Human ACE2 protein is a molecular switch controlling the mode of SARS-CoV-2 transmission.

BACKGROUND: Human angiotensin-converting enzyme 2 (hACE2) is the receptor mediating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. hACE2 expression is low in the lungs and is upregulated after SARS-CoV-2 infection. How such a hACE2-limited pulmonary environment supports efficient virus transmission and how dynamic hACE2 expression affects SARS-CoV-2 infection are unclear. METHODS: We generated stable cell lines with different expression levels of hACE2 to evaluate how the hACE2 expression level can affect SARS-CoV-2 transmission. RESULTS: We demonstrated that the hACE2 expression level controls the mode of SARS-CoV-2 transmission. The hACE2-limited cells have an advantage for SARS-CoV-2 shedding, which leads to cell-free transmission. By contrast, enhanced hACE2 expression facilitates the SARS-CoV-2 cell-to-cell transmission. Furthermore, this cell-to-cell transmission is likely facilitated by hACE2-containing vesicles, which accommodate numerous SARS-CoV-2 virions and transport them to neighboring cells through intercellular extensions. CONCLUSIONS: This hACE2-mediated switch between cell-free and cell-to-cell transmission routes provides SARS-CoV-2 with advantages for either viral spread or evasion of humoral immunity, thereby contributing to the COVID-19 pandemic and pathogenesis.

Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

A receptor-binding domain-based nanoparticle vaccine elicits durable neutralizing antibody responses against SARS-CoV-2 and variants of concern.

Numerous vaccines have been developed to address the current COVID-19 pandemic, but safety, cross-neutralizing efficacy, and long-term protectivity of currently approved vaccines are still important issues. In this study, we developed a subunit vaccine, ASD254, by using a nanoparticle vaccine platform to encapsulate the SARS-CoV-2 spike receptor-binding domain (RBD) protein. As compared with the aluminum-adjuvant RBD vaccine, ASD254 induced higher titers of RBD-specific antibodies and generated 10- to 30-fold more neutralizing antibodies. Mice vaccinated with ASD254 showed protective immune responses against SARS-CoV-2 challenge, with undetectable infectious viral loads and reduced typical lesions in lung. Besides, neutralizing antibodies in vaccinated mice lasted for at least one year and were effective against various SARS-CoV-2 variants of concern, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, particle size, polydispersity index, and zeta-potential of ASD254 remained stable after 8-month storage at 4°C. Thus, ASD254 is a promising nanoparticle vaccine with good immunogenicity and stability to be developed as an effective vaccine option in controlling upcoming waves of COVID-19.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

BACKGROUND: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. METHODS: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. RESULTS: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. CONCLUSIONS: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

Rapid generation of mouse model for emerging infectious disease with the case of severe COVID-19.

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.

Multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon IMPRESS.

In vivo two-photon imaging is a valuable technique for studies of viral pathogenesis and host responses to infection in vivo. In this protocol, we describe a methodology for analyzing influenza virus-infected lung in vivo by two-photon imaging microscopy. We describe the surgical procedure, how to stabilize the lung, and an approach to analyzing the data. Further, we provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (Color-flu) for multicolor analysis. Setup of this model typically takes ~30 min and enables the observation of influenza virus-infected lungs for >4 h during the acute phase of the inflammation and at least 1 h in the lethal phase. This imaging system, which we termed two-photon IMPRESS (imaging pathophysiology research system), is broadly applicable to analyses of other respiratory pathogens and reveals disease progression at the cellular level in vivo.

Antigenic drift originating from changes to the lateral surface of the neuraminidase head of influenza A virus.

Influenza viruses possess two surface glycoproteins, haemagglutinin and neuraminidase (NA). Although haemagglutinin plays a major role as a protective antigen, immunity to NA also contributes to protection. The NA protein consists of a stalk and a head portion, the latter of which possesses enzymatic NA (or sialidase) activity. Like haemagglutinin, NA is under immune pressure, which leads to amino acid alterations and antigenic drift. Amino acid changes accumulate around the enzymatic active site, which is located at the top of the NA head. However, amino acid alterations also accumulate at the lateral surface of the NA head. The reason for this accumulation remains unknown. Here, we isolated seven anti-NA monoclonal antibodies (mAbs) from individuals infected with A(H1N1)pdm09 virus. We found that amino acid mutations on the lateral surface of the NA head abolished the binding of all of these mAbs. All seven mAbs activated Fcγ receptor (FcγR)-mediated signalling pathways in effector cells and five mAbs possessed NA inhibition activity, but the other two did not; however, all seven protected mice from lethal challenge infection through their NA inhibition activity and/or FcγR-mediated antiviral activity. Serological analysis of individuals infected with A(H1N1)pdm09 virus revealed that some possessed or acquired the anti-NA-lateral-surface antibodies following infection. We also found antigenic drift on the lateral surface of the NA head of isolates from 2009 and 2015. Our results demonstrate that anti-lateral-surface mAbs without NA inhibition activity can provide protection by activating FcγR-mediated antiviral activity and can drive antigenic drift at the lateral surface of the NA head. These findings have implications for NA antigenic characterization in that they demonstrate that traditional NA inhibition assays are inadequate to fully characterize NA antigenicity.

In vivo imaging of the pathophysiological changes and neutrophil dynamics in influenza virus-infected mouse lungs.

The pathophysiological changes that occur in lungs infected with influenza viruses are poorly understood. Here we established an in vivo imaging system that combines two-photon excitation microscopy and fluorescent influenza viruses of different pathogenicity. This approach allowed us to monitor and correlate several parameters and physiological changes including the spread of infection, pulmonary permeability, pulmonary perfusion speed, number of recruited neutrophils in infected lungs, and neutrophil motion in the lungs of live mice. Several physiological changes were larger and occurred earlier in mice infected with a highly pathogenic H5N1 influenza virus compared with those infected with a mouse-adapted human strain. These findings demonstrate the potential of our in vivo imaging system to provide novel information about the pathophysiological consequences of virus infections.

Imaging, Tracking and Computational Analyses of Virus Entry and Egress with the Cytoskeleton.

Viruses have a dual nature: particles are “passive substances” lacking chemical energy transformation, whereas infected cells are “active substances” turning-over energy. How passive viral substances convert to active substances, comprising viral replication and assembly compartments has been of intense interest to virologists, cell and molecular biologists and immunologists. Infection starts with virus entry into a susceptible cell and delivers the viral genome to the replication site. This is a multi-step process, and involves the cytoskeleton and associated motor proteins. Likewise, the egress of progeny virus particles from the replication site to the extracellular space is enhanced by the cytoskeleton and associated motor proteins. This overcomes the limitation of thermal diffusion, and transports virions and virion components, often in association with cellular organelles. This review explores how the analysis of viral trajectories informs about mechanisms of infection. We discuss the methodology enabling researchers to visualize single virions in cells by fluorescence imaging and tracking. Virus visualization and tracking are increasingly enhanced by computational analyses of virus trajectories as well as in silico modeling. Combined approaches reveal previously unrecognized features of virus-infected cells. Using select examples of complementary methodology, we highlight the role of actin filaments and microtubules, and their associated motors in virus infections. In-depth studies of single virion dynamics at high temporal and spatial resolutions thereby provide deep insight into virus infection processes, and are a basis for uncovering underlying mechanisms of how cells function.

The nuclear export factor CRM1 controls juxta-nuclear microtubule-dependent virus transport.

Transport of large cargo through the cytoplasm requires motor proteins and polarized filaments. Viruses that replicate in the nucleus of post-mitotic cells use microtubules and the dynein-dynactin motor to traffic to the nuclear membrane and deliver their genome through nuclear pore complexes (NPCs) into the nucleus. How virus particles (virions) or cellular cargo are transferred from microtubules to the NPC is unknown. Here, we analyzed trafficking of incoming cytoplasmic adenoviruses by single-particle tracking and super-resolution microscopy. We provide evidence for a regulatory role of CRM1 (chromosome-region-maintenance-1; also known as XPO1, exportin-1) in juxta-nuclear microtubule-dependent adenovirus transport. Leptomycin B (LMB) abolishes nuclear targeting of adenovirus. It binds to CRM1, precludes CRM1-cargo binding and blocks signal-dependent nuclear export. LMB-inhibited CRM1 did not compete with adenovirus for binding to the nucleoporin Nup214 at the NPC. Instead, CRM1 inhibition selectively enhanced virion association with microtubules, and boosted virion motions on microtubules less than ∼2 µm from the nuclear membrane. The data show that the nucleus provides positional information for incoming virions to detach from microtubules, engage a slower microtubule-independent motility to the NPC and enhance infection.

Plaque2.0-A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion.

Classical plaque assay measures the propagation of infectious agents across a monolayer of cells. It is dependent on cell lysis, and limited by user-specific settings and low throughput. Here, we developed Plaque2.0, a broadly applicable, fluorescence microscopy-based high-throughput method to mine patho-biological clonal cell features. Plaque2.0 is an open source framework to extract information from chemically fixed cells by immuno-histochemistry or RNA in situ hybridization, or from live cells expressing GFP transgene. Multi-parametric measurements include infection density, intensity, area, shape or location information at single plaque or population levels. Plaque2.0 distinguishes lytic and non-lytic spread of a variety of DNA and RNA viruses, including vaccinia virus, adenovirus and rhinovirus, and can be used to visualize simultaneous plaque formation from co-infecting viruses. Plaque2.0 also analyzes clonal growth of cancer cells, which is relevant for cell migration and metastatic invasion studies. Plaque2.0 is suitable to quantitatively analyze virus infections, vector properties, or cancer cell phenotypes.

Tracking viral genomes in host cells at single-molecule resolution.

Viral DNA trafficking in cells has large impacts on physiology and disease development. Current methods lack the resolution and accuracy to visualize and quantify viral DNA trafficking at single-molecule resolution. We developed a noninvasive protocol for accurate quantification of viral DNA-genome (vDNA) trafficking in single cells. Ethynyl-modified nucleosides were used to metabolically label newly synthesized adenovirus, herpes virus, and vaccinia virus vDNA, without affecting infectivity. Superresolution microscopy and copper(I)-catalyzed azide-alkyne cycloaddition (click) reactions allowed visualization of infection at single vDNA resolution within mammalian cells. Analysis of adenovirus infection revealed that a large pool of capsid-free vDNA accumulated in the cytosol upon virus uncoating, indicating that nuclear import of incoming vDNA is a bottleneck. The method described here is applicable for the entire replication cycle of DNA viruses and offers opportunities to localize cellular and viral effector machineries on newly replicated viral DNA, or innate immune sensors on cytoplasmic viral DNA.

Kinesin-1-mediated capsid disassembly and disruption of the nuclear pore complex promote virus infection.

Many viruses deliver their genomes into the host cell nucleus for replication. However, the size restrictions of the nuclear pore complex (NPC), which regulates the passage of proteins, nucleic acids, and solutes through the nuclear envelope, require virus capsid uncoating before viral DNA can access the nucleus. We report a microtubule motor kinesin-1-mediated and NPC-supported mechanism of adenovirus uncoating. The capsid binds to the NPC filament protein Nup214 and kinesin-1 light-chain Klc1/2. The nucleoporin Nup358, which is bound to Nup214/Nup88, interacts with the kinesin-1 heavy-chain Kif5c to indirectly link the capsid to the kinesin motor. Kinesin-1 disrupts capsids docked at Nup214, which compromises the NPC and dislocates nucleoporins and capsid fragments into the cytoplasm. NPC disruption increases nuclear envelope permeability as indicated by the nuclear influx of large cytoplasmic dextran polymers. Thus, kinesin-1 uncoats viral DNA and compromises NPC integrity, allowing viral genomes nuclear access to promote infection.

Identification of the cellular prohibitin 1/prohibitin 2 heterodimer as an interaction partner of the C-terminal cytoplasmic domain of the HIV-1 glycoprotein.

Our studies aim to elucidate the functions carried out by the very long, and in its length highly conserved, C-terminal cytoplasmic domain (Env-CT) of the HIV-1 glycoprotein. Mass spectrometric analysis of cellular proteins bound to a tagged version of the HIV Env-CT led to the identification of the prohibitin 1 and 2 proteins (Phb1 and Phb2). These ubiquitously expressed proteins, which exist as stable heterodimers, have been shown to have multiple functions within cells and to localize to multiple cellular and extracellular compartments. The specificity of binding of the Phb1/Phb2 complex to the Env-CT was confirmed in various manners, including coimmunoprecipitation with authentic provirally encoded, full-length Env. Strong binding was dependent on Env residues 790 to 800 and could be severely inhibited by the double mutation L799R/L800Q but not by mutation of these amino acids individually. Analysis of the respective mutant virions revealed that their different abilities to bind Phb1/Phb2 correlated with their replicative properties. Thus, mutated virions with single mutations [HIV-Env-(L799R) and HIV-Env-(L800Q)] replicated similarly to wild-type HIV, but HIV-Env-(L799R/L800Q) virions, which cannot bind Phb1/Phb2, exhibited a cell-dependent replicative phenotype similar to that of HIV-Env-Tr712, lacking the entire Env-CT domain. Thus, replicative spread was achieved, although somewhat delayed, in "permissive" MT-4 cells but failed to occur in "nonpermissive" H9 T cells. These results point to binding of the Phb1/Phb2 complex to the Env-CT as being of importance for replicative spread in nonpermissive cells, possibly by modulating critical Phb-dependent cellular process(es).