Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.IMPORTANCE Human astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.KEYWORDS antibody, astrovirus, capsid, receptor binding, structure, vaccines, virus neutralization T he Astroviridae family is comprised of two genera, Mamastrovirus and Avastrovirus, which infect mammalian and avian species, respectively (1). Members of the Avastrovirus genus cause a variety of disease manifestations, growth defects, and mortality in poultry (2). Members of the Mamastrovirus genus cause infections in humans and a wide range of mammals (3). Human astroviruses (HAstVs) are classified into eight canonical serotypes (HAstV-1 to ) within the Mamastrovirus genogroup 1 (4), where HAstV-1 is the predominant serotype worldwide (5, 6). HAstV is a leading cause of viral diarrhea in children, immunocompromised individuals, and the elderly (7). There are approximately 3.9 million cases of viral diarrhea due to HAstV in the United States every year (8). In addition, highly divergent strains of HAstV have recently been attributed to encephalitis in immunocompromised individuals (9-11).

Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpetuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs. As a proof of concept, these PNAs were tested against five MDR clinical isolates: carbapenem-resistant Escherichia coli, extended-spectrum beta-lactamase Klebsiella pneumoniae, New Delhi Metallo-beta-lactamase-1 carrying Klebsiella pneumoniae, and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of treatments leading to greater than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, the FAST platform offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells by repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellular Salmonella infection in human epithelial cells.

Proliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations in Escherichia coli, identifying 90 synergistic interactions. Identified gene targets were subsequently tested for antibiotic synergy on the transcriptomic level via multiplexed CRISPR-dCas9 and showed successful sensitization of E. coli without a separate fitness cost. These fitness neutral gene perturbations worked as co-therapies in reducing a Salmonella enterica intracellular infection in HeLa. Finally, these results informed the design of four antisense peptide nucleic acid (PNA) co-therapies, csgD, fnr, recA and acrA, against four MDR, clinically isolated bacteria. PNA combined with sub-minimal inhibitory concentrations of trimethoprim against two isolates of Klebsiella pneumoniae and E. coli showed three cases of re-sensitization with minimal fitness impacts. Our results highlight a promising approach for extending the utility of current antibiotics.

1Multidrug-resistant (MDR) bacteria pose a grave concern to global health. This 2 problem is further aggravated by a lack of new and effective antibiotics and 3 countermeasure platforms that can sustain the creation of novel antimicrobials in 4 the wake of new outbreaks or evolution of resistance to antibiotics. To address 5 this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform 6 that can develop effective therapies against MDR bacteria within a week. Our FAST 7 platform combines four essential modules-design, build, test, and delivery-of drug 8 development cycle. The design module comprises a bioinformatics toolbox that 9 predicts sequence-specific peptide nucleic acids (PNAs) that target non-traditional 10 pathways and genes of bacteria in minutes. The build module constitutes in-situ 11 synthesis and validation of selected PNAs in less than four days and efficacy 12 testing within a day. As a proof of concept, these PNAs were tested against MDR 13 clinical isolates. Here we tested Enterobacteriaceae including carbapenem-14 resistant Escherichia coli, extended-spectrum beta-lactamase (ESBL) Klebsiella 15 pneumoniae, New Delhi Metallo-beta-lactamase-1 carrying Klebsiella pneumoniae 16 and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% 17 of treatments, with nearly 18% of the treatments leading to more than 97% 18 decrease. Further, these PNAs are capable of potentiating antibiotic activity in the 19 clinical isolates despite presence of cognate resistance genes. Finally, FAST offers 20 a novel delivery approach to overcome limited transport of PNAs into mammalian 21 cells to clear intracellular infections. This method relies on repurposing the 22 bacterial Type III secretion system in conjunction with a kill switch that is effective 23 at eliminating 99.6% of an intracellular Salmonella infection in human epithelial 24 cells. Our findings demonstrate the potential of the FAST platform in treating MDR 25 bacteria in a rapid and effective manner.26 27 Keywords: Multi-drug resistant bacteria, Peptide nucleic acids, rationally designed 28 therapy, Type III secretion system.29

Traditional antibiotics are reaching obsolescence as a consequence of antibiotic resistance; therefore novel antibiotic approaches are needed. A recent non-traditional approach involves formation of protein aggregates as antimicrobials to disrupt bacterial homeostasis.Previous work on protein aggregates has focused on genome mining for aggregation-prone sequences in bacterial genomes rather than on rational design of aggregating antimicrobial peptides. Here, we use a synthetic biology approach to design an artificial gene encoding the first de novo aggregating antimicrobial peptide. This artificial gene, opaL (overexpressed protein aggregator Lipophilic), disrupts bacterial homeostasis by expressing extremely hydrophobic peptides. When this hydrophobic sequence is disrupted by acidic residues, consequent aggregation and antimicrobial effect decreases. Further, to deliver this artificial gene, we developed a probiotic approach using RK2, a broad host range conjugative plasmid, to transfer opaL from donor to recipient bacteria. We utilize RK2 to mobilize a shuttle plasmid carrying the opaL gene by adding the RK2 origin of transfer. We show that opaL is non-toxic to the donor, allowing for maintenance and transfer since its expression is under control of a promoter with a recipient-specific T7 RNA polymerase. Upon mating of donor and recipient Escherichia coli, we observe selective growth repression in T7 polymerase expressing recipients. This technique could be used to target desired pathogens by selecting pathogen-specific promoters to control opaL expression. This system provides a basis for the design and delivery of novel antimicrobial peptides.