Summary Ebolavirus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection1 and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV2. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann-Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that the anti-viral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the N-terminal domain3–7, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit8. Thus, NPC1 is essential for EboV entry and a target for anti-viral therapy.

SARS-CoV-2 is responsible for the coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoproteins of SARS-CoV-2 mediate viral entry and are the main targets for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic, and public health interventions. Here, we perform a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against SARS-CoV-2 Spike. Most infected individuals elicit anti-Spike antibodies within 2 weeks of the onset of symptoms. The levels of receptor binding domain (RBD)-specific immunoglobulin G (IgG) persist over time, and the levels of anti-RBD IgM decrease after symptom resolution. Although most individuals develop neutralizing antibodies within 2 weeks of infection, the level of neutralizing activity is significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.

, we found that virus-specific differences in the requirement for cathepsin B are correlated with sequence polymorphisms at residues 47 in GP1 and 584 in GP2. We applied these findings to the analysis of additional ebolavirus isolates and correctly predicted that the newly identified ebolavirus species Bundibugyo, containing D47 and I584, is cathepsin B dependent and that ebolavirus Zaire-1995, the single known isolate of ebolavirus Zaire that lacks D47, is not. We also obtained evidence for virusspecific differences in the role of cathepsin L, including cooperation with cathepsin B. These studies strongly suggest that the use of endosomal cysteine proteases as host factors for entry is a general property of members of the family Filoviridae. E bdaviruses and the closely related marburgvirus comprise the family Filoviridae (6,8,9,16). Several lines of recent investigation have elucidated key steps in the pathway for ebolavirus entry into cells. Ebolavirus particles attach to cells through the binding of their glycoprotein (GP) to cell surface receptors or lectins, such as TIM-1 and DC-SIGN, expressed on the plasma membrane (1,22,27,29,37). Membrane-bound particles are taken up into cells by a macropinocytosis-like mechanism and transported to late endosomes/lysosomes (LE/LY) (20,30,31,34), which contain essential entry host factors. We previously showed that cleavage of ebolavirus Zaire-Mayinga (EBOV-May) GP by endosomal cysteine proteases is required for infection (7). More recent work has revealed a second host factor in LE/LY that is broadly required by filoviruses: Niemann-Pick C1 (NPC1) (5, 10), a multipass transmembrane protein that resides in the limiting membrane (44). According to a recently proposed model, virus GP is cleaved by endosomal cysteine proteases and binds to NPC1 (10).Several studies have examined the role of protease cleavage in more detail for EBOV-May. They show that cathepsin L functions in concert with cathepsin B to cleave the GP1 subunit of virus GP (7,35). Structural and functional studies reveal that proteases remove the heavily glycosylated carboxyl-terminal domain of GP1 to expose a more conserved domain that is closely associated with GP2 (12,19,25) and that is proposed to contain the binding site for the filovirus receptor (4,13,24,28). Further, we recently showed that cleaved, but not uncleaved, GP1 binds to purified LE/LY membranes in an NPC1-dependent manner and coimmunoprecipitates with NPC1 (10). We identified small molecules that target NPC1, inhibit infection, and block the binding of cleaved GP1 to NPC1-containing membranes (10), strongly suggesting that the conserved N-terminal domain of GP1 is a ligand for NPC1. Taken together, these previous findings suggest a model in which proteolytic cleavage of GP to remove the carboxylterminal domain of GP1 and expose its N-terminal domain may be functionally analogous to the role of CD4 binding to HIV gp120 to displace highly variable loops and create the coreceptorbinding site (18). Our recent studies show that NPC1 expression...

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remain effective against emerging variants of concern in vitro and in vivo. CV3-1 binds to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the “RBD-up” position and triggers potent shedding of the S1 subunit. In contrast, CV3-25 inhibits membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among β-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

Highlights d One mRNA vaccine dose induces robust humoral responses in convalescent donors d An extended interval between doses leads to high humoral responses in naive donors d These responses are stronger than in naive donors vaccinated with a short interval d Vaccine-elicited antibodies decline more rapidly in naive than convalescent donors Authors

Wildlife reservoirs of broad-host-range viruses have the potential to enable evolution of viral variants that can emerge to infect humans. In North America, there is phylogenomic evidence of continual transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer (Odocoileus virginianus) through unknown means, but no evidence of transmission from deer to humans. We carried out an observational surveillance study in Ontario, Canada during November and December 2021 (n = 300 deer) and identified a highly divergent lineage of SARS-CoV-2 in white-tailed deer (B.1.641). This lineage is one of the most divergent SARS-CoV-2 lineages identified so far, with 76 mutations (including 37 previously associated with non-human mammalian hosts). From a set of five complete and two partial deer-derived viral genomes we applied phylogenomic, recombination, selection and mutation spectrum analyses, which provided evidence for evolution and transmission in deer and a shared ancestry with mink-derived virus. Our analysis also revealed an epidemiologically linked human infection. Taken together, our findings provide evidence for sustained evolution of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.

42 43 Word Count for Summary: 168 44 45 Word Count for the Body of the Text: 1149 46 47 SUMMARY 48 The SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 49 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of 50 deaths. The Spike glycoprotein of SARS-CoV-2 mediates viral entry and is the main target for 51 neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is 52 crucial for the development of vaccine, therapeutic and public health interventions. Here we 53 performed a cross-sectional study on 98 SARS-CoV-2-infected individuals to evaluate humoral 54 responses against the SARS-CoV-2 Spike. The vast majority of infected individuals elicited anti-55 Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding 56 domain (RBD)-specific IgG persisted overtime, while the levels of anti-RBD IgM decreased 57 after symptoms resolution. Some of the elicited antibodies cross-reacted with other human 58 coronaviruses in a genus-restrictive manner. While most of individuals developed neutralizing 59 antibodies within the first two weeks of infection, the level of neutralizing activity was 60 significantly decreased over time. Our results highlight the importance of studying the 61 persistence of neutralizing activity upon natural SARS-CoV-2 infection. 62 63 convalescent patients (Figure 3g,h). Cross-reactive neutralizing antibodies against SARS-CoV S 131 protein (Figure 2b) were also detected in some SARS-CoV-2-infected individuals, but with 132

The prevention and treatment of cardiovascular diseases (CVD) has largely focused on lowering circulating LDL cholesterol, yet a significant burden of atherosclerotic disease remains even when LDL is low. Recently, microRNAs (miRNAs) have emerged as exciting therapeutic targets for cardiovascular disease. miRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression by degradation or translational inhibition of target mRNAs. A number of miRNAs have been found to modulate all stages of atherosclerosis, particularly those that promote the efflux of excess cholesterol from lipid-laden macrophages in the vessel wall to the liver. However, one of the major challenges of miRNA-based therapy is to achieve tissue-specific, efficient, and safe delivery of miRNAs in vivo. We sought to develop chitosan nanoparticles (chNPs) that can deliver functional miRNA mimics to macrophages and to determine if these nanoparticles can alter cholesterol efflux and reverse cholesterol transport in vivo. We developed chNPs with a size range of 150−200 nm via the ionic gelation method using tripolyphosphate (TPP) as a cross-linker. In this method, negatively charged miRNAs were encapsulated in the nanoparticles by ionic interactions with polymeric components. We then optimized the efficiency of intracellular delivery of different formulations of chitosan/TPP/miRNA to mouse macrophages. Using a well-defined miRNA with roles in macrophage cholesterol metabolism, we tested whether chNPs could deliver functional miRNAs to macrophages. We find chNPs can transfer exogenous miR-33 to nai ̈ve macrophages and reduce the expression of ABCA1, a potent miR-33 target gene, both in vitro and in vivo, confirming that miRNAs delivered via nanoparticles can escape the endosomal system and function in the RISC complex. Because miR-33 and ABCA1 play a key role in regulating the efflux of cholesterol from macrophages, we also confirmed that macrophages treated with miR-33-loaded chNPs exhibited reduced cholesterol efflux to apolipoprotein A1, further confirming functional delivery of the miRNA. In vivo, mice treated with miR33-chNPs showed decreased reverse cholesterol transport (RCT) to the plasma, liver, and feces. In contrast, when efflux-promoting miRNAs were delivered via chNPs, ABCA1 expression and cholesterol efflux into the RCT pathway were improved. Over all, miRNAs can be efficiently delivered to macrophages via nanoparticles, where they can function to regulate ABCA1 expression and cholesterol efflux, suggesting that these miRNA nanoparticles can be used in vivo to target atherosclerotic lesions.