Identification of diphenylurea derivatives as novel endocytosis inhibitors that demonstrate broad-spectrum activity against SARS-CoV-2 and influenza A virus both in vitro and in vivo.

Rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) poses enormous challenge in the development of broad-spectrum antivirals that are effective against the existing and emerging viral strains. Virus entry through endocytosis represents an attractive target for drug development, as inhibition of this early infection step should block downstream infection processes, and potentially inhibit viruses sharing the same entry route. In this study, we report the identification of 1,3-diphenylurea (DPU) derivatives (DPUDs) as a new class of endocytosis inhibitors, which broadly restricted entry and replication of several SARS-CoV-2 and IAV strains. Importantly, the DPUDs did not induce any significant cytotoxicity at concentrations effective against the viral infections. Examining the uptake of cargoes specific to different endocytic pathways, we found that DPUDs majorly affected clathrin-mediated endocytosis, which both SARS-CoV-2 and IAV utilize for cellular entry. In the DPUD-treated cells, although virus binding on the cell surface was unaffected, internalization of both the viruses was drastically reduced. Since compounds similar to the DPUDs were previously reported to transport anions including chloride (Cl-) across lipid membrane and since intracellular Cl- concentration plays a critical role in regulating vesicular trafficking, we hypothesized that the observed defect in endocytosis by the DPUDs could be due to altered Cl- gradient across the cell membrane. Using in vitro assays we demonstrated that the DPUDs transported Cl- into the cell and led to intracellular Cl- accumulation, which possibly affected the endocytic machinery by perturbing intracellular Cl- homeostasis. Finally, we tested the DPUDs in mice challenged with IAV and mouse-adapted SARS-CoV-2 (MA 10). Treatment of the infected mice with the DPUDs led to remarkable body weight recovery, improved survival and significantly reduced lung viral load, highlighting their potential for development as broad-spectrum antivirals.

Brevicillin, a novel lanthipeptide from the genus Brevibacillus with antimicrobial, antifungal, and antiviral activity.

AIM: This study was aimed to determine antimicrobial and antiviral activity of a novel lanthipeptide from a Brevibacillus sp. for disinfectant application. METHODS AND RESULTS: The antimicrobial peptide (AMP) was produced by a bacterial strain AF8 identified as a member of the genus Brevibacillus representing a novel species. Whole genome sequence analysis using BAGEL identified a putative complete biosynthetic gene cluster involved in lanthipeptide synthesis. The deduced amino acid sequence of lanthipeptide named as brevicillin, showed >30% similarity with epidermin. Mass determined by MALDI-MS and Q-TOF suggested posttranslational modifications like dehydration of all Ser and Thr amino acids to yield Dha and Dhb, respectively. Amino acid composition determined upon acid hydrolysis is in agreement with core peptide sequence deduced from the putative biosynthetic gene bvrAF8. Biochemical evidence along with stability features ascertained posttranslational modifications during formation of the core peptide. The peptide showed strong activity with 99% killing of pathogens at 12 µg ml-1 within 1 minute. Interestingly, it also showed potent anti-SARS-CoV-2 activity by inhibiting ∼99% virus growth at 10 µg ml-1 in cell culture-based assay. Brevicillin did not show dermal allergic reactions in BALB/c mice. CONCLUSION: This study provides detailed description of a novel lanthipeptide and demonstrates its effective antibacterial, antifungal and anti-SARS-CoV-2 activity.

Neutralizing Efficacy of Encapsulin Nanoparticles against SARS-CoV2 Variants of Concern.

Rapid emergence of the SARS-CoV-2 variants has dampened the protective efficacy of existing authorized vaccines. Nanoparticle platforms offer a means to improve vaccine immunogenicity by presenting multiple copies of desired antigens in a repetitive manner which closely mimics natural infection. We have applied nanoparticle display combined with the SpyTag-SpyCatcher system to design encapsulin-mRBD, a nanoparticle vaccine displaying 180 copies of the monomeric SARS-CoV-2 spike receptor-binding domain (RBD). Here we show that encapsulin-mRBD is strongly antigenic and thermotolerant for long durations. After two immunizations, squalene-in-water emulsion (SWE)-adjuvanted encapsulin-mRBD in mice induces potent and comparable neutralizing antibody titers of 105 against wild-type (B.1), alpha, beta, and delta variants of concern. Sera also neutralizes the recent Omicron with appreciable neutralization titers, and significant neutralization is observed even after a single immunization.

Changes in Lifestyle, Health Practices, and Perceived Anxiety amidst the COVID-19 Pandemic: A Cross-Sectional Study Conducted on the General Population of Pakistan.

Background: The emergence of the coronavirus disease 2019 (COVID-19) pandemic has created unprecedented challenges across the globe. In addition to its debilitating impacts on health, the pandemic has also resulted in sudden changes in the quality of life. Our study aims to assess and highlight the alterations in lifestyle, health practices, and perceived anxiety in amongst the Pakistani denizens during the categorical lockdown across the country. Methods: A cross-sectional study was conducted during April 2020 through an online self-administered questionnaire using the snowball sampling technique. The online survey included a diversified set of questions ranging from the demographics, participants' sleeping routine, physical activity, hygiene habits, daily routine, and dietary habits during the quarantine period. It also assessed their anxiety through a series of questions, stretching from their own apprehension of their mental health to their assumption regarding the uncertainty of the future. SPSS v23 was used for data analysis, and chi-square test was applied. Results: A total of 384 respondents were included in the study. The mean age of the participants was 21.26 ± 4.267 years. It was observed that 203 (52.9%) individuals spent most of their time in self-isolation on social media, and 167 (43.5%) of participants claimed to be undertaking online classes or watching television. Furthermore, half of the participants noted that their sleep duration had increased 194 (50.5%), along with increased levels of perceived anxiety 242 (63%). Conclusion: The imposed nationwide lockdown due to COVID-19 has extensively affected the daily routine of the people living in Pakistan, eliciting profound changes in their sleeping patterns, dietary habits, mental health, and physical activity. Therefore, addressing the issues that arise amidst the lockdown remains pivotal.

Recent advances in the vaccine development for the prophylaxis of SARS Covid-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-caused Coronavirus Disease 2019 (COVID-19) is currently a global pandemic that has wreaked havoc on public health, lives, and the global economy. The present COVID-19 outbreak has put pressure on the scientific community to develop medications and vaccinations to combat COVID-19. However, according to highly optimistic forecasts, we could not have a COVID-19 vaccine until September 2020. This is due to the fact that a successful COVID-19 vaccine will necessitate a careful validation of effectiveness and adverse reactivity given that the target vaccine population includes high-risk people over 60, particularly those with severe co-morbid conditions, frontline healthcare professionals, and those involved in essential industrial sectors. For passive immunization, which is being considered for Covid-19, there are several platforms for vaccine development, each with its own advantages and disadvantages. The COVID-19 pandemic, which is arguably the deadliest in the last 100 years after the Spanish flu, necessitates a swift assessment of the various approaches for their ability to incite protective immunity and safety to prevent unintended immune potentiation, which is crucial to the pathogenesis of this virus. Considering the pandemic's high fatality rate and rapid spread, an efficient vaccination is critical for its management. As a result, academia, industry, and government are collaborating in unprecedented ways to create and test a wide range of vaccinations. In this review, we summarize the Covid-19 vaccine development initiatives, recent trends, difficulties, comparison between traditional vaccines development and Covid-19 vaccines development also listed the approved/authorized, phase-3 and pre-clinical trials Covid-19 vaccines in different countries.

A dimeric proteomimetic prevents SARS-CoV-2 infection by dimerizing the spike protein.

Protein tertiary structure mimetics are valuable tools to target large protein-protein interaction interfaces. Here, we demonstrate a strategy for designing dimeric helix-hairpin motifs from a previously reported three-helix-bundle miniprotein that targets the receptor-binding domain (RBD) of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Through truncation of the third helix and optimization of the interhelical loop residues of the miniprotein, we developed a thermostable dimeric helix-hairpin. The dimeric four-helix bundle competes with the human angiotensin-converting enzyme 2 (ACE2) in binding to RBD with 2:2 stoichiometry. Cryogenic-electron microscopy revealed the formation of dimeric spike ectodomain trimer by the four-helix bundle, where all the three RBDs from either spike protein are attached head-to-head in an open conformation, revealing a novel mechanism for virus neutralization. The proteomimetic protects hamsters from high dose viral challenge with replicative SARS-CoV-2 viruses, demonstrating the promise of this class of peptides that inhibit protein-protein interaction through target dimerization.

A novel plant lectin, NTL-125, interferes with SARS-CoV-2 interaction with hACE2.

COVID-19 caused by SARS-CoV-2 virus has had profound impact on the world in the past two years. Intense research is going on to find effective drugs to combat the disease. Over the past year several vaccines were approved for immunization. But SARS-CoV-2 being an RNA virus is continuously mutating to generate new variants, some of which develop features of immune escape. This raised serious doubts over the long-term efficacy of the vaccines. We have identified a unique mannose binding plant lectin from Narcissus tazetta bulb, NTL-125, which effectively inhibits SARS-CoV-2 replication in Vero-E6 cell line. In silico docking studies revealed that NTL-125 has strong affinity to viral Spike RBD protein, preventing it from attaching to hACE2 receptor, the gateway to cellular entry. Binding analyses revealed that all the mutant variants of Spike protein also have stronger affinity for NTL-125 than hACE2. The unique α-helical tail of NTL-125 plays most important role in binding to RBD of Spike. NTL-125 also interacts effectively with some glycan moieties of S-protein in addition to amino acid residues adding to the binding strength. Thus, NTL-125 is a highly potential antiviral compound of natural origin against SARS-CoV-2 and may serve as an important therapeutic for management of COVID-19.

A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern.

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.

Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Impact of COVID-19 lockdown on air quality in Chandigarh, India: Understanding the emission sources during controlled anthropogenic activities.

The variation in ambient air quality during COVID-19 lockdown was studied in Chandigarh, located in the Indo-Gangetic plain of India. Total 14 air pollutants, including particulate matter (PM10, PM2.5), trace gases (NO2, NO, NOx, SO2, O3, NH3, CO) and VOC's (benzene, toluene, o-xylene, m,p-xylene, ethylbenzene) were examined along with meteorological parameters. The study duration was divided into four parts, i.e., a) 21 days of before lockdown b) 21 days of the first phase of lockdown c) 19 days of the second phase of lockdown d) 14 days of the third phase of lockdown. The results showed significant reductions during the first and second phases for all pollutants. However, concentrations increased during the third phase. The concentrations of SO2, O3, and m,p-xylene kept on increasing throughout the study period, except for benzene, which continuously decreased. The percentage decrease in the concentrations during consecutive periods of lockdown were 28.8%, 23.4% and 1.1% for PM2.5 and 36.8%, 22.8% and 2.4% for PM10 respectively. The Principal Component Analysis (PCA) and characteristic ratios identified vehicular pollution as a primary source during different phases of lockdown. During the lockdown, residential sources showed a significant adverse impact on the air quality of the city. Regional atmospheric transfer of pollutants from coal-burning and stubble burning were identified as secondary sources of air pollution. The findings of the study offer the potential to plan air pollution reduction strategies in the extreme pollution episodes such as during crop residue burning period over Indo-Gangetic plain.