Virological characteristics of the SARS-CoV-2 BA.2.86 variant (preprint)

In late 2023, a lineage of SARS-CoV-2 emerged and was named the BA.2.86 variant. BA.2.86 is phylogenetically distinct from other Omicron sublineages identified so far, displaying an accumulation of over 30 amino acid mutations in its spike protein. Here, we performed multiscale investigations to reveal the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Experimental studies showed that four clinically-available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 is significantly lower than that of BA.2 in both in vitro cell cultures and in vivo, it is suggested that the attenuated pathogenicity of BA.2.86 is due to its decreased replication capacity.

Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant (preprint)

In middle-late 2023, a sublineage of SARS-CoV-2 Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. Here, we performed multiscale investigations to reveal virological features of newly emerging EG.5.1 variant. Our phylogenetic-epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T, are critical to the increased viral fitness. Experimental investigations addressing the growth kinetics, sensitivity to clinically available antivirals, fusogenicity and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 is comparable to that of XBB.1.5. However, the cryo-electron microscopy reveals the structural difference between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible at least in our experimental setup. Our multiscale investigations provide the knowledge for understanding of the evolution trait of newly emerging pathogenic viruses in the human population.

Akaluc bioluminescence offers superior sensitivity to track in vivo dynamics of SARS-CoV-2 infection (preprint)

Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. For positive-sense, single-stranded RNA viruses, several studies have attempted to monitor viral kinetics in vivo using reporter genomes. The application of such recombinant viruses can be limited by challenges in accommodating bioluminescent reporter genes in the viral genome. Conventional luminescence also exhibits relatively low tissue permeability and thus less sensitivity for visualization in vivo. Here we show that unlike NanoLuc bioluminescence, the improved method, termed AkaBLI, allows visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Syrian hamsters. By successfully incorporating a codon-optimized Akaluc luciferase gene into the SARS-CoV-2 genome, we visualized in vivo infection, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of neutralizing antibodies and mRNA vaccination by monitoring changes in Akaluc signals. Overall, AkaBLI is an effective technology for monitoring viral dynamics in live animals.

Safety and immunogenicity of VLPCOV-02, a SARS-CoV-2 self-amplifying RNA vaccine with a modified base, 5-methylcytosine, in healthy individuals (preprint)

Continuing emergence of variants of concern resulting in reduced SARS-CoV-2 vaccine efficacy necessitates additional prevention strategies. The structure of VLPCOV-01, a lipid nanoparticle-encapsulated, self-amplifying RNA COVID-19 vaccine with a comparable immune response to BNT162b2, was revised by incorporating a modified base, 5-methylcytosine to reduce reactogenicity, and an updated receptor-binding domain derived from Brazil (gamma) variant. Interim analyses of a phase 1 dose-escalation booster vaccination study with the resulting construct, VLPCOV-02, in healthy, previously vaccinated Japanese individuals (N=96) are reported (jRCT2051230005). A dose-related increase in solicited local and systemic adverse events was observed, which were generally rated mild or moderate. The most commonly occurring events were tenderness, pain, fatigue, and myalgia. Serum SARS-CoV-2 immunoglobulin titers increased during the 4 weeks post-immunization. VLPCOV-02 demonstrated a favorable safety profile compared with VLPCOV-01, with a lower frequency of adverse events and fewer fever events at an equivalent dose. These findings support further study of VLPCOV-02.

Virological characteristics of the SARS-CoV-2 XBB.1.5 variant (preprint)

Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the F486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determined the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. The intrinsic pathogenicity of XBB.1.5 in hamsters is lower than that of XBB.1. Importantly, we found that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC expression. In vivo experiments using recombinant viruses revealed that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, these data suggest that the mutations in ORF8 and S could enhance spreading of XBB.1.5 in humans.

Neutralizing antibody responses and cellular responses against severe acute respiratory syndrome coronavirus 2 omicron subvariant BA.5 after an mRNA severe acute respiratory syndrome coronavirus 2 vaccine dose in kidney transplant recipients (preprint)

We examined the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein IgG antibody and neutralizing antibody titers and cellular immunity in 73 uninfected recipients and 17 uninfected healthy controls who received three doses of a coronavirus 2019 mRNA vaccine. Neutralizing antibody titers were evaluated using GFP-carrying recombinant SARS-CoV-2 with spike protein of B.1.1, omicron BA.1, or BA.5. For cellular immunity, peripheral blood mononuclear cells were stimulated with peptides corresponding to spike protein antigens of B.1.1, BA.1, and BA.5; spike-specific CD4/CD8 memory T cells were evaluated using intracellular cytokine staining. The median IgG antibody titers were 7.8 AU/mL in recipients and 143.0 AU/mL in healthy controls (p < 0.0001). Neutralizing antibody titers against all three viral variants were significantly lower in recipients (p < 0.0001). The number of spike-specific CD8 + memory T cells significantly decreased in recipients (p < 0.0001). Twenty recipients and seven healthy controls additionally received a bivalent omicron-containing booster vaccine, and IgG antibody and neutralizing antibody titers increased in both groups; however, the increase was significantly lower in recipients. Recipients did not gain sufficient immunity with a third dose of vaccine, suggesting a need to explore methods other than vaccines.

Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants (preprint)

In late 2022, the SARS-CoV-2 Omicron subvariants have highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged by recombination of two co-circulating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022 around India. In vitro experiments revealed that XBB is the most profoundly resistant variant to BA.2/5 breakthrough infection sera ever and is more fusogenic than BA.2.75. Notably, the recombination breakpoint is located in the receptor-binding domain of spike, and each region of recombined spike conferred immune evasion and augmented fusogenicity to the XBB spike. Finally, the intrinsic pathogenicity of XBB in hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provided evidence suggesting that XBB is the first documented SARS-CoV-2 variant increasing its fitness through recombination rather than single mutations.

Convergent evolution of the SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant (preprint)

In late 2022, although the SARS-CoV-2 Omicron subvariants have highly diversified, some lineages have convergently acquired amino acid substitutions at five critical residues in the spike protein. Here, we illuminated the evolutionary rules underlying the convergent evolution of Omicron subvariants and the properties of one of the latest lineages of concern, BQ.1.1. Our phylogenetic and epidemic dynamics analyses suggest that Omicron subvariants independently increased their viral fitness by acquiring the convergent substitutions. Particularly, BQ.1.1, which harbors all five convergent substitutions, shows the highest fitness among the viruses investigated. Neutralization assays show that BQ.1.1 is more resistant to breakthrough BA.2/5 infection sera than BA.5. The BQ.1.1 spike exhibits enhanced binding affinity to human ACE2 receptor and greater fusogenicity than the BA.5 spike. However, the pathogenicity of BQ.1.1 in hamsters is comparable to or even lower than that of BA.5. Our multiscale investigations provide insights into the evolutionary trajectory of Omicron subvariants.

Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing anchored RBD: a randomised, observer-blind, phase 1 study (preprint)

BACKGROUND VLPCOV-01 is a lipid nanoparticle-encapsulated self-amplifying (sa) RNA vaccine that expresses a membrane-anchored receptor-binding domain (RBD) derived from the SARS-CoV-2 spike protein. METHODS A phase 1 study of VLPCOV-01 was conducted at Medical Corporation Heishinkai OPHAC Hospital, Japan. The investigational vaccines were administered to participants, between February 16, 2022, and March 17, 2022. Participants aged 18 to 55 or [≥]65 years who had completed two doses of the BNT162b2 mRNA vaccine 6 to 12 months previously were randomised to receive one intramuscular vaccination of 0{middle dot}3, 1{middle dot}0, or 3{middle dot}0 {micro}g VLPCOV-01, 30 {micro}g BNT162b2, or placebo. Solicited adverse events were collected up to 6 days post-administration, with follow-up on all adverse events until week 4. Interim immunogenicity analyses following data cutoff at day 29 included SARS-CoV-2 IgG and neutralising antibody titres. (The trial is registered: jRCT2051210164). FINDINGS 92 healthy adults were enrolled, with 60 participants receiving VLPCOV-01. No serious adverse events were reported up to 26 weeks, and no prespecified trial-halting events were met. VLPCOV-01 induced robust IgG titres against SARS-CoV-2 RBD protein that were maintained up to 26 weeks in non-elderly participants, with geometric means ranging from 5037 (95% CI 1272-19,940) at 0{middle dot}3 {micro}g to 12,873 (95% CI 937-17,686) at 3 {micro}g, in comparison to 3166 (95% CI 1619-6191) with 30 {micro}g BNT162b2. Among elderly participants, IgG titres at 26 weeks post-vaccination with 3 {micro}g VLPCOV-01 were 9865 (95% CI 4396-22138) compared to 4183 (95% CI 1436-12180) following vaccination with 30 {micro}g BNT162b2. Pseudovirus-Neutralising antibody responses were observed against multiple SARS-CoV-2 variants and strongly correlated with anti-SARS-CoV-2 IgG (r=0{middle dot}950, p<0{middle dot}001). INTERPRETATION VLPCOV-01 is immunogenic following low dose administration, with anti-SARS-CoV-2 immune responses comparable to BNT162b2. These findings support further development of VLPCOV-01 as a COVID-19 booster vaccine and the potential for saRNA vectors as a vaccine platform. FUNDING Supported by AMED, Grant No. JP21nf0101627.

Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 (preprint)

SARS-CoV-2 Omicron BA.2.75 emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically different from BA.5, the currently predominant BA.2 descendant. Here, we showed that the effective reproduction number of BA.2.75 is greater than that of BA.5. While the sensitivity of BA.2.75 to vaccination- and BA.1/2 breakthrough infection-induced humoral immunity was comparable to that of BA.2, the immunogenicity of BA.2.75 was different from that of BA.2 and BA.5. Three clinically-available antiviral drugs were effective against BA.2.75. BA.2.75 spike exhibited a profound higher affinity to human ACE2 than BA.2 and BA.5 spikes. The fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were comparable to those of BA.5 but were greater than those of BA.2. Our multiscale investigations suggest that BA.2.75 acquired virological properties independently of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.

Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5 (preprint)

Unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants imposes us to continuous control measurement. Given the rapid spread, new Omicron subvariant named BA.5 is urgently required for characterization. Here we analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1 comprehensively. Although in vitro growth kinetics of BA.5 is comparable among the Omicron subvariants, BA.5 become much more fusogenic than BA.1 and BA.2. The airway-on-a-chip analysis showed that the ability of BA.5 to disrupt the respiratory epithelial and endothelial barriers is enhanced among Omicron subvariants. Furthermore, in our hamster model, in vivo replication of BA.5 is comparable with that of the other Omicrons and less than that of the ancestral B.1.1. Importantly, inflammatory response against BA.5 is strong compared with BA.1 and BA.2. Our data suggest that BA.5 is still low pathogenic compared to ancestral strain but evolved to induce enhanced inflammation when compared to prior Omicron subvariants.

Virological characteristics of the novel SARS-CoV-2 Omicron variants including BA.2.12.1, BA.4 and BA.5 (preprint)

After the global spread of SARS-CoV-2 Omicron BA.2 lineage, some BA.2-related variants that acquire mutations in the L452 residue of spike protein, such as BA.2.9.1 and BA.2.13 (L452M), BA.2.12.1 (L452Q), and BA.2.11, BA.4 and BA.5 (L452R), emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these L452R/M/Q-bearing BA.2-related Omicron variants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1 and BA.2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. Furthermore, infection experiments using hamsters indicated that BA.4/5 is more pathogenic than BA.2. Altogether, our multiscale investigations suggest that the risk of L452R/M/Q-bearing BA.2-related Omicron variants, particularly BA.4 and BA.5, to global health is potentially greater than that of original BA.2.

Characterization of various remdesivir-resistant mutations of SARS-CoV-2 by mathematical modeling and molecular dynamics simulation. (preprint)

Mutations continue to accumulate within the SARS-CoV-2 genome, and the ongoing epidemic has shown no signs of ending. It is critical to predict problematic mutations that may arise in clinical environments and assess their properties in advance to quickly implement countermeasures against future variant infections. In this study, we identified mutations resistant to remdesivir, which is widely administered to SARS-CoV-2-infected patients, and discuss the cause of resistance. First, we simultaneously constructed eight recombinant viruses carrying the mutations detected in in vitro serial passages of SARS-CoV-2 in the presence of remdesivir. Time course analyses of cellular virus infections showed significantly higher infectious titers and infection rates in mutant viruses than wild type virus under treatment with remdesivir. Next, we developed a mathematical model in consideration of the changing dynamic of cells infected with mutant viruses with distinct propagation properties and defined that mutations detected in in vitro passages canceled the antiviral activities of remdesivir without raising virus production capacity. Finally, molecular dynamics simulations of the NSP12 protein of SARS-CoV-2 revealed that the molecular vibration around the RNA-binding site was increased by the introduction of mutations on NSP12. Taken together, we identified multiple mutations that affected the flexibility of the RNA binding site and decreased the antiviral activity of remdesivir. Our new insights will contribute to developing further antiviral measures against SARS-CoV-2 infection.

Virological characteristics of SARS-CoV-2 BA.2 variant (preprint)

Soon after the emergence and global spread of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron lineage, BA.1 (ref1, 2), another Omicron lineage, BA.2, has initiated outcompeting BA.1. Statistical analysis shows that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralisation experiments show that the vaccine-induced humoral immunity fails to function against BA.2 like BA.1, and notably, the antigenicity of BA.2 is different from BA.1. Cell culture experiments show that BA.2 is more replicative in human nasal epithelial cells and more fusogenic than BA.1. Furthermore, infection experiments using hamsters show that BA.2 is more pathogenic than BA.1. Our multiscale investigations suggest that the risk of BA.2 for global health is potentially higher than that of BA.1.

Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant (preprint)

The emergence of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, Omicron, is the most urgent concern in the global health in December 2021. Our statistical modelling estimates that Omicron is >3.0-fold and >5.6-fold more transmissible than Delta in South Africa and the UK, respectively. Intriguingly, cell culture experiments show that Omicron is less fusogenic than Delta and ancestral SARS-CoV-2. Although the spike (S) protein of Delta is efficiently cleaved into the two subunits, which facilitates cell-cell fusion, Omicron S is faintly cleaved. Further, in hamster model, Omicron shows decreased lung infectivity and is less pathogenic compared to Delta and ancestral SARS-CoV-2. Our data suggest that the efficacy of SARS-CoV-2 S cleavage and viral fusogenicity are closely associated with viral pathogenicity, and Omicron evolved to exhibit increased transmissibility and attenuated pathogenicity.

SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion (preprint)

During the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and at least five variants of concerns (VOCs) have been considered as the hazardous SARS-CoV-2 variants to the human society. The newly emerging VOC, the B.1.617.2 lineage (delta variant), closely associates with a huge COVID-19 surge in India in Spring 2021. However, its virological property remains unclear. Here, we show that the B.1.617 variants are highly fusogenic and form prominent syncytia. Bioinformatic analyses reveal that the P681R mutation in the spike protein is highly conserved in this lineage. Although the P681R mutation decreases viral infectivity, this mutation confers the neutralizing antibody resistance. Notably, we demonstrate that the P681R mutation facilitates the furin-mediated spike cleavage and enhances and accelerates cell-cell fusion. Our data suggest that the P681R mutation is a hallmark characterizing the virological phenotype of this newest VOC, which may associate with viral pathogenicity. HighlightsO_LIP681R mutation is highly conserved in the B.1.617 lineages C_LIO_LIP681R mutation accelerates and enhances SARS-CoV-2 S-mediated fusion C_LIO_LIPromotion of viral fusion by P681R mutation is augmented by TMPRSS2 C_LI

An emerging SARS-CoV-2 mutant evading cellular immunity and increasing viral infectivity (preprint)

During the current SARS-CoV-2 pandemic that is devastating the modern societies worldwide, many variants that naturally acquire multiple mutations have emerged. Emerging mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has recently been investigated, that to human leukocyte antigen (HLA)-restricted cellular immunity remains unaddressed. Here we demonstrate that two recently emerging mutants in the receptor binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429) and Y453F (in B.1.298), can escape from the HLA-24-restricted cellular immunity. These mutations reinforce the affinity to viral receptor ACE2, and notably, the L452R mutation increases protein stability, viral infectivity, and potentially promotes viral replication. Our data suggest that the HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes, and the escape from cellular immunity can be a further threat of the SARS-CoV-2 pandemic.