Gravity-perfused airway-on-a-chip optimized for quantitative BSL-3 studies of SARS-CoV-2 infection: barrier permeability, cytokine production, immunohistochemistry, and viral load assays.

Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism of action of drugs, to evaluate the efficacy of treatment options for human disease and impairment, and to assess drug toxicity. By using human cells co-cultured in three-dimensional constructs, organ chips can provide greater fidelity to the human cellular condition than their two-dimensional predecessors. However, with the rise of SARS-CoV-2 and the global COVID-19 pandemic, it became clear that many microphysiological systems were not compatible with or optimized for studies of infectious disease and operation in a Biosafety Level 3 (BSL-3) environment. Given that one of the early sites of SARS-CoV-2 infection is the airway, we created a human airway organ chip that could operate in a BSL-3 space with high throughput and minimal manipulation, while retaining the necessary physical and physiological components to recapitulate tissue response to infectious agents and the immune response to infection.

Exogenous Klotho Extends Survival in COVID-19 Model Mice.

A striking feature of COVID-19 disease is the broad spectrum of risk factors associated with case severity, as well as the diversity of clinical manifestations. While no central agent has been able to explain the pathogenesis of SARS-CoV-2 infection, the factors that most robustly correlate with severity are risk factors linked to aging. Low serum levels of Klotho, an anti-aging protein, strongly correlate with the pathogenesis of the same risk factors and manifestations of conditions similar to those expressed in severe COVID-19 cases. The current manuscript presents original research on the effects of the exogenous application of Klotho, an anti-aging protein, in COVID-19 model mice. Klotho supplementation resulted in a statistically significant survival benefit in parametric and non-parametric models. Further research is required to elucidate the mechanistic role Klotho plays in COVID-19 pathogenesis as well as the possible modulation SARS-CoV-2 may have on the biological aging process.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Interactions of Equine Viruses with the Host Kinase Machinery and Implications for One Health and Human Disease.

Zoonotic pathogens that are vector-transmitted have and continue to contribute to several emerging infections globally. In recent years, spillover events of such zoonotic pathogens have increased in frequency as a result of direct contact with livestock, wildlife, and urbanization, forcing animals from their natural habitats. Equines serve as reservoir hosts for vector-transmitted zoonotic viruses that are also capable of infecting humans and causing disease. From a One Health perspective, equine viruses, therefore, pose major concerns for periodic outbreaks globally. Several equine viruses have spread out of their indigenous regions, such as West Nile virus (WNV) and equine encephalitis viruses (EEVs), making them of paramount concern to public health. Viruses have evolved many mechanisms to support the establishment of productive infection and to avoid host defense mechanisms, including promoting or decreasing inflammatory responses and regulating host machinery for protein synthesis. Viral interactions with the host enzymatic machinery, specifically kinases, can support the viral infectious process and downplay innate immune mechanisms, cumulatively leading to a more severe course of the disease. In this review, we will focus on how select equine viruses interact with host kinases to support viral multiplication.

Inhibitors of the Ubiquitin-Mediated Signaling Pathway Exhibit Broad-Spectrum Antiviral Activities against New World Alphaviruses.

New World alphaviruses including Venezuelan Equine Encephalitis Virus (VEEV) and Eastern Equine Encephalitis Virus (EEEV) are mosquito-transmitted viruses that cause disease in humans and equines. There are currently no FDA-approved therapeutics or vaccines to treat or prevent exposure-associated encephalitic disease. The ubiquitin proteasome system (UPS)-associated signaling events are known to play an important role in the establishment of a productive infection for several acutely infectious viruses. The critical engagement of the UPS-associated signaling mechanisms by many viruses as host-pathogen interaction hubs led us to hypothesize that small molecule inhibitors that interfere with these signaling pathways will exert broad-spectrum inhibitory activity against alphaviruses. We queried eight inhibitors of the UPS signaling pathway for antiviral outcomes against VEEV. Three of the tested inhibitors, namely NSC697923 (NSC), bardoxolone methyl (BARM) and omaveloxolone (OMA) demonstrated broad-spectrum antiviral activity against VEEV and EEEV. Dose dependency and time of addition studies suggest that BARM and OMA exhibit intracellular and post-entry viral inhibition. Cumulatively, our studies indicate that inhibitors of the UPS-associated signaling pathways exert broad-spectrum antiviral outcomes in the context of VEEV and EEEV infection, supporting their translational application as therapeutic candidates to treat alphavirus infections.

DNA origami presenting the receptor binding domain of SARS-CoV-2 elicit robust protective immune response.

Effective and safe vaccines are invaluable tools in the arsenal to fight infectious diseases. The rapid spreading of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the coronavirus disease 2019 pandemic has highlighted the need to develop methods for rapid and efficient vaccine development. DNA origami nanoparticles (DNA-NPs) presenting multiple antigens in prescribed nanoscale patterns have recently emerged as a safe, efficient, and easily scalable alternative for rational design of vaccines. Here, we are leveraging the unique properties of these DNA-NPs and demonstrate that precisely patterning ten copies of a reconstituted trimer of the receptor binding domain (RBD) of SARS-CoV-2 along with CpG adjuvants on the DNA-NPs is able to elicit a robust protective immunity against SARS-CoV-2 in a mouse model. Our results demonstrate the potential of our DNA-NP-based approach for developing safe and effective nanovaccines against infectious diseases with prolonged antibody response and effective protection in the context of a viral challenge.

Venezuelan equine encephalitis virus E1 protein interacts with PDIA6 and PDI inhibition reduces alphavirus production.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus transmitted by mosquitos that can cause a febrile illness and induce severe neurological complications in humans and equine populations. Currently there are no FDA approved vaccines or antiviral treatments to combat VEEV. Proteomic techniques were utilized to create an interactome of the E1 fusion glycoprotein of VEEV. VEEV E1 interacted with a number of cellular chaperone proteins including protein disulfide isomerase family A member 6 (PDIA6). PDI inhibition through LOC14 and/or nitazoxanide treatment effectively decreased production of VEEV and other alphaviruses in vitro, including eastern equine encephalitis virus, Sindbis virus, and chikungunya virus. Decreased oxidoreductive capabilities of PDIs through LOC14 or nitazoxanide treatment impacted both early and late events in viral replication, including the production of non-infectious virions and decreased VEEV E1 disulfide bond formation. Results from this study identified PDIs as critical regulators of alphavirus replication and potential therapeutic targets.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Brilacidin as a Broad-Spectrum Inhibitor of Enveloped, Acutely Infectious Viruses.

Alphaviruses, belonging to the Togaviridae family, and bunyaviruses, belonging to the Paramyxoviridae family, are globally distributed and lack FDA-approved vaccines and therapeutics. The alphaviruses Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV) are known to cause severe encephalitis, whereas Sindbis virus (SINV) causes arthralgia potentially persisting for years after initial infection. The bunyavirus Rift Valley Fever virus (RVFV) can lead to blindness, liver failure, and hemorrhagic fever. Brilacidin, a small molecule that was designed de novo based on naturally occurring host defensins, was investigated for its antiviral activity against these viruses in human small airway epithelial cells (HSAECs) and African green monkey kidney cells (Veros). This testing was further expanded into a non-enveloped Echovirus, a Picornavirus, to further demonstrate brilacidin's effect on early steps of the viral infectious cycle that leads to inhibition of viral load. Brilacidin demonstrated antiviral activity against alphaviruses VEEV TC-83, VEEV TrD, SINV, EEEV, and bunyavirus RVFV. The inhibitory potential of brilacidin against the viruses tested in this study was dependent on the dosing strategy which necessitated compound addition pre- and post-infection, with addition only at the post-infection stage not eliciting a robust inhibitory response. The inhibitory activity of brilacidin was only modest in the context of the non-enveloped Picornavirus Echovirus, suggesting brilacidin may be less potent against non-enveloped viruses.

Application of a Human Blood Brain Barrier Organ-on-a-Chip Model to Evaluate Small Molecule Effectiveness against Venezuelan Equine Encephalitis Virus.

The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae. An effective therapeutic intervention strategy should ideally not only control viral load in the host, but also prevent and/or reverse deleterious events at the BBB. Two dimensional monocultures, including trans-well models that use endothelial cells, do not recapitulate the intricate multicellular environment of the BBB. Complex in vitro organ-on-a-chip models (OOC) provide a great opportunity to introduce human-like experimental models to understand the mechanistic underpinnings of the disease state and evaluate the effectiveness of therapeutic candidates in a highly relevant manner. Here we demonstrate the utility of a neurovascular unit (NVU) in analyzing the dynamics of infection and proinflammatory response following VEEV infection and therapeutic effectiveness of omaveloxolone to preserve BBB integrity and decrease viral and inflammatory load.

Hyperimmunized Chickens Produce Neutralizing Antibodies against SARS-CoV-2.

The novel severe acute respiratory syndrome (SARS) coronavirus, SARS-CoV-2, is responsible for the global COVID-19 pandemic. Effective interventions are urgently needed to mitigate the effects of COVID-19 and likely require multiple strategies. Egg-extracted antibody therapies are a low-cost and scalable strategy to protect at-risk individuals from SARS-CoV-2 infection. Commercial laying hens were hyperimmunized against the SARS-CoV-2 S1 protein using three different S1 recombinant proteins and three different doses. Sera and egg yolk were collected at three and six weeks after the second immunization for enzyme-linked immunosorbent assay and plaque-reduction neutralization assay to determine antigen-specific antibody titers and neutralizing antibody titers, respectively. In this study we demonstrate that hens hyperimmunized against the SARS-CoV-2 recombinant S1 and receptor binding domain (RBD) proteins produced neutralizing antibodies against SARS-CoV-2. We further demonstrate that antibody production was dependent on the dose and type of antigen administered. Our data suggests that antibodies purified from the egg yolk of hyperimmunized hens can be used as immunoprophylaxis in humans at risk of exposure to SARS-CoV-2.

Inhibition of Venezuelan Equine Encephalitis Virus Using Small Interfering RNAs.

Acutely infectious new world alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) pose important challenges to the human population due to a lack of effective therapeutic intervention strategies. Small interfering RNAs that can selectively target the viral genome (vsiRNAs) has been observed to offer survival advantages in several in vitro and in vivo models of acute virus infections, including alphaviruses such as Chikungunya virus and filoviruses such as Ebola virus. In this study, novel vsiRNAs that targeted conserved regions in the nonstructural and structural genes of the VEEV genome were designed and evaluated for antiviral activity in mammalian cells in the context of VEEV infection. The data demonstrate that vsiRNAs were able to effectively decrease the infectious virus titer at earlier time points post infection in the context of the attenuated TC-83 strain and the virulent Trinidad Donkey strain, while the inhibition was overcome at later time points. Depletion of Argonaute 2 protein (Ago2), the catalytic component of the RISC complex, negated the inhibitory effect of the vsiRNAs, underscoring the involvement of the siRNA pathway in the inhibition process. Depletion of the RNAi pathway proteins Dicer, MOV10, TRBP2 and Matrin 3 decreased viral load in infected cells, alluding to an impact of the RNAi pathway in the establishment of a productive infection. Additional studies focused on rational combinations of effective vsiRNAs and delivery strategies to confer better in vivo bioavailability and distribution to key target tissues such as the brain can provide effective solutions to treat encephalitic diseases resulting from alphavirus infections.

Brilacidin, a Non-Peptide Defensin-Mimetic Molecule, Inhibits SARS-CoV-2 Infection by Blocking Viral Entry.

Brilacidin (PMX-30063), a non-peptide defensin-mimetic small molecule, inhibits SARS-CoV-2 viral infection but the anti-viral mechanism is not defined. Here we determined its effect on the specific step of the viral life cycle. Brilacidin blocked SARS-CoV-2 infection but had no effect after viral entry. Brilacidin inhibited pseudotyped SARS-CoV-2 viruses expressing spike proteins from the P.1 Brazil strain and the B.1.1.7 UK strain. Brilacidin affected viral attachment in hACE2-dependent and independent manners depending on the concentrations. The inhibitory effect on viral entry was not mediated through blocking the binding of either the spike receptor-binding domain or the spike S1 protein to hACE2 proteins. Taken together, brilacidin inhibits SARS-CoV-2 infection by blocking viral entry and is active against SARS-CoV-2 variants.

Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for antiviral strategies.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.

Junin Virus Activates p38 MAPK and HSP27 Upon Entry.

Junín virus (JUNV), a New World arenavirus, is a rodent-borne virus and the causative agent of Argentine hemorrhagic fever. Humans become infected through exposure to rodent host secreta and excreta and the resulting infection can lead to an acute inflammatory disease with significant morbidity and mortality. Little is understood about the molecular pathogenesis of arenavirus hemorrhagic fever infections. We utilized Reverse Phase Protein Microarrays (RPPA) to compare global alterations in the host proteome following infection with an attenuated vaccine strain, Candid#1 (CD1), and the most parental virulent strain, XJ13, of JUNV in a human cell culture line. Human small airway epithelial cells were infected with CD1 or XJ13 at an MOI of 10, or mock infected. To determine proteomic changes at early timepoints (T = 1, 3, 8 and 24 h), the JUNV infected or mock infected cells were lysed in compatible buffers for RPPA. Out of 113 proteins that were examined by RPPA, 14 proteins were significantly altered following JUNV infection. Several proteins were commonly phosphorylated between the two strains and these correspond to entry and early replication events, to include p38 mitogen-activated protein kinase (MAPK), heat shock protein 27 (HSP27), and nuclear factor kappa B (NFκB). We qualitatively confirmed the alterations of these three proteins following infection by western blot analysis. We also determined that the inhibition of either p38 MAPK, with the small molecule inhibitor SB 203580 or siRNA knockdown, or HSP27, by siRNA knockdown, significantly decreases JUNV replication. Our data suggests that HSP27 phosphorylation at S82 upon virus infection is dependent on p38 MAPK activity. This work sheds light on the nuances of arenavirus replication.

Development of a hybrid alphavirus-SARS-CoV-2 pseudovirion for rapid quantification of neutralization antibodies and antiviral drugs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S)-pseudotyped viruses are commonly used for quantifying antiviral drugs and neutralizing antibodies. Here, we describe the development of a hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) pseudovirion, which is a non-replicating SARS-CoV-2 virus-like particle composed of viral structural proteins (S, M, N, and E) and an RNA genome derived from a fast-expressing alphaviral vector. We validated Ha-CoV-2 for rapid quantification of neutralization antibodies, antiviral drugs, and viral variants. In addition, as a proof of concept, we used Ha-CoV-2 to quantify the neutralizing antibodies from an infected and vaccinated individual and found that the one-dose vaccination with Moderna mRNA-1273 greatly increased the anti-serum titer by approximately 6-fold. The post-vaccination serum can neutralize all nine variants tested. These results demonstrate that Ha-CoV-2 can be used as a robust platform for the rapid quantification of neutralizing antibodies against SARS-CoV-2 and its emerging variants.

Rift Valley fever virus Gn V5-epitope tagged virus enables identification of UBR4 as a Gn interacting protein that facilitates Rift Valley fever virus production.

Rift Valley fever virus (RVFV) is an arbovirus that was first reported in the Rift Valley of Kenya which causes significant disease in humans and livestock. RVFV is a tri-segmented, negative-sense RNA virus consisting of a L, M, and S segments with the M segment encoding the glycoproteins Gn and Gc. Host factors that interact with Gn are largely unknown. To this end, two viruses containing an epitope tag (V5) on the Gn protein in position 105 or 229 (V5Gn105 and V5Gn229) were generated using the RVFV MP-12 vaccine strain as a backbone. The V5-tag insertion minimally impacted Gn functionality as measured by replication kinetics, Gn localization, and antibody neutralization assays. A proteomics-based approach was used to identify novel Gn-binding host proteins, including the E3 ubiquitin-protein ligase, UBR4. Depletion of UBR4 resulted in a significant decrease in RVFV titers and a reduction in viral RNA production.

Maraviroc inhibits SARS-CoV-2 multiplication and s-protein mediated cell fusion in cell culture.

In an effort to identify therapeutic intervention strategies for the treatment of COVID-19, we have investigated a selection of FDA-approved small molecules and biologics that are commonly used to treat other human diseases. A investigation into 18 small molecules and 3 biologics was conducted in cell culture and the impact of treatment on viral titer was quantified by plaque assay. The investigation identified 4 FDA-approved small molecules, Maraviroc, FTY720 (Fingolimod), Atorvastatin and Nitazoxanide that were able to inhibit SARS-CoV-2 infection. Confocal microscopy with over expressed S-protein demonstrated that Maraviroc reduced the extent of S-protein mediated cell fusion as observed by fewer multinucleate cells in the context of drug-treatment. Mathematical modeling of drug-dependent viral multiplication dynamics revealed that prolonged drug treatment will exert an exponential decrease in viral load in a multicellular/tissue environment. Taken together, the data demonstrate that Maraviroc, Fingolimod, Atorvastatin and Nitazoxanide inhibit SARS-CoV-2 in cell culture.

Inhibitors of Venezuelan Equine Encephalitis Virus Identified Based on Host Interaction Partners of Viral Non-Structural Protein 3.

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections.