Efficacy and Safety of a Recombinant Plant-Based Adjuvanted Covid-19 Vaccine.

BACKGROUND: Coronavirus-like particles (CoVLP) that are produced in plants and display the prefusion spike glycoprotein of the original strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are combined with an adjuvant (Adjuvant System 03 [AS03]) to form the candidate vaccine. METHODS: In this phase 3, multinational, randomized, placebo-controlled trial conducted at 85 centers, we assigned adults (≥18 years of age) in a 1:1 ratio to receive two intramuscular injections of the CoVLP+AS03 vaccine or placebo 21 days apart. The primary objective of the trial was to determine the efficacy of the CoVLP+AS03 vaccine in preventing symptomatic coronavirus disease 2019 (Covid-19) beginning at least 7 days after the second injection, with the analysis performed after the detection of at least 160 cases. RESULTS: A total of 24,141 volunteers participated in the trial; the median age of the participants was 29 years. Covid-19 was confirmed by polymerase-chain-reaction assay in 165 participants in the intention-to-treat population; all viral samples that could be sequenced contained variants of the original strain. Vaccine efficacy was 69.5% (95% confidence interval [CI], 56.7 to 78.8) against any symptomatic Covid-19 caused by five variants that were identified by sequencing. In a post hoc analysis, vaccine efficacy was 78.8% (95% CI, 55.8 to 90.8) against moderate-to-severe disease and 74.0% (95% CI, 62.1 to 82.5) among the participants who were seronegative at baseline. No severe cases of Covid-19 occurred in the vaccine group, in which the median viral load for breakthrough cases was lower than that in the placebo group by a factor of more than 100. Solicited adverse events were mostly mild or moderate and transient and were more frequent in the vaccine group than in the placebo group; local adverse events occurred in 92.3% and 45.5% of participants, respectively, and systemic adverse events in 87.3% and 65.0%. The incidence of unsolicited adverse events was similar in the two groups up to 21 days after each dose (22.7% and 20.4%) and from day 43 through day 201 (4.2% and 4.0%). CONCLUSIONS: The CoVLP+AS03 vaccine was effective in preventing Covid-19 caused by a spectrum of variants, with efficacy ranging from 69.5% against symptomatic infection to 78.8% against moderate-to-severe disease. (Funded by Medicago; ClinicalTrials.gov number, NCT04636697.).

A small-molecule inhibitor of C5 complement protein.

The complement pathway is an important part of the immune system, and uncontrolled activation is implicated in many diseases. The human complement component 5 protein (C5) is a validated drug target within the complement pathway, as an anti-C5 antibody (Soliris) is an approved therapy for paroxysmal nocturnal hemoglobinuria. Here, we report the identification, optimization and mechanism of action for the first small-molecule inhibitor of C5 complement protein.

Citron-N is a neuronal Rho-associated protein involved in Golgi organization through actin cytoskeleton regulation.

The actin cytoskeleton is best known for its role during cellular morphogenesis. However, other evidence suggests that actin is also crucial for the organization and dynamics of membrane organelles such as endosomes and the Golgi complex. As in morphogenesis, the Rho family of small GTPases are key mediators of organelle actin-driven events, although it is unclear how these ubiquitously distributed proteins are activated to regulate actin dynamics in an organelle-specific manner. Here we show that the brain-specific Rho-binding protein Citron-N is enriched at, and associates with, the Golgi apparatus of hippocampal neurons in culture. Suppression of the whole protein or expression of a mutant form lacking the Rho-binding activity results in dispersion of the Golgi apparatus. In contrast, high intracellular levels induce localized accumulation of RhoA and filamentous actin, protecting the Golgi from the rupture normally produced by actin depolymerization. Biochemical and functional analyses indicate that Citron-N controls actin locally by assembling together the Rho effector ROCK-II and the actin-binding, neuron-specific, protein Profilin-IIa (PIIa). Together with recent data on endosomal dynamics, our results highlight the importance of organelle-specific Rho modulators for actin-dependent organelle organization and dynamics.

Discovery of 4-((2S,4S)-4-Ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic Acid (LNP023), a Factor B Inhibitor Specifically Designed To Be Applicable to Treating a Diverse Array of Complement Mediated Diseases.

The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.

Recovery of cell cycle delay following targeted gene repair by oligonucleotides.

We have previously shown that activation of the homologous recombinational repair pathway leads to a block of cell division in corrected cells, possibly through the activity of checkpoint proteins Chk1 and Chk2. In this study, we examine the long-term impact of this stalling on the growth of cells that have enabled gene repair events. Using a mutated eGFP gene as an episomal reporter, we show that corrected (eGFP-positive) cells contain only a few active replication templates 2 weeks after electroporation, yet do not display an apoptotic or senescent phenotype. By 6 weeks after electroporation, cells resume active replication with a cell cycle profile that is comparable to that of the non-corrected (eGFP-negative) population. These results indicate that the initial stalling is transient and eGFP-positive cells eventually resume a normal phenotypic growth pattern, allowing for passaging and expansion in vitro.

A Novel Selective Soluble Guanylate Cyclase Activator, MGV354, Lowers Intraocular Pressure in Preclinical Models, Following Topical Ocular Dosing.

Purpose: The nitric oxide/soluble guanylate cyclase/protein kinase G (NO/sGC/PKG) is known to be involved in the regulation of intraocular pressure (IOP) and may be dysregulated in glaucoma. The purpose is to demonstrate that the sGC activator MGV354 lowers IOP in a monkey model of glaucoma and could be considered as a possible new clinical drug candidate. Methods: Changes to cGMP were assessed in primary human trabecular meshwork (hNTM) cells and binding studies were conducted using human sGC full-length protein. Ocular safety tolerability, exposure, and efficacy studies were conducted in rabbit and monkey models following topical ocular dosing of MGV354. Results: sGC was highly expressed in the human and cynomolgus monkey outflow pathways. MGV354 had a 7-fold greater Bmax to oxidized sGC compared to that of reduced sGC and generated an 8- to 10-fold greater cGMP compared to that of a reduced condition in hTM cells. A single topical ocular dose with MGV354 caused a significant dose-dependent reduction of 20% to 40% (versus vehicle), lasting up to 6 hours in pigmented rabbits and 24 hours postdose in a cynomolgus monkey model of glaucoma. The MGV354-induced IOP lowering was sustained up to 7 days following once-daily dosing in a monkey model of glaucoma and was greater in magnitude compared to Travatan (travoprost)-induced IOP reduction. Mild to moderate ocular hyperemia was the main adverse effect noted. Conclusions: MGV354 represents a novel class of sGC activators that can lower IOP in preclinical models of glaucoma. The potential for sGC activators to be used as effective IOP-lowering drugs in glaucoma patients could be further determined in clinical studies.

The Discovery of ( S)-1-(6-(3-((4-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-2-methylphenyl)amino)-2,3-dihydro-1 H-inden-4-yl)pyridin-2-yl)-5-methyl-1 H-pyrazole-4-carboxylic Acid, a Soluble Guanylate Cyclase Activator Specifically Designed for Topical Ocular Delivery as a Therapy for Glaucoma.

Soluble guanylate cyclase (sGC), the endogenous receptor for nitric oxide (NO), has been implicated in several diseases associated with oxidative stress. In a pathological oxidative environment, the heme group of sGC can be oxidized becoming unresponsive to NO leading to a loss in the ability to catalyze the production of cGMP. Recently a dysfunctional sGC/NO/cGMP pathway has been implicated in contributing to elevated intraocular pressure associated with glaucoma. Herein we describe the discovery of molecules specifically designed for topical ocular administration, which can activate oxidized sGC restoring the ability to catalyze the production of cGMP. These efforts culminated in the identification of compound (+)-23, which robustly lowers intraocular pressure in a cynomolgus model of elevated intraocular pressure over 24 h after a single topical ocular drop and has been selected for clinical evaluation.

Targeted gene repair activates Chk1 and Chk2 and stalls replication in corrected cells.

Oligonucleotides (ODNs) can direct the exchange of single nucleotides at specific sites in the mammalian genome. It is generally believed that the ODN aligns in homologous register with its complementary site in the target gene and provides a template for the endogenous repair machinery to alter the sequence of the gene. We have been studying the initial phase of the reaction with particular emphasis on the cellular events that occur when the oligonucleotide enters the cell. Our results show that, following introduction of the oligonucleotide, the DNA-damage response pathway is activated, evidenced by the presence of phosphorylated p53, Chk1 and Chk2, respectively. As a result, progression of some of these cells through the cell cycle is slowed and those bearing corrected genes all contain phosphorylated Chk1 and Chk2. In contrast, uncorrected cells contain much lower levels of these proteins in the activated state and pass through the cell cycle in a normal fashion. We suggest that gene repair directed by oligonucleotides activates a pathway that prevents corrected cells from proliferating in cell culture through the activation of Chk1 and Chk2. Our results impact the future use of gene repair for ex vivo gene therapy applications.

Camptothecin enhances the frequency of oligonucleotide-directed gene repair in mammalian cells by inducing DNA damage and activating homologous recombination.

Camptothecin (CPT) is an anticancer drug that promotes DNA breakage at replication forks and the formation of lesions that activate the processes of homologous recombination (HR) and nonhomologous end joining. We have taken advantage of the CPT-induced damage response by coupling it to gene repair directed by synthetic oligonucleotides, a process in which a mutant base pair is converted into a wild-type one. Here, we show that pretreating DLD-1 cells with CPT leads to a significant stimulation in the frequency of correction of an integrated mutant enhanced green fluorescent protein gene. The stimulation is dose-dependent and coincident with the formation of double-strand DNA breaks. Caffeine, but not vanillin, blocks the enhancement of gene repair suggesting that, in this system, HR is the pathway most responsible for elevating the frequency of correction. The involvement of HR is further proven by studies in which wortmannin was seen to inhibit gene repair at high concentrations but not at lower levels that are known to inhibit DNA-PK activity. Taken together, our results suggest that DNA damage induced by CPT activates a cellular response that stimulates gene repair in mammalian cells.

Role of citron kinase in dendritic morphogenesis of cortical neurons.

Small GTPases of the rho family regulate the extensive rearrangements of the cytoskeleton that characterize neuronal differentiation. Citron kinase is a target molecule for activated rhoA, previously implicated in control of cytokinesis. We have found that, in addition, it could play an important role in modulating the extension of neuronal processes. Using constitutively active and dominant negative mutants, we showed that citron kinase is involved in the morphologic differentiation of N1E-115 neuroblastoma cells induced by serum starvation. More importantly, quantitative analysis of citron kinase knockout cerebral cortex displayed that this molecule may differentially regulate the morphology of the dendritic compartment in corticocollicular versus callosally-projecting pyramidal neurons.

Enhanced oligonucleotide-directed gene targeting in mammalian cells following treatment with DNA damaging agents.

Targeted gene repair, a form of oligonucleotide-directed mutagenesis, employs end-modified single-stranded DNA oligonucleotides to mediate single-base changes in chromosomal DNA. In this work, we use a specific 72-mer to direct the repair of a mutated eGFP gene stably integrated in the genome of DLD-1 cells. Corrected cells express eGFP that can be identified and quantitated by FACS. The repair of this mutant gene is dependent on the presence of a specifically designed oligonucleotide and the frequency with which the mutation is reversed is affected by the induction of DNA damage. We used hydroxyurea, VP16 (etoposide), and thymidine to modulate the rate of DNA replication through the stalling of the replication forks or the introduction of lesions. Addition of hydroxyurea or VP16 before the electroporation of the oligonucleotide, results in an accumulation of double-strand breaks (DSB) whose repair is facilitated by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The addition of thymidine results in DNA damage within replication forks, damage that is repaired through the process of homologous recombination. Our data suggest that gene repair activity is elevated when DNA damage induces or activates the homologous recombination pathway.