Genomic Diversity of SARS-CoV-2 Omicron Variant in South American Countries

Genomic surveillance of SARS-CoV-2 is one of the tools that provide genomic information on circulating variants. Given the recent emergence of the Omicron (B.1.1.529) variant, this tool has provided data about this lineage’s genomic and epidemiological characteristics. However, in South America, this variant’s arrival and genomic diversity are scarcely known. Therefore, this study determined the genomic diversity and phylogenetic relationships of 21,615 Omicron genomes available in public databases. We found that in South America, BA.1 (n = 15,449, 71%) and BA.1.1 (n = 6257, 29%) are the dominant sublineages, with several mutations that favor transmission and antibody evasion. In addition, these lineages showed cryptic transmission arriving on the continent in late September 2021. This event may have contributed to the dispersal of Omicron sublineages and the acquisition of new mutations. Considering the genomic and epidemiological characteristics of these lineages, especially those with a high number of mutations in their genome, it is important to conduct studies and surveillance on the dynamics of these lineages to identify the mechanisms of mutation acquisition and their impact on public health.     

Isolation and Genomic Characterization of SARS-CoV-2 Omicron Variant Obtained from Human Clinical Specimens

Due to the failure of virus isolation of the Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, an initial in vivo and subsequent in vitro approach was utilized for the isolation of the virus. A total of 74 oropharyngeal/nasopharyngeal specimens were collected from SARS-CoV-2 positive international travellers and a contact case at Delhi and Mumbai, India. All the specimens were sequenced using next-generation sequencing and simultaneously inoculated onto Vero CCL-81 cells for virus isolation. Subsequently, two omicron positive specimens were inoculated into Syrian hamsters for two passages. The initial passage of the positive hamster specimens was inoculated onto Vero CCL-81 cells. The clinical specimens, hamster specimens, and Vero CCL-81 passages were sequenced to assess the mutational changes in different host species. The replication of the Omicron variant in hamsters was confirmed with the presence of a high viral load in nasal turbinate and lung specimens of both passages. The successful isolation of the virus from hamster specimens with Vero CCL-81 was observed with cytopathic effect in infected cells and high viral load in the cell suspension. The genome analysis revealed the presence of L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene of hamster passage sequences and an absence of V17I mutation in E gene in hamster passage sequences, unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequences, suggesting intact pathogenicity of the virus isolate. Our data demonstrated successful isolation of the Omicron variant with the in vivo method first followed by in vitro method. The virus isolate could be used in the future to explore different aspects of the Omicron variant.     

Analytical Sensitivity of Six SARS-CoV-2 Rapid Antigen Tests for Omicron versus Delta Variant

Rapid antigen detection (RAD) tests are commonly used for the diagnosis of SARS-CoV-2 infections. However, with the continuous emergence of new variants of concern (VOC), presenting various mutations potentially affecting the nucleocapsid protein, the analytical performances of these assays should be frequently reevaluated. One hundred and twenty samples were selected and tested with both RT-qPCR and six commercial RAD tests that are commonly sold in Belgian pharmacies. Of these, direct whole-genome sequencing identified the strains present in 116 samples, of which 70 were Delta and 46 were Omicron (BA.1 and BA.1.1 sub-lineages, respectively). The sensitivity across a wide range of Ct values (13.5 to 35.7; median = 21.3) ranged from 70.0% to 92.9% for Delta strains and from 69.6% to 78.3% for Omicron strains. When taking swabs with a low viral load (Ct > 25, corresponding to <4.9 log₁₀ copies/mL), only the Roche RAD test showed acceptable performances for the Delta strains (80.0%), while poor performances were observed for the other RAD tests (20.0% to 40.0%). All the tested devices had poor performances for the Omicron samples with a low viral load (0.0% to 23.1%). The poor performances observed with low viral loads, particularly for the Omicron strain, is an important limitation of RAD tests, which is not sufficiently highlighted in the instructions for use of these devices.     

SARS-CoV-2 Omicron Variant,Lineage BA.1, Is Associated with Lower Viral Load in Nasopharyngeal Samples Compared to Delta Variant

Objectives: High viral load in upper respiratory tract specimens observed for Delta cases might contribute to its increased infectivity compared to the other variant. However, it is not yet documented if the Omicron variant’s enhanced infectivity is also related to a higher viral load. Our aim was to determine if the Omicron variant’s spread is also related to higher viral loads compared to the Delta variant. Methods: Nasopharyngeal swabs, 129 (Omicron) and 85 (Delta), from Health Care Workers were collected during December 2021 at the University Hospital of Lyon, France. Cycle threshold (Ct) for the RdRp target of cobas^® 6800 SARS-CoV-2 assay was used as a proxy to evaluate SARS-CoV-2 viral load. Variant identification was performed using a screening panel and confirmed by whole genome sequencing. Results: Herein, we showed that the RT-PCR Ct values in Health Care Workers sampled within 5 days after symptom onset were significantly higher for Omicron cases than Delta cases (21.7 for Delta variant and 23.8 for Omicron variant, p = 0.008). This difference was also observed regarding patient with complete vaccination. Conclusions: This result supports the studies showing that the increased transmissibility of Omicron is related to other mechanisms than higher virus excretion.     

The Evolutionary Landscape of SARS-CoV-2 Variant B.1.1.519 and Its Clinical Impact in Mexico City

The SARS-CoV-2 pandemic is one of the most concerning health problems around the globe. We reported the emergence of SARS-CoV-2 variant B.1.1.519 in Mexico City. We reported the effective reproduction number (Rt) of B.1.1.519 and presented evidence of its geographical origin based on phylogenetic analysis. We also studied its evolution via haplotype analysis and identified the most recurrent haplotypes. Finally, we studied the clinical impact of B.1.1.519. The B.1.1.519 variant was predominant between November 2020 and May 2021, reaching 90% of all cases sequenced in February 2021. It is characterized by three amino acid changes in the spike protein: T478K, P681H, and T732A. Its Rt varies between 0.5 and 2.9. Its geographical origin remain to be investigated. Patients infected with variant B.1.1.519 showed a highly significant adjusted odds ratio (aOR) increase of 1.85 over non-B.1.1.519 patients for developing a severe/critical outcome (p = 0.000296, 1.33–2.6 95% CI) and a 2.35-fold increase for hospitalization (p = 0.005, 1.32–4.34 95% CI). The continuous monitoring of this and other variants will be required to control the ongoing pandemic as it evolves.     

上海某方舱医院新型冠状病毒奥密克戎变异株感染者流行病学特征分析

[摘要]　目的　探讨上海某方舱医院新型冠状病毒奥密克戎变异株感染者的发病情况及流行病学特征。方法　以2022年4月9日—5月5日上海国家会展中心方舱医院收治的新型冠状病毒奥密克戎变异株感染者为研究对象,对感染者年龄、性别、地区、疫苗接种等疫情数据进行流行病学特征分析。结果　122 151例新型冠状病毒感染者均为奥密克戎变异株BA.2或BA.2.2亚型感染,其流行病学特征结果显示：患者男女比例为1.51:1;平均年龄为（44.91±15.38）岁;0～17岁、18～30岁、31～60岁和≥61岁感染者分别占4.74%、20.80%、62.52%和11.94%;无症状感染者占80.80%,轻型患者占19.20%;平均住院时间为（7.00±2.77）d;未接种和完成1、2和3次疫苗接种的感染者分别占20.30%、3.18%、31.30%和45.22%,其中≥61岁且完成3次疫苗接种的感染者仅占10.10%。结论　各个年龄段人群对于新型冠状病毒奥密克戎变异株普遍易感。无症状感染者是本次疫情的主体人群,临床症状不典型,早期隐匿传播,积极加强核酸检测是早期发现疫情的必要手段。     

Clinical Evaluation of a Fully-Automated High-Throughput Multiplex Screening-Assay to Detect and Differentiate the SARS-CoV-2 B.1.1.529 (Omicron) and B.1.617.2 (Delta) Lineage Variants

Background: The recently emerged SARS-CoV-2 B.1.1.529 lineage and its sublineages (Omicron variant) pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available monoclonal antibody therapies. RT-PCR-based variant tests can be used to screen large sample-sets rapidly and accurately for relevant variants of concern (VOC). The aim of this study was to establish and validate a multiplex assay on the cobas 6800/8800 systems to allow discrimination between the two currently circulating VOCs, Omicron and Delta, in clinical samples. Methods: Primers and probes were evaluated for multiplex compatibility. Analytic performance was assessed using cell culture supernatant of an Omicron variant isolate and a clinical Delta variant sample, normalized to WHO-Standard. Clinical performance of the multiplex assay was benchmarked against NGS results. Results: In silico testing of all oligos showed no interactions with a high risk of primer-dimer formation or amplification of human DNA/RNA. Over 99.9% of all currently available Omicron variant sequences are a perfect match for at least one of the three Omicron targets included in the multiplex. Analytic sensitivity was determined as 19.0 IU/mL (CI95%: 12.9–132.2 IU/mL) for the A67V + del-HV69-70 target, 193.9 IU/mL (CI95%: 144.7–334.7 IU/mL) for the E484A target, 35.5 IU/mL (CI95%: 23.3–158.0 IU/mL) for the N679K + P681H target and 105.0 IU/mL (CI95%: 80.7–129.3 IU/mL) for the P681R target. All sequence variances were correctly detected in the clinical sample set (225/225 Targets). Conclusion: RT-PCR-based variant screening compared to whole genome sequencing is both rapid and reliable in detecting relevant sequence variations in SARS-CoV-2 positive samples to exclude or verify relevant VOCs. This allows short-term decision-making, e.g., for patient treatment or public health measures.     

Early Emergence Phase of SARS-CoV-2 Delta Variant in Florida,US

SARS-CoV-2, the causative agent of COVID-19, emerged in late 2019. The highly contagious B.1.617.2 (Delta) variant of concern (VOC) was first identified in October 2020 in India and subsequently disseminated worldwide, later becoming the dominant lineage in the US. Understanding the local transmission dynamics of early SARS-CoV-2 introductions may inform actionable mitigation efforts during subsequent pandemic waves. Yet, despite considerable genomic analysis of SARS-CoV-2 in the US, several gaps remain. Here, we explore the early emergence of the Delta variant in Florida, US using phylogenetic analysis of representative Florida and globally sampled genomes. We find multiple independent introductions into Florida primarily from North America and Europe, with a minority originating from Asia. These introductions led to three distinct clades that demonstrated varying relative rates of transmission and possessed five distinct substitutions that were 3–21 times more prevalent in the Florida sample as compared to the global sample. Our results underscore the benefits of routine viral genomic surveillance to monitor epidemic spread and support the need for more comprehensive genomic epidemiology studies of emerging variants. In addition, we provide a model of epidemic spread of newly emerging VOCs that can inform future public health responses.     

Comparative Dynamics of Delta and Omicron SARS-CoV-2 Variants across and between California and Mexico

Evolutionary analysis using viral sequence data can elucidate the epidemiology of transmission. Using publicly available SARS-CoV-2 sequence and epidemiological data, we developed discrete phylogeographic models to interrogate the emergence and dispersal of the Delta and Omicron variants in 2021 between and across California and Mexico. External introductions of Delta and Omicron in the region peaked in early July (2021-07-10 [95% CI: 2021-04-20, 2021-11-01]) and mid-December (2021-12-15 [95% CI: 2021-11-14, 2022-01-09]), respectively, 3 months and 2 weeks after first detection. These repeated introductions coincided with domestic migration events with no evidence of a unique transmission hub. The spread of Omicron was most consistent with gravity centric patterns within Mexico. While cross-border events accounted for only 5.1% [95% CI: 4.3–6] of all Delta migration events, they accounted for 20.6% [95% CI: 12.4–29] of Omicron movements, paralleling the increase in international travel observed in late 2021. Our investigations of the Delta and Omicron epidemics in the California/Mexico region illustrate the complex interplay and the multiplicity of viral and structural factors that need to be considered to limit viral spread, even as vaccination is reducing disease burden. Understanding viral transmission patterns may help intra-governmental responses to viral epidemics.     

Sequencing during Times of Change: Evaluating SARS-CoV-2 Clinical Samples during the Transition from the Delta to Omicron Wave

The pandemic of SARS-CoV-2 is characterized by the emergence of new variants of concern (VOCs) that supplant previous waves of infection. Here, we describe our investigation of the lineages and host-specific mutations identified in a particularly vulnerable population of predominantly older and immunosuppressed SARS-CoV-2-infected patients seen at our medical center in Chicago during the transition from the Delta to Omicron wave. We compare two primer schemes, ArticV4.1 and VarSkip2, used for short read amplicon sequencing, and describe our strategy for bioinformatics analysis that facilitates identifying lineage-associated mutations and host-specific mutations that arise during infection. This study illustrates the ongoing evolution of SARS-CoV-2 VOCs in our community and documents novel constellations of mutations that arise in individual patients. The ongoing evaluation of the evolution of SARS-CoV-2 during this pandemic is important for informing our public health strategies.     

Characterization of a Lineage C.36 SARS-CoV-2 Isolate with Reduced Susceptibility to Neutralization Circulating in Lombardy,Italy

SARS-CoV-2 spike is evolving to maximize transmissibility and evade the humoral response. The massive genomic sequencing of SARS-CoV-2 isolates has led to the identification of single-point mutations and deletions, often having the recurrence of hotspots, associated with advantageous phenotypes. We report the isolation and molecular characterization of a SARS-CoV-2 strain, belonging to a lineage (C.36) not previously associated with concerning traits, which shows decreased susceptibility to vaccine sera neutralization.     

The Spread of SARS-CoV-2 Variant Omicron with a Doubling Time of 2.0–3.3 Days Can Be Explained by Immune Evasion

Omicron, the novel highly mutated SARS-CoV-2 Variant of Concern (VOC, Pango lineage B.1.1.529) was first collected in early November 2021 in South Africa. By the end of November 2021, it had spread and approached fixation in South Africa, and had been detected on all continents. We analyzed the exponential growth of Omicron over four-week periods in the two most populated of South Africa’s provinces, Gauteng and KwaZulu-Natal, arriving at the doubling time estimates of, respectively, 3.3 days (95% CI: 3.2–3.4 days) and 2.7 days (95% CI: 2.3–3.3 days). Similar or even shorter doubling times were observed in other locations: Australia (3.0 days), New York State (2.5 days), UK (2.4 days), and Denmark (2.0 days). Log–linear regression suggests that the spread began in Gauteng around 11 October 2021; however, due to presumable stochasticity in the initial spread, this estimate can be inaccurate. Phylogenetics-based analysis indicates that the Omicron strain started to diverge between 6 October and 29 October 2021. We estimated that the weekly growth of the ratio of Omicron to Delta is in the range of 7.2–10.2, considerably higher than the growth of the ratio of Delta to Alpha (estimated to be in in the range of 2.5–4.2), and Alpha to pre-existing strains (estimated to be in the range of 1.8–2.7). High relative growth does not necessarily imply higher Omicron infectivity. A two-strain SEIR model suggests that the growth advantage of Omicron may stem from immune evasion, which permits this VOC to infect both recovered and fully vaccinated individuals. As we demonstrated within the model, immune evasion is more concerning than increased transmissibility, because it can facilitate larger epidemic outbreaks.     

Epidemiological characterisation of the first 785 SARS-CoV-2 Omicron variant cases in Denmark,December 2021

By 9 December 2021, 785 SARS-CoV-2 Omicron variant cases have been identified in Denmark. Most cases were fully (76%) or booster-vaccinated (7.1%); 34 (4.3%) had a previous SARS-CoV-2 infection. The majority of cases with available information reported symptoms (509/666; 76%) and most were infected in Denmark (588/644; 91%). One in five cases cannot be linked to previous cases, indicating widespread community transmission. Nine cases have been hospitalised, one required intensive care and no deaths have been registered.     

An ACE2-Based Decoy Inhibitor Effectively Neutralizes SARS-CoV-2 Omicron BA.5 Variant

The recently circulating SARS-CoV-2 Omicron BA.5 is rampaging the world with elevated transmissibility compared to the original SARS-CoV-2 strain. Immune escape of BA.5 was observed after treatment with many monoclonal antibodies, calling for broad-spectrum, immune-escape-evading therapeutics. In retrospect, we previously reported Kansetin as an ACE2 mimetic and a protein antagonist against SARS-CoV-2, which proved potent neutralization bioactivity on the Reference, Alpha, Beta, Delta, and Omicron strains of SARS-CoV-2. Since BA.5 is expected to rely on the interaction of the Spike complex with human ACE2 for cell entry, we reasonably assumed the lasting efficacy of the ACE2-mimicking Kansetin for neutralizing the new SARS-CoV-2 variant. The investigation was accordingly performed on in vitro Kansetin-Spike binding affinity by SPR and cell infection inhibition ability with pseudovirus and live virus assays. As a result, Kansetin showed dissociation constant K_D and half inhibition concentration IC₅₀ at the nanomolar to picomolar level, featuring a competent inhibition effect against the BA.5 sublineage. Conclusively, Kansetin is expected to be a promising therapeutic option against BA.5 and future SARS-CoV-2 sublineages.     

Dominance of Three Sublineages of the SARS-CoV-2 Delta Variant in Mexico

In this study, we analyzed the sequences of SARS-CoV-2 isolates of the Delta variant in Mexico, which has completely replaced other previously circulating variants in the country due to its transmission advantage. Among all the Delta sublineages that were detected, 81.5 % were classified as AY.20, AY.26, and AY.100. According to publicly available data, these only reached a world prevalence of less than 1%, suggesting a possible Mexican origin. The signature mutations of these sublineages are described herein, and phylogenetic analyses and haplotype networks are used to track their spread across the country. Other frequently detected sublineages include AY.3, AY.62, AY.103, and AY.113. Over time, the main sublineages showed different geographical distributions, with AY.20 predominant in Central Mexico, AY.26 in the North, and AY.100 in the Northwest and South/Southeast. This work describes the circulation, from May to November 2021, of the primary sublineages of the Delta variant associated with the third wave of the COVID-19 pandemic in Mexico and highlights the importance of SARS-CoV-2 genomic surveillance for the timely identification of emerging variants that may impact public health.     

Impact of the Omicron variant on SARS-CoV-2 reinfections in France,March 2021 to February 2022

Since the first reports in summer 2020, SARS-CoV-2 reinfections have raised concerns about the immunogenicity of the virus, which will affect SARS-CoV-2 epidemiology and possibly the burden of COVID-19 on our societies in the future. This study provides data on the frequency and characteristics of possible reinfections, using the French national COVID-19 testing database. The Omicron variant had a large impact on the frequency of possible reinfections in France, which represented 3.8% of all confirmed COVID-19 cases since December 2021.     

Omicron mutations enhance infectivity and reduce antibody neutralization of SARS-CoV-2 virus-like particles

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant contains extensive sequence changes relative to the earlier-arising B.1, B.1.1, and Delta SARS-CoV-2 variants that have unknown effects on viral infectivity and response to existing vaccines. Using SARS-CoV-2 virus-like particles (VLPs), we examined mutations in all four structural proteins and found that Omicron and Delta showed 4.6-fold higher luciferase delivery overall relative to the ancestral B.1 lineage, a property conferred mostly by enhancements in the S and N proteins, while mutations in M and E were mostly detrimental to assembly. Thirty-eight antisera samples from individuals vaccinated with Pfizer/BioNTech, Moderna, or Johnson & Johnson vaccines and convalescent sera from unvaccinated COVID-19 survivors had 15-fold lower efficacy to prevent cell transduction by VLPs containing the Omicron mutations relative to the ancestral B.1 spike protein. A third dose of Pfizer vaccine elicited substantially higher neutralization titers against Omicron, resulting in detectable neutralizing antibodies in eight out of eight subjects compared to one out of eight preboosting. Furthermore, the monoclonal antibody therapeutics casirivimab and imdevimab had robust neutralization activity against B.1 and Delta VLPs but no detectable neutralization of Omicron VLPs, while newly authorized bebtelovimab maintained robust neutralization across variants. Our results suggest that Omicron has similar assembly efficiency and cell entry compared to Delta and that its rapid spread is due mostly to reduced neutralization in sera from previously vaccinated subjects. In addition, most currently available monoclonal antibodies will not be useful in treating Omicron-infected patients with the exception of bebtelovimab.

Understanding the molecular determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral fitness is central to effective vaccine and therapeutic development. The emergence of viral variants including Delta and Omicron underscores the need to assess both infectivity and antibody neutralization, but biosafety level 3 handling requirements slow the pace of research on intact SARS-CoV-2. Although vesicular stomatitis virus and lentivirus pseudotyped with the SARS-CoV-2 spike (S) protein enable evaluation of S-mediated cell binding and entry via the ACE2 and TMPRSS2 receptors, they cannot determine effects of mutations outside the S gene (1, 2). To address these challenges, we developed SARS-CoV-2 virus-like particles (SC2-VLPs) comprising the S, N, M, and E structural proteins and a packaging signal-containing messenger RNA (mRNA) that together form RNA-loaded capsids capable of spike-dependent cell transduction (3). This system faithfully reflects the impact of mutations in structural proteins that are observed in infections with virus isolates, enabling rapid testing of SARS-CoV-2 structural gene variants for their impact on both infection efficiency and antibody or antiserum neutralization.     

珠海市新型冠状病毒奥密克戎变异株感染者心肌损害情况及心电图特征

目的]探索珠海市新型冠状病毒(SARS-CoV-2)奥密克戎变异株感染者心肌损害情况及异常心电图特征。 [方法]纳入2022年1月13日—3月20日中山大学附属第五医院收治的84例SARS-CoV-2奥密克戎变异株感染者(奥密克戎变异株组),以及2020年1月17日—2月17日收治的88例SARS-CoV-2非奥密克戎变异株感染者(非奥密克戎变异株组),采用回顾性研究比较两组患者的心肌损害情况、异常心电图情况及临床特征差异。 [结果]奥密克戎变异株组患者平均年龄小于非奥密克戎变异株组[(36.6±15.6)岁比(49.8±14.3)岁,P＜0.01],入院时体温、入院时收缩压及发热患者比例均低于非奥密克戎变异株组(P＜0.05)。奥密克戎变异株组患者中性粒细胞/淋巴细胞比值[(2.93(3.03,5.81)比7.06(2.2,1.27),P＜0.001]、白细胞介素2(IL-2)及白细胞介素6(IL-6)水平均明显低于非奥密克戎变异株组(P＜0.01)。奥密克戎变异株组心肌肌钙蛋白I(cTnI)均为阴性,cTnI、肌酸激酶同工酶(CK-MB)、N末端脑钠肽前体(NT-proBNP)浓度及其升高患者比例均明显低于非奥密克戎变异株组(P＜0.01)。奥密克戎变异株组患者异常心电图发生率亦明显低于非奥密克戎变异株组(25.0%比42.0%,P＝0.001),以窦性心动过速、房性早搏及T波改变为主,非奥密克戎变异株组患者以房性早搏、T波改变、ST段压低及束支传导阻滞为主。 [结论]珠海市SARS-CoV-2奥密克戎变异株感染者可能因绝大部分患者已接种新型冠状病毒疫苗而没有出现明显心肌损害,异常心电图发生率亦明显低于非奥密克戎变异株组,主要以窦性心动过速、房性早搏及T波改变为主。     

Effect of Previous COVID-19 Vaccination on Humoral Immunity 3 Months after SARS-CoV-2 Omicron Infection and Booster Effect of a Fourth COVID-19 Vaccination 2 Months after SARS-CoV-2 Omicron Infection

In this study, we aimed to determine the effect of COVID-19 vaccination on 3-month immune response and durability after natural infection by the Omicron variant and to assess the immune response to a fourth dose of COVID-19 vaccination in patients with prior natural infection with the Omicron variant. Overall, 86 patients aged ≥60 years with different vaccination histories and 39 health care workers (HCWs) vaccinated thrice before Omicron infection were enrolled. The sVNT50 titer was significantly lower in patients with incomplete vaccination before SARS-CoV-2 infection with the S clade (p < 0.001), Delta variant (p < 0.001), or Omicron variant (p = 0.003) than in those vaccinated thrice. The sVNT results against the Omicron variant did not differ significantly in patients aged ≥60 years (p = 0.49) and HCWs (p = 0.17), regardless of the recipient receiving the fourth dose 2 months after COVID-19. Incomplete COVID-19 vaccination before Omicron infection for individuals aged ≥60 years conferred limited protection against homologous and heterologous virus strains, whereas two or three doses of the vaccine provided cross-variant humoral immunity against Omicron infection for at least 3 months. However, a fourth dose 2 months after Omicron infection did not enhance immunity against the homologous strain. A future strategy using the bivalent Omicron-containing booster vaccine with appropriate timing will be crucial.