SARS-CoV-2 Variants and Vaccination

1Institute of Infectious Disease, Shenzhen Bay Laboratory, Shenzhen, Guangdong, China 2Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA 3Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, USA 4Institute for Translational Sciences, University of Texas Medical Branch, Galveston, TX, USA 5Sealy Institute for Drug Discovery, University of Texas Medical Branch, Galveston, TX, USA 6Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, USA

Severe acute respiratory syndrome corona virus 2 (SARSCoV2), the pathogen responsible for coronavirus disease 2019 (COVID19), has caused a global pandemic since its emergence in Wuhan, China in late 2019 [1]. As of January 5, 2022, it has caused more than 296 million infections leading to more than 5.4 million fatalities [2]. As a positive singlestranded RNA virus whose polymerase lacks proofread ing capability, SARSCoV2 frequently mutates during viral replication, thus generating numerous viral variants with altered viral infectivity, transmission, and disease severity [3]. The original Wuhan isolate sequence of the spike glycoprotein on SARSCoV2, which binds the cellular receptor angiotensin converting enzyme 2 (ACE2), was used for vaccine develop ment. New mutations in the spike gene have continually emerged in variants over time. This Commentary (i) summarizes the key variants and their spike mutations, and (ii) discusses their implications in viral replication, transmission, and immune evasion.

DOMINANT SARS-COV-2 VARIANTS
Among all SARSCoV2 strains, some notable variants have fitness advantages and have outcompeted other variants during viral evolution. The first dominant spike (S) protein D614G substitution increases viral replication in the human upper respiratory tract; this mutant rapidly replaced nearly all prior SARSCoV2 strains from June 2020 onward [4] [1113]. According to previous studies on known mutations and clinical observations, the Omicron variant is transmitted much more rapidly than the Delta variant, with a case doubling time as short as 1.5-3 days. In addition, Omicron can escape many monoclonal antibodies and evade vaccineelicited neutralization [1416]. Furthermore, infection with previ ous non Omicron variants does not appear to elicit robust neutralization against Omicron [17]. However, booster vac cination (e.g., a third dose of the Pfizer/BioNTech vaccine or a booster dose of the Moderna vaccine) elicits good neu tralization against Omicron [18], thus supporting a booster vaccination strategy. The durability of the protective immu nity of booster vaccination against Omicron remains to be determined. Omicron's high transmissibility and immune evasion have enabled it to become the new dominant SARSCoV2 variant. More than 1 million cases in a single day have been recorded in the United States (Fig 1).

NOTABLE MUTATIONS IN THE S PROTEIN
The trimeric S glycoprotein of coronaviruses is the major surface protein present on the viral envelope [19]. Mature S protein, formed through cleavage by furin and transmem brane serine protease 2 (TMPRSS2), mediates viral binding to the ACE2 receptor, entry, and immune escape. Thus, S protein is considered the key determinant of viral infectivity and transmissibility [20]. The D614G substitution is the first dominant mutation that occurred in the S protein. Elevated quantities of D614G virus have been detected in the respira tory tracts in both patients and animal models, thus indicating this mutant's higher infectivity and transmissibility [21,22]. Another mutation, N501Y, located at the receptorbinding domain in the S protein, has been demonstrated to be the most important amino acid substitution in the Alpha vari ant. The N501Y substitution markedly increases the bind ing affinity to the human ACE2 receptor, thus leading to rapid infection in the upper respiratory tract and higher viral transmission [7,23]. The Delta variant has the spike mutation P681R, which is located at a furincleavage site in the S protein. Experimental results in human primary airway epi thelial culture and animal models have demonstrated that the P681R substitution enhances viral replication and infec tion, possibly through increasing the furin cleavage of the fulllength S protein into S1 and S2 subunits [10,24]. Many other amino acid changes in the S protein, including S13I, L18F, 69-70 deletion, W152C, K417N/T, N439K, N440K, L452R, Y453F, S477G/N/R, E484K/Q/P, S494P, and H655Y, have been reported to decrease vaccinated serum neutralization and monoclonalantibody inhibition, and/ or increase viral infection and transmission ( Table 1). The Omicron variant contains more than 30 mutations in the S protein, several of which are present in other variants of con cern, including Alpha, Beta, Gamma, and Delta [39]. Some of these mutations, including 69-70 deletion, K417N, N440K, S477N, E484A, N501Y, D614G, H655Y, and P681H, have been well studied and are known to enhance viral infectiv ity, transmissibility, and immune escape, thus leading to high concern regarding the pandemic's potential severity resulting from ongoing Omicron surges.

NEUTRALIZATION OF SARS-COV-2 VARIANTS AND VACCINE STRATEGIES
Several COVID19 vaccines have been approved and used for immunization globally to develop herd immunity against COVID19 [40]. The Pfizer/BioNTech BNT162b2 nucle osidemodified mRNA vaccine is one of the most common vaccines, which has been widely used in North America and Europe [41]. Because of the high mutation frequency of SARSCoV2 S protein, some newly emerged variants have diminished susceptibility to neutralization by antibod ies generated by vaccination or natural infection. Several approaches have been used to measure the neutralization sensitivity of variants to human sera, including pseudotype virus for expression of SARSCoV2 S protein, clinical viral isolates, and chimeric SARSCoV2 bearing the S protein from different variants [13,14,42]. Among these approaches, the use of chimeric SARSCoV2 bearing variant S has two major advantages. First, this approach does not require waiting for the isolation of clinical viral strains; as soon as the S sequence is available, the variant S sequence can be synthesized and engineered into the original SARSCoV2 backbone [43]. Second, in this approach, in contrast to the pseudovirus approach, the chimeric virus is an authentic SARSCoV2. The 50% plaquereduction neutralization titer against various recombinant viruses can be easily tested and accurately compared among all variants. The Alpha variants have higher infection and transmission efficiency, but the neutralization titers are approximately equivalent to those of the WT strain [42]. The D614G and representative Delta variants exhibit modestly lower neutralization titers than the WT virus [44,45]. Unfortunately, two doses of the Pfizer vaccine are insufficient to induce robust antibody neutralization against the Omicron variant [46]. The above studies were all performed on the same set of human sera collected 2 or 4 weeks after the administration of two doses of the Pfizer vaccine (Fig 2). Many other studies have also demonstrated that the neutralization titers of infected and vaccinated people are significantly lower against Omicron than the WT virus [17,18]. However, as described above, a third dose of the Pfizer vaccine increases the magnitude and breadth of neutralization, thus leading to robust neutrali zation against the Omicron variant [18,46,47]. To date, the

D614G
The C-terminal of S1 domain All lineages Increased infectivity [22] H655Y Near S1/S2 cleavage site Gamma, Omicron Immune escape [38] P681H/R Near S1/S2 cleavage site Alpha, Kappa, Mu, Omicron Increased cleavage efficiency [10] *Omicron is underlined to indicate multiple mutations and deletions with respect to other variants. neutralization level has been found to remain robust as long as 4 months after the third dose of the Pfizer vaccine; how ever, the durability of neutralization beyond 4 months after the third dose remains to be determined [46]. These results support a twopronged vaccine strategy against Omicron and other newly emerged variants, involving (i) booster vac cination with the currently approved safe vaccines and (ii) modification of the vaccine S sequences to match those of Omicron and new variants. The mRNA vaccine techno logy allows for rapid modification of the S sequence. Real world vaccine effectiveness data and laboratory studies are needed to guide the implementation of this twopronged vaccine strategy. Two years have elapsed since the onset of the COVID19 pandemic, and various SARSCoV2 variants, in turns, have dominated viral transmission and surges. The current Omicron surge may not be the last. Although some vari ants have become able to escape immune protection from vaccination and/or natural infection [4345,48], compelling evidence demonstrates that vaccination minimizes the risk of severe disease, and lowers the rates of hospitalization and death [49,50]. Thus, mass immunization and administration of booster shots with highly effective and safe vaccines, together with mask wearing and social distancing, will con tinue to be the most effective strategies to finally end the COVID19 pandemic.