A single-dose of intranasal vaccination with a live-attenuated SARS-CoV-2 vaccine candidate promotes protective mucosal and systemic immunity.

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (∆3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of ∆3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the ∆3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest ∆3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2.

A single-dose of intranasal vaccination with a live-attenuated SARS-CoV-2 vaccine candidate promotes protective mucosal and systemic immunity.

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (∆3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of ∆3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the ∆3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest ∆3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2.

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.

SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

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.

SARS-CoV-2 Uses Nonstructural Protein 16 to Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'- O methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'- O MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo , using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive to type I interferon (IFN-I) in vitro . Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'- O methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, a methyltransferase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a possible target for future antiviral therapies. Importance: Similar to other coronaviruses, disruption of SARS-CoV-2 NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo , our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1, but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'- O methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Evaluations of rationally designed rift valley fever vaccine candidate RVax-1 in mosquito and rodent models.

Rift Valley fever (RVF) is a mosquito-borne zoonosis endemic to Africa and the Arabian Peninsula, which causes large outbreaks among humans and ruminants. Single dose vaccinations using live-attenuated RVF virus (RVFV) support effective prevention of viral spread in endemic countries. Due to the segmented nature of RVFV genomic RNA, segments of vaccine strain-derived genomic RNA could be incorporated into wild-type RVFV within co-infected mosquitoes or animals. Rationally designed vaccine candidate RVax-1 displays protective epitopes fully identical to the previously characterized MP-12 vaccine. Additionally, all genome segments of RVax-1 contribute to the attenuation phenotype, which prevents the formation of pathogenic reassortant strains. This study demonstrated that RVax-1 cannot replicate efficiently in orally fed Aedes aegypti mosquitoes, while retaining strong immunogenicity and protective efficacy in an inbred mouse model, which were indistinguishable from the MP-12 vaccine. These findings support further development of RVax-1 as the next generation MP-12-based vaccine for prevention of Rift Valley fever in humans and animals.

Engineering SARS-CoV-2 specific cocktail antibodies into a bispecific format improves neutralizing potency and breadth.

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce emergence of antibody resistance. Here we engineer two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv)2 design (14-H-06) but not the CrossMAb design (14-crs-06) shows increased antigen-binding and virus-neutralizing activities against multiple SARS-CoV-2 variants as well as increased breadth of neutralizing activity compared to the cocktail. X-ray crystallography and cryo-EM reveal distinct binding models for individual cocktail antibodies, and computational simulations suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and multiple variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth.

An antibody that neutralizes SARS-CoV-1 and SARS-CoV-2 by binding to a conserved spike epitope outside the receptor binding motif.

The rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as the Omicron variants that are highly transmissible and immune evasive, underscores the need to develop therapeutic antibodies with broad neutralizing activities. Here, we used the LIBRA-seq technology, which identified SARS-CoV-2-specific B cells via DNA barcoding and subsequently single-cell sequenced BCRs, to identify an antibody, SW186, which could neutralize major SARS-CoV-2 variants of concern, including Beta, Delta, and Omicron, as well as SARS-CoV-1. The cryo-EM structure of SW186 bound to the receptor binding domain (RBD) of the viral spike protein showed that SW186 interacted with an epitope of the RBD that is not at the interface of its binding to the ACE2 receptor but is highly conserved among SARS coronaviruses. This epitope encompasses a glycosylation site (N343) of the viral spike protein. Administration of SW186 in mice after they were infected with SARS-CoV-2 Alpha, Beta, or Delta variants reduced the viral loads in the lung. These results demonstrated that SW186 neutralizes diverse SARS coronaviruses by binding to a conserved RBD epitope, which could serve as a target for further antibody development.

A live-attenuated SARS-CoV-2 vaccine candidate with accessory protein deletions.

We report a live-attenuated SARS-CoV-2 vaccine candidate with (i) re-engineered viral transcription regulator sequences and (ii) deleted open-reading-frames (ORF) 3, 6, 7, and 8 (∆3678). The ∆3678 virus replicates about 7,500-fold lower than wild-type SARS-CoV-2 on primary human airway cultures, but restores its replication on interferon-deficient Vero-E6 cells that are approved for vaccine production. The ∆3678 virus is highly attenuated in both hamster and K18-hACE2 mouse models. A single-dose immunization of the ∆3678 virus protects hamsters from wild-type virus challenge and transmission. Among the deleted ORFs in the ∆3678 virus, ORF3a accounts for the most attenuation through antagonizing STAT1 phosphorylation during type-I interferon signaling. We also developed an mNeonGreen reporter ∆3678 virus for high-throughput neutralization and antiviral testing. Altogether, the results suggest that ∆3678 SARS-CoV-2 may serve as a live-attenuated vaccine candidate and a research tool for potential biosafety level-2 use.

Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis.

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral 'RG' motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2's continued adaptation to human infection.

Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly.

Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating VP35's engagement in different functions are not well-understood. We previously showed that the host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate virus replication. However, how K309 ubiquitination regulates the function of VP35 as the viral polymerase co-factor and the precise stage(s) of the EBOV replication cycle that require VP35 ubiquitination are not known. Here, we generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The K309R mutant retains dsRNA binding and efficient type-I Interferon (IFN-I) antagonism due to the basic residue conservation. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant's suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This disruption of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. In addition, lack of ubiquitination on K309 results in enhanced interactions with the viral nucleoprotein and premature nucleocapsid packaging, leading to dysregulation of virus assembly. Overall, we identified a novel role of VP35 ubiquitination in coordinating viral transcription and assembly.

Engineering SARS-CoV-2 cocktail antibodies into a bispecific format improves neutralizing potency and breadth.

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce antibody resistance. We engineered two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv) 2 design (14-H-06) but not the CrossMAb design (14-crs-06) increases antigen-binding and virus-neutralizing activities and spectrum against multiple SARS-CoV-2 variants including the Omicron, than the cocktail. X-ray crystallography and computational simulations reveal distinct neutralizing mechanisms for individual cocktail antibodies and suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and the Beta, Gamma, and Delta variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth.

Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis.

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo . Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral 'RG' motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2's continued adaptation to human infection. AUTHOR SUMMARY: Since its emergence, SARS-CoV-2 has continued to adapt for human infection resulting in the emergence of variants with unique genetic profiles. Most studies of genetic variation have focused on spike, the target of currently available vaccines, leaving the importance of variation elsewhere understudied. Here, we characterize a highly variable motif at residues 203-205 in nucleocapsid. Recreating the prominent nucleocapsid R203K+G204R mutation in an early pandemic background, we show that this mutation is alone sufficient to enhance SARS-CoV-2 replication and pathogenesis. We also link augmentation of SARS-CoV-2 infection by the R203K+G204R mutation to its modulation of nucleocapsid phosphorylation. Finally, we characterize an analogous alanine double substitution at positions 203-204. This mutant was found to mimic R203K+G204R, suggesting augmentation of infection occurs by disrupting the ancestral sequence. Together, our findings illustrate that mutations outside of spike are key components of SARS-CoV-2's adaptation to human infection.

Rift Valley fever virus 78kDa envelope protein attenuates virus replication in macrophage-derived cell lines and viral virulence in mice.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus with a wide host range including ruminants and humans. RVFV outbreaks have had devastating effects on public health and the livestock industry in African countries. However, there is no approved RVFV vaccine for human use in non-endemic countries and no FDA-approved antiviral drug for RVFV treatment. The RVFV 78kDa protein (P78), which is a membrane glycoprotein, plays a role in virus dissemination in the mosquito host, but its biological role in mammalian hosts remains unknown. We generated an attenuated RVFV MP-12 strain-derived P78-High virus and a virulent ZH501 strain-derived ZH501-P78-High virus, both of which expressed a higher level of P78 and carried higher levels of P78 in the virion compared to their parental viruses. We also generated another MP-12-derived mutant virus (P78-KO virus) that does not express P78. MP-12 and P78-KO virus replicated to similar levels in fibroblast cell lines and Huh7 cells, while P78-High virus replicated better than MP-12 in Vero E6 cells, fibroblast cell lines, and Huh7 cells. Notably, P78-High virus and P78-KO virus replicated less efficiently and more efficiently, respectively, than MP-12 in macrophage cell lines. ZH501-P78-High virus also replicated poorly in macrophage cell lines. Our data further suggest that inefficient binding of P78-High virus to the cells led to inefficient virus internalization, low virus infectivity and reduced virus replication in a macrophage cell line. P78-High virus and P78-KO virus showed lower and higher virulence than MP-12, respectively, in young mice. ZH501-P78-High virus also exhibited lower virulence than ZH501 in mice. These data suggest that high levels of P78 expression attenuate RVFV virulence by preventing efficient virus replication in macrophages. Genetic alteration leading to increased P78 expression may serve as a novel strategy for the attenuation of RVFV virulence and generation of safe RVFV vaccines.

STAT-1 Knockout Mice as a Model for Wild-Type Sudan Virus (SUDV).

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014-2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 101 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures.

Discovery of a novel highly potent broad-spectrum heterocyclic chemical series of arenavirus cell entry inhibitors.

We identified and explored the structure-activity relationship (SAR) of a novel heterocyclic chemical series of arenavirus cell entry inhibitors. Optimized lead compounds, including diphenyl-substituted imidazo[1,2-a]pyridines, benzimidazoles, and benzotriazoles exhibited low to sub-nanomolar potency against both pseudotyped and infectious Old and New World arenaviruses, attractive metabolic stability in human and most nonhuman liver microsomes as well as a lack of hERG K + channel or CYP enzyme inhibition. Moreover, the straightforward synthesis of several lead compounds (e.g., the simple high yield 3-step synthesis of imidazo[1,2-a]pyridine 37) could provide a cost-effective broad-spectrum arenavirus therapeutic that may help to minimize the cost-prohibitive burdens associated with treatments for emerging viruses in economically challenged geographical settings.