Omicron Variant Could Be an Antigenic Shift of SARS-CoV-2

Abstract

In the past 5 years, the COVID-19 pandemic has experienced frequently changing variants contextualizing immune evasion. The emergence of Omicron with >30–50 mutations on the spike gene has shown a sharp divergence from its relative VOCs, such as WT, Alpha, Beta, Gamma, and Delta. The requisition of prime boosting was essential within 3–6 months to improve the Nab response that had been not lasted for longer. Omicron subvariant BA.1.1 was less transmissible, but with an extra nine mutations in next variant BA.2 made it more transmissible. This remarkable heterogeneity was reported in ORF1ab or TRS sites, ORF7a, and 10 regions in the genomic sequences of Omicron BA.2 and its evolving subvariants BA.4.6, BF.7, BQ.2, BF. 7, BA.2.75.2, and BA.5 (BQ.1 and BQ.1.1). The mutational stability of subvariants XBB, XBB 1, XBB 1.5, and XBB 1.6 conferred a similar affinity towards ACE-2. This phenomenon has been reported in breakthrough infections and after booster vaccinations producing hybrid immunity. The reduced pathogenic nature of Omicron has implicated its adaptation either through immunocompromised individuals or other animal hosts. The binding capacity of RBD and ACE-2, including the proteolytic priming via TMPRSS2, reveals its (in-vitro) transmissibility behavior. RBD mutations signify transmissibility, S1/S2 enhances virulence, while S2 infers the effective immunogenic response. Initial mutations D614G, E484A, N501Y, Q493K, K417N, S477N, Y505H, and G496S were found to increase the Ab escape. Some mutations such as, R346K, L452R, and F486Vwere seen delivering immune pressure. HR2 region (S2) displayed mutations R436S, K444T, F486S, and D1199N with altered spike positions. Later on, the booster dose or breakthrough infections contributed to elevating the immune profile. Several other mutations in BA.1.1-N460K, R346T, K444T, and BA.2.75.2-F486S have also conferred the neutralization resistance. The least studied T-cell response in SARS-CoV-2 affects HLA- TCR interactions, thus, it plays a role in limiting the virus clearance. Antigenic cartographic analysis has also shown Omicron’s drift from its predecessor variants. The rapidly evolving SARS-CoV-2 variants and subvariants have driven the population-based immunity escape in fully immunized individuals within short period. This could be an indication that Omicron is heading towards endemicity and may evolve in future with subvariants could lead to outbreaks, which requires regular surveillance.

1. Introduction

We are almost in the fifth year following the eruption of the COVID-19 pandemic, which was fueled by the abrupt emergence of many variants and natural viral recombinants in different geographies around the world. The implementation of regular surveillance systems and vaccination programs over time has significantly slowed down the virus’s evolution. In early November 2024, the death rate decline had reached 41 per 1000 hospitalization cases, and is continuing to decline [1]. The staggering number of long COVID-19 cases (90%) due to pandemic fatigue and mild COVID has been an increasing mid- pandemic concern. However, the number of post-COVID-19 cases is declining (now at 6%) due to regular vaccination, but it still poses a risk of leading to an outbreak. The pandemic fatigue has created sociological, economic, and psychological repercussions [2,3]. Mutants and recombinants, especially “variants of concern”, were aggressively transmissible and virulent, and not only posed a challenge to the treatment modalities, but also took a significant toll on human health. Indeed, the vaccines have remained important prophylactic tools to control the spread of virus in large populations. The reported Delta variant was 97–100% more virulent than the original Wuhan strain and previous variants due to its heavy mutations in the RBD region of SARS-CoV-2, and has lowered the immune response produced through vaccinations in a short time. Therefore, the concept of prime-boosting has been established as an applicable strategy to vaccinate in six months to achieve an adequate level of immune response in a short period of time [1,4,5,6].

The efficacy of Adenovirus vectored (ChAdOx1 and Ad26.CoV-2-S) and mRNA (BNT162b2) vaccines has shown around a 1.4 to 9-fold decline in Ab (antibody) levels against Delta variant compared to Alpha and WT/D614G. Notably, Coronavac vaccine efficacy was concluded with ~17–22 times decrease in Ab titre against Alpha and Beta, which extended up to 31.64 times against Delta [6]. The limited protection was achieved with two primary doses of ChAdOx1 nCoV-19 or BNT162b2 vaccines, which improved the neutralization activity subsequently with a third booster dose using either BNT162b2 or mRNA-1273. The booster dose was discovered to overcome the waning immunity over time. Omicron surge had drastically reduced the full protection in vaccinated people, that was first reported in South Africa. The vaccinated individuals were discovered to contract the new subvariants, which conferred early immune escape; thus, a booster dose within three months was found to substantiate the immune response outcomes. The extended activities on variant detection, testing, and sequencing for surveillance have truly enabled the efforts to use rapid response vaccination against Omicron subvariants, and even its predecessor variants. Notably, the prime boosting strategy has remained in place. The right combination of sequences for reported variants could have provided the long-lasting immunity for one year [7,8]. The origins of the variants of concern are depicted in

Table 1. The five most prevalent VOCs of SARS-CoV-2.

Name of VOC	Lineage Status	Average Number of Spike Mutations	Location First Identified
Alpha	B.1.1.7	29.7	UK in late 2020
Beta	B.1.351	28.4	South Africa in late 2020
Gamma	P.1	29.1	Brazil in late 2020
Delta	B.1.617.2	35.4	India in late 2020 Became dominant worldwide
Omicron	B.1.1529	>50	South Africa in late 2021—rapidly disseminated worldwide

.

The subvariants of omicron emerged from subvariant BA.1 (B.1.1.529.1), which was classified into several sub-lineages, such as BA.1.1 (B.1.1.529.1.1) (which is less transmissible than its predecessor BA.1), BA.2 (B.1.1.529.2—which has nine spike mutations and was more contagious—subdivided into BA.2.74, BA.2.75, and BA.2.76), BA.3 (B.1.1.529.3—less transmissible), BA.4 (B.1.1.529.4—evolved from BA.2 and less transmissible than BA.2), and BA.5 (B.1.1.529.5—evolved from BA.2 and more transmissible than BA.2 and BA.4). BF.7 was also a sub lineage of BA.5 found prevalent during 2023 [6,9,10]. Risks of reinfection with BA.2 are far greater than BA.1 [11]. BA.5 and BA.2.75 are further diversified into several other variants: BA.4.6, BF.7, BQ.1, and BQ 1.1 (originated from BA.5) and BA 2.75.2 (originated from BA.2.75) [10,11,12,13,14]. The 157 genomic sequences of SARS-CoV-2 variants showed most heterogeneity discovered with exceptional standard error rate in Brazil (1.395), whereas Nei gene diversity ratio was 0.01 in Japan. Additionally, the highest mutations reported in ORF1ab or TRS sites, ORF7a and ORF10 regions of genome. Only the nucleocapsid, ORF9, corona nucleoca region and DUF5515 were observed to be the most conserved domains [15].

The XBB* recombinant, derived from BA.2.10.1 and BA.2.75, is capable of infecting individuals. BQ.1* is a sub-lineage of BA.5 (K444T and N460K mutations). The BQ1.1 sub-lineage has an antigenic site with an additional mutation (R 346 T). Its transmissibility patterns, immune escape status, and impact on vaccination have not been analyzed to date [16].

XBB.1.5 is a descendant of the XBB offshoot from BA.2 and has been nicknamed ‘Kraken’. It is described as more transmissible and contagious [17]. Immunocompromised individuals become infected easily and develop resistance to drugs and vaccines, which not only can lead to long COVID or post-acute COVID syndrome, but also leads to the evolution of more contagious variants. Omicron appears to have evolved separately from all the previous mutational variants. Therefore, it was speculated to have the potential to lead to another pandemic by generating more and more resistant strains/variants that could easily escape from vaccine-driven immunity. The emergence of variants resulted from the possible antigenic drift of the early strain, which helped these variants to become more transmissible during the evolutionary process [9] (Figure 1). The XBB.1.6 variant (Arcturus) became dominant in June 2023. A comprehensive genome-based survey showed that XBB 1.16 variant exhibited limited diversifications that propagated at slower substitution rate of 3.95 × 10⁻⁴ sub/site/year. This was a direct progenitor of XBB and XBB.1.5, and remained in circulation for several months. Its maximum variability was noted until May 2023. Later on, the NTD and RBD surface charge variability characteristics of the surging four variants, such as XBB, XBB1, and XBB 1.5, were negative, as well as that of XBB1.6 NTD, with a higher electrostatic charge, but were also shown to be comparatively neutral. These differences were crossed-checked and the ACE-2 affinity to all of them was found to be similar, conferring mutational stability [18].

The changes in Omicron, with numerous mutations, and its persistent domination over other variants, contribute to its high level of viral fitness. Hence, subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 were predominant, and these variants exhibited the potential to escape neutralization, especially XBB, XBD, and XBF [19]. The XE variant (stealth Omicron) is a combination of BA.1 and BA.2, the XD variant is the recombinant of BA.1 and Delta, and the XF variant, similar to XD, was shown to be a combination of sub-lineage BA.1 and Delta (UK), as shown in Figure 1C. The stealth variant displayed 10 times higher infectivity than lineage BA.2 [19]. By January 2024, more than 80% of the circulatory variants were reported to be XBB.1, XBB 1.5, XBB.1.9.1, XBB.1.16.1, EG.5.1.1, EG.5.1.3, XBF, BA.2.86.1, or JN.1 [20]. The BA.2.86.1 and JN.1 variants were reported to attain >30 additional changes, whereas XBB attained some recurrent mutations. The sub-variants were grown and tested on a human carcinoma cancer cell line (IGROV-1) and Vero E6 showed a limited fusogenic capability. Nasal epithelial cells are highly susceptible to all the circulatory variants. There were fewer BA.1/BA.2-infected individuals than those infected with BA.1. However, breakthrough infections with XBB were found to increase the immune response. Hence, the viral evolution has largely led to less infective and attenuated attributes [20].

Figure 1. (A) Evolutionary landscape of various VOCs of SARS-CoV-2 and the divergence of Omicron based on S-glycoprotein spike mutations [21]. (B) Notable Omicron mutations in the spike region [22]. (C) Various Omicron sublineages that diverge from their predecessor emerged during 2022–2024. Omicron variants BA.1, BA.2, and BA.5 were substantially diversified, building a trajectory in different directions.

Figure 1. (A) Evolutionary landscape of various VOCs of SARS-CoV-2 and the divergence of Omicron based on S-glycoprotein spike mutations [21]. (B) Notable Omicron mutations in the spike region [22]. (C) Various Omicron sublineages that diverge from their predecessor emerged during 2022–2024. Omicron variants BA.1, BA.2, and BA.5 were substantially diversified, building a trajectory in different directions.

The two most important theories regarding the development of the Omicron variant are as follows: (a) The intra-host environment of an immunocompromised host/or a population harboring the infection for long period of time instigate an increase in mutation numbers, which enhances the transmissibility required for their survival and thus reduced their virulence or severity; (b) Epizootic viral infection, in which animals such as mice live in close proximity to humans causes viruses to affect and adapt to different hosts within a short time and to pass through immune surveillance mechanisms in different animal hosts before jumping back to humans. Therefore, Omicron could be the result of “reverse zoonosis”, building its own molecular trajectory to continue its survival [9].

2. Neutralization-Resistant Mutations

The new variants were disseminated through the population more vigorously than ever, despite the availability of more advanced vaccines and molecular therapeutics. This impacted (a) vaccine efficacy, leading to a less protective immunogenic response, (b) controlled transmissibility, and (c) diagnostic accuracy, due to the variability of the S-antigen, which often was not detected following the arrival of the alpha variant [23]. Therefore, the most significant feature of Omicron was its immune escape from vaccine-generated Nabs, which can be contextualized through looking at its evolutionary process, which involved enhancements in the S-protein binding with ACE-2 and the initiation of effective proteolytic priming via TMPRSS2 [9]. Most of the mutations in the RBD region are reported to have increased transmissibility and an increased infection rate. Additionally, the NTD region is also linked increases in transmissibility patterns and virus binding affinity, S1/S2 also increases infectiousness and transmissibility, and S2 signifies the development of an immunogenic response [9]. Some known mutations, such as D614G (B.1), N501Y, E484K (Eek), K 417, and L452R, allow the virus to bind more tightly to human cells and help spread the virus faster than ever. Kumar et al., 2022 [24] through computational analysis, predicted that mutations such as Y505H, N786K, T95I, N211I, N856K, and V213R could increase pathogenicity using positive electrostatic effects that interact with RBD and hACE-2 to increase transmission as compared to the wild type.

Eight significant mutations—D614G, E484A, N501Y, Q493K, K417N, S477N, Y505H, and G496S—were involved in antibody escape, infectivity quotient, and increased and decreased stability regarding the molecular flexibility of S-glycoprotein’s interactions with ACE-2. These RBD-region mutations were investigated via liberated Gibbs Free Energy (ΔΔG) scores to analyze their potential stability. The stability of the mutational outcomes of D614G, Q493K, and S477N achieved ΔΔG scores of 0.351 kcal/mol, 0.470 kcal/mol, and 0.628 kcal/mol, respectively, for the consensus prediction of mutational stability. Moreover, the D614G, Q493K, and S477N mutations have molecular flexibility with S-glycoprotein, allowing for interactions with ACE-2 and enhancing the virulent nature of these variants [4,25,26]. New evidence has shown that substitutions of R346K (BA.1.1), L452R, and F486V mutations exert more immunological pressure, triggering immune evasion [9]. A mutation like T478K is close to mutation E484K, which is also involved in antibody escape in the epitope region. This variant is resistant to bamlanivimab, anti-RBD, and anti-NTD monoclonal antibodies [5]. D614G was the first mutation reported during the SARS-CoV-1 and SARS-CoV-2 outbreaks, and features a unique viral fitness to promote transmission. S-glycoprotein clearly affects the cleavage pattern responsible for infection and reinfection [12,13,14,27]. Various mutational patterns were observed over time to be responsible for neutralization resistance, as depicted in

Table 2. The pattern of mutations reported to be neutralization-resistant to monoclonal antibodies, convalescent sera, and sera from vaccinated individuals.

Mutations Evading the Variants Neutralization	References
D614G, E484A, N501Y, Q493K, K417N, S477N, Y505H, G496S	Chakraborty et al., 2022 [4]
D614G, T478K, E484K, E484A, N501Y, Q493K, K417N, S477N, Y505H, G496S	Planas et al., 2022 [5]
R346T, K444T, N460K, F486S	Qu et al., 2022 [12,13,14]
S371F, S373P, S375F, D614G	Park et al., 2022 [28]
Q183E, K444T, V445P, F490S, R346T, N460K, F486S	Wang et al., 2023 [29]
K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H	Hoffman et al., 2022 [30]
R346K, S371L, N440K, G446S, Q493R	Liu et al., 2022 [31]
Y505H, N786K, T951, N211I, N856K, V213R	Kumar et al., 2022 [24]
Q493R, N501Y, S371L, S373P, S375F, Q498R, T478K	Dhawan et al., 2022 [9]
L455S	Wang et al., 2024 [32]
Y144 (YY144-145TSN), Y144 del, FF486S, F486V	Tamura et al., 2023 [33]

.

Important mutations of the Omicron sub-lineage, such as R436S, K444T, F486S, and D1199N (the HR2 region of S2), were shown to be involved in antibody recognition, with altered spike positions on the cellular membrane (as observed in a structural modeling study). Mutations like N460K, N658S, F486S, and D1199N displayed fusogenicity and the S processing of Omicron subvariants BQ.1, BQ.1.1, and BA 2.75.2. R346T, K444T, N460K, and F486S mutations were found to represent key neutralization escape positions [12,13,14]. The emerging Omicron subvariants were tested against the sera obtained from vaccinated healthcare workers (three doses), hospitalized BA.1-wave patients, and BA.5-wave patients. The subvariants BQ.1 and BQ.1.1, harboring N460K, R346T, and K444T mutations, and BA.2.75.2, with F486S mutations, showed enhanced neutralization resistance. The N460K mutation in BQ.1 and BQ.1.1 exhibited enhanced fusogenicity and S-processing. The F486S mutation may enhance fusogenicity and S processing, while the D1199N mutation interestingly reduces this pattern [13,14]. Since November 2021, many sublineages of SARS-CoV-2, including Omicron, have been reported to evade the Ab response and become resistant to vaccination or infection-induced immunity. Immune evasion was recovered if a booster dose was administered within six months of vaccination [12,13,14,34,35,36,37]. Omicron BA.1 was the subject of experiments using K18-hACE-2 mice, and it was discovered to be less controlled via mRNA vaccination [38].

All the sub-lineages of Omicron have significantly challenged the efficacies of vaccines and a substantial decline in neutralizing antibodies was found for BA.1, via R346K mutational alterations, and B.1.1.529.2 (BA.2). BA.2 also exhibited marked resistance against most monoclonals, including sotrovimab [39]. BA.2 was noted to have eight unique spike alterations, but lacked the thirteen spike alterations found in BA.1.

Omicron variants have been shown to have reduced infection efficiency in lung-derived CaLu-3 cells. Lung tropism is less likely due to the shift in TMPRSS2-mediated plasma membrane towards cathepsin B/L-mediated endosomal entry [12,13,14,38,40,41]. Vaccine boosters or breakthrough infections can produce a potent neutralizing response to combat Omicron infection. Moreover, breakthrough infection also elicited a better response locally, in the nasal cavity, to control virus transmission.

A potent neutralizing antibody, S2X324 with pan-variant potency, was effective against all the variants of SARS-CoV-2. These S2X324 (IgG2) antibodies developed protective response within 4 days of SARS-CoV-2 infection in a dose-dependent manner in a Syrian hamster model with 0.1–5 mg/kg body weight [28]. Polyclonal sera tested for serum neutralization were obtained from five cohorts of vaccinated people who received three shots of the WT (wild type) vaccine or four shots of the WT bivalent vaccine (WT and BA.5), in people who received three shots of the COVID-19 vaccine plus bivalent vaccine, and in people who experienced BA.2 and BA.4 or BA.5 breakthrough infection after vaccination. BA.2 and BA.4/5 were shown to have high resistance to serum neutralization compared to D614G for all the polyclonal sera. Therefore, the neutralization titre was decreased in the cases of BQ.1, BQ.1.1, XBB, and XBB.1 compared to D614G. Polyclonals from the BA.2 and BA.4/5 breakthrough cohorts responded to the new emerging variants in terms of antibody induction. One limitation of the study is that the T-cell response was not considered to better understand the immune status. The BQ.1 and BQ 1.1 variants were also resistant to class I and class IV epitope-mapped monoclonal antibodies like tixagevimab, bebtelovimab, sotrovimab, cilgavimab, non-RBD mapped, and NTD-SD2 monoclonal antibodies. The loss of neutralization in RBD class I and NTDs was due to the N460K mutation, and RBD class II monoclonal efficacy was reduced due to the R346T and K444T mutations. Evidently, they displayed no greater affinity towards ACE-2 [29]. Since these variants are predominant worldwide and are distantly placed in the phylogenetic tree of sarbeco viruses, as shown in Figure 1, the discovery of antigenic drift would be alarming, suggesting frequent immune evasion, despite the low hospitalization rates and reduced risk of post-acute sequelae of COVID-19 or long COVID [29].

The neutralizing antibody level was reported to be weakened in unvaccinated, convalescent, and naïve individuals who received two doses of the mRNA vaccine. Therefore, two doses of the vaccine were not sufficient to establish an effective humoral response that was regained following reinfection or with a booster dose, although it still provided short-term protection. Interestingly, higher titers in convalescent vaccinated individuals were noticed when the mRNA-1273 was received compared to the BNT-162b2 vaccine. However, it was not clear which antigenic epitope was subjected to the antibodies involved in the residual neutralization of Omicron [42].

The reported vaccine efficacies in preventing symptomatic infection with Omicron were 73% for vaccinated plus boosted individuals and 35% for only vaccinated individuals. This suggests that vaccination is not sufficient to protect against the highly transmissible Omicron variant [43]. ChAdOx1 nCoV-19 (AZ) vaccine efficacy remained at 66.5% after two doses. However, primary immunization against symptomatic COVID-19 Omicron remained limited [6]. Pre-existing cellular and humoral immunity, and even non-neutralizing antibodies of vaccinated and convalescent individuals, appear to protect against severe disease. Breakthrough infections serve as attenuated infections, boosting the production of antibodies. This is a clear explanation of antigenic drift.

Anti-RBD IgM (297 mAbs) protects against pseudoviruses Beta and Omicron BA.1. It was also effective against SARS-CoV-2 WA 1 when epithelial cells were infected in vitro [44]. A cocktail of monoclonal antibodies (297), used alongside Regeneron REGN 10,987/10,933 mAbs, were unable to neutralize Omicron.

However, mAbs 297 possesses neutralizing activity against Omicron BA.1 and BA.2, although this activity is reduced against BA.4 and BA.5. Monoclonal antibodies are only effective if the respective variant is active [45]. This provides the clues needed to develop pan-variant monoclonal antibodies that are potent against the RBD-, NTD-, and S2-conserved regions. There are 11 relevant mutations: six deletions and one insertion with N211∆ and ins 214 EPE, which are unique in the NTD region. Fifteen unique mutations, including G339D, S371C, S373P, and S375F, are mutations responsible for antibody evasion in the RBD region. The T547K and P681H mutations modulate the cleavage S1/S2 in the RBD-S1/S2 site. Omicron robustly binds to orthologous ACE-2 from different animals for efficient cell entry. Its effective cell invasion is indicative of its zoonotic potential. Interestingly, the spike was inhibited by soluble ACE-2, but resistant against monoclonal antibodies bamlanivimab, etesevimab, imdevimab, and casirivimab (selective against the RBD and NTD regions), which inhibit spike entry in a concentration-dependent manner. A cocktail of bamlanivimab and etesevimab was inefficient to stop pseudovirus replication (Omicron B.1.1.529). Similarly, casirivimab and imdevimab were also incapable of generating a response. However, sotrovimab was less inhibitory [30].

If H655Y and N679K mutations occur near the furin cleavage site, this can make the virus more contagious, blocking the T-cell response and increasing the chances of re-infection in the case of BA.5. Tomato Flu or HFMD (Hand, Foot, and Mouth Disease) outbreak, caused by coxsackievirus A-16, spread frequently during COVID-19 and in monkeypox patients, producing more complications for human health during 2021–2022 in India [6].

Liu et al. revealed a reduction in potency for four classes of RBD and NTD monoclonal antibodies in clinical use. Omicron might be a couple of mutations away from becoming pan-resistant to all currently available antibodies [31]. Omicron BA.2.86, with >30 mutations, escapes neutralization in cell culture (Vero E-6-TMPRSS2 and H1299-ACE-2), while XBB. 1.5 was observed to have the highest level of escape [46]. The immune escape of BA.2.75, BQ, XBB, and XBF in bivalently boosted individuals show that cross neutralization improved for those with hybrid immunity [47]. The convergent evolution of Omicron subvariants, including five substitutional mutations, R346, K444, L452, N460, and N486, increases the viral fitness in pandemic dynamics. Breakthrough infections BA.5 and BQ.1.1, as compared to BA.5 and BQ.1.1, can evade neutralization, and BQ.1.1 is less pathogenic than BA.5 [48].

2.1. T-Cell Response and Its Implications

The thymus-dependent pathway often involves antigen- or protein-derived co-stimulation, especially for T-cell-derived CD40L and macrophages that confer the antigen through MHC-II to activate B-cells. The thymus-independent pathway cognates the functions of CD8+ cells via APCs to process the antigen through proteasome and peptidase activation to degrade the viral antigen via MHC-I activation [25]. T-cell exhaustion is described as dysregulated pathways producing an impaired protective immune response, which causes massive destruction in the organs, increasing regular inflammation due the excessive secretion of proinflammatory cytokines and leading to the accumulation of a large amount of CD8+ cells. Therefore, the cytokine storm eventually spreads from one place to another, resulting in lymphopenia [49]. The worst disease outcome was reported in elderly individuals due to the depletion in their T-cell response as compared to the healthy, mildly infected, and recovered individuals [25]. Most immune escape studies lacked a determination of the robust T-cell response.

The robust CD8+ T-cell response against the SARS-CoV-2 wild strain was reported to activate the memory T-cells, and is consequently involved with viral clearance. On the other hand, the continuous eruption of different Omicron variants, VoCs, increases the CD8+ T-cell’s passive immune response as highly active macrophages frequently engulf the viral variants, and MHC epitopes must downregulate TLRs due to the excessive release of proinflammatory cytokines and activation of MAPK pathways. These activities impair CD8+ cells’ activities and they are frequently killed by natural killer cells, causing lymphopenia, resulting in immune escape, and facilitating infection, inflammation, and immunopathogenesis [25,49]. The non-synonymous mutations in MHC-I-restricted epitope CD8+ T-cells reduce their binding with the mutated version of peptide-specific antigenic epitopes (in vitro), activating T-cells, IFN-γ, and cytotoxic CD8+ T-cells in COVID-19 patients [50].

Additionally, most COVID-19 patients had robust CD8+/CD4+ T-cells with plasmablasts and a robust B-cell response, although ~20% cases remained undetectable. A deep immune profiling of T-cells led to the following classifications: immunotype 1, with highly activated CD4+ T-cells; immunotype 2, with effector CD8+ T-cells but less robust CD4+ T-cells, with plasmablasts and memory B-cells; immunotype 3, with an undetectable T-cell response. These responses regarded as highly antigenic-specific. Therefore, plasmablast studies are needed to determine the accuracy of the antigenic response and humoral response. Furthermore, patients identified as having a moderate etiology could have an interventional immune response [51]. Cytotoxic T-lymphocytes and natural killer cells control the viral infection; however, overactive and exhausted T-cells are normally linked to a poor disease prognosis. A reduction in NK and CD8+ T-cells, suggesting a severe SARS-CoV-2 case, can mean that anti-viral immunity is reduced at the beginning of infection [52]. The acute phase of SARS-CoV-2 involves an activated cytotoxic T-cell response. Convalescent and even, although less frequently, seronegative individuals present with a SARS-CoV-2-specific T-cell response. Therefore, natural infection can control recurrent episodes of severe COVID-19 [53]. In mild COVID-19 cases, the CD8+ and CD4+ ratio was found to be >90% higher than that of neutrophils. The reactive CD4+, CD154+, CD137+, CD154+, and CD137+ T-cells displayed a robust response in convalescent individuals [54].

HLA and TCR interactions are important to confer the CD8+ T-cell response and viral pathogenesis to improve prognosis. An NYN viral spike epitope with HLA restrictions elicits the broad CD8+ T-cell immune response. The four natural mutations—N450K, L452Q, L452R, and Y453F—that are linked to the NYN epitope were found to lose affinity for the public TCR-HLA epitope, and thus abrogate the bond between the CDR loop and peptides, reducing the activation of TCR HYN-I T-cells. Therefore, the viral evolution can impact CD8+ T-cell immunity [26]. The efficacy of CD4+ cells in fighting various mutants, including WT, was sufficient for their use in interventions [55]. The molecular docking (structural and physical analyses) of mutations such as S24L, L84S, V62L, and W45L in the ORF8 region of SARS-CoV-2 causes them to strongly bind to the IRF3, thus halting the production of IRF3, which could help them to evade immune response [56]. This degrades MHC-I activity, disrupts HLA-I recognition, and reduces the cytotoxic T-lymphocytes (CTL) response. These mutations increase the transmission of viral variants, as well as increasing their severity and resistance to immune responses [25,57].

2.2. Antigenic Cartography

Antigenic cartography, in the past 20 years, has become a routine practice to determine the antigenic behavior and variations in the constantly emerging influenza strains, which have the potential to cause outbreaks. The viral variants’ binding with compatible epitopes or specific neutralization antibodies is often visualized through quantitative neutralization methods and was first described to map the antigenic properties of influenza-A virus (H3N2) through a hemagglutinin inhibition (HI) test [58]. Single-exposure human sera are normally considered the gold standard; these are obviously quite rare, especially in endemic situations. As SARS-CoV-2 may become endemic in the near future, single-exposure sera will become increasingly difficult to procure. The selection of suitable and sustainable animal models is a standard requisite to fill the gaps in single-human exposure sera, to increase the study of specific antisera against the emerging endemic strains [47,59]. Therefore, non-human hosts/animals that can be immunized against the selective variants, for reasons of cartography, are the most accepted and reliable method to check antigenic variability [60].

Due to vaccination and breakthrough infections, a large number of people have already developed immunity against SARS-CoV-2 variants. Muhlemann et al., 2024 [60] developed sera against the 16 prevalent variants in Syrian hamsters to build an antigenic cartography map. A diversified level of sera reaction was measured for pre-Omicron variants such as D614G, Alpha, Delta, Beta, Mu, and engineered B.1 + E484K. This was extended to BA.1, BA.2, BA.1.3.1 or JN. 1, and XBB.2 variants. However, the increase in titres against XBB.2 homologous sera also increased reactivity against EG5.1, BA.5, and JN.1 variants. XBB.2 homologous sera was eventually discovered to be non-reactive to the very first recombinant, “D614G”, which appeared to cause the initial phase of the pandemic, just after the outbreak of the original Wuhan strain. The serum-detectable titres ranged from <1:160 to <1:20 [60]. Sera obtained from vaccinated individuals with hybrid immunity due to repeated breakthrough Omicron infections (except JN.1 and JN.2) lowered the neutralization patterns of the BA.5 and BA.2.75 variants but failed to neutralize the BQ.1.1 and XBB lineages. Moreover, further breakthrough infection with JN.1 eventually shortened the neutralization distance in comparison to the aforementioned variants, showing the needs for antigenic boosts, either through weak infection or a vaccine-based immunogenic boosts, to keep immunity up to date [61]. A diversified antigenic mapping from sera collected from sputnik V vaccinated, unvaccinated, and convalescent individuals after six months, and from individuals who received a booster dose after one year, was carried out after sera were tested against a panel of six lab-prepared pseudoviruses: WT, Alpha, Beta, Delta, Omicron, BA.1, and BA.4/BA.5. Pre-booster-dose sera neutralized all the variants after six months, but the titres remained at the baseline, and BA.4/BA.5 was emergent [62]. The booster-dose sera from vaccinated individuals who were observed to be weakly or non-reactive to the subsequent Omicron subvariants could yield the relevant data. Nevertheless, the significance of using animal models, such as hamsters, mice, and non-primates, who are immunized against the originating authentic strains or pseudovirus types, to produce evolutionary antigenic cartography maps has clearly been stated in the literature [47,58,59,60,63,64,65]. Additionally, non-human primates with physiological and genetical similarities to humans were used in an experiment by Rossler et al., 2025 [59], to study variant-specific neutralization activities. A total of 23 SARS-CoV-2 variants were used in a study to understand the impacts on sera reactivities, as detailed in

Table 3. Antigenic cartography mapping of SARS-CoV-2 VoCs to evaluate the sharp antigenic shift in the Omicron subvariant.

Models for Cartography Mapping	SARS-CoV-2 VoCs and Omicron Subvariants	Sera Reactivity Under Various Circumstances	References
Syrian hamsters (serum against pre-Omicron and Omicron)	D614G, Alpha, Delta and B.1+E484K	Anti-D614G—reactive	Muhlemann B. et al., 2024 [60]
	Alpha (B.1.1.7), Beta (B.1.351), Mu (B.1.621) and D614G recombinant	Anti-Alpha B.1.1.7, Anti-B.1+E484K, Anti-Beta B.1.351, Anti-Delta B.1.617.2, and anti-D614G—highly reactive
	Omicron BA.1, BA.2, BQ.1.18, BF.7, and XBB.2	Ant-Omicron BA.1, BA.2, BA.4/BA.5 and XBB.2—highly reactive
	Omicron EG.5.1, JN.1 and BN.1.3.1	Anti-Omicron BA.1, BA.2, BA.4/BA.5 and XBB.2—non-reactive
Hamsters (serum developed against pre-Omicron)	Pre-Omicron variants	Closely reactive	Mykytyn A.Z. et al., 2022 [63]
	Omicron BA.1 and BA.2	Faintly reactive to pre-Omicron variants’ sera Reactive to Anti-BA.1
Humans (6 months prior to booster vaccine and 1 year post-booster with 1 year of sera) with Sputnik V vaccine	WT, Alpha, Beta, Delta, Omicron BA.1 and BA.4/BA.5	Vaccinated sera (pre-boost) remained highly reactive to pre-Omicron variants	Astakhova E.A. et al., 2023 [62]
	BA.1 and BA.4/BA.5	Remained at baseline reactivity to Omicron BA.1 and BA.4/BA.5
Human serum from subjects who received an early vaccination	Pre-Omicron variants Wuhan, D614G, Alpha, Beta, Gamma, Delta	Sera strongly reactive to WT, D614G, Alpha, Beta, Gamma, and Delta (with a mutational profile including N501Y, E484K, K417N/T, L452R)	Mykytyn A.Z. et al., 2023 [64,65]
Humans following vaccination after Omicron infection	Early Omicron variants BA.1, BA.2, BA 2.12.1, BA4/5, BA.2.75	Sera highly reactive to early Omicron variants
Following vaccination after Omicron infection	Emerged Omicron variants B.Q. and X.B.B	Sera faintly reactive to emerged Omicron variants
Sera from vaccinated and Omicron-infected individuals	SARS-CoV, Pangolin and bat Sarbecoviruses	Obtained sera were not reactive
Sera collected from infected and immunized primates, with animals immunized with monovalent XBB.1.5 vaccine	Pre-Omicron (Wuhan, Alpha, Beta and Delta)	Highly Reactive to Alpha	Rossler A. 2025 [59]
	Early Omicron (BA.1, BA.2, BA.2.12.1, CH.1.1, DV.7.1, BA.5, and BQ.1.1	BA.5 and BQ.1.1 located near to XBB.1 descendants. BA.2.75 and DV.7.1 were a similar distance from BA.1 and BA.2, but more distant from XBB.1. BQ.1.1 and DV.7.1 were distinct from earlier variants on the scale. CH.1.1 had the most undetectable titre with all sera cohorts (excluded, as it made map unstable). Sera reactive to other variants before saltation of BA.2.75.
	XBC.1.6, and XBB.1—descendent variants (XBB.1, XBB.1.5, FL.1.5.1, HV.1, HK.3, and EG.5)	Sera from vaccinated (Wuhan strain and XBB; one strain) individuals were shown to decline Nabs against XBB.1.5
	Recent Omicron variants (BA.2.86, JN.1, KP.2, KP.3, and KZ.1.1.1	A strong neutralization from earlier variants’ sera
Hamster model (sera developed against BA.5, which is genetically close to BA.2 (with the exception of three substitutions and two deletions)	BA.1, BA.2, BA.5, BQ.1.1, XBB.1 and BM 1.1.1	Efficient neutralization of BA.2 and BQ.1.1. BA.1 and BM1.1.1 poorly neutralized. XBB.1 not neutralized.	Mykytyn A.Z. et al., 2023, 2025 [64,65]
Human model (First-exposure Sera; first-infection sera + two doses of BNT162b2, BNT-vaccinated subjects who received three doses; vivalent booster against BA.5, CK2.1.1, and BA.4/5 + with and without infection)	Representative variants: BA.2.75, BA.5, recombinant XBB and XBF lineages: BA.2.75 (CB.1, BR.3, CH.1.1). Six variants of BA.5 (BA.5.2.1, BE.1.1, Bf.7, BQ.1.3, BQ.1.1, BQ. 1.18. 2 XBB recombinant (XBB.1, XBB.1.5.1). XBF one recombinant variant (XBF3).	Poorly reactive and strong immune escape in BA.2.75, CB.1, BR.3, and CH. L1 with first-exposure sera. With first-exposure sera + two doses of Bivalent BNT. Similar neutralization pattern in BA.5 with BA.5.2.1, B.E. 1.1, and B.F. 7. B.Q. variants with a greater drop in neutralization Abs. First-exposure Sera (ancestral, Alpha, Delta, BA.1, Omicron, and BA.2 Omicron variants) + new Sera of BA.5 and 2 CK.2.11 eiyh XBF3, XBB.1, and XBB. 1.51 had a higher rate of escape. CK.2.1.1 convalescent sera > neutralization pattern with B.Q.1.18 + R346T and B.Q.1.3 + E619Q. No neutralization of the BQ variant using BA.5 convalescent sera. XBB and XBF recombinants poorly neutralized by single-infection sera +3. BNT doses against pre-Omicron variants BA.1, BA.2, and BA.5	Rössler A. et al., 2023 [47,59]
	Panel variants: D614G, Beta, Delta, BA.1, BA.2, CB.1, BR.3, CH.1.1, BA.5 (BA.5.3.2), BF.7, BQ.1.3, BQ.1.3, BQ.1.1, BQ.1.18, XBB.1, XBB.1.5.1, and XBF.3 Broad N-Antigen	The Biv.alent vaccinated group was show to have a higher level of neutralizing abs. BA4/5 bivalent booster with or without infection; antibodies were reactive against the N-antigen due to the previous infection.	Rössler A. et al., 2023 [47]

Cartography map analysis is displayed based on the 2D and 3D analyses in all the studies.

. Non-human primates often elicit a homologous response in comparison to humans regarding the antigenic variability of variants [59].

An antigenic cartography carried out in a hamster model revealed that BA.1 and BA.2 were very removed from pre-Omicron variants. Pre-Omicron variants were 10–38-fold away from emerging variants like BA.2, with the level of neutralization patterns being appx. 7–114 times more reduced in sera from hamsters without A.2. There was only a two-fold difference in dilution between similar isolated authentic SARS-CoV-2 variants and lab-prepared pseudoviral variants [63]. The antigenic susceptibility patterns that are analyzed in any given animal model may not precisely show the robust functionality, but could explain the structural differences in the originating stains. The initial antigenic variations are often analyzed using sera from single-exposure or vaccinated human subjects.

The present study evaluates a body of evidence revealing the clear antigenic drift in Omicron SARS-CoV-2 VoC from the previous VoCs as the pandemic evolved, and was also adapted to other animal hosts. Then, the effect on humans was studied, revealing a reduction in severity but signifying the continued presence of SARS-CoV-2 infection in humans [63,64,65].

2.3. New Approaches

Nanobodies are under clinical investigation for their medicinal use in cancer and various infectious diseases. Caplacizumab (bivalent nanobodies) have been authorized by the EU and FDA for the first time to treat patients with thrombotic thrombocytopenic purpura and thrombosis. Often, biparatropic nanobodies offer the best alternative to current monoclonals [66]. Nanobodies can be nebulized to improve their potency in the lungs compared to intravenous administration. These nanobodies have specific neutralizing capabilities and bind the RBD region efficiently. Their effective range was found to range from 2 to 22 nmol in relation to the binding affinity equilibrium constant to neutralize SARS-CoV-2 infection/or challenge in amounts ranging from 48 to 185 nmol via 50% plaque reduction assay. Specific neutralizing nanobodies bind to the RBD of the virus. At approximately 22 dissociation constants, these nanomolecules can neutralize the virus in plaque reduction assays. The biparatropic nanobodies were more efficient and effective against SARS-CoV-2, irrespective of the different known virulent mutations. They induce the premature transition of the flexible spike conformation to an irreversible post-fusion conformation, inhibiting its attachment to ACE-2 [67]. The next-generation vaccines that were introduced orally or intranasally in hamsters showed a robust mucosal antibody response against SARS-CoV-2. This strategy can be applied to reduce the transmission of the virus during outbreaks [68,69].

The spike in the Omicron variant harnessed the richness of the mutations, providing a direct indication of its immune evasion, monoclonal antibody resistance, and higher transmissibility [30]. This variant showed the ability to reinfect convalescent and vaccinated individuals, suggesting waning immunity. However, the Beta and Delta variants were rarely reported to cause reinfection, as Omicron’s emergence had already indicated the need for significant upgrades to the vaccines and monoclonal antibodies. A large body of evidence revealed that Omicron harbors the capacity for immune evasion at a population-wide scale, unlike the Beta and Delta variants. A stable nanoparticle-based platform encapsulating monomeric forms of RBD amino acids was developed for future vaccines. mRBD is encapsulated with Myxomonas xanthus, displaying receptor-binding derivatives [70] that provide long-term thermostability. It elicited a ~100-fold immune response after one immunization shot, which was increased approximately 42 times after a booster when challenged against all variants of a pseudovirus. These types of nano-based platforms could considerably reduce viral loads and associated lung pathologies [70].

The FINLAY-FR-1A vaccine, a recombinant protein and dimer of the RBD (with a Cys5p8 to Cys 538 disulfide bridge) 319–541 sequence obtained from CHO cells, produced >31 anti-RBD antibodies against Alpha, Beta, and Delta VOCs in clinical trials [6]. In recent studies, the monoclonal antibodies appeared to bind against antigenic determinants outside of the RBD motif in sites IV–V, and sites I–II, containing rare antibodies, which partially overlap the RNB, are also involved to some extent [31,71]. The decrease in immunity within this short time span is unique, and the utmost attention should be paid to developing new methods of virus detection, including regular surveillance to measure vaccine-driven immunity and the increasing risk of re-infection. These will provide important tools to improve our preparedness for another pandemic [72].

The human adenovirus serotype S (Ad5) vectored Omicron spike induced a robust 3 log sIgA response, which was more effective than that of the serum antibodies. sIgA was found to block the spike-mediated cell-to-cell transmission and protect hACE-2 mice from XBB challenge. An intranasal vaccine could be effectively elicit an early and local immune defense [73]. An avirulent new castle disease virus linked to spike antigens (NDV-HXP-S) was taken as an antigenic candidate for an Omicron variant vaccine. This led to the development of high-mucosal IgA and serum IgG titres against = SARS-CoV-2 VoC (in mice. Inducing memory B cells and local T-cell response in lungs can substantially reduce infection [74]. Therefore, the precise induction of a cellular immune response also determines the level of local protective immunity. However, the NDV response was not measured. The long-term effects of intranasal vaccines have yet to be revealed.

3. Discussion and Conclusions

Protection against the virus in boosted individuals increased by 75%, but the presence of long-lasting protection was not verified at the time. NAb binding was preserved in convalescent, vaccinated, and boosted individuals against NTD, RBD, and other spike-specific regions. Most of the antibodies cross-reacted with other specific spike regions, revealing that the conserved region reacted with the S2 subunit of Omicron [42]. The non-neutralizing Abs attained binding capacity in cell culture, which may contribute to protection against viral infections. In connection with T-cell-based immunity, non-neutralizing Abs can target the S2 domain along with RBD and NTD. The cross-reactive antibodies can attach to S2 or any conserved region. The mRNA vaccine induced a permanent B-cell germinal cell response. In boosted individuals, the germinal B-cell response and plasmablast activity in lymph node drain fluid induced an S-binding that was sustained for 12 weeks, while the circulating plasmablast cells appeared to peak one week after the booster dose [75].

The cognate function of Abs through B-cells is still present, even if lower activity is observed in RBD and NTD mutations. B-cell activity is also observed during original or variant infection or vaccination, and is capable of producing a strong plasmablast response that can control the spread of the virus. Generally, antigenic proteins can enter the lymph nodes to engage with germinal cells producing Abs via affinity maturation. Moreover, Abs can also protect the Fc-mediated effector function, even if the actual neutralization activity is reduced, as reported in influenza. Broadly neutralizing MAbs against the stalk region of hemagglutinin interact with Fc (FcγRs), conferring protection against the lethal challenge of the H1N1 strain of influenza [76]. In normal practice, however, the actual titre of reactive Abs directly confers protection against virus infection.

Silent polymorphism and synonymous mutations do not affect the amino acid but can contribute to the transmissibility and infectivity of the phenotype. The low 5′ stability in synonymous changes may cause instability in mRNA, affecting the translation mechanism and lowering or increasing the severity of infection. A low abundance of tRNA can enhance translation in rich conditions. Any process involved in changes in translational capacity can alter the protein, the translational accuracy, of the co-translational protein folding [9].

Regular environmental surveillance is needed to prevent the emergence of new variants and recombinants from unknown origins and to stop them from propagating in the population and causing havoc. Therefore, more research is needed to map the trajectories of viral resurgence and monitor their biological functionality, which could help to guide the preparation of effective medicines. Accordingly, new challenges could be controlled easily and pandemics could be prevented.

Sarbecovirus monoclonal antibodies (including sotrovimab 2, S2X2593, and S2H974) and broadly neutralizing antibodies are capable of recognizing antigenic sites outside the receptor-binding motif. They play a key role in neutralizing Omicron, despite the observation of an antigenic shift in this particular strain, which may pave the way for dealing with the ongoing pandemic and future zoonotic spillovers [71]. Various recombinants can be formed during infection, especially in immunocompromised hosts, which can pose a threat to the resurgence of virulent strains. The continuous environmental surveillance of emerging variants from unknown origins and the increasing trend of Nab evasion, as well as surveillance of their biological activities, are necessary to prepare vaccine candidates (pan-variant vaccine) [9]. Additionally, the virus’ evolutionary trajectory should be monitored [77], pandemic preparedness programs and associated policies should be developed, and the general population should be educated to avoid complacency when following containment measures. These measures could cease the transmission and emergence of new outbreaks in a timely manner. Reassortment of the virus can lead to drastic changes that change its phenotype; such reassortments are often referred to as antigenic shift. A typical example is the influenza H1N1 outbreak in 2009, which was a result of antigenic shift and the reassortment of antigens among avian, human, and swine viruses [67].

Most of the studies were conducted using a pseudovirus instead of the original Omicron strain, and there was no analysis of the actual T-cell response; these could be considered significant limitations. Furthermore, the heterologous immunity obtained with AZ/BNT might provide a better response, along with the implementation of public health measures like face masks and social distancing, etc. [30].

The booster dose significantly improved the humoral response against Omicron, which was necessary to counteract virus transmission. An update of the pharmacopeia of monoclonal and vaccine effects is also required. Due to the reduced and impaired activity of serum against Omicron, booster vaccines are required shortly after the initial dose to maintain a protective level of neutralizing antibodies [78].

Smallpox is the only example of the vaccine-mediated eradication of a disease in humans, achieved through massive global initiatives and massive efforts to achieve a high immunization coverage. Sustained containment measures are required for effective vaccine and infection control surveillance, as well as rapid molecular diagnostics (using the current version of the isolated variant and active surveillance). It is worth noting that virus elimination strategies were designed while taking into account the example of the failure to control polio due to the lack of an animal reservoir [79].

GISAID and NCBI collect and store a database of various sequences and mutational trajectories for SARS-CoV-2. The occurrence and spread of variants are tracked by these websites on frequent basis around the globe. This genetic epidemiology provides knowledge on clades and lineages (nucleotide- and AA-based information) based on the marker mutations [27].

Mutations track an SNP (single-nucleotide polymorph) or multiple SNPs, but they will not signify whether a particular mutation could lead to an outbreak. Therefore, these mutations must be compared with the WT strain and other dominant variants. For this, the sequencing information obtained from the GISAID database is used to determine the mutation’s alignment with different clades, like G and V, as well as various subclades. An annotated algorithm reveals the mutational events. The average mutation rate or eruptions of variants could be different in different geographies. This helps to determine how fast-paced the outbreak is going to be. Amino acid sequences were obtained from SNPs from 5′ to 3′ UTR to analyze the mutational pattern. These mutations can be studied in cell cultures and animal hosts to check their infectivity in comparison to the authentic variants [27].

GISAID data showed that Omicron is different from other VOCs. This means that the monophyletic group of Omicron might be aligned with the gamma variant. This divergence of Omicron supports the hypothesis that it could have evolved in animals, and after attaining a high number of mutations in the spike, may have been passed back to humans from animals (reverse zoonosis) [77].

In conclusion, it is fair to say that the emergence of Omicron significantly reduced COVID hospitalizations as it has a less severe clinical presentation compared to other VOCs, although the therapeutics in clinical use face a reduction in their efficiency within a short time span. Gene sequencing and genomic platforms are still under standardization to ensure they provide robust results for many diagnostic and therapeutic platforms, which may warrant further research on these portfolios. The rapid-response nucleic acid vaccines, nevertheless, has saved millions of lives in a short period of time and the widespread immunity developed in the population undoubtedly diminished the fierce virulent response of previous VoCs. The emergence of Omicron and its subvariants, with a mild immunostimulatory response, could be a direct indication that SARS-CoV-2 is shifting to become an endemic in the near future. This will require regular surveillance and updates to the population’s immunity, as a future variant may erupt and lead to another outbreak.