SARS-CoV-2 Omicron XBB lineage spike structures, conformations, antigenicity, and receptor recognition.

A recombinant lineage of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant, named XBB, appeared in late 2022 and evolved descendants that successively swept local and global populations. XBB lineage members were noted for their improved immune evasion and transmissibility. Here, we determine cryoelectron microscopy (cryo-EM) structures of XBB.1.5, XBB.1.16, EG.5, and EG.5.1 spike (S) ectodomains to reveal reinforced 3-receptor binding domain (RBD)-down receptor-inaccessible closed states mediated by interprotomer RBD interactions previously observed in BA.1 and BA.2. Improved XBB.1.5 and XBB.1.16 RBD stability compensated for stability loss caused by early Omicron mutations, while the F456L substitution reduced EG.5 RBD stability. S1 subunit mutations had long-range impacts on conformation and epitope presentation in the S2 subunit. Our results reveal continued S protein evolution via simultaneous optimization of multiple parameters, including stability, receptor binding, and immune evasion, and the dramatic effects of relatively few residue substitutions in altering the S protein conformational landscape.

Protection of the receptor binding domain (RBD) dimer against SARS-CoV-2 and its variants.

IMPORTANCE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants achieved immune escape and became less virulent and easily transmissible through rapid mutation in the spike protein, thus the efficacy of vaccines on the market or in development continues to be challenged. Updating the vaccine, exploring compromise vaccination strategies, and evaluating the efficacy of candidate vaccines for the emerging variants in a timely manner are important to combat complex and volatile SARS-CoV-2. This study reports that vaccines prepared from the dimeric receptor-binding domain (RBD) recombinant protein, which can be quickly produced using a mature and stable process platform, had both good immunogenicity and protection in vivo and could completely protect rodents from lethal challenge by SARS-CoV-2 and its variants, including the emerging Omicron XBB.1.16, highlighting the value of dimeric recombinant vaccines in the post-COVID-19 era.

Integrative Genome-Based Survey of the SARS-CoV-2 Omicron XBB.1.16 Variant.

The XBB.1.16 SARS-CoV-2 variant, also known as Arcturus, is a recent descendant lineage of the recombinant XBB (nicknamed Gryphon). Compared to its direct progenitor, XBB.1, XBB.1.16 carries additional spike mutations in key antigenic sites, potentially conferring an ability to evade the immune response compared to other circulating lineages. In this context, we conducted a comprehensive genome-based survey to gain a detailed understanding of the evolution and potential dangers of the XBB.1.16 variant, which became dominant in late June. Genetic data indicates that the XBB.1.16 variant exhibits an evolutionary background with limited diversification, unlike dangerous lineages known for rapid changes. The evolutionary rate of XBB.1.16, which amounts to 3.95 × 10-4 subs/site/year, is slightly slower than that of its direct progenitors, XBB and XBB.1.5, which have been circulating for several months. A Bayesian Skyline Plot reconstruction suggests that the peak of genetic variability was reached in early May 2023, and currently, it is in a plateau phase with a viral population size similar to the levels observed in early March. Structural analyses indicate that, overall, the XBB.1.16 variant does not possess structural characteristics markedly different from those of the parent lineages, and the theoretical affinity for ACE2 does not seem to change among the compared variants. In conclusion, the genetic and structural analyses of SARS-CoV-2 XBB.1.16 do not provide evidence of its exceptional danger or high expansion capability. Detected differences with previous lineages are probably due to genetic drift, which allows the virus constant adaptability to the host, but they are not necessarily connected to a greater danger. Nevertheless, continuous genome-based monitoring is essential for a better understanding of its descendants and other lineages.

Distinctive Features of the XBB.1.5 and XBB.1.16 Spike Protein Receptor-Binding Domains and Their Roles in Conformational Changes and Angiotensin-Converting Enzyme 2 Binding.

The emergence and the high transmissibility of the XBB.1.5 and XBB.1.16 subvariants of the SARS-CoV-2 omicron has reignited concerns over the potential impact on vaccine efficacy for these and future variants. We investigated the roles of the XBB.1.5 and XBB.1.16 mutations on the structure of the spike protein's receptor-binding domain (RBD) and its interactions with the host cell receptor ACE2. To bind to ACE2, the RBD must transition from the closed-form to the open-form configuration. We found that the XBB variants have less stable closed-form structures that may make the transition to the open-form easier. We found that the mutations enhance the RBD-ACE2 interactions in XBB.1.16 compared to XBB.1.5. We observed significant structural changes in the loop and motif regions of the RBD, altering well-known antibody-binding sites and potentially rendering primary RBD-specific antibodies ineffective. Our findings elucidate how subtle structural changes and interactions contribute to the subvariants' fitness over their predecessors.

Virological and Mitochondriopathogical Characteristics of the SARS-CoV-2 Omicron XBB.1.16 Spike.

The emergence of XBB.1.16 has gained rapid global prominence. Previous studies have elucidated that the infection of SARS-CoV-2 induces alterations in the mitochondrial integrity of host cells, subsequently influencing the cellular response to infection. In this study, we compared the differences in infectivity and pathogenicity between XBB.1.16 and the parental Omicron sublineages BA.1 and BA.2 and assessed their impact on host mitochondria. Our findings suggest that, in comparison with BA.1 and BA.2, XBB.1.16 exhibits more efficient spike protein cleavage, more efficient mediating syncytia formation, mild mitochondriopathy, and less pathogenicity. Altogether, our investigations suggest that, based on the mutation of key sites, XBB.1.16 exhibited enhanced infectivity but lower pathogenicity. This will help us to further investigate the biological functions of key mutation sites.

XBB.1.16-RBD-based trimeric protein vaccine can effectively inhibit XBB.1.16-included XBB subvariant infection.

The newly identified XBB.1.16-containing sublineages, including XBB.1.5, have become the prevailing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant in circulation. Unlike previous Omicron XBB variants (e.g., XBB.1.5 and XBB.1.9) harboring the F486P substitution, XBB.1.16 also carries a T478R substitution in the receptor-binding domain (RBD). Numerous researchers have delved into the high transmissibility and immune evasion of XBB.1.16 subvariant. Therefore, developing a new vaccine targeting XBB.1.16, including variants of concern (VOCs), is paramount. In our study, we engineered a recombinant protein by directly linking the S-RBD sequence of the XBB.1.16 strain of SARS-CoV-2 to the sequences of two heptad repeat sequences (HR1 and HR2) from the SARS-CoV-2 S2 subunit. Named the recombinant RBDXBB.1.16-HR/trimeric protein, this fusion protein autonomously assembles into a trimer. Combined with an MF59-like adjuvant, the RBDXBB.1.16-HR vaccine induces a robust humoral immune response characterized by high titers of neutralizing antibodies against variant pseudovirus and authentic VOCs and cellular immune responses. Additionally, a fourth heterologous RBDXBB.1.16-HR vaccine enhances both humoral and cellular immune response elicited by three-dose mRNA vaccines. These findings demonstrate that the recombinant RBDXBB.1.16-HR protein, featuring the new T478R mutation, effectively induces solid neutralizing antibodies to combat newly emerged XBB variants.

Impact of mutations in SARS-CoV-2 recombinant sub-variant XBB.1.16 on the binding affinity with human ACE2 receptor.

Despite the waning threat of the COVID-19 pandemic, its detrimental impact on global health persists. Regardless of natural immunity or immunity obtained through vaccination, emerging variants of the virus continue to undergo mutations and propagate globally. The persistent mutations in SARS-CoV-2, along with the subsequent formation of recombinant sub-variants has become a challenge for researchers and health professionals, raising concerns about the efficacy of current vaccines. Gaining a better understanding of the biochemical interactions between the Spike Protein (RBD) of SARS-CoV-2 variants and the human ACE2 receptor can prove to be beneficial in designing and developing antiviral therapeutics that are equally effective against all strains and emerging variants. Our objective in this study was to investigate the interfacial binding pattern of the SARS-CoV-2 RBD-ACE2 complex of the Wild Type (WT), Omicron, and the Omicron recombinant sub-variant XBB.1.16. We aimed to examine the atomic level factors and observe how mutations influence the interaction between the virus and its host using Molecular Dynamics simulation, MM/GBSA energy calculations, and Principal Component Analysis. Our findings reveal a higher degree of structural deviation and flexibility in XBB.1.16 compared to WT and Omicron. PCA indicated a wider cluster and significant flexibility in the movements of XBB.1.16 which can also be observed in free energy landscapes, while the normal mode analysis revealed converging motions within the RBD-ACE2 complexes which can facilitate the interaction between them. A pattern of decreased binding affinity was observed in case of XBB.1.16 when compared to the WT and Omicron. These observed deviations in XBB.1.16 when compared to its parent lineage Omicron, and WT can be attributed to the mutations specific to it. Collectively, these results enhance our understanding of the impact of mutations on the interaction between this strain and the host, taking us one step closer to designing effective antiviral therapeutics against the continually mutating strains.

Genomic surveillance of SARS-CoV-2 and emergence of XBB.1.16 variant in Rajasthan.

PURPOSE: Genomic surveillance of positive SARS-CoV-2 samples is important to monitor the genetic changes occurring in virus, this was enhanced after the WHO designation of XBB.1.16 as a variant under monitoring in March 2023. From 5th February till May 6, 2023 all positive SARS-CoV-2 samples were monitored for genetic changes. METHODS: A total of 1757 samples having Ct value <25 (for E and ORF gene) from different districts of Rajasthan were processed for Next Generation Sequencing (NGS). The FASTA files obtained on sequencing were used for lineage determination using Nextclade and phylogenetic tree construction. RESULTS AND CONCLUSIONS: Sequencing and lineage identification was done in 1624 samples. XBB.1.16 was the predominant lineage in 1413 (87.0%) cases while rest was other XBB (207, 12.74%) and other lineages (4, 0.2%). Of the 1413 XBB.1.16 cases, 57.47% were males and 42.53% were females. Majority (66.53%) belonged to 19-59 year age. 84.15% of XBB.1.16 cases were infected for the first time. Hospitalization was required in only 2.2% cases and death was reported in 5 (0.35%) patients. Most of the cases were symptomatic and the commonest symptoms were fever, cough and rhinorrhea. Co-morbidities were present in 414 (29.3%) cases. Enhanced genomic surveillance helped to rapidly identify the spread of XBB variant in Rajasthan. This in turn helped to take control measures to prevent spread of virus and estimate public health risks of the new variant relative to the previously circulating lineages. XBB variant was found to spread rapidly but produced milder disease.

Comprehensive Analysis of Omicron Subvariants: EG.5 Rise, Vaccination Strategies, and Global Impact.

The emergence of new variants of the SARS-CoV-2 virus during the COVID-19 pandemic has prompted significant developments in the understanding, monitoring, and response to these strains. This comprehensive review focuses on two prominent variants of interest (VoI), XBB. 1.5 (Kraken) and XBB.1.16 ("Arcturus"), along with seven variants under observation (VuM), including EG.5. The World Health Organization (WHO) identified these variants in July 2023, highlighting EG.5's noteworthy rise in prevalence. EG.5, also known as "Eris," has exhibited an increased effective reproductive rate, prompting concerns about its contagiousness and immune evasion capabilities. With an altered spike protein in the Receptor-Binding Domain (RBD), EG.5 shares similarities with XBB.1.5 but surpasses it in prevalence, constituting 20% of COVID-19 cases in the United States by late August. EG.5's subvariant, EG.5.1, poses challenges with mutations like Q52H and F456L, contributing to its ability to bypass neutralizing antibodies. The global distribution of SARS-CoV-2 variants presents a dynamic landscape, with XBB.1.16 and other strains gaining prominence. The advent of the BA.2.86 variant further complicates the scenario, with its notable spread in regions lacking robust viral surveillance. A thorough analysis of mutations reveals the evolving nature of the Omicron variant, with distinct amino acid changes characterizing XBB.1.5, XBB.1.16, and EG.5. The WHO designates EG.5 as a "variant of interest" due to its increased contagiousness and potential immune evasion, emphasizing the need for vigilant monitoring. The risk assessment of EG.5 underscores its rapid development and growing prevalence globally. While booster vaccines targeting XBB.1.5 are in development, antiviral medications like nirmatrelvir/ritonavir (Paxlovid) continue to exhibit efficacy. In the context of the evolving variants, the FDA has granted emergency use authorization for updated COVID-19 vaccines targeting circulating strains, reflecting the adaptability of vaccination strategies to address emerging challenges. This comprehensive overview provides a nuanced understanding of the diverse Omicron subvariants, their global impact, and the ongoing efforts to combat their spread through vaccination and therapeutic interventions.

Validation of a Pseudovirus Neutralization Assay for Severe Acute Respiratory Syndrome Coronavirus 2: A High-Throughput Method for the Evaluation of Vaccine Immunogenicity.

The evaluation of coronavirus disease 2019 (COVID-19) vaccine immunogenicity remains essential as the severe acute respiratory syncytial virus 2 (SARS-CoV-2) pandemic continues to evolve and as additional variants emerge. Neutralizing antibodies are a known correlate of protection for SARS-CoV-2 vaccines. A pseudovirus neutralization (PNT) assay was developed and validated at Novavax Clinical Immunology Laboratories to allow for the detection of neutralizing antibodies in vaccine clinical trial sera. The PNT assay was precise, accurate, linear, and specific in measuring SARS-CoV-2 neutralization titers in human serum for ancestral strain and the Omicron subvariants BA.5 and XBB.1.5, with an overall geometric coefficient of variation of ≤43.4%, a percent relative bias within the expected range of -60% to 150%, and a linearity value of R2 > 0.98 for all three strains. This pseudovirus assay will be useful for the analysis of vaccine clinical trial samples to assess vaccine immunogenicity. Future work will focus on modifying the assay for emerging variants, including XBB.1.16, EG.5.1, BA.2.86, and any other variants that emerge in the ongoing pandemic.

New variants of COVID-19 (XBB.1.5 and XBB.1.16, the "Arcturus"): A review of highly questioned concerns, a brief comparison between different peaks in the COVID-19 pandemic, with a focused systematic review on expert recommendations for prevention, vaccination, and treatment measures in the general population and at-risk groups.

INTRODUCTION: The COVID-19 pandemic has taken many forms and continues to evolve, now around the Omicron wave, raising concerns over the globe. With COVID-19 being declared no longer a "public health emergency of international concern (PHEIC)," the COVID pandemic is still far from over, as new Omicron subvariants of interest and concern have risen since January of 2023. Mainly with the XBB.1.5 and XBB.1.16 subvariants, the pandemic is still very much "alive" and "breathing." METHODS: This review consists of five highly concerning questions about the current state of the COVID Omicron peak. We searched four main online databases to answer the first four questions. For the last one, we performed a systematic review of the literature, with keywords "Omicron," "Guidelines," and "Recommendations." RESULTS: A total of 31 articles were included. The main symptoms of the current Omicron wave include a characteristically high fever, coughing, conjunctivitis (with itching eyes), sore throat, runny nose, congestion, fatigue, body ache, and headache. The median incubation period of the symptoms is shorter than the previous peaks. Vaccination against COVID can still be considered effective for the new subvariants. CONCLUSION: Guidelines recommend continuation of personal protective measures, third and fourth dose boosters, along with administration of bivalent messenger RNA vaccine boosters. The consensus antiviral treatment is combination therapy using Nirmatrelvir and Ritonavir, and the consensus for pre-exposure prophylaxis is Tixagevimab and Cilgavimab combination. We hope the present paper raises awareness for the continuing presence of COVID and ways to lower the risks, especially for at-risk groups.

A Pseudovirus-Based Neutralization Assay for SARS-CoV-2 Variants: A Rapid, Cost-Effective, BSL-2-Based High-Throughput Assay Useful for Vaccine Immunogenicity Evaluation.

Neutralizing antibody responses from COVID-19 vaccines are pivotal in conferring protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Effective COVID-19 vaccines and assays measuring neutralizing antibodies against emerging variants (i.e., XBB.1.5, XBB.1.16, and XBB.2.3) are needed. The use of biosafety level (BSL)-3 laboratories for live virus assays results in higher costs and a longer turnaround time; therefore, a BSL-2-based pseudovirus neutralization assay (PNT) was developed. The pseudoviruses were produced by cotransfecting cells with plasmids encoding a lentiviral backbone-expressing luciferase reporter; non-surface proteins for lentiviral production; and ancestral or Omicron (BA.1 and BA.5) SARS-CoV-2 spike (S) proteins. The PNT was developed and optimized in dose and kinetics experiments. The representative serum samples (COVID-19-convalescent or NVX-CoV2373-vaccinated participants enrolled in the 2019nCoV-101 trial) demonstrated a wide dynamic range. The neutralization data showed robust correlation with validated anti-recombinant spike IgG levels and angiotensin-converting enzyme 2 inhibition titers (ancestral). This assay is suitable for measurement of the neutralization ability in clinical samples from individuals infected with SARS-CoV-2 or immunized with a COVID-19 vaccine. The results suggest that this PNT provides a lower cost, high-throughput, rapid turnaround alternative to BSL-3-based microneutralization assays and enables the discovery and development of effective vaccines against emerging variants.

Immune Evasion of SARS-CoV-2 Omicron Subvariants XBB.1.5, XBB.1.16 and EG.5.1 in a Cohort of Older Adults after ChAdOx1-S Vaccination and BA.4/5 Bivalent Booster.

The recently emerged SARS-CoV-2 Omicron sublineages, including the BA.2-derived XBB.1.5 (Kraken), XBB.1.16 (Arcturus), and EG.5.1 (Eris), have accumulated several spike mutations that may increase immune escape, affecting vaccine effectiveness. Older adults are an understudied group at significantly increased risk of severe COVID-19. Here we report the neutralizing activities of 177 sera samples from 59 older adults, aged 62-97 years, 1 and 4 months after vaccination with a 4th dose of ChAdOx1-S (Oxford/AstraZeneca) and 3 months after a 5th dose of Comirnaty Bivalent Original/Omicron BA.4/BA.5 vaccine (Pfizer-BioNTech). The ChAdOx1-S vaccination-induced antibodies neutralized efficiently the ancestral D614G and BA.4/5 variants, but to a much lesser extent the XBB.1.5, XBB.1.16, and EG.5.1 variants. The results showed similar neutralization titers between XBB.1.16 and EG.5.1 and were lower compared to XBB.1.5. Sera from the same individuals boosted with the bivalent mRNA vaccine contained higher neutralizing antibody titers, providing a better cross-protection against Omicron XBB.1.5, XBB.1.16 and EG.5.1 variants. Previous history of infection during the epidemiological waves of BA.1/BA.2 and BA.4/BA.5, poorly enhanced neutralization activity of serum samples against XBBs and EG.5.1 variants. Our data highlight the continued immune evasion of recent Omicron subvariants and support the booster administration of BA.4/5 bivalent vaccine, as a continuous strategy of updating future vaccine booster doses to match newly emerged SARS-CoV-2 variants.

Chasing SARS-CoV-2 XBB.1.16 Recombinant Lineage in India and the Clinical Profile of XBB.1.16 Cases in Maharashtra, India.

Background SARS-CoV-2 has evolved rapidly, resulting in the emergence of lineages with a competitive advantage over one another. Co-infections with different SARS-CoV-2 lineages can give rise to recombinant lineages. To date, the XBB lineage is the most widespread recombinant lineage worldwide, with the recently named XBB.1.16 lineage causing a surge in the number of COVID-19 cases in India. Methodology The present study involved retrieval of SARS-CoV-2 genome sequences from India (between December 1, 2022 and April 8, 2023) through GISAID; sequences were curated, followed by lineage and phylogenetic analysis. Demographic and clinical data from Maharashtra, India were collected telephonically, recorded in Microsoft® Excel, and analyzed using IBM® SPSS statistics, version 29.0.0.0 (241). Results A total of 2,944 sequences were downloaded from the GISAID database, of which 2,856 were included in the study following data curation. The sequences from India were dominated by the XBB.1.16* lineage (36.17%) followed by XBB.2.3* (12.11%) and XBB.1.5* (10.36%). Of the 2,856 cases, 693 were from Maharashtra; 386 of these were included in the clinical study. The clinical features of COVID-19 cases with XBB.1.16* infection (XBB.1.16* cases, 276 in number) showed that 92% of those had a symptomatic disease, with fever (67%), cough (42%), rhinorrhea (33.7%), body ache (14.5%) and fatigue (14.1%) being the most common symptoms. The presence of comorbidity was found in 17.7% of the XBB.1.16* cases. Among the XBB.1.16* cases, 91.7% were vaccinated with at least one dose of vaccine against COVID-19. While 74.3% of XBB.1.16* cases were home-isolated; 25.7% needed hospitalization/institutional quarantine, of these, 33.8% needed oxygen therapy. Out of 276 XBB.1.16* cases, seven (2.5%) cases succumbed to the disease. The majority of XBB.1.16* cases who died belonged to an elderly age group (60 years and above), had underlying comorbid condition/s, and needed supplemental oxygen therapy. The clinical features of COVID-19 cases infected with other co-circulating Omicron variants were similar to XBB.1.16* cases. Conclusion The study reveals that XBB.1.16* lineage has become the most predominant SARS-CoV-2 lineage in India. The study also shows that the clinical features and outcome of XBB.1.16* cases were similar to those of other co-circulating Omicron lineage infected cases in Maharashtra, India.

Continued evasion of neutralizing antibody response by Omicron XBB.1.16.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to challenge the efficacy of vaccination efforts against coronavirus disease 2019 (COVID-19). The Omicron XBB lineage of SARS-CoV-2 has presented dramatic evasion of neutralizing antibodies stimulated by mRNA vaccination and COVID-19 convalescence. XBB.1.16, characterized by two mutations relative to the dominating variant XBB.1.5, i.e., E180V and K478R, has been on the rise globally. In this study, we compare the immune escape of XBB.1.16 with XBB.1.5, alongside ancestral variants D614G, BA.2, and BA.4/5. We demonstrate that XBB.1.16 is strongly immune evasive, with extent comparable to XBB.1.5 in bivalent-vaccinated healthcare worker sera, 3-dose-vaccinated healthcare worker sera, and BA.4/5-wave convalescent sera. Interestingly, the XBB.1.16 spike is less fusogenic than that of XBB.1.5, and this phenotype requires both E180V and K478R mutations to manifest. Overall, our findings emphasize the importance of the continued surveillance of variants and the need for updated mRNA vaccine formulations.

XBB.1.16 Omicron subvariant rise to a variant of interest: Implications for global alertness and preparedness.

The emergence of the XBB.1.16 Omicron subvariant of COVID-19 has been a cause for concern for the WHO and health authorities globally. This subvariant, which originated from a hybrid of two BA.2 progeny pedigree, has two amino acid mutations in its spike protein and shares a genetic makeup similar to the XBB.1.5 variant. The WHO initially labeled it as a variant under monitoring before elevating it to a variant of interest after it was found to have caused a surge of COVID-19 cases in India for seven months. The XBB.1.16 subvariant has a proliferative edge and can evade the immune system. It has been spreading rapidly on a global scale and has been linked with a higher effective reproductive number than other subvariants. As such, a concerted international effort to prevent and contain its transmission has been recommended. Health authorities must strengthen their health systems, surveillance, and data collection systems to enable them to detect, track, and respond to emerging and reemerging strains of the virus in a timely and effective manner. Research into the XBB.1.16 subvariant is crucial for alerting and preparing the global populace for a potential outbreak, developing treatment options, and potential vaccines. Implementing the One Health approach can promote greater collaboration between diverse disciplines and societal levels to build a more resilient and sustainable future for all.

Coding-complete genomes of XBB.1.16, XBB.2.3, FL.4 (alias of XBB.1.9.1.4), and XBB.3 of SARS-CoV-2 Omicron isolated from Bangladesh.

We announce the coding-complete genomes of four different strains of SARS-CoV-2 Omicron lineages, XBB.1.16, XBB.2.3, FL.4 (alias of XBB.1.9.1.4), and XBB.3. These strains were obtained between October 2022 and May 2023 from nasopharyngeal swabs of four Bangladeshi individuals, while one of them had a travel history. Genomic data were produced by implementing ARTIC Network-based amplicon sequencing using the Oxford Nanopore Technology.