DNA Origami Presenting the Receptor Binding Domain of SARS-CoV-2 Elicit Robust Protective Immune Response (preprint)

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 of 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.

Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for therapy (preprint)

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. Keywords: SARS-CoV-2, Numb-associated kinases, kinase inhibitors, host-targeted antivirals

Novel lipid nanoparticle provides potent SARS-CoV-2 mRNA vaccine at low dose with low local reactogenicity, high thermostability and limited systemic biodistribution (preprint)

Concerns with current mRNA Lipid Nanoparticle (LNP) systems include dose-limiting reactogenicity, adverse events that may be partly due to systemic off target expression of the immunogen, and a very limited understanding of the mechanisms responsible for the frozen storage requirement. We applied a new rational design process to identify a novel multiprotic ionizable lipid, called C24, as the key component of the mRNA LNP delivery system. We show that the resulting C24 LNP has a multistage protonation behavior resulting in greater endosomal protonation and greater translation of an mRNA-encoded luciferase reporter after intramuscular (IM) administration compared to the standard reference MC3 LNP. Off-target expression in liver after IM administration was reduced 6 fold for the C24 LNP compared to MC3. Neutralizing titers in immunogenicity studies delivering a nucleoside-modified mRNA encoding for the diproline stabilized spike protein immunogen were 10 fold higher for the C24 LNP versus MC3, and protection against viral challenge in a SARS-CoV-2 mouse model occurred at a very low 0.25 µg prime/boost dose of the same immunogen in the C24 LNP. Injection site inflammation was notably reduced for C24 compared to MC3. In addition, we found the C24 LNP to be entirely stable in bioactivity and mRNA integrity when stored at 4 ºC for at least 19 days. Storage at higher temperatures reduced both bioactivity and mRNA integrity, but less so for C24 than MC3, and in a manner consistent with the phosphodiester transesterification reaction mechanism of mRNA cleavage. The higher potency, lower injection site inflammation, and higher stability of the C24 LNP present important advancements in the evolution mRNA vaccine delivery.

Pan-ErbB inhibition protects from SARS-CoV-2 replication, inflammation, and injury (preprint)

Effective therapies are needed to combat emerging viruses. Seventeen candidates that rescue cells from SARS-CoV-2-induced lethality and target diverse functions emerged in a screen of 4,413 compounds. Among the hits was lapatinib, an approved inhibitor of the ErbB family of receptor tyrosine kinases. Lapatinib and other pan-ErbB inhibitors suppress replication of SARS-CoV-2 and unrelated viruses with a high barrier to resistance. ErbB4, but not lapatinib's cancer targets ErbB1 and ErbB2, is required for SARS-CoV-2 entry and Venezuelan equine encephalitis virus infection and is a molecular target mediating lapatinib's antiviral effect. In human lung organoids, lapatinib protects from SARS-CoV-2-induced activation of pathways implicated in acute and chronic lung injury downstream of ErbBs (p38-MAPK, MEK/ERK, and AKT/mTOR), pro-inflammatory cytokine production, and epithelial barrier injury. These findings reveal regulation of viral infection, inflammation, and tissue injury via ErbBs and propose approved candidates to counteract these effects with implications for coronaviruses and unrelated viruses.

Hyperimmunized Chickens Produce Neutralizing Antibodies Against SARS-CoV-2 (preprint)

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 suggest 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.

Development of a novel hybrid alphavirus-SARS-CoV-2 particle for rapid in vitro screening and quantification of neutralization antibodies, antiviral drugs, and viral mutations (preprint)

Timely development of vaccines and antiviral drugs are critical to control the coronavirus disease 2019 (COVID-19) global pandemic. Current methods for validation of vaccine efficacy involve the use of pseudoviruses, such as the SARS-CoV-2 spike protein (S) pseudotyped lentivirus or vesicular stomatitis virus (VSV), to quantify neutralizing antibodies for blocking viral infection. The process of pseudovirus infection and quantification is time consuming and can take days to complete. In addition, pseudoviruses contain structural proteins not native to SARS-CoV-2, which may alter particle properties in receptor binding and responses to antibody neutralization. Here we describe the development of a new hybrid alphavirus-SARS-CoV-2 particle (Ha-CoV-2) for rapid screening and quantification of neutralization antibodies and antiviral drugs. Ha-CoV-2 is a non-replicating SARS-CoV-2 virus-like particle, composed of only SARS-CoV-2 structural proteins (S, M, N, and E) and a RNA genome derived from a fast expressing alphavirus vector. We demonstrate that Ha-CoV-2 can rapidly and robustly express reporter genes in target cells within 3-5 hours following viral entry. We further validate the Ha-CoV-2 system for rapid quantification of neutralization antibodies and antiviral drugs. In addition, we assembled a Ha-CoV-2 particle bearing the D614G mutant spike protein, and found that the mutation led to an approximately 200% increase in virion infectivity. These results demonstrate that Ha-CoV-2 can also be applied for rapid monitoring and quantification of viral mutations for effects on neutralizing antibodies induced by vaccines.

Brilacidin, a COVID-19 Drug Candidate, Exhibits Potent In Vitro Antiviral Activity Against SARS-CoV-2 (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than one million deaths worldwide since it was first detected in 2019. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality. Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options. In this manuscript, we describe the potent antiviral activity exerted by brilacidin-a de novo designed synthetic small molecule that captures the biological properties of HDPs-on SARS-CoV-2 in a human lung cell line (Calu-3) and a monkey cell line (Vero). These data suggest that SARS-CoV-2 inhibition in these cell culture models is primarily a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin has demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 and thus supports brilacidin as a promising COVID-19 drug candidate. Highlights: Brilacidin potently inhibits SARS-CoV-2 in an ACE2 positive human lung cell line. Brilacidin achieved a high Selectivity Index of 426 (CC50=241{micro}M/IC50=0.565{micro}M). Brilacidin's main mechanism appears to disrupt viral integrity and impact viral entry. Brilacidin and remdesivir exhibit excellent synergistic activity against SARS-CoV-2. Significance Statement: SARS-CoV-2, the emergent novel coronavirus, has led to the current global COVID-19 pandemic, characterized by extreme contagiousness and high mortality rates. There is an urgent need for effective therapeutic strategies to safely and effectively treat SARS-CoV-2 infection. We demonstrate that brilacidin, a synthetic small molecule with peptide-like properties, is capable of exerting potent in vitro antiviral activity against SARS-CoV-2, both as a standalone treatment and in combination with remdesivir, which is currently the only FDA-approved drug for the treatment of COVID-19.

Amino acid transporter B0AT1 influence on ADAM17 interactions with SARS-CoV-2 receptor ACE2 putatively expressed in intestine, kidney, and cardiomyocytes (preprint)

SARS-CoV-2 exhibits significant experimental and clinical gastrointestinal, renal, and cardiac muscle tropisms responsible for local tissue-specific and systemic pathophysiology capriciously occurring in about half of COVID-19 patients. The underlying COVID-19 mechanisms engaged by these extra-pulmonary organ systems are largely unknown. We approached this knowledge gap by recognizing that neutral amino acid transporter B0AT1 (alternately called NBB, B, B0 in the literature) is a common denominator expressed nearly exclusively by three particular cell types: intestinal epithelia, renal proximal tubule epithelium, and cardiomyocytes. B0AT1 provides uptake of glutamine and tryptophan. The gut is the main depot expressing over 90% of the body's entire pool of SARS-CoV-2 receptor angiotensin converting enzyme-2 (ACE2) and B0AT1. Recent cryo-EM studies established that ACE2 forms a thermodynamically favored dimer-of-heterodimers complex with B0AT1 assembled in the form of a dimer of two ACE2:B0AT1 heterodimers anchored in plasma membranes. Prior epithelial cell studies demonstrated ACE2 chaperone trafficking of B0AT1. This contrasts with monomeric expression of ACE2 in lung pneumocytes, in which B0AT1 is undetectable. The cell types in question also express a disintegrin and metalloproteinase-17 (ADAM17) known to cleave and shed the ectodomain of monomeric ACE2 from the cell surface, thereby relinquishing protection against unchecked renin-angiotensin-system (RAS) events of COVID 19. The present study employed molecular docking modeling to examine the interplaying assemblage of ACE2, ADAM17 and B0AT1. We report that in the monomer form of ACE2, neck region residues R652-N718 provide unimpeded access to ADAM17 active site pocket, but notably R708 and S709 remained >10-15 [A] distant. In contrast, interference of ADAM17 docking to ACE2 in a dimer-of-heterodimers arrangement was directly correlated with the presence of a neighboring B0AT1 subunit complexed to the partnering ACE2 subunit of the 2ACE2:2B0AT1 dimer of heterodimers, representing the expression pattern putatively exclusive to intestinal, renal and cardiomyocyte cell types. The monomer and dimer-of-heterodimers docking models were not influenced by the presence of SARS-CoV-2 receptor binding domain (RBD) complexed to ACE2. The results collectively provide the underpinnings for understanding the role of B0AT1 involvement in COVID-19 and the role of ADAM17 steering ACE2 events in intestinal and renal epithelial cells and cardiomyocytes, with implications useful for consideration in pandemic public hygiene policy and drug development.

Development and pre-clinical characterization of two therapeutic equine formulations towards SARS-CoV-2 proteins for the potential treatment of COVID-19 (preprint)

In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited high in vitro neutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.

Maraviroc inhibits SARS-CoV-2 multiplication and s-protein mediated cell fusion in cell culture (preprint)

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 screen of 19 small molecules and 3 biologics was conducted in cell culture and the impact of treatment on viral titer was quantified by plaque assay. The screen 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 drug-treated cells. 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. HighlightsO_LIMaraviroc, FTY720, Nitazoxanide and Atorvastatin inhibit SARS-CoV-2 multiplication in cell culture. C_LIO_LIMaraviroc does not interfere with the interaction between SARS-CoV-2 spike protein and ACE2 receptor. C_LIO_LIMaraviroc exhibits only modest synergistic activities with FTY720, Nitazoxanide or Atorvastatin. C_LIO_LIMaraviroc reduces the extent of SARS-CoV-2 S-protein mediated cell fusion. C_LIO_LIMathematical modeling reveals that Maraviroc treatment will elicit an exponential decrease in viral load in a multicellular tissue environment. C_LI

Rapid community-driven development of a SARS-CoV-2 tissue simulator (preprint)

The 2019 novel coronavirus, SARS-CoV-2, is an emerging pathogen of critical significance to international public health. Knowledge of the interplay between molecular-scale virus-receptor interactions, single-cell viral replication, intracellular-scale viral transport, and emergent tissue-scale viral propagation is limited. Moreover, little is known about immune system-virus-tissue interactions and how these can result in low-level (asymptomatic) infections in some cases and acute respiratory distress syndrome (ARDS) in others, particularly with respect to presentation in different age groups or pre-existing inflammatory risk factors like diabetes. Given the nonlinear interactions within and among each of these processes, multiscale simulation models can shed light on the emergent dynamics that lead to divergent outcomes, identify actionable "choke points" for pharmacologic interventions, screen potential therapies, and identify potential biomarkers that differentiate patient outcomes. Given the complexity of the problem and the acute need for an actionable model to guide therapy discovery and optimization, we introduce and iteratively refine a prototype of a multiscale model of SARS-CoV-2 dynamics in lung tissue. The first prototype model was built and shared internationally as open source code and an online interactive model in under 12 hours, and community domain expertise is driving rapid refinements with a two-to-four week release cycle. In a sustained community effort, this consortium is integrating data and expertise across virology, immunology, mathematical biology, quantitative systems physiology, cloud and high performance computing, and other domains to accelerate our response to this critical threat to international health.