Emerging SARS-CoV-2 variants of concern and potential intervention approaches

Spike protein mediates the virus attachment to human cell surface angiotensin converting enzyme 2 (ACE2) receptor, thus facilitating viral entry during infection [10‐12]. It is split into two subunits, S1 and S2. The S1 unit possess the receptor-binding domain (RBD) which can directly bind to ACE2 receptor and is also the dominant target of neutralizing antibodies (Ab) against SARS-CoV-2. S1 is thus considered a hotspot for mutations that may have high clinical relevance in terms of virulence, transmissibility, and host immune evasion [13‐16] (Table 2).

The Alpha variant has an N501Y mutation: at the 501 residue, N asparagine has been replaced with Y tyrosine, as well as K417N—lysine K replaced with asparagine N [9]. An emerging variant derived from B.1.1.7 also carries E484K mutation—glutamic acid E replaced with lysine K [9]. Both Beta and Gamma variants have more substitutions other than N501Y [9]. The Beta variant has E484K, while the Gamma variant has the E484K and the K417T mutations [9]. The latest major variants, Delta and Kappa, sharing two mutations E484Q (glutamic acid E substituted by glutamine Q) and L452R (leucine L altered by arginine R) were identified in India’s second COVID-19 wave. Other than the two mutations above, Delta also harbours a unique mutation, T478K (threonine T replaced by lysine K) [9].

The S1 mutations significantly increases the binding affinity to ACE2 while showing lower affinity to neutralizing antibodies [17‐21], suggesting a possible explanation for their occurring higher transmissibility and virulence [22, 23].

Another mutation at non-RBD sites, named D614G, is the most spreading mutation carried by over 99% of prevalent variants since early 2020 [23, 24]. Such mutation does not change the binding affinity to ACE2 or neutralizing Abs for the virion, yet it may increase spike density by preserving the integrity of spike and avoiding S1 shedding [25]. With more functional spikes available, D614G variants are armed with increased infectivity and hence increased replication in vitro while earlier transmission in vivo [23, 25, 26]. Recently, increasing deletions are observed in the neutralizing Ab-recognizing domain, namely recurrent deletion regions (RDRs), in the N-terminus of S1 subunit [27]. Deletions in RDRs wipe out the epitopes, and eventually aiding the virus evading host’s immune supervision and potentially defecting certain neutralizing Abs or vaccines. A majority of Alpha derived variants (ΔRDR1, S: ΔHV 69–70, & ΔRDR2, S: ΔY144), Beta derived variants (ΔRDR4, S: ΔLAL 242–244) and B.1.36 (ΔRDR3, S: ΔI210) carry this kind of mutation [27].

Two mutation hot-spots, NSP1 of ORF1a/ORF1ab, and ORF8, have been found related to the virulence and transmissibility. NSP1 is a key protein to antagonize type I interferon induction in the host and benefit the replication of the virus itself [28, 29]. ORF8 is known as an immune-evasive protein that downregulates major histocompatibility complex class I (MHC-I) in host cells [30, 31]. Recently, the Alpha variant, identified from a single immunocompromised individual, was shown to contains a premature stop codon at position 27 of ORF8[32].

Variants with partial deletion of NSP1 and ORF8 have been identified (e.g., the NSP1: Δ500-532 variant in Sichuan, China, and the ORF8: Δ382 variant in Singapore) [29, 31]. Despite that truncated NSP1 and ORF8 both contribute to milder infections [29‐31] and account for less than 5% of infections worldwide, they have become the major variants in Africa since late 2020[9].

It was shown that S-protein mutation D614G may impact SARS-CoV-2 transmissibility rate due to higher affinity for olfactory epithelium and it was shown to have higher transmissibility in animal models [33, 34]. It was also shown that it has a higher virion stability and was shown to be more resistant to proteolytic cleave as well as higher viral titer in upper airways [35, 36] suggesting that it may potentially affect virus transmissibility and virulence. Yet, it showed increased susceptibility for neutralizing antibodies and no difference in clinical severity nor hospitalization outcomes and mortality was observed [37, 38].

Evidence suggest that the VOCs Alpha and Beta increased transmissibility rate at ~ 50% especially in younger group ages and children [39, 40]. Alpha variant was shown to increase hospitalizations and mortality that may be attributed to their escape from neutralizing Abs due to their RBD mutations [41].

The Epsilon variant (B.1.427/B.1.429, California variants) increased transmissibility up to 24% with higher viral shedding, which is attributed to the of L452R spike mutation that was shown to stabilize spike-ACE2 receptor interaction [42, 43].

Although it is also suggested that other variants such as Gamma, Epsilon variants and recent Iota variants (B.1526, New York variant) may also have increased virulence due to spike mutations that increase affinity to ACE2, there is still no data available regarding viral virulence.

The new VOCs can reduce the detection sensitivity of RT-PCR based diagnostic tools especially when mutations occur in locations where probes and primers may bind [44]. Reports suggest that 79% of the primer binding sites used in the RT-PCR assay are already mutated in at least one genome with the highest significance of the GGG → AAC substitution [45]. Recent analysis which mapped primers or probes binding sites showed a cumulative variants frequency of ≥ 1% in the global SARS-CoV-2 genomes [46]. The Alpha lineage was shown to have higher false-negative results when using specific commercial kits directed to the spike (S) gene but not when using standard protocols such as Berlin-Cherite protocol since it does not involve the S protein-encoding gene as target [47]. Another concern is a variant detected in France of a S deletion (ΔH69-V70) which has shown to be associated with S-gene target gene detection failure in three-target RT-PCR [48]. Several reports have targeted mutations in different open reading frames (ORFs) especially ORF8 position which was found in some isolates from Mexico, Belize and Guatemala as potentially leading to epitope loss and reduced sensitivity for serological testing [49‐51].

On the other hand, other studies showed that although mismatches in the primer/probes binding regions of SARS-CoV-2 diagnostic assays can be detected in different SARS-CoV-2 variants, they were tolerated and did not result in reduced assay performance and false-negative results [52, 53]. Moreover, according to bioinformatic analysis performed, the known variability occurring in the SARS-CoV-2 population have minimal or no effect on the sensitivity existing diagnostic tools for viral detection [54, 55].

Still, the continuous emergence of SARS-CoV-2 variants and possible mismatches highlight the importance of global molecular surveillance and designing diagnostic strategies such as combining diagnostic methods during future outbreaks or perform assays that target two or more positions in highly conserved regions of the viral genome to promote higher specificity and sensitivity results as well as developing highly specific diagnostic tools using CRISPR [56, 57].

Currently, all vaccines are based on introducing spike protein, which is the major superficial virulence of SARS-CoV-2, using the reference genome isolates early in the pandemic. As there is no sufficient evidence to support the effect of vaccines against Delta and Kappa variants, we’ll focus on the Alpha, Beta and Kappa variants.

Whether specifically targeting spike proteins using small peptide-based therapies or using single-domains neutralizing antibodies against any of those targets, these therapeutic strategies efficiency may be compromised by the emergence of SARS-CoV-2 variants especially those possessing spike proteins and RBD mutations that increase affinity to ACE2 such as Alpha, and Iota variant, by potentially escaping neutralizing antibodies and competing with those agents for the same binding targets [73‐75]. In order to avoid antibody escape, strategies to combine different neutralizing antibody cocktail have been suggested as a therapeutic approach against the emerging variants [76]. Other treatments such as anti-RBD nanobodies isolated from llamas were shown to neutralize RBD variants suggesting they might be a promising tool against new SARS-CoV-2 VOCs as well [77, 78].

Different engineered variants of human recombinant soluble ACE2 (hrACE2), were reported to significantly inhibit SARS-CoV-2 infection in vitro and causing sustained viral entry blockade upon engagement of hrACE2 with the RBD in SARS-CoV-2 S protein with high affinity [79‐81]. This is a potentially powerful treatment against SARS-CoV-2 VOCs as it can exploit the increase S-protein host receptor-binding affinity caused by S-mutations, toward increasing S-protein affinity to hrACE2. Moreover, no mutations that limit receptor-binding affinity were discovered as this will decrease affinity to native ACE2 receptor and may likely to attenuate virulence [82], suggesting that viral escape from hrACE is very unlikely.

Targeting endosomal formation of SARS-CoV-2 to block entry to host cells such as antimalarial drugs and macrolides, and us of drugs targeting host cell transmembrane protease serine 2 (TMPRSS2) such as Camostat [83‐85] or A disintegrin and metalloprotease 17 (ADAM17) inhibitors [86].

Promising antiviral drugs such as the FDA-approved Remdesivir and its metabolites, Ribaverin and Galidesivir have been shown to inhibit viral replication in vitro and in vivo studies due to their effect on inhibiting RNA dependent RNA polymerase (RdRp) [87, 88]. The discovery of RdRp hotspot mutations in SARS-CoV-2, found mostly in European strains may lead to drug-resistance of to RdRp inhibitors in a similar mechanism found in Influenza and Hepatitis C [89‐91]. However, it has been shown currently that those variants have minimal impact for pre-existing resistance to Remdesivir.

Another potential approach is Prophylactic Antiviral CRISPR in Human Cells (PAC-MAN), which is a Cas13d-based strategy that target reserved regions such as nucleocapsid protein and RdRp in SARS-CoV-2 viral genome and may serve as pan-coronavirus strategy for any future coronaviruses and variant that may emerge [92].