Superiority of intranasal over systemic administration of bioengineered soluble ACE2 for survival and brain protection against SARS-CoV-2 infection (preprint)

The present study was designed to investigate the effects of a soluble ACE2 protein termed ACE2 618-DDC-ABD, bioengineered to have long duration of action and high binding affinity to SARS-CoV-2, when administered either intranasally (IN) or intraperitoneally (IP) and before or after SARS-CoV-2 inoculation. K18hACE2 mice permissive for SARS-CoV-2 infection were inoculated with 2x104 PFU wildtype SARS-CoV-2. In one protocol, ACE2 618-DDC-ABD was given either IN or IP, pre- and post-viral inoculation. In a second protocol, ACE2 618-DDC-ABD was given either IN, IP or IN+IP but only post-viral inoculation. In addition, A549 and Vero E6 cells were used to test neutralization of SARS-CoV-2 variants by ACE2 618-DDC-ABD at different concentrations. Survival by day 5 was 0% in infected untreated mice, and 40% in mice from the ACE2 618-DDC-ABD IP-pre treated group. By contrast, in the IN-pre group survival was 90%, histopathology of brain and kidney was essentially normal and markedly improved in the lungs. When ACE2 618-DDC-ABD was administered only post viral inoculation, survival was 30% in the IN+IP group, 20% in the IN and 0% in the IP group. Brain SARS-CoV-2 titers were high in all groups except for the IN-pre group where titers were undetectable in all mice. In cells permissive for SARS-CoV-2 infection, ACE2 618-DDC-ABD neutralized wildtype SARS-CoV-2 at high concentrations, whereas much lower concentrations neutralized omicron BA.1. We conclude that ACE2 618-DDC-ABD provides much better survival and organ protection when administered IN and prior to viral inoculation than when given IP or after inoculation and that lowering brain titers is a critical determinant of survival and organ protection.

A comprehensive SARS-CoV-2-human protein-protein interactome network identifies pathobiology and host-targeting therapies for COVID-19 (preprint)

Physical interactions between viral and host proteins are responsible for almost all aspects of the viral life cycle and the host’s immune response. Studying viral-host protein-protein interactions is thus crucial for identifying strategies for treatment and prevention of viral infection. Here, we use high-throughput yeast two-hybrid and affinity purification followed by mass spectrometry to generate a comprehensive SARS-CoV-2-human protein-protein interactome network consisting of both binary and co-complex interactions. We report a total of 739 high-confidence interactions, showing the highest overlap of interaction partners among published datasets as well as the highest overlap with genes differentially expressed in samples (such as upper airway and bronchial epithelial cells) from patients with SARS-CoV-2 infection. Showcasing the utility of our network, we describe a novel interaction between the viral accessory protein ORF3a and the host zinc finger transcription factor ZNF579 to illustrate a SARS-CoV-2 factor mediating a direct impact on host transcription. Leveraging our interactome, we performed network-based drug screens for over 2,900 FDA-approved/investigational drugs and obtained a curated list of 23 drugs that had significant network proximities to SARS-CoV-2 host factors, one of which, carvedilol, showed promising antiviral properties. We performed electronic health record-based validation using two independent large-scale, longitudinal COVID-19 patient databases and found that carvedilol usage was associated with a significantly lowered probability (17%-20%, P < 0.001) of obtaining a SARS-CoV-2 positive test after adjusting various confounding factors. Carvedilol additionally showed anti-viral activity against SARS-CoV-2 in a human lung epithelial cell line [half maximal effective concentration (EC50) value of 4.1 µM], suggesting a mechanism for its beneficial effect in COVID-19. Our study demonstrates the value of large-scale network systems biology approaches for extracting biological insight from complex biological processes.

SARS-CoV-2 diverges from other betacoronaviruses in only partially activating the IRE1α/XBP1 ER stress pathway in human lung-derived cells (preprint)

Despite the efficacy of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 5 million individuals worldwide and continues to spread in countries where the vaccines are not yet widely available or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed essential for viral replication. We examined the activation status and requirement of the master UPR sensor IRE1 kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and the murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1 as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1 whereby it autophosphorylates, but its RNase fails to splice XBP1. Moreover, IRE1 was dispensable for optimal replication in human cells for all coronaviruses tested. Our findings demonstrate that IRE1 activation status differs upon infection with distinct betacoronaviruses and is not essential for efficient replication of any of them. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1 through an unknown mechanism, perhaps as a strategy to eliminate detection by the host immune system.

Generation of potent cellular and humoral immunity against SARS-CoV-2 antigens via conjugation to a polymeric glyco-adjuvant (preprint)

The SARS-CoV-2 virus has caused an unprecedented global crisis, and curtailing its spread requires an effective vaccine which elicits a diverse and robust immune response. We have previously shown that vaccines made of a polymeric glyco-adjuvant conjugated to an antigen were effective in triggering such a response in other disease models and hypothesized that the technology could be adapted to create an effective vaccine against SARS-CoV-2. The core of the vaccine platform is the copolymer p(Man-TLR7), composed of monomers with pendant mannose or a toll-like receptor 7 (TLR7) agonist. Thus, p(Man-TLR7) is designed to target relevant antigen-presenting cells (APCs) via mannose-binding receptors and then activate TLR7 upon endocytosis. The p(Man-TLR7) construct is amenable to conjugation to protein antigens such as the Spike protein of SARS-CoV-2, yielding Spike-p(Man-TLR7). Here, we demonstrate Spike-p(Man-TLR7) vaccination elicits robust antigen-specific cellular and humoral responses in mice. In adult and elderly wild-type mice, vaccination with Spike-p(Man-TLR7) generates high and long-lasting titers of anti-Spike IgGs, with neutralizing titers exceeding levels in convalescent human serum. Interestingly, adsorbing Spike-p(Man-TLR7) to the depot-forming adjuvant alum, amplified the broadly neutralizing humoral responses to levels matching those in mice vaccinated with formulations based off of clinically-approved adjuvants. Additionally, we observed an increase in germinal center B cells, antigen-specific antibody secreting cells, activated T follicular helper cells, and polyfunctional Th1-cytokine producing CD4+ and CD8+ T cells. We conclude that Spike-p(Man-TLR7) is an attractive, next-generation subunit vaccine candidate, capable of inducing durable and robust antibody and T cell responses.

Polymersomes decorated with SARS-CoV-2 spike protein receptor binding domain elicit robust humoral and cellular immunity (preprint)

A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

A novel soluble ACE2 protein totally protects from lethal disease caused by SARS-CoV-2 infection (preprint)

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) uses full-length angiotensin converting enzyme 2 (ACE2), which is membrane bound, as its initial cell contact receptor preceding viral entry. Here we report a human soluble ACE2 variant fused with a 5kD albumin binding domain (ABD) and bridged via a dimerization motif hinge-like 4-cysteine dodecapeptide, which we term ACE2 1-618-DDC-ABD. This protein is enzymatically active, has increased duration of action in vivo conferred by the ABD-tag, and displays 20-30-fold higher binding affinity to the SARS-CoV-2 receptor binding domain than its des-DDC monomeric form (ACE2 1-618-ABD) due to DDC-linked dimerization. ACE2 1-618-DDC-ABD was administered for 3 consecutive days to transgenic k18-hACE2 mice, a model that develops lethal SARS-CoV-2 infection, to evaluate the preclinical preventative/ therapeutic value for COVID-19. Mice treated with ACE2 1-618-DDC-ABD developed a mild to moderate disease for the first few days assessed by a clinical score and modest weight loss. The untreated control animals, by contrast, became severely ill and had to be sacrificed by day 6/7 and lung histology revealed extensive pulmonary alveolar hemorrhage and mononuclear infiltrates. At 6 days, mortality was totally prevented in the treated group, lung histopathology was improved and viral titers markedly reduced. This demonstrates for the first time in vivo the preventative/ therapeutic potential of a novel soluble ACE2 protein in a preclinical animal model.

Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response (preprint)

The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARS-CoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.

Enhanced immunogenicity of a synthetic DNA vaccine expressing consensus SARS-CoV-2 Spike protein using needle-free immunization (preprint)

The ongoing global pandemic of Coronavirus Disease 2019 (COVID-19) calls for an urgent development of effective and safe prophylactic and therapeutic measures. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is a major immunogenic and protective protein, and plays a crucial role in viral pathogenesis. In this study, we successfully constructed a synthetic codon-optimized DNA-based vaccine as a countermeasure against SARS-CoV-2; denoted as VIU-1005. The design was based on the synthesis of codon-optimized coding sequence for optimal mammalian expression of a consensus full-length S glycoprotein. The successful construction of the vaccine was confirmed by restriction digestion and sequencing, and the protein expression of the S protein was confirmed by western blot and immunofluorescence staining in mammalian cells. The immunogenicity of the vaccine was tested in two mouse models (BALB/c and C57BL/6J). Th1-skewed systemic S-specific IgG antibodies and neutralizing antibodies (nAbs) were significantly induced in both models four weeks post three injections with 100 g of the VIU-1005 vaccine via intramuscular needle injection but not intradermal or subcutaneous routes. Importantly, such immunization induced long-lasting IgG response in mice that lasted for at least 6 months. Interestingly, using a needle-free system, we showed an enhanced immunogenicity of VIU-1005 in which lower doses such as 25-50 g or less number of doses were able to elicit significantly high levels of Th1-biased systemic S-specific IgG antibodies and nAbs via intramuscular immunization compared to needle immunization. Compared to the intradermal needle injection which failed to induce any significant immune response, intradermal needle-free immunization elicited robust Th1-biased humoral response similar to that observed with intramuscular immunization. Furthermore, immunization with VIU-1005 induced potent S-specific cellular response as demonstrated by the significantly high levels of IFN-{gamma}, TNF and IL-2 cytokines production in memory CD8+ and CD4+ T cells in BALB/c mice. Together, our results demonstrate that the synthetic VIU-1005 candidate DNA vaccine is highly immunogenic and capable of inducing long-lasting and Th1-skewed immune response in mice. Furthermore, we show that the use of needle-free system could enhance the immunogenicity and minimize doses needed to induce protective immunity in mice, supporting further preclinical and clinical testing of this candidate vaccine.

In vitro inactivation of SARS-CoV-2 with 0.5% povidone iodine nasal spray (Nasodine) at clinically relevant concentrations and timeframes using tissue culture and PCR based assays (preprint)

BACKGROUND: There has been considerable speculation regarding the potential of PVP-I nasal disinfection as an adjunct to other countermeasures during the ongoing SARS-CoV-2 pandemic. Nasodine is a commercial formulation of 0.5% PVP-I that has been evaluated for safety and efficacy in human trials as a treatment for the common cold, including a Phase III trial (ANZCTR: ACTRN12619000764134). This study presents the first report of the in vitro efficacy of this formulation against SARS-CoV-2. METHODS: We conducted in vitro experiments to determine if the PVP-I formulation inactivated SARS-CoV-2 using two independent assays and virus isolates, and incorporating both PCR-based detection and cell culture methods to assess residual virus after exposure to the formulation. RESULTS: Based on cell culture results, the PVP-I formulation was found to rapidly inactivate SARS-CoV-2 isolates in vitro in short timeframes (15 seconds to 15 minutes) consistent with the minimum and maximum potential residence time in the nose. The Nasodine formula was found to be more effective than 0.5% PVP-I in saline. Importantly, it was found that the formulation inactivated culturable virus but had no effect on PCR-detectable viral RNA. CONCLUSIONS: The PVP-I formulation eliminated the viability of SARS-CoV-2 virus with short exposure times consistent with nasal use. PCR alone may not be adequate for viral quantification in nasal PVP-I studies; future studies should incorporate cell culture to assess viral viability. Nasal disinfection with PVP-I may be a useful intervention for newly-diagnosed COVID-19 patients to reduce transmission risk and disease progression to the lower respiratory tract.

Nanotraps for the containment and clearance of SARS-CoV-2 (preprint)

SARS-CoV-2 enters host cells through its viral spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors on the host cells. Here we show functionalized nanoparticles, termed "Nanotraps", completely inhibited SARS-CoV-2 infection by blocking the interaction between the spike protein of SARS-CoV-2 and the ACE2 of host cells. The liposomal-based Nanotrap surfaces were functionalized with either recombinant ACE2 proteins or anti-SARS-CoV-2 neutralizing antibodies and phagocytosis-specific phosphatidylserines. The Nanotraps effectively captured SARS-CoV-2 and completely blocked SARS-CoV-2 infection to ACE2-expressing human cell lines and primary lung cells; the phosphatidylserine triggered subsequent phagocytosis of the virus-bound, biodegradable Nanotraps by macrophages, leading to the clearance of pseudotyped and authentic virus in vitro. Furthermore, the Nanotraps demonstrated excellent biosafety profile in vitro and in vivo. Finally, the Nanotraps inhibited pseudotyped SARS-CoV-2 infection in live human lungs in an ex vivo lung perfusion system. In summary, Nanotraps represent a new nanomedicine for the inhibition of SARS-CoV-2 infection.

Host PDZ-containing proteins targeted by SARS-Cov-2 (preprint)

Small linear motif targeting protein interacting domains called PDZ have been identified at the C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins E, 3a, and N. Using a high-throughput approach of affinity-profiling against the full human PDZome, we identified sixteen human PDZ binders of SARS-CoV-2 proteins E, 3A and N showing significant interactions with dissociation constants values ranging from 3 M to 82 M. Six of them (TJP1, PTPN13, HTRA1, PARD3, MLLT4, LNX2) are also recognized by SARS-CoV while three (NHERF1, MAST2, RADIL) are specific to SARS-CoV-2 E protein. Most of these SARS-CoV-2 protein partners are involved in cellular junctions/polarity and could be also linked to evasion mechanisms of the immune responses during viral infection. Seven of the PDZ-containing proteins among binders of the SARS-CoV-2 proteins E, 3a or N affect significantly viral replication under knock-down gene expression in infected cells. This PDZ profiling identifying human proteins potentially targeted by SARS-CoV-2 can help to understand the multifactorial severity of COVID19 and to conceive effective anti-coronaviral agents for therapeutic purposes.

Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Infection as an Innate Antiviral Mechanism (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes, but not the ACE2-negative controls, blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50–150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Furthermore, a dramatic increase in plasma ACE2+ exosome levels were detected in patients with severe COVID-19 pathogenesis. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 in vitro (preprint)

There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.

From Face-to-Face to Online Modality: Implications for Undergraduate Learning While the World is Temporarily Closed in the Age of COVID-19 (preprint)

The second half of the Spring 2020 semester has been an unprecedented time globally due to the ongoing coronavirus disease 2019 (COVID-19). COVID-19 pandemic has forced more than one billion students out of school, has disrupted the world and led all university courses switched to online instruction social distancing actions taken to limit the spread of the virus. The aim of the present study is to evaluate the pandemic related changes for undergraduate students, to assess their perspectives related to their learning, experiences in two courses, and to discuss the far-reaching potential implications for the upcoming summer and fall semesters. An electronic survey was conducted to gather data on the student perceptions and learning characteristics of this transition from face-to-face (F2F) to online at a medium-sized university in the Southeast in the Spring 2020 semester. Nearly 88% of the participants indicated that the COVID-19 pandemic impacted their education, while 19% indicated that they prefer online over F2F learning. Furthermore, the online modality significantly increased attendance in General Biology I. Our study also showed that the usage of live conferencing and digital applications increased due to the pandemic. The current research fills the gap in the existing literature by providing the first study on the effects of the ongoing COVID-19 pandemic on undergraduate learning and experiences in the most unique dual modality of the Spring 2020 semester.

Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors (preprint)

The number of new cases world-wide for the COVID-19 disease is increasing dramatically, while efforts to contain Severe Acute Respiratory Syndrome Coronavirus 2 is producing varied results in different countries. There are three key SARS-CoV-2 enzymes potentially targetable with antivirals: papain-like protease (PLpro), main protease (Mpro), and RNA-dependent RNA polymerase. Of these, PLpro is an especially attractive target because it plays an essential role in several viral replication processes, including cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex (RTC), and disruption of host viral response machinery to facilitate viral proliferation and replication. Moreover, this enzyme is conserved across different coronaviruses and promising inhibitors have already been discovered for its SARS-CoV variant. Here we report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the enzyme from SARS-CoV-2 in both wild-type and mutant forms. These efforts include the first structures of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors, determined at 1.60-2.70 Angstroms. This collection of structures provides fundamental molecular and mechanistic insight to PLpro, and critically, illustrates details for inhibitors recognition and interactions. All presented compounds inhibit the peptidase activity of PLpro in vitro, and some molecules block SARS-CoV-2 replication in cell culture assays. These collated findings will accelerate further structure-based drug design efforts targeting PLpro, with the ultimate goal of identifying high-affinity inhibitors of clinical value for SARS-CoV-2.

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2 (preprint)

ABSTRACTSARS-CoV-2 Nsp15 is a uridylate-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family. It degrades the polyuridine extensions in (-) sense strand of viral RNA and some non-translated RNA on (+) sense strand. This activity seems to be responsible for the interference with the innate immune response and evasion of host pattern recognition. Nsp15 is highly conserved in coronaviruses suggesting that its activity is important for virus replication. Here we report first structures with bound nucleotides and show that SARS-CoV-2 Nsp15 specifically recognizes U in a pattern previously predicted for EndoU. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. Inhibitors of Nsp15 have been reported but not actively pursued into therapeutics. The current COVID-19 pandemic brought to attention the repurposing of existing drugs and the rapid identification of new antiviral compounds. Tipiracil is an FDA approved drug that is used with trifluridine in the treatment of colorectal cancer. Here, we combine crystallography, biochemical and whole cell assays, and show that this compound inhibits SARS-CoV-2 Nsp15 and interacts with the uridine binding pocket of the enzyme’s active site, providing basis for the uracil scaffold-based drug development.Competing Interest StatementThe authors have declared no competing interest.View Full Text