Chimeric mRNA-based COVID-19 vaccine induces protective immunity against Omicron and Delta variants.

The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the effectiveness of currently approved mRNA vaccines. To achieve wider coverage of VOCs, we first constructed a cohort of mRNAs harboring a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). The mutation abolished the cleavage between the S1 and S2 subunits. Systematic evaluation in vaccinated mice discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. In particular, the neutralizing antibodies (nAb) produced by immunization with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs. However, neither mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original Omicron variant and some sublineages. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we engineered an mRNA-based chimeric immunogen by introducing the receptor-binding domain of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA induced potent and broadly nAbs against Omicron and Delta, which paves the way to developing new vaccine candidates to target emerging variants in the future.

Discovery and intranasal administration of a SARS-CoV-2 broadly acting neutralizing antibody with activity against multiple Omicron subvariants.

BACKGROUND: The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant and its BA.X lineages, has rendered ineffective a number of previously FDA emergency use authorized SARS-CoV-2 neutralizing antibody therapies. Furthermore, those approved antibodies with neutralizing activity against Omicron BA.1 are reportedly ineffective against the subset of Omicron subvariants that contain a R346K substitution, BA.1.1, and the more recently emergent BA.2, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. METHODS: Following a campaign of antibody discovery based on the vaccination of Harbor H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. FINDINGS: STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against each of the tested Omicron subvariants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. CONCLUSIONS: With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for intravenous or intranasal use in human clinical trials. FUNDING: Funded by CRIPT (no. 75N93021R00014), DARPA (HR0011-19-2-0020), and NCI Seronet (U54CA260560).

Unbiased approach to identify and assess efficacy of human SARS-CoV-2 neutralizing antibodies.

Coronavirus disease 2019 (COVID-19) continues to significantly impact the global population, thus countermeasure platforms that enable rapid development of therapeutics against variants of SARS-CoV-2 are essential. We report use of a phage display human antibody library approach to rapidly identify neutralizing antibodies (nAbs) against SARS-CoV-2. We demonstrate the binding and neutralization capability of two nAbs, STI-2020 and STI-5041, against the SARS-CoV-2 WA-1 strain as well as the Alpha and Beta variants. STI-2020 and STI-5041 were protective when administered intravenously or intranasally in the golden (Syrian) hamster model of COVID-19 challenged with the WA-1 strain or Beta variant. The ability to administer nAbs intravenously and intranasally may have important therapeutic implications and Phase 1 healthy subjects clinical trials are ongoing.

A systematic exploration of boceprevir-based main protease inhibitors as SARS-CoV-2 antivirals.

Boceprevir is an HCV NSP3 inhibitor that was explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (MPro) and contains an α-ketoamide warhead, a P1 ß-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butylglycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based MPro inhibitors including PF-07321332 and characterized their MPro inhibition potency in test tubes (in vitro) and 293T cells (in cellulo). Crystal structures of MPro bound with 10 inhibitors and cytotoxicity and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a ß-(S-2-oxopyrrolidin-3-yl)-alanyl (Opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the MPro active site cysteine. The P1 Opal residue, P2 dimethylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with MPro, explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains a P4 N-terminal isovaleramide. In its MPro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of MPro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency to inhibit ectopically expressed MPro in human 293T cells. In general, inhibitors with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to cytotoxicity tests on 293T cells and antiviral potency tests on three SARS-CoV-2 variants. They all have relatively low cytotoxicity and high antiviral potency with EC50 values around 1 µM. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.

Delivering an mRNA vaccine using a lymphatic drug delivery device improves humoral and cellular immunity against SARS-CoV-2.

The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic has captured the world's attention and remains an ongoing issue due to concerns of balancing protection against emerging variants of concern (VoCs) while also generating long lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid encoding the spike protein in a lipid nanoparticle formulation (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In an effort to improve immunity, a clinically-focused lymphatic drug delivery device (MuVaxx) was engineered to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) and tested to measure adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine-producing CD8 T cells, neutralizing antibodies against the Washington (wild type) strain and South African (Beta) variants, and LN-resident spike-specific memory B cells. Remarkably, a 4-fold elevated T cell response was observed in MuVaxx administered vaccination compared to that of IM administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatics-mediated delivery using MuVaxx for SARS-CoV-2 and VoC vaccines.

A multi-pronged evaluation of aldehyde-based tripeptidyl main protease inhibitors as SARS-CoV-2 antivirals.

As an essential enzyme of SARS-CoV-2, the COVID-19 pathogen, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 positions and the N-terminal protection group, we synthesized 18 tripeptidyl MPro inhibitors that contained also an aldehyde warhead and ß-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 position. Systematic characterizations of these inhibitors were conducted, including their in vitro enzymatic inhibition potency, X-ray crystal structures of their complexes with MPro, their inhibition of MPro transiently expressed in 293T cells, and cellular toxicity and SARS-CoV-2 antiviral potency of selected inhibitors. These inhibitors have a large variation of determined in vitro enzymatic inhibition IC50 values that range from 4.8 to 650 nM. The determined in vitro enzymatic inhibition IC50 values reveal that relatively small side chains at both P2 and P3 positions are favorable for achieving high in vitro MPro inhibition potency, the P3 position is tolerable toward unnatural amino acids with two alkyl substituents on the α-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. In all structures, the MPro active site cysteine interacts covalently with the aldehyde warhead of the bound inhibitor to form a hemithioacetal that takes an S configuration. For all inhibitors, election density around the N-terminal protection group is weak indicating possible flexible binding of this group to MPro. In MPro, large structural variations were observed on residues N142 and Q189. Unlike their high in vitro enzymatic inhibition potency, most inhibitors showed low potency to inhibit MPro that was transiently expressed in 293T cells. Inhibitors that showed high potency to inhibit MPro transiently expressed in 293T cells all contain O-tert-butyl-threonine at the P3 position. These inhibitors also exhibited relatively low cytotoxicity and high antiviral potency. Overall, our current and previous studies indicate that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for tripeptidyl aldehyde inhibitors of MPro.

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay.

As an essential enzyme of SARS-CoV-2, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of MPro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular MPro inhibition information to assess an MPro inhibitor. We used this assay to analyze 30 known MPro inhibitors. Contrary to their strong antiviral effects and up to 10 µM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests.

Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay

As an essential enzyme of SARS-CoV-2, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of MPro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular MPro inhibition information to assess an MPro inhibitor. We used this assay to analyze 30 known MPro inhibitors. Contrary to their strong antiviral effects and up to 10 μM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests. A cell sorting-based assay was developed to study about 30 SARS-CoV-2 main protease inhibitors, revealing MPI8 as the most potent one and others with likely different mechanisms.

Salicylanilides Reduce SARS-CoV-2 Replication and Suppress Induction of Inflammatory Cytokines in a Rodent Model.

SARS-CoV-2 virus has recently given rise to the current COVID-19 pandemic where infected individuals can range from being asymptomatic, yet highly contagious, to dying from acute respiratory distress syndrome. Although the world has mobilized to create antiviral vaccines and therapeutics to combat the scourge, their long-term efficacy remains in question especially with the emergence of new variants. In this work, we exploit a class of compounds that has previously shown success against various viruses. A salicylanilide library was first screened in a SARS-CoV-2 activity assay in Vero cells. The most efficacious derivative was further evaluated in a prophylactic mouse model of SARS-CoV-2 infection unveiling a salicylanilide that can reduce viral loads, modulate key cytokines, and mitigate severe weight loss involved in COVID-19 infections. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and a previously established favorable pharmacokinetic profile for the lead salicylanilide renders salicylanilides in general as promising therapeutics for COVID-19.

The role of afferent pulmonary innervation in ARDS associated with COVID-19 and potential use of resiniferatoxin to improve prognosis: A review.

Acute respiratory distress syndrome (ARDS) is one of the major causes of mortality associated with COVID-19 disease. Many patients will require intensive care with ventilatory support. Despite progress and best efforts, the mortality rates projected remain high. Historical data outlook points towards 80% expected fatality for patients progressing to advanced pulmonary disease, even when hospitalized in the intensive care unit. This is particularly true among the patient population over 65. Novel life-saving strategies are desperately needed to mitigate the high mortality that will be associated with the late stage SARS-CoV-2 viral infection associated with the fatal respiratory distress. We hypothesize that the morbidity, severity of the disease, and underlying physiological events leading to mortality are closely linked to the TRPV1 expressing neuronal system (afferent/efferent neurons) in the lungs. TRPV1 expressing cells are responsible for pain transmission, inflammation and immunomodulation throughout the entire pulmonary system and are modulating the processes associated with localized cytokine release (storm) and overall rapid disease progression. We suggest that therapeutic approaches targeting TRPV1 containing nerve fibers in the lungs will modulate the inflammatory and immune signal activity, leading to reduced mortality and better overall outcomes. We also propose to further explore the use of resiniferatoxin (RTX), an ultra-potent TRPV1 agonist currently in clinical trials for cancer and osteoarthritis pain, as a possible ablating agent of TRPV1 positive pulmonary pathways in patients with advanced COVID-19 disease.

Novel decoy cellular vaccine strategy utilizing transgenic antigen-expressing cells as immune presenter and adjuvant in vaccine prototype against SARS-CoV-2 virus.

A novel approach modifying cells to express viral markers to elicit protective immunity responses (decoy cellular vaccination) in the prevention of COVID-19 disease is currently being explored. Our approach entails utilizing SARS-CoV-2 Spike antigen-expressing, non-replicating cells as carriers and presenters of immunogenic antigens, so called "I-cells". By using irradiated cells as presenting vehicles of SARS-CoV-2 viral antigens(s) in a cellular context, these presented viral proteins can be recognized by the host immune system, thus, an efficient protective immune response might be elicited. Another advantage of this strategy is that the manufacturing process is scalable and yields uniform cell products allowing for "off-the-shelf" frozen supply availability. To prevent engraftment and proliferation of the cells after administration, the cells will be irradiated post-harvesting abolishing in vivo replication potential. Specifically, immunoreactive Spike-1 proteins from SARS-CoV-2 are expressed on the surface of irradiated target I-cells. Utilizing this innovative strategy, these viral antigen-displaying decoy cells will be developed as a vaccine to protect against COVID-19 disease.