CMPK2 restricts Zika virus replication by inhibiting viral translation.

Flaviviruses continue to emerge as global health threats. There are currently no Food and Drug Administration (FDA) approved antiviral treatments for flaviviral infections. Therefore, there is a pressing need to identify host and viral factors that can be targeted for effective therapeutic intervention. Type I interferon (IFN-I) production in response to microbial products is one of the host's first line of defense against invading pathogens. Cytidine/uridine monophosphate kinase 2 (CMPK2) is a type I interferon-stimulated gene (ISG) that exerts antiviral effects. However, the molecular mechanism by which CMPK2 inhibits viral replication is unclear. Here, we report that CMPK2 expression restricts Zika virus (ZIKV) replication by specifically inhibiting viral translation and that IFN-I- induced CMPK2 contributes significantly to the overall antiviral response against ZIKV. We demonstrate that expression of CMPK2 results in a significant decrease in the replication of other pathogenic flaviviruses including dengue virus (DENV-2), Kunjin virus (KUNV) and yellow fever virus (YFV). Importantly, we determine that the N-terminal domain (NTD) of CMPK2, which lacks kinase activity, is sufficient to restrict viral translation. Thus, its kinase function is not required for CMPK2's antiviral activity. Furthermore, we identify seven conserved cysteine residues within the NTD as critical for CMPK2 antiviral activity. Thus, these residues may form an unknown functional site in the NTD of CMPK2 contributing to its antiviral function. Finally, we show that mitochondrial localization of CMPK2 is required for its antiviral effects. Given its broad antiviral activity against flaviviruses, CMPK2 is a promising potential pan-flavivirus inhibitor.

Oral Supplementation with AHCC®, a Standardized Extract of Cultured Lentinula edodes Mycelia, Enhances Host Resistance against SARS-CoV-2 Infection.

The coronavirus disease 2019 (COVID-19) pandemic has significantly impacted global public health safety and the economy. Multiple antiviral drugs have been developed, and some have received regulatory approval and/or authorization. The use of nutraceuticals can be beneficial for preventing and treating COVID-19 complications. AHCC is a standardized, cultured extract of an edible mushroom Lentinula edodes of the Basidiomycete family of fungi that is enriched in acylated α-1,4-glucans. Here, we evaluated the effects of the oral administration of AHCC on the host response to SARS-CoV-2 infection in two murine models, K18-hACE2 transgenic mice and immunocompetent BALB/c mice. Oral administration of AHCC every other day for one week before and one day post SARS-CoV-2 infection in both strains of mice decreased the viral load and attenuated inflammation in the lungs. AHCC treatment also significantly reduced SARS-CoV-2-induced lethality in the K18-hACE2 mice. AHCC administration enhanced the expansion of γδ T cells in the spleen and lungs before and after viral infection and promoted T helper 1-prone mucosal and systemic T cell responses in both models. In AHCC-fed BALB/c mice, SARS-CoV-2 specific IgG responses were also enhanced. In summary, AHCC supplementation enhances host resistance against mild and severe COVID-19 infection primarily via the promotion of innate and adaptive T cell immune responses in mice.

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5.

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2'-methyl-2'-fluoro guanosine 5'-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2'-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2'-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 ≈ 0.50 µM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

Mutations in SARS-CoV-2 variant nsp6 enhance type-I interferon antagonism.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve after its emergence. Given its importance in viral infection and vaccine development, mutations in the viral Spike gene have been studied extensively; however, the impact of mutations outside the Spike gene are poorly understood. Here, we report that a triple deletion (ΔSGF or ΔLSG) in nonstructural protein 6 (nsp6) independently acquired in Alpha and Omicron sublineages of SARS-CoV-2 augments nsp6-mediated antagonism of type-I interferon (IFN-I) signaling. Specifically, these triple deletions enhance the ability of mutant nsp6 to suppress phosphorylation of STAT1 and STAT2. A parental SARS-CoV-2 USA-WA1/2020 strain containing the nsp6 ΔSGF deletion (ΔSGF-WA1) shows reduced susceptibility to IFN-I treatment in vitro, outcompetes the parental strain in human primary airway cultures, and increases virulence in mice; however, the ΔSGF-WA1 virus is less virulent than the Alpha variant (which has the nsp6 ΔSGF deletion and additional mutations in other genes). Analyses of host responses from ΔSGF-WA1-infected mice and primary airway cultures reveal activation of pathways indicative of a cytokine storm. These results provide evidence that mutations outside the Spike protein affect virus-host interactions and may alter pathogenesis of SARS-CoV-2 variants in humans.

The multiple roles of nsp6 in the molecular pathogenesis of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and adapt after its emergence in late 2019. As the causative agent of the coronavirus disease 2019 (COVID-19), the replication and pathogenesis of SARS-CoV-2 have been extensively studied by the research community for vaccine and therapeutics development. Given the importance of viral spike protein in viral infection/transmission and vaccine development, the scientific community has thus far primarily focused on studying the structure, function, and evolution of the spike protein. Other viral proteins are understudied. To fill in this knowledge gap, a few recent studies have identified nonstructural protein 6 (nsp6) as a major contributor to SARS-CoV-2 replication through the formation of replication organelles, antagonism of interferon type I (IFN-I) responses, and NLRP3 inflammasome activation (a major factor of severe disease in COVID-19 patients). Here, we review the most recent progress on the multiple roles of nsp6 in modulating SARS-CoV-2 replication and pathogenesis.

Immunogenicity and Safety of a Third COVID-19 BNT162b2 mRNA Vaccine Dose in 5- to 11-Year Olds.

In this ongoing study, substantially increased ancestral SARS-CoV-2 neutralizing responses were observed 1 month after a third 10-µg BNT162b2 dose given to 5 to 11-year olds versus neutralizing responses post-dose 2. After dose 3, increased neutralizing responses against Omicron BA.1 and BA.4/BA.5 strains were also observed. The safety/tolerability profile was acceptable. (NCT04816643).

Low neutralization of SARS-CoV-2 Omicron BA.2.75.2, BQ.1.1, and XBB.1 by parental mRNA vaccine or a BA.5-bivalent booster.

The newly emerged SARS-CoV-2 Omicron sublineages, including the BA.2-derived BA.2.75.2 and the BA.5-derived BQ.1.1 and XBB.1, have accumulated additional spike mutations that may affect vaccine effectiveness. Here we report neutralizing activities of three human serum panels collected from individuals 23-94 days after dose 4 of a parental mRNA vaccine, 14-32 days after a BA.5-bivalent-booster from individuals with 2-4 previous doses of parental mRNA vaccine, or 15-32 days after a BA.5-bivalent-booster from individuals with previous SARS-CoV-2 infection and 2-4 doses of parental mRNA vaccine. The results showed that a BA.5-bivalent-booster elicited a high neutralizing titer against BA.4/5 measured at 14- to 32-day post-boost; however, the BA.5-bivalent-booster did not produce robust neutralization against the newly emerged BA.2.75.2, BQ.1.1, or XBB.1. Previous infection significantly enhanced the magnitude and breadth of BA.5-bivalent-booster-elicited neutralization. Our data support a vaccine update strategy that future boosters should match newly emerged circulating SARS-CoV-2 variants.

Evaluation of BNT162b2 Covid-19 Vaccine in Children Younger than 5 Years of Age.

BACKGROUND: Safe and effective vaccines against coronavirus disease 2019 (Covid-19) are urgently needed in young children. METHODS: We conducted a phase 1 dose-finding study and are conducting an ongoing phase 2-3 safety, immunogenicity, and efficacy trial of the BNT162b2 vaccine in healthy children 6 months to 11 years of age. We present results for children 6 months to less than 2 years of age and those 2 to 4 years of age through the data-cutoff dates (April 29, 2022, for safety and immunogenicity and June 17, 2022, for efficacy). In the phase 2-3 trial, participants were randomly assigned (in a 2:1 ratio) to receive two 3-µg doses of BNT162b2 or placebo. On the basis of preliminary immunogenicity results, a third 3-µg dose (≥8 weeks after dose 2) was administered starting in January 2022, which coincided with the emergence of the B.1.1.529 (omicron) variant. Immune responses at 1 month after doses 2 and 3 in children 6 months to less than 2 years of age and those 2 to 4 years of age were immunologically bridged to responses after dose 2 in persons 16 to 25 years of age who received 30 µg of BNT162b2 in the pivotal trial. RESULTS: During the phase 1 dose-finding study, two doses of BNT162b2 were administered 21 days apart to 16 children 6 months to less than 2 years of age (3-µg dose) and 48 children 2 to 4 years of age (3-µg or 10-µg dose). The 3-µg dose level was selected for the phase 2-3 trial; 1178 children 6 months to less than 2 years of age and 1835 children 2 to 4 years of age received BNT162b2, and 598 and 915, respectively, received placebo. Immunobridging success criteria for the geometric mean ratio and seroresponse at 1 month after dose 3 were met in both age groups. BNT162b2 reactogenicity events were mostly mild to moderate, with no grade 4 events. Low, similar incidences of fever were reported after receipt of BNT162b2 (7% among children 6 months to <2 years of age and 5% among those 2 to 4 years of age) and placebo (6 to 7% among children 6 months to <2 years of age and 4 to 5% among those 2 to 4 years of age). The observed overall vaccine efficacy against symptomatic Covid-19 in children 6 months to 4 years of age was 73.2% (95% confidence interval, 43.8 to 87.6) from 7 days after dose 3 (on the basis of 34 cases). CONCLUSIONS: A three-dose primary series of 3-µg BNT162b2 was safe, immunogenic, and efficacious in children 6 months to 4 years of age. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04816643.).

Development of Highly Potent Noncovalent Inhibitors of SARS-CoV-2 3CLpro.

The 3C-like protease (3CLpro) is an essential enzyme for the replication of SARS-CoV-2 and other coronaviruses and thus is a target for coronavirus drug discovery. Nearly all inhibitors of coronavirus 3CLpro reported so far are covalent inhibitors. Here, we report the development of specific, noncovalent inhibitors of 3CLpro. The most potent one, WU-04, effectively blocks SARS-CoV-2 replications in human cells with EC50 values in the 10-nM range. WU-04 also inhibits the 3CLpro of SARS-CoV and MERS-CoV with high potency, indicating that it is a pan-inhibitor of coronavirus 3CLpro. WU-04 showed anti-SARS-CoV-2 activity similar to that of PF-07321332 (Nirmatrelvir) in K18-hACE2 mice when the same dose was administered orally. Thus, WU-04 is a promising drug candidate for coronavirus treatment.

Cross-neutralization and viral fitness of SARS-CoV-2 Omicron sublineages.

The rapid evolution of SARS-CoV-2 Omicron sublineages mandates a better understanding of viral replication and cross-neutralization among these sublineages. Here we used K18-hACE2 mice and primary human airway cultures to examine the viral fitness and antigenic relationship among Omicron sublineages. In both K18-hACE2 mice and human airway cultures, Omicron sublineages exhibited a replication order of BA.5 ≥ BA.2 ≥ BA.2.12.1 > BA.1; no difference in body weight loss was observed among different sublineage-infected mice. The BA.1-, BA.2-, BA.2.12.1-, and BA.5-infected mice developed distinguishable cross-neutralizations against Omicron sublineages, but exhibited little neutralization against the index virus (i.e. USA-WA1/2020) or the Delta variant. Surprisingly, the BA.5-infected mice developed higher neutralization activity against heterologous BA.2 and BA.2.12.1 than that against homologous BA.5; serum neutralizing titres did not always correlate with viral replication levels in infected animals. Our results revealed a distinct antigenic cartography of Omicron sublineages and support the bivalent vaccine approach.

Positive-strand RNA viruses-a Keystone Symposia report.

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium.

Determinants and Mechanisms of the Low Fusogenicity and High Dependence on Endosomal Entry of Omicron Subvariants.

The rapid spread and strong immune evasion of the SARS-CoV-2 Omicron subvariants has raised serious concerns for the global COVID-19 pandemic. These new variants exhibit generally reduced fusogenicity and increased endosomal entry pathway utilization compared to the ancestral D614G variant, the underlying mechanisms of which remain elusive. Here, we show that the C-terminal S1 mutations of the BA.1.1 subvariant, H655Y and T547K, critically govern the low fusogenicity of Omicron. Notably, H655Y also dictates the enhanced endosome entry pathway utilization. Mechanistically, T547K and H655Y likely stabilize the spike trimer conformation as suggested by increased molecular interactions in structural modeling and enhanced S1 shedding of their reversion mutants K547T and Y655H in viral producer cells. Importantly, the H655Y mutation also determines the low fusogenicity and enhanced dependence on the endosomal entry pathway of other Omicron subvariants, including BA.2, BA.2.12.1, BA.4/5, and BA.2.75. Together, these results uncover mechanisms governing Omicron subvariant entry and provide insights into altered Omicron tissue tropism and pathogenesis. IMPORTANCE Omicron has been shown to predominantly use the endosomal entry pathway, resulting in reduced lung tropism and reduced disease severity; however, the underlying mechanism is not fully understood. In addition, whether the most recent Omicron subvariants, including BA.5 and BA.2.75, use the same pathway as their ancestor for entry is currently not known. In this study, we show that T547K and H655Y mutations in the C terminus of the S1 subunit critically determine the enhanced dependence on the endosomal entry pathway as well as the reduced cell-cell fusion activity of Omicron BA.1, BA.1.1, and other subvariants. Further experiments and molecular modeling suggest that H655Y and K547T stabilize the spike trimer conformation, likely contributing to the decreased fusogenicity and endosomal entry. Our work uncovers novel mechanisms underlying the distinct entry pathway of Omicron subvariants and advances our understanding of their biological characteristics.

Bivalent Omicron BA.1-Adapted BNT162b2 Booster in Adults Older than 55 Years.

BACKGROUND: The emergence of immune-escape variants of severe acute respiratory syndrome coronavirus 2 warrants the use of sequence-adapted vaccines to provide protection against coronavirus disease 2019. METHODS: In an ongoing phase 3 trial, adults older than 55 years who had previously received three 30-µg doses of the BNT162b2 vaccine were randomly assigned to receive 30 µg or 60 µg of BNT162b2, 30 µg or 60 µg of monovalent B.1.1.529 (omicron) BA.1-adapted BNT162b2 (monovalent BA.1), or 30 µg (15 µg of BNT162b2 + 15 µg of monovalent BA.1) or 60 µg (30 µg of BNT162b2 + 30 µg of monovalent BA.1) of BA.1-adapted BNT162b2 (bivalent BA.1). Primary objectives were to determine superiority (with respect to 50% neutralizing titer [NT50] against BA.1) and noninferiority (with respect to seroresponse) of the BA.1-adapted vaccines to BNT162b2 (30 µg). A secondary objective was to determine noninferiority of bivalent BA.1 to BNT162b2 (30 µg) with respect to neutralizing activity against the ancestral strain. Exploratory analyses assessed immune responses against omicron BA.4, BA.5, and BA.2.75 subvariants. RESULTS: A total of 1846 participants underwent randomization. At 1 month after vaccination, bivalent BA.1 (30 µg and 60 µg) and monovalent BA.1 (60 µg) showed neutralizing activity against BA.1 superior to that of BNT162b2 (30 µg), with NT50 geometric mean ratios (GMRs) of 1.56 (95% confidence interval [CI], 1.17 to 2.08), 1.97 (95% CI, 1.45 to 2.68), and 3.15 (95% CI, 2.38 to 4.16), respectively. Bivalent BA.1 (both doses) and monovalent BA.1 (60 µg) were also noninferior to BNT162b2 (30 µg) with respect to seroresponse against BA.1; between-group differences ranged from 10.9 to 29.1 percentage points. Bivalent BA.1 (either dose) was noninferior to BNT162b2 (30 µg) with respect to neutralizing activity against the ancestral strain, with NT50 GMRs of 0.99 (95% CI, 0.82 to 1.20) and 1.30 (95% CI, 1.07 to 1.58), respectively. BA.4-BA.5 and BA.2.75 neutralizing titers were numerically higher with 30-µg bivalent BA.1 than with 30-µg BNT162b2. The safety profile of either dose of monovalent or bivalent BA.1 was similar to that of BNT162b2 (30 µg). Adverse events were more common in the 30-µg monovalent-BA.1 (8.5%) and 60-µg bivalent-BA.1 (10.4%) groups than in the other groups (3.6 to 6.6%). CONCLUSIONS: The candidate monovalent or bivalent omicron BA.1-adapted vaccines had a safety profile similar to that of BNT162b2 (30 µg), induced substantial neutralizing responses against ancestral and omicron BA.1 strains, and, to a lesser extent, neutralized BA.4, BA.5, and BA.2.75 strains. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04955626.).

Reverse genetic systems of SARS-CoV-2 for antiviral research.

Reverse genetic systems are widely used to engineer recombinant viruses with desired mutations. In response to the COVID-19 pandemic, four types of reverse genetic systems have been developed for SARS-CoV-2: (i) a full-length infectious clone that can be used to prepare recombinant SARS-CoV-2 at biosafety level 3 (BSL3), (ii) a trans-complementation system that can be used to produce single-round infectious SARS-CoV-2 at BSL2, (iii) an attenuated SARS-CoV-2 vaccine candidate (with deletions of viral accessory genes) that may be developed for veterinary use as well as for antiviral screening at BSL2, and (iv) replicon systems with deletions of viral structural genes that can be used at BSL2. Each of these genetic systems has its advantages and disadvantages that can be used to address different questions for basic and translational research. Due to the long genomic size and bacteria-toxic sequences of SARS-CoV-2, several experimental approaches have been established to rescue recombinant viruses and replicons, including (i) in vitro DNA ligation, (ii) bacterial artificial chromosome (BAC) system, (iii) yeast artificial chromosome (YAC) system, and (iv) circular polymerase extension reaction (CPER). This review summarizes the current status of SARS-CoV-2 genetic systems and their applications for studying viral replication, pathogenesis, vaccines, and therapeutics.