Biology subjects

Moss, B.

Publications and source records attributed to Moss, B..

11 recordsLinked to original sources

bioRxiv2025

Single cell transcriptomics in a treatment-segregated cohort exposes a STAT3-regulated therapeutic gap in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic pulmonary disease of unknown etiology. Since approved IPF drugs only slow disease progression, novel therapeutics are required that improve clinical outcomes. Here we report a single cell lung RNA-Seq and gene regulatory network analysis of the largest IPF cohort assembled to date. Segregating this cohort based on status of treatment with approved first-generation IPF antifibrotics (untreated, nintedanib- and pirfenidone-treated), we describe for the first time the transcriptional landscape of untreated IPF across 40 lung cell types, and the elements of this program that are impacted by these antifibrotics. On average, nearly 60% of the untreated IPF-dysregulated transcriptome is refractory to treatment with these drugs, a transcriptional deficit we refer to as the IPF therapeutic gap. Gene regulatory network analysis indicated a dominant functional footprint for the transcription factor STAT3 in both untreated IPF and the IPF therapeutic gap. Validating our analysis in a translational precision cut lung slice platform that recapitulates IPF explants, pharmacological inhibition of STAT3 reduced the IPF therapeutic gap in numerous lung cell types. Finally, we resolved a STAT3-anchored master regulatory network comprising numerous profibrotic transcription factors in IPF alveolar fibroblasts, a critical fibrotic lineage. Our study represents a comprehensive resource for translational lung fibrosis research and introduces a strategy for drug discovery that is adaptable to human disease more broadly.

cell biology↗

bioRxiv2025

Antibody Binding and Neutralizing Targets within the Predicted Structure of the Poxvirus Multiprotein Entry-Fusion Complex

The increased incidence of mpox emphasizes a need for new and improved vaccines. Poxviruses rely on a highly conserved but poorly characterized 11-protein entry-fusion complex (EFC), providing numerous potential targets. Here, we demonstrate that antibodies induced by six of 10 EFC proteins are neutralizing. Protein targets of the neutralizing and non-neutralizing antibodies are located within discrete regions of a model of the EFC predicted by AlphaFold3. Two newly identified targets, A16 and G9, at the apex of the EFC induced cross-neutralizing orthopoxvirus antibodies and protected female mice against a lethal VACV infection. Unexpectedly, antibodies to A16 and G9 were not detected following infection by attenuated or pathogenic VACV, likely due to physical sequestration of the proteins in the viral membrane. Our findings provide a model for the physical, immunogenic and antigenic structure of the EFC, new immunogens for incorporation into recombinant vaccines and suggest a novel poxvirus immune evasion strategy. 150 words.

microbiology↗

bioRxiv2025

The 2.3 A Structure of A21, a Protein Component of the Conserved Poxvirus Entry-Fusion Complex

Poxviruses are exceptional in having an entry-fusion complex (EFC) consisting of eleven conserved proteins embedded in the membrane of mature virions. With the goal of understanding the function of the EFC, extensive efforts have been made to determine the structures and roles of its components, and to date, structures have been determined for nine of the eleven proteins. Here, we report the crystal structure of A21, the 10th EFC protein, comprising two -helices clasping a twisted antiparallel {beta}-sheet stabilized by two conserved disulfide bonds. The stability of each of the three A21 loops is provided by hydrogen bonds between main-chain atoms and several highly conserved residues, making the overall fold of A21 and its orthologs resilient to evolutionary change. Based on AlphaFold modeling and phylogenetic analysis of A21, we suggest that its highly conserved N-terminal transmembrane domain and C-terminal -helix enable A21 integration into EFC, where it primarily interacts with the G3/L5 subcomplex and the smallest of EFC components, the O3 protein. HighlightsO_LIWith the structure of A21 determined by us, the structures of 10 out of the 11 proteins forming the entry-fusion complex (EFC) of the poxvirus are now known. C_LIO_LIA21 features a highly conserved transmembrane (TM) domain, which, along with the C--helix, facilitates the integration of A21 and its orthologs into the EFC. C_LIO_LIWithin the EFC, A21 primarily interacts with the G3/L5 subcomplex and the O3 peptide. C_LI

molecular biology↗

bioRxiv2024

Mpox mRNA-1769 Vaccine Inhibits Orthopoxvirus Replication at Intranasal, Intrarectal, and Cutaneous Sites of Inoculation

The increasing incidence of mpox in Africa and the recent global outbreak with evidence of sexual transmission have stimulated interest in new vaccines and therapeutics. Our previous study demonstrated that mice immunized twice with a quadrivalent lipid nanoparticle vaccine comprising four monkeypox virus mRNAs raised neutralizing antibodies and antigen-specific T cells and were protected against a lethal intranasal challenge with vaccinia virus. Here we extended these findings by using live animal imaging to demonstrate that the mRNA vaccine greatly reduced virus replication and spread from an intranasal site of inoculation and prevented detectable replication at intrarectal and cutaneous inoculation sites. Moreover, considerable protection was achieved with a single vaccination and a booster vaccination enhanced protection for at least 4 months. Protection was related to the amount of mRNA inoculated, which correlated with neutralizing antibody levels. The role of antibody in protection was demonstrated by passive transfer of immune serum pre- or post-challenge to immunocompetent and immunodeficient mice lacking mature B and T cells and therefore unable to mount an adaptive response. These findings provide insights into the mechanism and extent of mRNA vaccine induced protection of orthopoxviruses and support clinical testing.

microbiology↗

bioRxiv2023

Human FAM111A inhibits vaccinia virus replication by degrading viral uncoating factor I3 and is antagonized by poxvirus host range factor SPI-1

Poxviruses are large double-stranded DNA viruses that infect a wide range of animals including humans. Since the eradication of smallpox, other members of the poxvirus family, such as monkeypox virus (MPXV) are still posing a great threat to public health. Vaccinia virus (VACV) is a prototypic poxvirus used as the vaccine strain for smallpox eradication. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified vaccinia virus Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A, a nuclear protein that regulates host DNA replication, was shown to restrict the replication of VACV-{Delta}SPI-1 in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-{Delta}SPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-{Delta}SPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like protease domain and DNA binding domain but not the PCNA-interacting motif. We further discovered that FAM111A translocated into the cytoplasm upon VACV infection and this process was mediated by the cGAS-STING signaling pathway. Infection-triggered FAM111A degraded the nuclear pore complex via its protease activity, translocated to the cytoplasm, and interacted with and promoted the degradation of virus DNA binding protein I3 in a DNA-dependent manner. Interestingly, the protease activity of FAM111A was only needed for nuclear export but not I3 degradation as further analysis showed I3 was degraded through autophagy. Moreover, VACV SPI-1 was found primarily in the nucleus of infected cells and antagonized FAM111A by prohibiting its nuclear export. MPXV and lumpy skin disease virus SPI-1s also inhibited human FAM111A. Our findings reveal the detailed mechanism by which FAM111A functions to restrict a cytoplasmic DNA virus and provide explanations for the immune evasive function of VACV SPI-1.

microbiology↗

bioRxiv2022

A monkeypox mRNA-lipid nanoparticle vaccine targeting virus binding, entry, and transmission drives protection against lethal orthopoxviral challenge

Monkeypox virus (MPXV) caused a global outbreak in 2022, fueled by behaviorally-altered and enhanced human-to-human transmission. While smallpox vaccines were rapidly deployed to curb spread and disease among those at highest risk, breakthrough disease was noted after complete immunization. Given the imminent threat of additional zoonotic events as well as the virus evolving ability to drive human-to-human transmission, there is an urgent need for the development of a MPXV-specific vaccine that is able to also confer broad protection against evolving strains and related orthopoxviruses. Here, we demonstrate that an mRNA-lipid nanoparticle vaccine encoding a set of four highly conserved MPXV surface proteins involved in virus attachment, entry and transmission can induce MPXV-specific immunity and heterologous protection against a lethal vaccinia virus (VACV) challenge. Compared to Modified Vaccinia Virus Ankara (MVA), which forms the basis for the current MPXV vaccine, mRNA-vaccination generated superior neutralizing and cellular spread-inhibitory activities against MPXV and VACV as well as greater Fc-effector Th1-biased humoral immunity to the four MPXV antigens and the four VACV homologs. Single MPXV antigen mRNA vaccines provided partial protection against VACV challenge, while combinations of two, three or four MPXV antigen expressing mRNAs protected against disease-related weight loss and death. Remarkably, the cross-protection by multivalent MPXV mRNAs was superior to the homologous protection by MVA, associated with a combination of neutralizing and non-neutralizing antibody functions. These data reveal robust protection against VACV using an mRNA-based vaccine targeting four highly conserved viral surface antigens, linked to the induction of highly functional antibodies able to rapidly control viral infection.

immunology↗

bioRxiv2022

Enhanced Protection from SARS-CoV-2 Variants by MVA-Based Vaccines Expressing Matched or Mismatched S Proteins Administered Intranasally to hACE2 Mice

The continuous evolution of SARS-CoV-2 strains is contributing to the prolongation of the global pandemic. We previously reported the prevention or more rapid clearance of SARS-CoV-2 from the nasal turbinates and lungs of susceptible K18-hACE2 mice that had been vaccinated intranasally (IN) rather than intramuscularly (IM) with a recombinant MVA (rMVA) expressing a modified S protein of the ancestor SARS-CoV-2 strain. Here, we constructed additional rMVAs and pseudoviruses expressing modified S protein of SARS-CoV-2 variants and compared the ability of vaccines with S proteins that were matched or mismatched to neutralize variants, bind to S proteins and protect K18-hACE2 mice against SARS-CoV-2 challenge. Although vaccines with matched S proteins induced higher neutralizing antibodies, vaccines with mismatched S proteins still protected against severe disease and reduced virus and mRNAs in the lungs and nasal turbinates, though not as well as vaccines with matched S proteins. In mice earlier primed and boosted with rMVA expressing ancestral S, antibodies to the latter increased after one immunization with rMVA expressing Omicron S, but neutralizing antibody to Omicron required a second immunization. Passive transfer of Wuhan immune serum with Omicron S binding but undetectable neutralizing activities reduced infection of the lungs by the variant. Notably, the reduction in infection of the nasal turbinates and lungs was significantly greater when the rMVAs were administered IN rather than IM and this held true for vaccines that were matched or mismatched to the challenge SARS-CoV-2.

microbiology↗

bioRxiv2022

Virulence Differences of Monkeypox Virus Clades 1, 2a and 2b.1 in a Small Animal Model

Human monkeypox, a disease with similarities to smallpox, is endemic in Africa where it has persisted as a zoonosis with limited human-to-human spread. Unexpectedly, the disease expanded globally in 2022 driven by human-to-human transmission outside of Africa. It is not yet known whether the latter is due solely to behavioral and environmental factors or whether the monkeypox virus is adapting to a new host. Genome sequencing has revealed differences between the current outbreak strains, classified as clade 2b, and the prior clade 2a and clade 1 viruses but whether these differences contribute to virulence or transmission has not been determined. We demonstrate that the wild-derived inbred CAST/EiJ mouse provides an exceptional animal model for investigating clade differences in monkeypox virus virulence and show that the order is clade 1 > clade 2a > clade 2b.1. The greatly reduced replication of the clade 2b.1 major outbreak strain in mice and absence of lethality at 100-times the lethal dose of a closely related clade 2a virus, despite similar multiplication in cell culture, suggest that clade 2b is evolving diminished virulence or adapting to other species. SIGNIFICANCEThree clades of monkeypox virus are recognized: clade 1 is present in the Congo Basin, causes 10% human mortality and is transmitted by rodents with little human-to-human spread; clade 2a exists in West Africa, has a low mortality and is also a zoonosis; clade 2b is currently spreading globally by human transmission. The genetic basis for differences in virulence and transmission have not been determined. A major roadblock is the need for a small animal model that can be studied under the stringent safety conditions required. Here we demonstrate that the three clades exhibit highly significant differences in CAST/EiJ mice in the order clade 1 > clade 2a > clade 2b, similar to the severity of clinical disease.

microbiology↗

bioRxiv2022

Exaptation of inactivated host enzymes for structural roles in orthopoxviruses and novel protein folds revealed by protein structure modeling

Viruses with large double-stranded DNA genomes appear to have captured the majority of their genes from the hosts at different stages of evolution. The origin of many virus genes is readily detected through highly significant sequence similarity with cellular homologs. This is the case, in particular, for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs so that their origin remains enigmatic. We sought to explore potential origins of proteins of unknown provenance encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus which includes major human pathogens. To this end, we used AlphaFold2, to predict the structures of all 214 proteins encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded a clear indication of origin for 14, along with validating several inferences previously made by sequence analysis. The major trend that emerges from these findings is the exaptation of enzymes from cellular organisms for non-enzymatic, structural roles in virus reproduction which is accompanied by disruption of catalytic sites and overall drastic divergence which precludes detection of homology at the sequence level. Among the 16 orthopoxvirus proteins found to be inactivated enzyme derivatives, are the poxvirus replication processivity factor A20, an inactivated derivative of bacterial NAD-dependent DNA ligase; major core protein A3, an inactivated deubiquitinase; F11, an inactivated prolyl hydroxylase; and more similar cases. However, for nearly one third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement, yielding novel protein folds.

microbiology↗

bioRxiv2022

Inhibition of vaccinia virus L1 N-myristoylation by the host N-myristoyltransferase inhibitor IMP-1088 generates non-infectious virions defective in cell entry

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we reveal the role of N-myristoylation during vaccinia virus (VACV) infection in human host cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors.

microbiology↗

bioRxiv2021

MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease

Replication-restricted modified vaccinia virus Ankara (MVA) is a licensed smallpox vaccine and numerous clinical studies investigating recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases have been completed or are in progress. Two rMVA COVID-19 vaccine trials are at an initial stage, though no animal protection studies have been reported. Here, we characterize rMVAs expressing the S protein of CoV-2. Modifications of full length S individually or in combination included two proline substitutions, mutations of the furin recognition site and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected human cells and was recognized by anti-RBD antibody and by soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced S-binding and pseudovirus-neutralizing antibodies. Boosting occurred following a second homologous rMVA but was higher with adjuvanted purified RBD protein. Weight loss and lethality following intranasal infection of transgenic hACE2 mice with CoV-2 was prevented by one or two immunizations with rMVAs or by passive transfer of serum from vaccinated mice. One or two rMVA vaccinations also prevented recovery of infectious CoV-2 from the lungs. A low amount of virus was detected in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of subgenomic mRNA in turbinates on day 2 only indicated that replication was abortive in immunized animals. SignificanceVaccines are required to control COVID-19 during the pandemic and possibly afterwards. Recombinant nucleic acids, proteins and virus vectors that stimulate immune responses to the CoV-2 S protein have provided protection in experimental animal or human clinical trials, though questions remain regarding their ability to prevent spread and the duration of immunity. The present study focuses on replication-restricted modified vaccinia virus Ankara (MVA), which has been shown to be a safe, immunogenic and stable smallpox vaccine and a promising vaccine vector for other infectious diseases and cancer. In a transgenic mouse model, one or two injections of recombinant MVAs that express modified forms of S inhibited CoV-2 replication in the upper and lower respiratory tracts and prevented severe disease.

microbiology↗