that time, the Gamma variant (also known as P.1, B.1.1.248, 501Y.V3, or Brazil variant) spread in Manaus, Brazil. The prevalence of the Gamma variant raises significant concerns because it carries 12 mutations in the spike, including K417T, E484K, and N501Y. 34 Later, in the early year of 2021, considerable attention was drawn to the emergence of the Delta variant, which first appeared in India in October 2020 and has spread into over 21 countries. 35,36 The Delta variant (also named B.1.617.2), a sublineage of B.1.617, harbors T19R, G142D, one deletion (Δ156-Δ157), R158G, L452R, T478K, D614G, P681R, and D950N substitutions. 37,38 It has replaced the pre-existing lineages and induced intense transmission in India, resulting in 17 million COVID-19 cases, including reinfections from March to May of 2021. 37 Since a traveler returned to the United Kingdom from India, a rapidly increasing proportion of Delta infections was observed. 39 40 It also shares several mutations with other VOCs (Figure 3A), and a more detailed description of the five VOCs is shown in Table 1 Figure 3B). In the first few months of the Omicron wave, BA.1 was dominant, followed by BA.2 replaced BA.1. Presently, BA.5 continues to be dominant globally, which indicates faster and more frequent mutations of SARS-CoV-2.

| Clinical features of SARS-CoV-2 variant-induced COVID-19
The reproductive numbers (R0) of the SARS-CoV-2 ancestor and Alpha variant are approximately 2.3-5.7 and 5-8, respectively. 53 Delta has an R0 of 3.2-8, which is about 55%, 60%, and 34% higher than the Alpha, Beta and Gamma variants, respectively. 37,38,54,55 Omicron's R0 is 3.19-fold more elevated than that of Delta, 56 indicating that the Omicron variant has higher transmissibility than the previous VOCs. The enhanced transmissibility of Omicron makes it the dominant variant worldwide within a short time. Correspondingly, a surge in confirmed COVID-19 cases was observed after the Omicron prevalence. According to the data from the WHO, over 300 million new cases were reported from January to September 2022, which was more than the total number of COVID-19 patients from 2019 to the end of 2021 (https://apps.who.int/iris/discover?rpp=10&etal=0&query=COVID-19+weekly+epidemiological+update&group_by=none&page=2). Fortunately, the majority of Omicron infections displayed symptoms more akin to an upper respiratory tract infection rather than pneumonia. 57

Data from South
Africa and England demonstrated that the proportion of Omicron-infected patients requiring hospitalization was 4.9%. 58 This is remarkably lower than the hospitalization rates of Beta (13.7%) and Delta (18.9%). 58 In addition, the risk of severe disease or death is relatively low in hospitalized patients with Omicron. In Canada, Omicron infected cases have a 65% lower risk of hospitalization or death than Delta infections. 59 In vitro studies revealed that Omicron replicated faster than Alpha, Beta, and Delta. 60 Interestingly, the replication competence of Omicron in human alveolar cells was significantly lower than that of Delta, 60 which might explain the reduced severity of Omicron.

| Spike mutation characteristics of the SARS-CoV-2 variants
The binding affinity between the spike and receptor ACE2 on the host cells is one of the major factors determining the infectivity and transmissibility of SARS-CoV-2. 61 Studies have reported that there is a single salt bridge formed by Glu30 (ACE2)-Lys417 (RBD) with 11 hydrogen bonds and 125 nonbonded contacts between ACE2 and wildtype RBD. 62 Mutations in Alpha, Beta, and Gamma variants notably enhance the affinity of ACE2 for RBD by approximately 7-fold for Alpha variant and 19-fold for Beta and Gamma variants, which might play a role in their increased transmissibility. 32 As shown in Figures 3 and 4, almost all prevalent SARS-CoV-2 variants, including the five VOCs, are based on the D614G substitution caused by an A-to-G nucleotide mutation. D614G substitution disrupts the interprotomer hydrogen bond with T859 in the adjacent protomer of the spike trimer, which allosterically shifts the RBD to more than one "up" conformation in the spike trimer and results in an ACE2-binding fusion-competent state to increase transmissibility and infectivity. 63 D614 interacted with K848 in S2 rather than T859 to stabilize the closed spike conformation. 14,64,65 Nevertheless, the D614G substitution could enhance SARS-CoV-2 replication by up to 13.9-fold in a competition assay, 63,66 and produced higher viral loads than D614 viruses in the infected human upper respiratory tract. [66][67][68][69] The enhanced flexibility in the conformation of the G614 spike may explain why the present variants have advantages and exert higher infectivity. Plante et al. 63 verified that mutagenesis of D614 to G614 in the spike strengthened the stability of SARS-CoV-2 and increased the genomic RNA/plaque-forming unit (PFU) ratios up to 1.9-to 3.0-fold in the human lung epithelial cell line Calu-3. Moreover, the variant harboring the D614G mutation began in Europe and became the globally dominant form of the SARS-CoV-2 variant over just 1 month, which is consistent with the contribution of this mutation to the increased transmissibility. 63 Approximately 50% increased transmission. 29,30 No impact on susceptibility to EUA mAb treatments. 41 Minimal effects on neutralization by convalescent and postvaccination sera. 31 Reduced neutralization by convalescent and postvaccination sera. 51,52 Note: Data were collected from WHO and PubMed.
In addition to the D614G mutation, the N501Y, E484K, L452R, T478K, P681R, and D950N substitutions in the spike were also correlated with increased infectivity and transmissibility. The N501Y substitution was found in Alpha, Beta, Gamma, and Omicron variants. It was identified as an adaptive mutation potentially associated with the serial passage of the clinically isolated SARS-CoV-2 in the respiratory tract of mice. 70 N501Y is located within the RBD and is a key residue to enhance the binding affinity of SARS-CoV-2 RBD and ACE2. 71 N501Y introduces a favorable π-π interaction between Y501 and Y41 of ACE2, and forms an extra H bond with K353 of ACE2, which could enhance the RBD-ACE2 binding affinity. 62,[70][71][72][73] In addition, Leung et al. 74 found that the transmissibility of 501Y lineage without amino acid deletion Δ69/Δ70 was 10% (6%-13%) higher than that of the 501N lineage.
Conversely, this rate elevated dramatically up to 75% (70%-80%) when the 501Y lineage exhibited the amino acid deletion Δ69/Δ70. 74 The Alpha variant has both mutations of N501Y and deletion Δ69/Δ70, the enhanced transmissibility of which resulted in a significantly increased percentage of the infected cases from 0.1% in early October 2020 to 91.8% in late January 2021 in the United Kingdom according to the data on GISAID (www.gisaid. org). 74 E484 is located within the RBD and forms weak contact with K31 of ACE2. 71 E484K mutation appears in Beta and Gamma variants. This mutation generates a new site for ACE2 binding via amino acid 75, which leads to a stronger binding interaction, even stronger than that of the N501Y mutation. 75,76 As analyzed by molecular interaction, the substitution E484K contributes two salt bridges, 12 hydrogen bonds, and 144 nonbonded contacts between ACE2 and RBD complex. 62 The extra salt bridge formed by Glu35 (ACE2)-Lys484 (RBD with E484K mutation) and an additional hydrogen bond formulated by the substituted residue Lys484 enhanced the binding of the ACE2-E484K mutant RBD complex. 62,75 The combination of N501Y and E484K enhanced the stability of the complex with ACE2 compared with that of a single mutant. 33,77 Moreover, although the K417N or K417T mutation is expected to reduce affinity to ACE2, molecular docking investigation on the variants of K417N-E484K-N501Y (reported in Beta variant) and K417T-E484K-N501Y (reported in Gamma variant) with triple mutations showed that these variants shared higher electrostatic energy interactions for an increased affinity to the receptor and substantially exhibited higher infectivity than the prototype RBD. 33,62,75 Therefore, the double substitutions of E484K and N501Y might be critical for the fast transmission and high infectivity of SARS-CoV-2 variants, such as Beta and Gamma variants, which are mediated by salt bridge formation and hydrogen bonding interactions to alter their binding ability.
Unlike Alpha, Beta, and Gamma variants, the Delta RBD exhibits a comparable binding affinity to ACE2 compared to the prototype RBD, much weaker than that of the Alpha variant. 78 As shown in Figure 4, the critical mutation N501Y that enhances the binding of RBD with ACE2 does not exist in Delta. L452R, T478K, P681R, and D950N substitutions appear in Delta but not in Alpha and Beta variants. Studies have shown that the L452R and T478K substitutions located on the Delta RBD do not participate in the RBD-ACE2 interaction, 78,79 but the L452R substitution is a significant adaptive mutation to SARS-CoV-2. 80,81 According to GISAID, over 90% of the isolates reported between December 2020 and February 2021 were L452R-carrying lineages. 80 The L452 residue contributes to forming a hydrophobic patch on the surface of the RBD with L492 and F490. 47,81 The L452R substitution abolishes hydrophobic contact and results in increased spike stability, viral infectivity and viral fusogenicity. 61,80 P681 is located at the "PRRAR" furin cleavage sites. P681R mutation significantly improves the efficacy of spike cleavage by furin, promoting syncytium formation and increasing infectivity. 50,82 D950N is located in the S2 subunit and could enhance the fusogenicity of the spike. 22,79 Although the Delta RBD-ACE2 binding affinity is weaker than Alpha RBD-ACE2 binding, the Delta variant is 60% more infectious than the Alpha variant. 37,38,79 Studies have shown that Delta spike can enter low ACE2-expression cells more efficiently than other variants. 79 Besides, the Delta variant infects Vero cells with the highest efficiency among the five VOCs. 50,71 These data suggest that efficient fusion may account for the high transmissibility of Delta.
Specifically, for the Omicron variant, the mutations K417N, S477N, E484A, Q493R, Q498R, N501Y, and Y505H are located on the RBD-ACE2 binding interface ( Figure 4). 83 The results of the binding affinity of the Omicron RBD to ACE2 are slightly different. Some studies reported that the binding affinity of Omicron RBD to ACE2 is approximately 2.4-fold higher than that of the prototype RBD. 83,84 However, Han et al. 78 analyzed the binding properties between ACE2 and VOC RBDs. They found that the binding affinity between Omicron RBD and hACE2 is comparable to that of the prototype RBD. As the most mutated variant, Omicron harbors a large number of mutations, including a unique cluster of mutations in the RBD ( Figure 4). In addition to N501Y, T478K, and substitutions may stabilize the "up" conformation of the RBD that promotes interactions with ACE2. 85 S477N substitution forms two new H bonds with S19 of ACE2, strengthening the ACE2-RBD interaction. 78 The deep mutational scanning (DMS) results have shown that the Q498R mutation affects RBD stability and binding affinity to ACE2. 86 The combination of Q498R and N501Y substitutions could enhance the binding affinity of the RBD to ACE2 approximately fourfold above that of N501Y alone. 87 Interestingly, some RBD mutations decrease the binding affinity of Omicron RBD to ACE2. E484A substitution makes the side chain too short to contact ACE2, resulting in reduced binding affinity. 71 H505 forms fewer interactions with ACE2 than Y505. 78 In summary, the S477N, Q498R, and N501Y mutations in the Omicron RBD remarkably enhance the binding affinity of Omicron RBD with ACE2; however, the K417N, E484A, and Y505H substitutions decrease their binding affinity, which might be due to compensation for both transmissibility and immune escape.

| THE EFFECTS OF SPIKE MUTATIONS ON CURRENT THERAPEUTICS
The spike of SARS-CoV-2 plays a critical role in mediating virus entry into host cells. Therefore, the majority of therapeutic recombinant neutralizing mAbs against SARS-CoV-2 and most vaccine candidates utilize the spike of SARS-CoV-2 as a target. 88 However, the exposure of spike to selective pressure from the host immune system might drive antigenic drift and evolution to increased adaptability. SARS-CoV-2 might develop resistance to neutralizing mAbs under these pressures, resulting in a significant reduction in therapeutic effects, including the host immune response or drug treatment. 89 4.1 | Effects of mutations on the neutralization capability of sera from convalescent Convalescent serum has been used to prevent and treat infectious diseases for decades. 90 In 1893, therapeutic serum was first used in Berlin, where 220 patients with diphtheria were treated with standardized serum by Ehrlich. 91 Transfusing serum collected from recovered patients with an infectious disease could transfer neutralizing mAbs and confer passive immunity to the recipients. 91 Therefore, convalescent serum has been used to treat a wide range of viral infections, including parvovirus B19, H1N1, Ebola and MERS-CoV, and SARS-CoV. [92][93][94][95] At the beginning of the SARS-CoV-2 pandemic, few evidence-based treatments were available for COVID-19 patients, which made convalescent plasma therapy a promising strategy. 96 An extensive analysis of antibody responses in 647 SARS-CoV-2-infected individuals revealed that more than 90% of the serum neutralizing activity targets RBD. 97 showed about 2.5-fold and 7.7-fold reductions in antibodies against Delta and Omicron pseudoviruses, respectively, compared to Alpha pseudovirus. 108 Half of the serum samples from COVID-19 convalescent patients lost neutralizing activity against Delta and Omicron pseudoviruses. 108 This can explain the phenomenon that individuals exposed and recovered from previous SARS-CoV-2 variants would be reinfected by Delta or Omicron variants. The results of a case-control design showed that, compared with uninfected and unvaccinated subjects, the previous infection induced different protection rates against reinfection: approximately 90% against Alpha, 86% against Beta, 92% against Delta, but only a 56% protection rate against reinfection with Omicron, 109 implying the high immune escape of Omicron.

| Effect of mutations on monoclonal antibody treatment
Pharmaceutical-grade neutralizing mAbs emerged as the first SARS-CoV-2-specific treatment in clinical therapy.
Spike is a key target for the treatment of COVID-19 because of its essential role in host binding and viral entry. 21 To date, most licensed or developing anti-COVID-19 neutralizing mAbs target the SARS-CoV-2 spike to inhibit the interaction between SARS-CoV-2 and ACE2. Among them, the majority of mAbs target the RBD, with a few of them targeting the NTD. Therefore, mutations in the spike, especially in the RBD, could affect the treatment of  51 Additionally, variants carrying E484K and K417N/T substitutions escape the neutralization of Etesevimab. 112 The Delta variant is resistant to some anti-NTD and anti-RBD mAbs such as Bamlanivimab, which is consistent with previous results demonstrating that the L452R substitution is an escape mutation for Bamlanivimab. 39,112 But Etesevimab, Casirivimab and Imdevimab preserve neutralization to the Delta variant. 39 Mutants with S494P and E484Q result in approximately five-and nine-fold lower susceptibility to Casirivimab in neutralization assays. 110,111 It has also been reported that two RBD-binding antibodies (F61 and HE ET AL. | 941 H121) of 12 high-affinity antibodies generated from convalescent donors show high neutralization capability against Alpha and Beta variants, whereas three S2-target antibodies display abolished neutralization ability. 113 Currently, although studies on how SARS-CoV-2 mutations induce resistance to neutralizing antibodies remain limited, there are still some experiences to minimize the risk of escape mutations. The design of monoclonal antibodies that target the highly conserved regions of the spike might be an effective strategy as the highly conserved epitopes usually overlap with regions critical for viral function. Thus, mutations in this region could impair viral fitness and are more likely to be eliminated in the viral population. 114  studies. 118 Furthermore, using a cocktail of neutralizing mAbs rather than monotherapy has been found to prevent the generation of escape mutants in passaging experiments in vitro. 119 This strategy has been used for the treatment or prophylaxis of COVID-19, such as cocktails of Casirivimab/Imdevimab and Bamlanivimab/ Etesevimab. 89 Haslwanter et al. 120 also found that the combination of RBD and NTD neutralizing mAbs alleviated the occurrence of neutralization-escape mutants in vitro. Collectively, the effects of mutations on the neutralization activity of monoclonal neutralizing antibodies are still not comprehensive, and the susceptibility of emerging SARS-CoV-2 variants to neutralizing mAbs may need to be updated promptly. Furthermore, the safety and efficacy of different combinations of neutralizing mAbs should be investigated, which may better prevent treatment-emergent resistance.

| Impaired vaccine effectiveness toward SARS-CoV-2 variants
The SARS-CoV-2 virus shares extensive sequence, structural and functional homologies with SARS-CoV and MERS-CoV. 122 Studies revealed that anti-SARS-CoV spike antibodies possessed the ability to inhibit the binding of SARS-CoV-2 to ACE2. 123  Except for the inactivated vaccine, most vaccine candidates are constituted by variations in antigen fragments from the spike. In brief, the antigen fragments may contain full-length spike, different lengths of RBD, or even synthesized peptides as engineered multiepitope to induce high neutralizing antibodies to neutralize viruses. [124][125][126] Therefore, mutations in the spike of several SARS-CoV-2 variants impaired the protective efficacy of COVID-19 vaccines that were developed based on the spike.
Several COVID-19 vaccines have been approved for use and exert adequate protection when wild-type SARS- illness. 127,128 The adenovirus vector vaccine ChAdOx1 (AZD1222) also shows 90% efficacy if vaccinated at a low dose followed by a standard dose. 129 Besides, the viral vectored and inactivated virus vaccines also exhibited effective protection against COVID-19. [129][130][131] However, these vaccine candidates are designed based on wild-type SARS-CoV-2, and the vaccine effectiveness against SARS-CoV-2 variants is of particular concern.
It has been reported that sera from individuals who have received one dose of Pfizer (BNT162b2) or AstraZeneca (ChAdOx1) vaccine could barely neutralize the Delta variant. 39 After being vaccinated with two doses of BNT162b2, 3% (6/159) and 5% (9/159) of participants' sera lack neutralizing activity against Delta and Beta variants, respectively. 104 Consistent with these results, complete or partial loss of neutralization against the Beta variant was observed in most of the serum samples from individuals who received two doses of inactivated-virus vaccines. 132 The neutralizing activity of sera from recipients of a single dose of Ad26.COV2.S vaccine against the Beta, Gamma and Delta variants were reduced by 5.0-, 3.3-, and 1.6-fold as compared to the ancestor, respectively. 133 The effectiveness of the COVID-19 vaccine against Delta and Omicron variant transmission is a great concern.
As shown in Table 2, 134-139 the effectiveness of BNT162b2 or mRNA-1273 vaccines in preventing contact transmission of Delta is 9%-38%. Even if fully vaccinated, the effectiveness is 27%-65%. 134,136 The effectiveness of the ChAdOx1 vaccine against Delta transmission is 36%~42%. 135,137 Notably, the protection of full vaccination against Omicron infection of close contacts was significantly reduced compared to that of Delta. The effectiveness of vaccination with COVID-19 mRNA vaccines against contact transmission decreased to 22.5%. 138 Another study reported that vaccination or prior infection showed little protection against Omicron transmission to household contacts. 139 These results indicate that physical protection, such as wearing a mask, is still necessary.
Here we also summarized the vaccine effectiveness of current authorized COVID-19 vaccines against symptomatic infection, hospitalization and death induced by SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants (Table 3). 134 showed that boosting with mRNA-1273 had higher neutralizing antibody titers against Omicron than boosting with mRNA-Omicron. 175 Consistent with these data, our previous results revealed that recombinant protein vaccine based on Omicron S1 induced decreased neutralizing activity against Omicron-included variants.  Meanwhile, the Omicron S1 recombinant protein vaccine elicited a significantly weaker T-cell response compared to the prototype S1-based recombinant protein vaccine candidate, 176 which indicates that the Omicron S1 subunit may not be an appropriate selection for developing specific vaccines against Omicronincluded SARS-CoV-2 variants. Some other studies found that mRNA vaccines containing several pivotal mutations of the Omicron and Delta spike could elicit high neutralizing antibodies against the Omicron variant, 177 suggesting the consideration of the essential mutations of SARS-CoV-2 virus spike, not limited to the currently prevalent VOCs. Taken together, the ongoing pandemic of SARS-CoV-2 variants calls for continuous surveillance of vaccine efficacy against emerging mutations, which is an extremely important factor for vaccine optimization.

| Spike mutations and immune evasion
Numerous studies have been carried out to elaborate on the underlying mechanisms for the impaired protective effects of neutralizing mAbs and COVID-19 vaccines against SARS-CoV-2 variants. In the early days, the D614G mutation raised concerns since all the following prevalent variants were based on it. Fortunately, several subsequent studies have revealed that the D614G substitution had a modest effect on the neutralization capability of sera from COVID-19 convalescent or vaccinated individuals. Plante et al. 63 reported that sera from hamsters infected with D614 virus exert higher neutralization titers against G614 virus than D614 virus. D614G mutation presented approximately 1.7-fold higher susceptibility to serum neutralization and was unlikely to reduce the vaccine efficacy for protection against COVID-19 in clinical trials. In addition to D614G, N501Y also has little effect on vaccine efficacy. 72,178 High ambiguity-driven protein-protein docking (HADDOCK) analysis found that the docking score of Y501 is similar to that of N501. 178 Additionally, N501Y possesses a similar interaction pattern with ACE2 to wild-type SARS-CoV-2, indicating that the already developed vaccines could work against the variants carrying N501Y. In a neutralizing study, Shi et al. 178 found that sera from 20 participants vaccinated with the mRNA vaccine BNT162b2 had neutralizing titers equivalent to the N501 and Y501 viruses. These data suggest that D614G and N501Y are not the key culprits of impaired vaccine efficacy in clinical trials and indicate the possibility of other mutations in reducing vaccine efficacy.
Studies have revealed that residues at sites 417, 452, 477, 484, and 493 of the RBD are critical for the binding of neutralizing mAbs. 33,62,83 DMS further identified E484 as the most remarkable one in RBD mutation-mediated recognition and neutralization by convalescent sera. 179,180 Structurally, residue E484 forms H-bonds with N33 and Y34 of the light chain as well as a salt bridge, and the H-bond interacts with the R112 side chain of the light chain. 75 Mutations at E484 (E484K, E484A, or E484Q) were correlated with the most significant drop in neutralization capability. 47,75,[181][182][183] The SARS-CoV-2 variants carrying the E484K or E484A mutation, including Beta, Gamma, P.2, Omicron and their sublineages, are associated with resistance to neutralizing mAbs and impaired vaccine efficacy. 77,173,184,185 Xie et al. 186 reported that combined mutations of E484K, N501Y and D614G led to even lower neutralization titers compared with only N501Y, Δ69/70, N501Y, or D614G mutations. Introducing an additional E484K mutation in the Alpha variant background led to a further decline in neutralizing activity by the BNT162b2 mRNA vaccine-elicited antibodies compared to the Alpha variant alone. 173 Our previous study also found that sera from mice immunized with prototype spike exhibited a significantly reduced neutralizing effect against pseudovirus with E484K mutation. 187 Consistent with these findings, several studies have demonstrated that the Beta variant decreases the neutralization activity of antibodies elicited by non-Beta variant infection or vaccination. 33,46,47,107 Similar results were also observed in the cases of Gamma and P.2 variants. 119,179,188 K417N substitution broke the salt bridge with D104 from HCDR3 of class I antibodies, resulting in the reduced binding ability to the Omicron spike. 83 SARS-CoV-2 variants harboring a combination of K417N, E484K, and N501Y could escape most RBD-targeting antibodies. 189 Interestingly, the Beta, Gamma, and Omicron variants share substitutions at sites 417 and 484, while the Omicron variant has additional S477N and Q493R mutations. S477N and Q493R mutations could reduce the binding affinity of neutralizing antibodies to the Omicron RBD, leading to the loss of neutralization. 190 Q493R mutation abolishes the hydrogen bonds due to the long side chain of arginine and therefore affects the binding of LY-CoV555 and polyclonal sera to the Omicron spike. 191 These mutations confer greater neutralizing antibody resistance on Omicron than previous SARS-CoV-2 variants.
The L452R mutation appears in the Delta, Omicron BA.4 and BA.5 variants. L452R mutation abolishes the neutralizing potential of 14 out of 35 RBD-specific mAbs. 121 The intermolecular interaction results revealed that the L452R mutation breaks the hydrophobicity with both residues I103 and V105 of the antibody's heavy chain as well as the binding force of the H-bond and electrostatic interaction with R112. 81 Furthermore, the L452R mutation changes the conformation of epitope peptides with either reduced binding affinity or suboptimal recognition by the T cell receptor, which contributes to the escape of the L452-carrying variants from human leukocyte antigen-restricted cellular immunity. 192 (Table 4). 164,[195][196][197][198][199][200][201][202] These results showed that the vaccine effectiveness began to decline 6 months after the completion of vaccination. 164,[195][196][197]201 This decline occurs more frequently in older people. 198 Moreover, vaccine efficacy varies among people of different ages.
Several studies have reported that the efficacy of COVID-19 vaccines in older adults is lower than that of the young group at a similar interval time after full vaccination. 149,151,156 The vaccine effectiveness against COVID-19 hospitalization and death is about 94% and 96%, respectively, in 65-79-year-old subjects, but this effectiveness for the elderly over 80 years decreases by approximately15%. 203 This may be because ageassociated changes in the function of T cells, B cells, natural killer cells and antigen-presenting cells lead to a decrease in cellular and humoral immune responses. 204,205 With the decline in vaccine effectiveness over time and the lower immune response of elderly individuals, different strategies for a booster dose of the COVID-19 vaccine to tackle the COVID-19 pandemic have been carried out worldwide in recent months. Several studies have shown that the booster dose contributes to a decrease in infection and severe disease, even in the elderly population. 206,207 As shown in Table 5, 207-217 the vaccine effectiveness of a BNT162b2 booster dose against Delta variant infection is over 80%, and the vaccine effectiveness against severe disease or death is about 95%. [207][208][209][210][211][212] The booster dose of Ad26.COV2.S vaccines also showed effective protection against Alpha, Delta and Mu variants. 216 It is noteworthy that Omicron drastically escapes vaccine-induced immunity. The  Notably, our team has been devoted to exploring the effect of a bivalent vaccine based on the S1 subunit of SARS-CoV-2. 187 We found that sera from wild-type S1 (S1-WT)-or mutant S1 (S1-Mut)-immunized mice could induce strong protective immunity to neutralize wild-type or mutant pseudoviruses, respectively. In the presence of the AdS03 adjuvant, the bivalent vaccine consisting of S1-WT and S1-Mut recombinant proteins exhibited ideal neutralization properties against both wild-type and variants of SARS-CoV-2, including Beta and Gamma variants.

| Exploration of combination vaccines as universal coronavirus vaccines
The combined vaccines contain two or more antigens that could be produced in different platforms and mixed subsequently by the manufacturer in advance or immediately before administration. 231 These vaccines may possess more advantages over different vaccine platform technologies (e.g., recombinant protein, mRNA vaccine, virus vector vaccine, etc.), such as higher vaccine efficacy, single inoculation, improved vaccine coverage, lower cost, timely design and manufacture toward a new variant. 231,232 The subunit vaccines deliver antigens as purified recombinant proteins, which exhibit advantages of high safety, large-scale production and minimized host immunopotentiation. 233,234 However, an obvious disadvantage of subunit vaccines is the low antigenicity, which requires the addition of adjuvants and repeated vaccination doses to increase their immunogenicity. 235 Nucleic acid vaccines and viral vector-based vaccines could elicit strong cellular immunity to deal with COVID-19. 236,237 Combining subunit vaccines with them may be an effective strategy to activate both arms of the immune system. Li et al. 221 vaccinated nonhuman primates with a DNA vaccine encoding the spike and a recombinant S1 subunit vaccine of SARS-CoV-2. They found that the combined vaccine elicited high levels of neutralizing mAbs as well as T cell immune responses to protect rhesus macaques against the intratracheal challenge of SARS-CoV-2. Moreover, the protective efficacy of the combined vaccine was higher than that of DNAbased or protein-based vaccines alone. 221 A combination of DNA vaccine and protein vaccine may be an effective method to induce durably immune response since the DNA vaccines facilitate the development of germinal center B cells, which is essential for high-affinity memory B cell responses. 221,238 The design of the antigens could be manipulated according to the SARS-CoV-2 variants. The combined vaccines might be potential universal vaccines to evoke both humoral and cellular immune responses ( Figure 6). The combination approach is not limited to subunit and DNA vaccines. Other types of vaccines may also be used to prepare combined vaccines to maximize the advantages of two or more types of vaccine platforms while overcoming their potential drawbacks. Taken together, the combined vaccine approach may be an appealing and promising strategy against all circulating variants. Omicron sequence-based vaccines are not inspiring. In contrast, the broad neutralizing ability generated by the Delta sequence-specific vaccine suggests the potential for developing Delta-based vaccines, which indicates that a large number of mutations in spike may change its epitope and reduce its immunogenicity. Some important mutations, such as E484K, L452R, S477N, and F486V, should be carefully considered when designing spike-based COVID-19 vaccines in the future, even not against Delta or Omicron variants. In the future, understanding the precise mechanism of Omicron's immune evasion and host immune responses and discovering critical viral epitopes will also contribute to developing better vaccines and neutralizing mAbs against SARS-CoV-2 variants. In

CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.