Antiviral Drugs and Vaccines for Omicron Variant: A Focused Review.

The Omicron variant of concern (VOC) replaced the delta variant rapidly and became the predominant strain due to more mutations in spike protein and receptor-binding domain (RBD) enhancing its infectivity and binding affinity. The severity of the illness is less than that of the delta variant. Omicron is nonsusceptible to REGEN-COV™ and bamlanivimab with etesevimab. Drugs that are effective against the Omicron variant are oral antiviral drugs such as Paxlovid (nirmatrelvir/ritonavir), remdesivir, sotrovimab, and molnupiravir. The potency of sotrovimab is reduced to 3-fold against Omicron, and 8-fold reduction in potency with sotrovimab is found in a particular variant of Omicron with a R346K substitution in spike protein. There are neither clinical trials comparing the efficacy of these 4 therapies with each other nor any data on a combination of two or more therapies. The current recommendation for mild-moderate, nonhospitalized patients who are at a high risk of disease progression is to use Paxlovid as the first-line option. If Paxlovid is not available or cannot be administered due to drug interactions, then the next best choice is sotrovimab. The third choice is remdesivir if sotrovimab is also not available and molnupiravir is to be given if the other three options are not available or cannot be administered. For prevention, 2130 (cilgavimab) in combination with COV2-2196 (tixagevimab) has been effective against BA.2 only. LY-CoV1404 (bebtelovimab) is recently authorized as it is effective against all sublineages of the Omicron variant. Regarding vaccine efficacy (VE), the 3-dose VE with mRNA vaccines at 14-60 days was found to be 71.6%, and after 60 days, it is 47.4%. There is a 34-38-fold reduction of neutralizing activity with prebooster sera and a 19-fold reduction with booster sera for the Omicron variant. This probably explains the reason for worldwide breakthrough infections with the Omicron variant with waning immunity. The neutralizing antibody response against Omicron elicited by the bivalent vaccine is superior to that of the ancestral Wuhan strain, without any safety concerns. For future advances, the ribosome display technology can be applied for the generation of human single-chain fragment variable (scFv) antibodies from B cells of recovered patients against Omicron and other Coronavirus variants as they are easier and faster to produce and have high affinity and high specificity.

Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain.

A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of KD = 1.794 × 10-8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.

The interplay between MMP-12 and t-PA in the brain after ischemic stroke.

Tissue-type plasminogen activator (t-PA) expression is known to increase following transient focal cerebral ischemia and reperfusion. Previously, we reported downregulation of t-PA upon suppression of matrix metalloproteinase-12 (MMP-12), following transient focal cerebral ischemia and reperfusion. We now present data on the temporal expression of t-PA in the brain after transient ischemia, as well as the interaction between MMP-12 and t-PA, two proteases associated with the breakdown of the blood-brain barrier (BBB) and ischemic brain damage. We hypothesized that there might be reciprocal interactions between MMP-12 and t-PA in the brain after ischemic stroke. This hypothesis was tested using shRNA-mediated gene silencing and computational modeling. Suppression of t-PA following transient ischemia and reperfusion in rats attenuated MMP-12 expression in the brain. The overall effect of t-PA shRNA administration was to attenuate the degradation of BBB tight junction protein claudin-5, diminish BBB disruption, and reduce neuroinflammation by decreasing the expression of the microglia/macrophage pro-inflammatory M1 phenotype (CD68, iNOS, IL-1ß, and TNFα). Reduced BBB disruption and subsequent lack of infiltration of macrophages (the main source of MMP-12 in the ischemic brain) could account for the decrease in MMP-12 expression after t-PA suppression. Computational modeling of in silico protein-protein interactions indicated that MMP-12 and t-PA may interact physically. Overall, our findings demonstrate that MMP-12 and t-PA interact directly or indirectly at multiple levels in the brain following an ischemic stroke. The present findings could be useful in the development of new pharmacotherapies for the treatment of stroke.

Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice.

Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.

Environmental factors and their role in the transmission of SARS-CoV-2.

In December of 2019, several cases of atypical pneumonia caused by an unknown agent were reported in Wuhan, the capital city of Hubei Province in China. In early January 2020, it was announced that these cases were caused by a novel coronavirus. The virus was later named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which causes a disease associated with atypical pneumonia termed Corona virus disease 2019 (COVID-19). Several respiratory viruses, including coronaviruses and influenza viruses tend to have prominent peaks of infection during colder seasons, especially in temperate regions. The cold temperatures, along with accompanying dry conditions can drive respiratory tract infections by assisting with viral transmission, weakening the human immune system, and increasing viral molecular stability. Though the topic of SARS-CoV-2 transmission and warm weather has been associated with misinformation campaigns, it is worth investigating since an informative answer may give an indication of the future behavior of SARS-CoV-2.

Recent progress in the repurposing of drugs/molecules for the management of COVID-19.

Introduction: In the current scenario, COVID-19 is a clinical and public health problem globally. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains the causative agent, emerged in China and continuously spreading across the globe. Until now, no efficient therapeutics have been approved, which combat COVID-19. FDA approved broad-spectrum drugs/molecules could be repurposed against the COVID-19 and are under clinical trials, if the outcome of these trials proves positive, it could be used to manage COVID-19 pandemic.Areas covered: This article reviews the FDA approved drugs/molecules which could be repurposed in the combination or single to combat the COVID-19.Expert opinion: In this focused review, we suggested the repurposing of the pathogen-centric, host-centric, dual sword (act as pathogen-centric as well as host-centric), and the combinatorial (pathogen and host-centric) drugs against COVID-19 patients. These drugs singly or in combination could be effective for the management of COVID-19.

Therapeutic and Vaccine Options for COVID-19: Status after Six Months of the Disease Outbreak.

An outbreak of the coronavirus disease 2019 (COVID-19) caused by an infection of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred in Wuhan, China, in December 2019. This new virus belongs to the group of enveloped RNA beta-coronaviruses. Symptoms may differ in various infected persons, but major presentations include dry cough, nasal congestion, shortness of breath, fever, and general malaise. The disease appears to be more severe in patients above the age of 60 years and those with underlying conditions such as diabetes, cancer, cardiovascular diseases, chronic respiratory disease, and hypertension. There is still no approved vaccine against COVID-19, but more than a hundred are at different stages of development. It is known that the development of new drugs takes a relatively long time, so several known and already-approved drugs are being repurposed for the treatment of this disease. In this review, we explore the therapeutic and vaccine options that are available for COVID-19 6 months after its outbreak. Most noteworthy among the therapeutic options are dexamethasone, remdesivir, Avigan (favipiravir) and convalescent plasma.

Ribosome Display Technology: Applications in Disease Diagnosis and Control.

Antibody ribosome display remains one of the most successful in vitro selection technologies for antibodies fifteen years after it was developed. The unique possibility of direct generation of whole proteins, particularly single-chain antibody fragments (scFvs), has facilitated the establishment of this technology as one of the foremost antibody production methods. Ribosome display has become a vital tool for efficient and low-cost production of antibodies for diagnostics due to its advantageous ability to screen large libraries and generate binders of high affinity. The remarkable flexibility of this method enables its applicability to various platforms. This review focuses on the applications of ribosome display technology in biomedical and agricultural fields in the generation of recombinant scFvs for disease diagnostics and control.

Generation and Selection of a Panel of Pan-Filovirus Single-Chain Antibodies using Cell-Free Ribosome Display.

Filoviruses, which include ebolaviruses and marburgvirus, can cause outbreaks of highly lethal hemorrhagic fever. This disease causes significant morbidity and mortality in humans and non-human primates, with human fatality rates reaching 90% during some outbreaks. Currently, there is lack of licensed vaccines or antivirals for these viruses. Since early symptoms of filovirus infection mimic more common diseases, there is a strong unmet public health and biodefense need for broad-spectrum filovirus rapid diagnostics. We have generated a panel of mouse single-chain Fv-antibodies (scFvs) to filovirus glycoproteins (GPs) using cell-free ribosome display and determined their cross-reactivity profiles to all known filovirus species. Two scFvs (4-2 and 22-1) were able to detect all known Ebolavirus and Marburgvirus species. This is the first report on ribosome display scFvs that can detect a broad set of filovirus GPs, which demonstrates the potential for use in diagnostics.

Exosomes Secreted by the Cocultures of Normal and Oxygen-Glucose-Deprived Stem Cells Improve Post-stroke Outcome.

Emerging stroke literature suggests that treatment of experimentally induced stroke with stem cells offered post-stroke neuroprotection via exosomes produced by these cells. Treatment with exosomes has great potential to overcome the limitations associated with cell-based therapies. However, in our preliminary studies, we noticed that the exosomes released from human umbilical cord blood-derived mesenchymal stem cells (MSCs) under standard culture conditions did not improve the post-stroke neurological outcome. Because of this apparent discrepancy, we hypothesized that exosome characteristics vary with the conditions of their production. Specifically, we suggest that the exosomes produced from the cocultures of regular and oxygen-glucose-deprived (OGD) MSCs in vitro would represent the exosomes produced from MSCs that are exposed to ischemic brain cells in vivo, and offer similar therapeutic benefits that the cell treatment would provide. We tested the efficacy of therapy with exosomes secreted from human umbilical cord blood (HUCB)-derived MSCs under in vitro hypoxic conditions on post-stroke brain damage and neurological outcome in a rat model of transient focal cerebral ischemia. We performed the TTC staining procedure as well as the neurological tests including the modified neurological severity scores (mNSS), the modified adhesive removal (sticky-tape), and the beam walking tests before ischemia and at regular intervals until 7 days reperfusion. Treatment with exosomes obtained from the cocultures of normal and OGD-induced MSCs reduced the infarct size and ipsilateral hemisphere swelling, preserved the neurological function, and facilitated the recovery of stroke-induced rats. Based on the results, we conclude that the treatment with exosomes secreted from MSCs at appropriate experimental conditions attenuates the post-stroke brain damage and improves the neurological outcome.

Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies.

BACKGROUND: Zika virus (ZIKV) is an emerging pathogen with no approved therapeutics and only limited diagnostics available. To address this gap, six mouse single-chain antibodies (scFvs) to ZIKV envelope (E) protein were isolated rapidly and efficiently from a ribosome-displayed antibody library constructed from the spleens of five immunized mice. METHODOLOGY/RESULTS: In this report, we have generated a panel of mouse scFvs to ZIKV E protein using ribosome display. The six scFvs demonstrated no cross-reactivity with DENV2 NGC envelope protein, suggesting specificity for ZIKV E protein. These scFvs showed differences in their affinity: two (scFv45-3, scFv63-1) of them were dominant after four rounds of panning, and showed higher affinity (an apparent Kd values from 19 to 27 nM) than the other four (scFv5-1, scFv7-2, scFv38-1, and scFv51-2). All six scFvs showed ZIKV-neutralizing activity in the plaque reduction neutralization test (PRNT) assay and their neutralizing activity was positively correlated with their affinities. CONCLUSIONS/SIGNIFICANCE: The scFvs (45-3 and 63-1) with highest affinity may have dual utility as diagnostics capable of recognizing ZIKV E subtypes and may be further developed to treat ZIKV infection. Our approach has the added advantage of generating Fc receptor-deficient antibodies, minimizing concern of antibody-dependent enhancement (ADE) of infection.

Streptokinase-A Drug for Thrombolytic Therapy: A Patent Review.

Accumulation of fibrin in blood vessels significantly increases thrombosis, leading to myocardial infraction and other cardiovascular diseases. Microbial enzymes are one option for curing this pathological condition. Fibrinolytic enzymes such as urokinase (UK), tissue type plasminogen activator (t-PA) and streptokinase (SK) attracted much attention for thrombolytic therapy. Among them SK is preferable in low-resource settings because it is cost-effective. Therefore, the purpose of this review is to summarize recent patents related to the occurrence, mechanism of action, physico-chemical properties, cloning and expression, production, structure, immunogenicity, chemical modification, in vivo application and clinical trials of SK. This patent review considers the properties and characteristics of SK that make it a preferred agent for thrombolytic therapy.

Laccases and their applications: a patent review.

Laccases are an interesting group of multi copper enzymes, which have received much attention of researchers in last decades due to their ability to oxidize both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants. This makes these biocatalysts very useful for their application in several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, polymer synthesis, bioremediation of contaminated soils, wine and beverage stabilization. Laccases are also used as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. Recently, the utility of laccases has also been applied to nanobiotechnology. This paper reviews recent and important patents related to the properties, heterologous production, molecular cloning, and applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.

Engineering and Applications of fungal laccases for organic synthesis.

Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis.Thus, laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed.

Urokinase-a very popular cardiovascular agent.

Urokinase (UK) [EC 3.4.99.26] is a serine protease that activates plasminogen to plasmin, which in turn degrades fibrin clots. Hence, UK finds its value as an important anti-thromboembolic drug. Plasmin has diverse physiological roles apart from its fibrinolytic role in the regulation of blood clotting. It has been implicated in complement activation, cell migration, wound healing, and generation of localized extracellular proteolysis during tissue remodelling, pro-hormone conversion, carcinogenesis and neoplasia. Among the plasminogen activators, UK provides a superior alternative for the simple reasons of its being more potent as compared to tissue-plasminogen activator and non-antigenic by virtue of its human origin unlike streptokinase. Based on these observations, UK is a very popular cardiovascular agent. Hence, UK, as one of the most potent plasminogen activators is attracting a great deal of attention. We will summarize recent patents related to the occurrence, mechanism of action, structure and function, physico-chemical properties, in vitro production, cloning and expression, purification and applications of UK.

Streptokinase--the drug of choice for thrombolytic therapy.

Thrombosis, the blockage of blood vessels with clots, can lead to acute myocardial infarction and ischemic stroke, both leading causes of death. Other than surgical interventions to remove or by pass the blockage, or the generation of collateral vessels to provide a new blood supply, the only treatment available is the administration of thrombolytic agents to dissolve the blood clot. This article describes a comprehensive review of streptokinase (SK). We discuss the biochemistry and molecular biology of SK, describing the mechanism of action, structures, confirmational properties, immunogenecity, chemical modification, and cloning and expression. The production and physico-chemical properties of this SK are also discussed. In this review, considering the properties and characteristics of SK that make it the drug of choice for thrombolytic therapy.

Amylase production in solid state fermentation by the thermophilic fungus Thermomyces lanuginosus.

The production of extracellular amylase by the thermophilic fungus Thermomyces lanuginosus was studied in solid state fermentation (SSF). Solid substrates such as wheat bran, molasses bran, rice bran, maize meal, millet cereal, wheat flakes, barley bran, crushed maize, corncobs and crushed wheat were studied for enzyme production. Growth on wheat bran gave the highest amylase activity. The maximum enzyme activity obtained was 534 U/g of wheat bran under optimum conditions of an incubation period of 120 h, an incubation temperature of 50 degrees C, an initial moisture content of 90%, a pH of 6.0, an inoculum level of 10% (v/w), a salt solution concentration of 1.5:10 (v/w) and a ratio of substrate weight to flask volume of 1:100 with soluble starch (1% w/w) and peptone (1% w/w) as supplements.