The spike protein is 1273 amino acids long with a molecular weight of 180-200 kDa. It is a type 1 fusion membrane protein, consisting of an extracellular N-terminus domain (NTD), an RBD, a cleavage site, a fusion peptide, two heptad repeats, a transmembrane domain, and a cytosolic C-terminus domain ( Figure 2A ) (29). The extracellular region of the spike protein is highly glycosylated. The S protein has two subunits, S₁ (14-685 aa residues) and S₂ (686-1273 aa residues), that dissociate via a host furin protease, at its cleavage site (30). In the infection cycle, the S protein is responsible for binding to the host angiotensin-converting enzyme 2 receptor (ACE2), and mediates membrane fusion which induces viral entry (26, 29, 30).

The variability observed in the SARS-CoV-2 variants is dominated by mutations in the RBD region of the S₁ subunit (26). The RBD region reflects evolutionary relativeness among coronaviruses between humans, cats, and swine species, yet this region is the least conserved (26, 29, 30). Mutations in this region directly impact the binding affinity of the virus towards ACE2 and alters the interaction between the virus and target cell (31). RBD mutations are associated with changes in viral virulence, therefore some variants are more infectious than others. Mutations in the S₂ subunit have also been hypothesized to influence infectivity of SARS-CoV-2. The S₂ region relies on the efficiency of furin and serine proteases to access cleavage sites to mediates the fusion of viral and host membranes (30). Therefore, mutations in S₂ evolve to increase the number of furin and serine cleavage sites and/or increase susceptibility of cleavage events (31). The presence of multiple cleavage site in SARS-CoV-2 has significant implications on infectivity as the absence of these sites lead to a less infectious virus, such as SARS-CoV-1. Mutation in the Spike protein and their effects on virulence are discussed below.

In the highly mutating spike protein, the N501Y mutation, found in the B.1.1.7 (UK), B.1.128.1 (South Africa), B.1.351(Brazil), P.1 (Brazil) and B.1.1.529 (South Africa) variants, increased binding affinity of the RBD to ACE2 receptor (32–34). The amino acid substitution is located within the RBD region of the S₁ subunit and associated with increased infection rates. This mutation is one of the earliest changes detected in emerging SARS-CoV-2 variants mentioned. Similarly, the K417N substitution, found in the B.1.351 (South Africa, Beta variant), P.1 (Brazil), B.1.617 (India), and B.1.1.529 (South Africa) variants, significantly increased binding affinity of spike to ACE2 (35–38). In addition, K417N contributed to immune evasion by reducing antibody binding affinities against RBD (38). The E484K mutation was found in the B.1.351 (South Africa, Beta variant), P.1 (Brazil), P.2 (Zeta), B.1.525 (Eta), and B.1.526 (Iota) variants (36, 37). The amino acid change alters the electrostatic interactions between antibodies and RBD, decreasing the neutralization capability of SARS-CoV-2 polyclonal serum (39–41). L452R and L18F mutations were associated with similar effects as K417N, in evading the host humoral response against SARS-CoV-2 (39–41).

Additionally, the spike protein has mutations that influence the S protein dissociation and cleaving processes. For example, A570D was first found in the B.1.1.7 (UK) variant that induced a conformational change, along with D614G, that destabilizes the S protein (42, 43). Another mutation, S982A, found in the B.1.1.7(UK) and P.3 (Japan/Philippines) variants prevented the formation of hydrogen bonds between S₁ and S₂ subunits, increasing the likeliness of these subunits to dissociate (44). Mutations directly involved in the furin cleaving process were characterized by P681H and D614G changes and associated with higher infectivity and replication rates of SARS-CoV-2 (43, 45). All in all, the spike is a highly variable protein that significantly impacts SARS-CoV-2’s ability to infect target cells (28). The 1^st generation COVID-19 vaccines are spike-based which have been effective in mitigating COVID-19 associated pathologies, hospitalizations, and deaths. Generally, spike-based immunity has been defined as neutralizing as it prevents the virus from entering targeted host cells. Evidently, during the pandemic, the spike protein has acquired many mutations ( Figure 2B ), and this results in the decline of vaccine-induced immunity. For future vaccines and longer-lasting immunity, it may be important to consider other proteins and antigen targets that are conserved and have lower tendencies to acquire mutations.

The envelope protein is ~75 amino acid residues long with a molecular weight of 8-12 KDa, consisting of a hydrophilic extracellular N-terminus, followed by a hydrophobic transmembrane domain, and cytoplasmic C-terminus (46). The envelope protein is responsible for the activation of inflammasomes, and the budding and release of the viral progeny (47). In addition to these functions, the envelope protein forms a pentameric ion channel with low ion selectivity, called a viroporin (47, 48). Many animal studies have suggested that the viroporin plays an important role in virulence and have shown that its ion channel activity is directly proportional to SARS-CoV-2 pathogenesis (47–49). In mouse models, knocking out the envelope gene was found to have no impact on SARS-CoV-2 viral replication, but it reduced edema (47, 50). Many animal studies, where the virus lacks viroporin activity, show reduced IL-1β, TNF-α and IL-6 production in the lungs (47). It is suggested that viroporins and their ion channel activity are linked to the development of cytokine storm and acute respiratory distress syndrome (ARDS).

The envelope protein is a highly conserved protein among various members of coronaviruses, therefore, very few mutations are observed in the E protein (46). Surveying all the SARS-CoV-2 variants, B.1.351 (South Africa, Beta variant) had a P71L mutation in the envelope protein that associated with disease severity and death rate (51). The low variability of the envelope protein makes it an attractive target as a vaccine and therapeutic target, as many circulating viral variants would be vulnerable. Also, the ion channel activity associated with pathogenesis makes the protein immunogenic and potentially capable of inducing a robust immune response. A conserved, immunogenic protein, like the E protein, can be a potential target for immunotherapeutics or vaccine candidates.

The membrane protein is a glycoprotein, 222 amino acid residues long, and the most translated protein (22, 52). The membrane protein is a self-formulating protein that interacts with all structural proteins (S, E and N) to form virus-like particles (VLP) (46, 53). The membrane protein is involved in stabilizing the interaction between genomic mRNA and the N-protein complex to incorporate the SARS-CoV-2 genome into the virion (46). Protein-protein interactions between M and other structural proteins allow the trafficking of these proteins for assembly and release. Lastly, the membrane protein is involved in the budding and release of the newly synthesized virions.

Evolutionarily, the M protein has been conserved across Beta-coronaviruses providing a common viral structural protein for all viruses within the genus (54). Genomic sequencing reports have shown more than 97% homology between M sequences of SARS-CoV-2 variants sampled (55, 56). Troyano-Hernáez et al. surveyed 103,419 SARS-CoV-2 M sequences, and reported 99.99% conservation, with only 291aa changes found (55). Out of 291 aa changes, only two mutations were presented in significantly higher frequencies: D3G (0.7%, in 724 sequences) and T175M (1%, in 1026 sequences). The D3G mutation first emerged out of Africa and South Africa, and the T175M was discovered in Europe. Another study surveyed 5677 GenBank records and found that 5557 sequences had no mutations or synonymous mutations, while the other 120 sequences had non-synonymous mutations in the M protein (56). Furthermore, the study found 10 mutations (C64F, A69S, A69V, V70F, N113B, R158L, V170I, D190N, D209Y and S214I) in the M protein that are predicted to change secondary protein structure. All in all, the M protein has evolved and incorporated mutations, but it is largely conserved across Betacoronaviruses and SARS-CoV-2 variants. With the M protein being involved in integral parts of the infection cycle, such as virus assemble, protein trafficking, and dictating protein-protein interaction of SARS-CoV-2 structural proteins, any major changes can result in detrimental effects on the overall fitness of the virus. Vaccine and immunotherapeutic targeting the SARS-CoV-2 membrane protein can potentially induce immune responses that can cross-react with various SARS-CoV-2 variants and other Betacoronaviruses because of the high sequence homology in the M protein.

The nucleocapsid protein is 419 aa residues long (57). It consists of a N-(NTD) and C-terminus domain (CTD) connected by a serine/arginine linker region (LKR). The N protein helps package the viral RNA genome and ensures proper replication and viral assembly processes take place. The LKR allows the N protein to oligomerize with other N-proteins and exposes the RNA-binding domains, located within the NTD/CTD, to bind to the viral genome (58–60). In this process, serine/arginine amino acids will stabilize the interaction between the N protein and RNA genome forming a stable ribonucleoprotein complex, called a capsid (58). The role of the viral capsid is to protect the viral genome from environmental conditions and host immune responses. It also plays an important role in delivery of the genetic contents into targeted cells.

Among coronaviruses, the N protein has been largely conserved, except for certain regions. There is 90% sequence identity between the SARS-CoV-2 and SARS-CoV-1 N proteins (61). Within the N protein sequence, the LKR (182-247 aa residues) accounts for the majority of the mutations in the SARS-CoV-2 variants. These mutations are located around multiple, highly phosphorylated sites that influence N protein thermodynamic properties and have a major impact on the viral life cycle. The R203K mutation is located in the LKR region and was first discovered in the B.1.1.7 (UK) and P.1 (Brazil) variants (62). This mutation coincides with G204R and has been found to increase overall expression levels of the N protein and various sub-genomic RNA transcripts (63). Structurally, the 203/204 mutations increase entropy and intrinsic disorder causing changes to the N protein oligomerization process (62, 63). Next, the T205I mutation was first observed in the B.1.351(South Africa) variant (63). It is a highly phosphorylated site within the LKR. This mutation alters the activation of the N protein resulting in decreased fitness of the virus, however the T205I mutation was reported 43% of the time in B.1.351 variants. Lastly, the S235F mutation was reported in the B.1.1.7 (UK) variant (63). The S235F mutation is unique in that it is associated with immune evasion. The mutation alters a highly targeted B cell epitope, thereby decreasing the specificity of certain antibodies to mount a response against SARS-CoV-2 N protein (63). Immunity against the SARS-CoV-2 N protein is governed by CD4⁺ T cells, and they have been shown to cross-react with other endemic coronaviruses (28).

The B.1.1.7 variant was first discovered in the UK on September 20, 2020 (73). The virus consisted of several novel spike mutations, including 17 non-synonymous, six synonymous, and two deletions: ΔH69-V70, ΔY144, R203K, S235F, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H ( Figure 2B ) (34, 73). Three of the mutations in the S protein that were reported to be concerning due to an increase in viral transmission, pathogenicity, infectivity, and decreased susceptibility to neutralizing antibodies (34, 72–77). These mutations included the previously discussed N501Y and P681H mutations, and the ΔH69-V70 deletion (3, 34). The ΔH69-V70 deletion, located in the RBD, led to a conformational change in the S protein that evaded the host immune response (78). Additionally, a peer-reviewed study reported a 75% increase in transmissibility of the B.1.1.7 variant compared to the Wuhan-Hu-1 strain (74). Within three months, the B.1.1.7 variant was the predominant circulating strain in the UK and spreading across 114 countries (74–77). In terms of diagnosis, Public Health England reported that the B.1.1.7 mutations in the S gene alluded the RT-PCR detection assay—which amplifies the S gene to detect COVID-19 positivity in sick patients (79). In March 2021, Ontario’s COVID-19 Science Advisory Committee reported that 67% of COVID-19 cases were due to the B.1.1.7 variant (80, 81). These cases were associated with a 63% increase in hospitalization risk, 103% increase in intensive care admissions related to COVID-19 and 56% increase in mortalities (75, 81). With increased infectivity and transmissibility, the B.1.1.7 variant was reported to target younger age groups in Canada, between ages 20-30 y and up to 59 y (82).

The B.1.351 emerged from South Africa in October 2020 (83). From October 2020 to February 2021, the variant had spread to over 40 countries, including South Africa, Philippines, USA, Canada, and several EU countries. This lineage is defined by multiple mutations in Spike, including L18F, D80A, D215G, Δ241-243, K417N, E484K, N501Y, D614G and A701V ( Figure 2B ) (83, 84). Other mutations in N, E and orf1a proteins, include T205I, P71L and K1655N substitutions, respectively (83). In these B.1.351 variants, E484K and K417N mutations were concerning as they compromised neutralizing responses established from natural SARS-CoV-2 infections and/or vaccine-induced immune responses (85). Many studies reported that E484K, K417N and N501Y mutations can escape vaccine induced neutralizing antibody responses, as much as 3-5 folds (86, 87). In addition to immune evasion, the B.1.351 variant was 50% more infectious compared to its ancestor, as it dominated the second wave in South Africa, infecting more than 1.3 million people and causing 37,000 deaths (88, 89). Surges in B.1.351 cases demonstrated that the variant was more transmissible and infectious, but no evidence of increased severity was reported. In a molecular study, it was determined that sensitivity of the RT-PCR diagnostic test was not affected by the B.1.351 variant (90).

The P.1 lineage emerged out of Brazil in December 2020. It incorporated 17 non-synonymous mutations, 3 deletions, 4 synonymous mutations and one four-nucleotide insertion. Mutations located in the S protein were L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F ( Figure 2B ) (91). Other mutations included, P80R in the N protein, S1188L and K1795Q in orf1ab, G174C in orf3a, E92K in orf8, two-nucleotide deletions at positions 11288/9, deletion in the nsp6 protein (S106del, G107del, and F108del), and a four-nucleotide insertion at the orf8/N intergenic region (ins28263) (92–97). The triple mutation in Spike protein of K417T, E484K, and N501Y retained its function of evading both vaccine-induced and natural-infection acquired immunity. In its entirety, the spike mutations in the P.1 lineage associated with higher affinity for ACE2 receptor compared to non-P.1 lineages. This potentially led to a 2.5-fold increase in transmission compared to previously circulating variants and had a reinfection probability of 6.4% (93). From December 2020 to January 2021, the P.1 variant became the dominant variant in Manaus, Brazil as 85% of genotyped samples were found to be P.1 positive (93–95). The WHO announced the P.1 lineage as a VOC, due to its increased transmissibility and its propensity to re-infect individuals.

The B.1.617 lineage emerged out of India in December 2021 (98). The S protein lineage-defining mutations include, T19R, V70F, G142D, Δ156-157, R158G, A222V, W258L, K417N, L452R, T478K, D614G, P681R, and D950N ( Figure 2B ) (98). Researchers identified two potential mutations, L452R and E484Q, previously reported in B.1.1.7/B.1.351 and B.1.427/B.1.429 variants, respectively, as the reason for the exponential infectivity and immune evasion ability of B.1.617 variants (75, 99). Many viral fitness parameters were enhanced by the acquired mutations in the B.1.617 (Delta) variant. The onset of symptoms after exposure shortened from an average of 16.7 days to 10.3 days in unvaccinated individuals, indicating an enhanced replication ability of the B.1.617 lineage (99–102). A study in India reported that B.1.617 infections were associated with higher viral load and shedding as B.1.617 infections (n=47; Mean Ct: 16.5 cycles) has lower cycle thresholds compared to non-B.1.617 infections (n=22; Mean Ct: 19 cycles) (103). According to the Centre for Disease Control and Prevention (CDC), the reproduction number (R₀) was between 5-9 for the lineage; in other words, the variant could be more transmissible than other viruses, such as Smallpox, Influenza, MERS, and SARS (102, 104). Moreover, the P681R mutation, previously identified in B.1.1.7 variants, was reported to increase fusion activity via enhanced furin cleaving which could possibly be contributing to the increased infectivity and transmissibility seen in the B.1.617 variant (27–32). In relation to disease severity, one study showed infection with the B.1.617 variant in hamsters caused a higher viral load in the lungs, substantial lung lesions and weight loss, compared to other B.1 variants (105). The B.1.617 variants doubled the risk of hospitalizations compared to the B.1.1.7 variant, and patients infected with B.1.617 had severe disease outcomes and higher in-hospital morality rates (106, 107). Vaccine efficacy and neutralizing responses had significant fold-decreases with the introduction of the B.1.617 variant. The B.1.617 variant was the most devastating SARS-CoV-2 variant ever seen, in terms of its ability to replicate, transmit, infect, and inflict pathologies as well as mortality (107–109).

The first reports of B.1.1.529 emerged from South Africa, in November 2021 (98). Several studies confirmed that the B.1.1.529 spike protein had 32 mutations, including amino acid substitutions, insertions, and deletions (A67V, Δ69-70, T95I, Δ142-144, Y145D, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F) ( Figure 2B ) (98). The overwhelming number of mutations were predicted to result in the most virulent, infectious, transmissive, and pathogenic virus yet. However, the acquisition of these mutations reduced disease severity clinically (110). Hui et al. found that the B.1.1.529 variants had a lower replication rate compared to other SARS-CoV-2 variants (110). The paper suggests the reduced replication efficiency as a potential explanation for reduced disease severity in patients infected with B.1.1.529. In South Africa, hospital admissions lowered by 29%, compared to the first wave in 2020, highlighting less severe disease outcomes associated with the B.1.1.529 variant (111). The U.S. Food and Drug Administration and Public Health Ontario reported that Antigen Diagnostic Tests were less sensitive in detecting the B.1.1.529 variant as more false negative outcomes were registered (112, 113). Further analysis revealed a 9-nucleotide deletion resulting in failure to detect the SARS-CoV-2 B.1.1.529 variant (113, 114). According to Ontario’s Risk Assessment report, the B.1.1.529 variant dominated COVID cases with numbers increasing from 838 to 14449 within a two-week period in December 2021 (112). In South Africa, B.1.1.529 case numbers increased by 2.5 folds within November 2021 (75). The B.1.1.529 variants and its closely related sub-variants, BA.1 and BA.2, were reported to have alarming rates of transmission, making it a major concern for public health (115, 116).

The Oxford-AstraZeneca COVID-19 vaccine (ChAdOx1nCov-19) is a non-replicating viral vector vaccine (119). It utilizes a chimpanzee adenovirus vector encoding the wild-type SARS-CoV-2 S protein. The two-dose regimen of ChAdOx1nCov-19 induces a robust immune response composed of IgG₁/IgG₃ antibodies and T cell responses directed at Wuhan-Hu-1 spike protein. It also activates a T_H1 response via CD4⁺ T cell secretion of IFN-γ/TNF-α (117, 119). The immunity induced by ChAdOx1nCov-19 has been targeted towards the RBD as both antibody and T cells epitopes are specific for that region of spike. ChAdOx1nCov-19 vaccination trials conducted in the UK (11 636 participants) reported an overall vaccine efficacy rate of 70.4% (95.8% CI, 54.8-80.6) (120–122). Furthermore, 21 days after the 1^st dose, there were zero hospitalizations reported within the vaccinated group compared to 15 hospitalizations in the control group (121, 122). Their analysis showed that the two-dose regimen of ChAdOx1nCov-19 requires more than 14 days after the 2^nd dose to effectively protect against COVID-19 disease (122). ELISA-based virus neutralization assays revealed that the antibody response induced by ChAdOx1nCov-19 has preserved its effectiveness in protecting against severe disease outcomes of B.1.1.7 (Alpha) variants, however a marginal reduction was observed (119).

Following the B.1.1.7 (Alpha) wave, the B.1.351 (Beta) variant dominated the COVID-19 landscape in many countries around the world. Vaccine efficacy studies on ChAdOx1nCov-19 reported ineffectiveness against B.1.351 as antibody neutralizing ability was reduced against the D614G spike mutation (75, 119). Thus, the humoral response against SARS-CoV-2 is highly compromised by viral mutation. A single amino acid change has the ability make an antibody response ineffective as the study reported vaccine efficacies dropped to 10.4% against mild-to-moderate disease caused by B.1.351 variants. These results caused several countries to halt rollout plans for the AstraZeneca vaccine (75). A more positive experience occurred in Brazil where a two-dose regimen of AstraZeneca was implemented during the epidemic with the P.1 (Gamma) variant. Reports out of Brazil indicated that one dose of ChAdOx1nCov-19 had mitigating effects against COVID-19 as hospitalizations and deaths decreased by 55.1% (95% CI, 46.6-62.2) and 61.8% (95% CI, 48.9-71.4), respectively (123). Moreover, after two doses of ChAdOx1nCov-19, administered three months apart, the effectiveness against the P.1 variant was 77.9%, and hospitalizations and deaths were reduced to 87.6% (95% CI, 78.2-92.9) and 93.6% (95% CI, 81.9-97.7), respectively (123). Similarly, the AstraZeneca vaccine has shown effectiveness in reducing hospitalizations and risk of COVID infections against the B.1.617 (Delta) variant (75, 124). However, the vaccine effectiveness of ChAdOx1nCov-19 against B.1.617 is diminishing when compared to the B.1.1.7 (Alpha) variant (124–126). The effectiveness of the two-dose regimen was 74.5% (95% CI, 68.4 to 79.4) against the B.1.1.7 variant and 67.0% (95% CI, 61.3 to 71.8) against the B.1.617 variant (124). Compared to the Wuhan-Hu-1 SARS-CoV-2 strain, sera from AstraZeneca-vaccinated individuals have reduced neutralizing ability against B.1.617 variant by 9-fold (125). The overall trend observed as novel SARS-CoV-2 variants emerge is that the AstraZeneca vaccine is becoming less effective. Furthermore, vaccine effectiveness has significantly been compromised with the emergence of the B.1.1.529 (Omicron) variant (75). Andrews et al. found there was no protection after 15 weeks of completing the two-dose regimen of ChAdOx1nCov-19, and vaccine efficacy dropped to 34-37%, against the B.1.1.529 variant (127). The study also showed that AstraZeneca recipients, with a Pfizer (BNT162b2) booster restored effectiveness against the B.1.1.529 infections to 92.6% (127). Taken together, it appears that there are cross-reactive mechanisms induced by ChAdOx1nCov-19 that promote cross-protection against the B.1.1.7 and P.1 variants. Cross-reactive mechanisms are critical and desirable for heterologous immunity as these mechanisms will orchestrate and dictate the protective ability against SARS-CoV-2 variants and heterologous pathogens (117). The ChAdOx1nCov-19 vaccine is effective at inducing heterologous immunity as immune responses are providing a certain degree of effectiveness against B.1.1.7 (Alpha), P.1 (Gamma), and even B.1.617 (Delta) variants (117–127). In contrast, it is evident that ChAdOx1nCov-19 induced immunity was not cross-protective towards the B.1.1.529 (Omicron) variant (117–127).

The Johnson & Johnson (J&J) vaccine is a non-replicating viral vector vaccine composed of a human adenovirus type 26 incorporating a full-length wild type S protein (128). J&J vaccine, Ad26.CoV2-S, is a single-dose regimen that generates a robust antibody response targeting the spike protein and RBD (129). Spike and RBD-specific IgA₁, IgA₂, IgG₁, IgG₂, IgG₃, IgG₄, and IgM antibodies were generated from Ad26.CoV2-S and found to cross-react with SARS-CoV-1 S protein and other human coronaviruses such as CoV-229E, CoV-HKU1, CoV-NL63 and CoV-OC43, in vitro (129). The CoV1001 Phase I-IIa clinical trial reported humoral and cellular responses against B.1.1.7 (Alpha), B.1.351 (Beta), and P.1 (Gamma) variants in Ad26.CoV2-S vaccinated individuals (129–135). Regarding the B.1.1.7 (Alpha) variant, sera from Ad26.CoV2-S recipients were tested for their neutralizing activity with a pseudovirus inhibition neutralization assay, reporting that Ad26.CoV2-S has significant neutralization ability against B.1.1.7 (132–135). They found that antibody-dependent cellular phagocytosis, complement pathways and natural killer cells were activated against B.1.351 infections (131). In contrast, pseudovirus neutralization assay results indicated that the effectiveness of neutralizing antibody responses were lowered 5.0-fold and 3.3-fold against B.1.351 and P.1 variants, respectively (131). Although protection against infection was reduced significantly, a vaccine safety study conducted in South Africa reported a single dose of Ad26.CoV2-S protected against severe-to-critical disease outcomes, reducing COVID-19-related hospitalizations and deaths (130). The study reported a vaccine efficacy of 73.1% and 81.7% against moderate-to-critical COVID-19, on days 14 and 28 post-vaccination, respectively (130). The antibody response induced by Ad26.CoV2-S has cross-variant capabilities that recognized the B.1.1.7 (Alpha), B.1.351 (Beta), and P.1 (Gamma) variants, and clinically exhibited protection against VOCs, despite waning neutralization ability. Ad26.CoV2-S vaccination induced multiple antibody isotypes, including IgA, IgG, and IgM, which allows antibody protection at many sites of the body, including mucosal barriers, blood, lymphatic fluids, and other extracellular fluids. Inducing a diversified antibody response may be linked to better protective outcomes against SARS-CoV-2 variants. Collectively, an IgA/IgG/IgM response increases the coverage of the humoral immunity to multiple locations around the body and broadens the function of the antibody response, compared to an antibody response mediated by a single antibody isotype as seen with ChAdOx1nCov-19 (131). Notably, IgA antibodies play an important role in fighting infections in the respiratory tract and serve to defend against cell entry of respiratory viruses. Specifically with SARS-CoV-2 infections, a robust IgA response can potentially have neutralizing effects as the virus can be eliminated before reaching the respiratory epithelia and binding to ACE2. Early indicators of a robust IgA response and increased sampling of antibodies at the mucosal barriers may serve as a strong indicator of protection against SARS-CoV-2 infections. In addition to a diversified humoral response, non-neutralizing CD4⁺/CD8⁺ T effector and central memory cells were found to response to SARS-CoV-2 variants in vaccinated individuals. These memory populations may contribute to the protection against disease severity, but the role of these populations is still unclear (131).

Following the B.1.351 and P.1 variants, the B.1.617 (Delta) variant dominated the United States. A cohort study with 422 034 Ad26.CoV2-S vaccinated individuals reported vaccine efficacy rates of 76% against B.1.617 (Delta) variant infections, and 81% against COVID-19 related hospitalizations (136). These vaccine efficacies were maintained for 180 days after vaccination. Additionally, a neutralization study found that sera from Ad26.CoV2-S recipients was reduced 1.6-fold against the B.1.617 variant, while the most noteworthy reduction was observed against B.1.351(3.6-fold) and P.1(3.4-fold) variants (137). Mandy et al. suggests this lower neutralization by Ad26.CoV2-S-vaccinated sera may be associated with mutational differences in the RBD between variants. The RBD mutations associated with immune evasion, K417T, N501Y and E484K, have been identified in the B.1.351 and P.1 variants; whereas the B.1.617 variant has acquired mutations at, L452R and T478K, associated with infectivity (137). A repetitive theme has been observed with S-based antibody responses induced by vaccines: as novel mutations are acquired by variants, the neutralization capability declines. Mechanistically, antibodies recognize epitopes on pathogens to provide immunity against a given pathogen. However, the tendency of pathogens to evolve and mutate alters these epitopes to the point where antibodies are unable to recognize their targets, leading to an unprotective humoral response. Therefore, a vaccine that induces heterologous immunity cannot only depend on spike- and RBD-specific neutralizing antibodies to provide long term and cross-variant immunity. As novel SARS-CoV-2 variants are emerging, more mutations accumulate in the S protein and the antibody responses are expected to weaken. Many studies have reported sera from Ad26.CoV2-S recipients display a substantial fold decrease or no neutralization capability against the B.1.1.529 (Omicron) variant (138–140). With over 30 mutations in the spike, antibody responses have proven to be insufficient at protecting against Omicron infections. This shifted the focus towards looking at other immune parameters conferring vaccine induced immunity against the Omicron variant. A study with macaques immunized with Ad26.CoV2-S vaccine found the presence of cross-reactive CD8⁺ T cell responses against the Omicron variant (141–143). Further evaluation of the vaccinated group found that immunologic profiles with moderate omicron-specific neutralizing antibody (NAbs) titres and negligible CD8⁺ T cell responses, and low-to-moderate Omicron NAbs titres and low CD8⁺ T cell responses fail to establish viral control in the upper respiratory tract. Meanwhile, macaques with low NAbs titres and high CD8⁺ T cell responses, and high NAbs titre and low CD8⁺ T cell response, established virological control upon SARS-CoV-2 challenge. The paper suggests that CD8⁺ T cells responses play an important role in viral protection and control against SARS-CoV-2 variants. Other studies comparing cellular responses among COVID-19 vaccines have reported Ad26.CoV2-S recipients with a sustainable CD4⁺/CD8⁺ T cell response at 8 months post-vaccination. The median CD8⁺ T cell counts for Ad26.CoV2-S recipients (0.12%) were significantly higher compared to BNT162b2 (0.016%) and mRNA-1273 recipients (0.017%) (144, 145). All in all, the waning efficacy rates of Ad26.CoV2-S vaccine against the B.1.1.529 (Omicron) variants highlighted the susceptibilities of the vaccine-induced humoral responses and emphasized the importance of cellular responses.

Evidently, Ad26.CoV2-S recipients were not protected from Omicron infections, therefore, research into homologous (same as the initial vaccine) and heterologous (different from the initial vaccine) booster vaccines were explored to re-establish the lost immunity. It was determined that both, heterologous and homologous boosting regimens were safe and effective at restoring immunity against the Omicron variant (145). A primary Ad26.CoV2-S vaccination, followed by Pfizer vaccine booster shot increased NAbs titers by 6- to 73-fold, compared to a homologous booster shot which increase titers by 4- to 20-fold (146). Spike-specific CD8⁺ T cell counts were significantly increased with the heterologous boosting regimen, whereas homologous boosting induced a more long-lasting spike-specific CD8⁺ T cell response. In South Africa, 500 000 health care workers were given a homologous Ad26.CoV2-S boosting regimen to follow and found vaccine efficacy for hospitalization increased from 63% to 84% (147). Another clinical trial among health care workers, the SWITCH Trial, compared the homologous Ad26.CoV2-S regimen with a heterologous mRNA-vaccine based regimen, and found the strongest response occurred from the mRNA-based boosting regimen (145). Heterologous mRNA-based boosters generate a higher NAb titer compared to the homologous Ad26.CoV2-S regimen (145). Additionally, all recipients of a booster shot demonstrated spike-specific IFN-γ⁺ T cell responses. Moreover, the mRNA booster (mRNA-1273: 91.7%; BNT162b2: 91.5%) has a cellular recall response compared to the Ad26.CoV2-S (72.7%) booster shot (145). Data on vaccine efficacy against infection, transmission and severe disease are not available, but are predicted to increase based on the humoral and cellular response data.

The Moderna, mRNA-1273, vaccine is an mRNA lipid nanoparticle encoding the wild-type SARS-CoV-2 spike protein with a transmembrane anchor and cleavage site (148). It is an FDA-approved vaccine for all ages, consisting of a two-dose regimen, 28 days apart. Moderna (mRNA-1273) elicits a strong CD4⁺ T cell response and neutralizing antibody titre against the RBD (149). An evaluation of the mRNA vaccine-induced responses by Bezawit et al. revealed CD4⁺ T cells with cross-variant and cross-species reactivity as they recognize the B.1.1.7/B.1.351 S-protein as effectively as the wild-type SARS-CoV-2 and heterologous HCoV-HL63 S-protein (150). In addition, the antibody response had cross-reactive properties towards B.1.1.7/B.1.351 variants, leading to a robust NAb titer. The mRNA-1273-induced immunity was found effective as vaccine efficacies against B.1.1.7, B.1.351 and B.1.617 infections, after two doses of mRNA-1273, were 100%, 96.4% and 86.7%, respectively (151, 152). Moreover, mRNA-1273 recipients were protected from severe COVID-19 outcomes and hospitalizations against the B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617 (Delta) variants (122, 150–152). The emergence of the B.1.1.529 (Omicron) variant had negative implications for the Moderna vaccine. There was a significant reduction in effectiveness of the vaccine in protecting against infection, but protection against severe disease was still observed (153–156). Serum samples from mRNA-1273 recipients showed 41-to-84-fold reduction in neutralization ability, and vaccine efficacy declined against the B.1.1.529 (Omicron) infections to 44.0% (157, 158). With the marked reduction in protection against infection, an mRNA-1273 booster shot was encouraged. The administration of an mRNA-1273 booster restored vaccine efficacies against hospitalization and disease outcomes of COVID-19. Unfortunately, the efficacy of the booster shot against Omicron infections dropped from 71.6% to 47.4%, after 60 days (157). Research shows that the booster shots may be a temporary measure to mitigate the infectivity associated with emerging SARS-CoV-2 variants as the human population remains highly susceptible to SARS-CoV-2 infections.

The mRNA-based vaccines have proven to generate a more robust and durable memory response compared to the other COVID-19 vaccine types. The mRNA-1273 and BNT162b2, have been shown to induce spike- and RBD-specific memory B cells that cross-react with B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617 (Delta) variants and generated functional neutralizing antibodies (159). The study found that more than 50% of memory B cells cross-reacted with the three VOC (159). This cross-variant humoral response generated from the mRNA vaccines has shown to be effective as vaccine efficacy against the Alpha, Beta and Delta infections remains high (151, 152, 159). In terms of heterologous immunity, this is an ideal humoral response as it confers specificity against spike and RBD targets, establishes memory, cross-reactivity, and protects against infection. Regarding a sustainable, cross-reactive humoral response, these memory B cells increased in frequency from 3 to 6 months post-vaccination, and variant-binding memory B cells were reported to be more hypermutated compared to wild-type binding memory B cells (159). However, Choi et al. found that sera from mRNA-1273 recipients have reductions in neutralizing ability against B.1.351 (Beta), P.1(Gamma) and B.1.617 (Delta) variants, ranging from 2.1- to 8.4-fold (160). Despite the reduction in neutralizing ability, the mRNA vaccine efficacies against infections have remained intact, whereas protection against SARS-CoV-2 infections dropped with the Ad26.CoV2-S and ChAdOx1nCov-19 vaccines (75, 160). Goel et al. has associated this resilience to the early establishment of CD4⁺ T follicular cells, the presence of SARS-CoV-2-specific CD4⁺ and CD8⁺ memory T cells 3-to-6 months after vaccination, and rapid recall responses upon re-exposure of antigen (159). Notably, early CD4⁺ T follicular cells have been correlated to antibody production at 6 months post-vaccination, emphasising the role of early cellular responses in long term immunity against SARS-CoV-2. Second, the stabilization and rapid recall of SARS-CoV-2 specific CD4⁺ and CD8⁺ memory responses has been shown to correlate with long-term humoral immunity. Perhaps, as the antibody responses decline in neutralization ability, CD4⁺ T follicular cells and other T memory responses reactivate in germinal centres, and other mechanisms that facilitate memory B cells to undergo somatic hypermutation, produce functional cross-reactive antibodies that also come to play. Interestingly, T follicular cells and other populations influencing germinal centres may play an important role in adapting humoral memory responses against emerging SARS-CoV-2 variants. Moreover, inducing heterologous immunity against SARS-CoV-2 may manifest within these T cell populations that allows the adaptive immune system to repurpose their immune mechanisms to the changing landscape of SARS-CoV-2 variants.

Pfizer/BioNTech manufactured a lipid nanoparticle mRNA vaccine that encodes full-length wild type spike protein (120). The Pfizer/BioNTech (BNT162b2) vaccine induces a high neutralizing IgG/IgA antibody titre, activates CD4⁺/CD8⁺ T cells and memory B cell responses against the S-protein, including the RBD (149, 161). A two-dose regimen of BNT162b2 has an overall 95% effectiveness at protecting against COVID-19 disease outcomes, and vaccine efficacies against B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617 (Delta), and B.1.1.529 (Omicron) variants were 93.7%, 75.0%, 88.0%, and 70%, respectively (87, 124, 162, 163). Serum samples from BNT162b2 recipients showed neutralization ability declining as novel variants emerged. Antibody neutralization responses were reduced by 2.6-fold, 4.9-fold, 5.8-fold, and 41-fold against B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617 (Delta), and B.1.1.529 (Omicron) (164, 165). The Pfizer vaccine has been shown to be effective until the emergence of the B.1.1.529 (Omicron) variant when the vaccine-induced immunity against infection was significantly compromised (166). Subsequently, protection against hospitalizations and disease have also been negatively impacted. Fortunately, many studies have reported that a booster dose restored the neutralization ability of recipient-sera against the B.1.1.529 (Omicron) variant (167–170). The first dose of BNT162b2 generated an early S₂-specific IgA⁺ plasmablast response, which is an early indicator of IgA-secretion (167). Then development of an IgG⁺ memory B cell response, targeting the S₁ subunit of the spike protein, was observed three-weeks after the first shot (167). The second dose served to boost the established humoral responses and produce functional NAbs against SARS-CoV-2 and its variants (167). Furthermore, one week after the second dose, the antibody response efficiently blocked viral entry of the B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617 (Delta) variants (168). However, the antibody titers and entry inhibition proficiencies were reported to decline at 3- and 6-months after the second dose (168). The BNT162b2 vaccine-induced humoral response was robust and effective in the early stages, but from one week onwards, the humoral response began to wane.

The success of mRNA vaccines against SARS-CoV-2 has been attributed to its multi-dimensional approach to priming immune mechanisms to mitigate the pathologies, infections, hospitalizations, and deaths related to COVID-19. The role of the antibody response is to neutralize extracellular viruses entering the host from the environment or prevent newly released virions from infecting additional host cells. When the antibody responses weaken, the virus can infect host cells and it is the responsibility of CD8⁺ T cells to eliminate viruses intracellularly. In addition to blocking cell attachment and entry, antibodies can also lead to killing of virus infected cells via ADCC (antibody dependent cellular cytotoxicity) and CDCC (complement dependent cellular cytotoxicity). The CD4⁺ T cell response orchestrates cellular mechanisms to protect against infectious agents such as SARS-CoV-2, by providing B and T cell help, generating memory, and balancing T_H1 vs T_H2 responses (169). The T cell immunity elicited by the BNT162b2 vaccine shows longevity and protection against SARS-CoV-2 variants (159, 171). Guerrera et al. found spike-specific CD4⁺ and CD8⁺ T memory cells, two-weeks, and 6-months after vaccination (171). These CD4⁺/CD8⁺ T memory cells were poly-functional, inducing T_H1 cytokines (IFN-γ and IL-2), generating S-specific central memory (T_CM) and effector memory (T_EM) populations, and activating T stem cell memory (T_SCM) (171). The T_SCM populations were stable and deemed a predictor for activated spike-specific CD4⁺ and CD8⁺ T cells at 6-months post-vaccination (171). T_SCM cells have self-renewal ability and function to replenish memory and effector subsets populations (172). It has been theorized that immunological memory and decade-lasting protective immunity stems from T_SCM populations differentiating into antigen-specific memory T cells subsets, upon re-exposure of antigen (172–175). Guerrera et al. have emphasized the stabilization and rapid recall ability of T_SCM populations as a signature for a sustainable cellular response (171). Another study examined memory responses associated with the Pfizer booster. They reported a strong IFN-γ⁺ or IL-2⁺ response in CD4⁺/CD8⁺ T cells, and early differentiation of SARS-CoV-2 specific CD8⁺ T cells into T_EM populations, which accounted for 0.02-2.92% of circulating CD8⁺ T cells (169). These CD8⁺ T cells responded to epitopes that were frequently presented by MHC alleles and conserved across SARS-CoV-2 variants, which allowed the memory T cells to develop cross-variant specificity (169). It is evident that successful COVID-19 vaccines have an intricate induction of T cell pathways and mechanisms that are diverse in their functionality and ability to recognize variants. These attributes observed in the mRNA vaccines have led to protection against B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617 (Delta), and B.1.1.529 (Omicron) variants, despite waning antibody responses over time post-vaccination.