Development and characterization of three novel mouse monoclonal antibodies targeting spike protein S1 subunit of Middle East Respiratory Syndrome Corona Virus.

BACKGROUND: Middle East Respiratory Syndrome Coronavirus is a highly pathogenic virus that poses a significant threat to public health. OBJECTIVE: The purpose of this study is to develop and characterize novel mouse monoclonal antibodies targeting the spike protein S1 subunit of the Middle East Respiratory Syndrome Corona Virus (MERS-CoV). METHODS: In this study, three mouse monoclonal antibodies (mAbs) against MERS-CoV were generated and characterized using hybridoma technology. The mAbs were evaluated for their reactivity and neutralization activity. The mAbs were generated through hybridoma technology by the fusion of myeloma cells and spleen cells from MERS-CoV-S1 immunized mice. The resulting hybridomas were screened for antibody production using enzyme-linked immunosorbent assays (ELISA). RESULTS: ELISA results demonstrated that all three mAbs exhibited strong reactivity against the MERS-CoV S1-antigen. Similarly, dot-ELISA revealed their ability to specifically recognize viral components, indicating their potential for diagnostic applications. Under non-denaturing conditions, Western blot showed the mAbs to have robust reactivity against a specific band at 116 KDa, corresponding to a putative MERS-CoV S1-antigen. However, no reactive bands were observed under denaturing conditions, suggesting that the antibodies recognize conformational epitopes. The neutralization assay showed no in vitro reactivity against MERS-CoV. CONCLUSION: This study successfully generated three mouse monoclonal antibodies against MERS-CoV using hybridoma technology. The antibodies exhibited strong reactivity against MERS-CoV antigens using ELISA and dot ELISA assays. Taken together, these findings highlight the significance of these mAbs for potential use as valuable tools for MERS-CoV research and diagnosis (community and field-based surveillance and viral antigen detection).

Analysis of the nasopharyngeal microbiome and respiratory pathogens in COVID-19 patients from Saudi Arabia.

BACKGROUND: Infection with SARS-CoV-2 may perturb normal microbiota, leading to secondary infections that can complicate the viral disease. The aim of this study was to probe the alteration of nasopharyngeal (NP) microbiota in the context of SARS-CoV-2 infection and obesity and to identify other respiratory pathogens among COVID-19 cases that may affect patients' health. METHODS: A total of 107 NP swabs, including 22 from control subjects and 85 from COVID-19 patients, were processed for 6S amplicon sequencing. The respiratory pathogens causing secondary infections were identified by RT-PCR assay, using a kit that contained specific primers and probes combinations to amplify 33 known respiratory pathogens. RESULTS: No significant (p > 0.05) difference was observed in the alpha and beta diversity analysis, but specific taxa differed significantly between the control and COVID-19 patient groups. Genera of Sphingomonas, Kurthia, Microbacterium, Methylobacterium, Brevibacillus, Bacillus, Acinetobacter, Lactococcus, and Haemophilus was significantly abundant (p < 0.05) in COVID-19 patients compared with a healthy control group. Staphylococcus was found in relatively high abundance (35.7 %) in the COVID-19 patient groups, mainly those treated with antibiotics. A relatively high percentage of Streptococcus was detected in COVID-19 patient groups with obesity or other comorbidities. Respiratory pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Salmonella species, along with Pneumocystis jirovecii fungal species were detected by RT-PCR mainly in the COVID-19 patients. Klebsiella pneumoniae was commonly found in most of the samples from the control and COVID-19 patients. Four COVID-19 patients had viral coinfections with human adenovirus, human rhinovirus, enterovirus, and human parainfluenza virus 1. CONCLUSIONS: Overall, no substantial difference was observed in the predominant NP bacterial community, but specific taxa were significantly changed between the healthy control and COVID-19 patients. Comparatively, an increased number of respiratory pathogens were identified in COVID-19 patients, and NP colonization by K. pneumoniae was probably occurring in the local population.

Lack of Evidence on Association between Iron Deficiency and COVID-19 Vaccine-Induced Neutralizing Humoral Immunity.

Iron is a crucial micronutrient for immunity induction in response to infections and vaccinations. This study aimed to investigate the effect of iron deficiency on COVID-19-vaccine-induced humoral immunity. We investigated the effectiveness of COVID-19 vaccines (BNT162b2, mRNA-1273, and ChAdOx nCov-2019) in iron-deficient individuals (n = 63) and provide a side-by-side comparison to healthy controls (n = 67). The presence of anti-SARS-CoV-2 spike (S) and anti-nucleocapsid (NP) IgG were assessed using in-house S- and NP-based ELISA followed by serum neutralization test (SNT). High concordance between S-based ELISA and SNT results was observed. The prevalence of neutralizing antibodies was 95.24% (60/63) in the study group and 95.52% (64/67) in the controls with no significant difference. The presence/absence of past infection, period since vaccination, vaccine type, and being iron-deficient or having iron-deficiency anemia did not exert any significant effect on the prevalence or titer of anti-SARS-CoV-2 neutralizing antibodies. NP-based ELISA identified individuals unaware of exposure to SARS-CoV-2. Moreover, absence of anti-NP IgG was noted in participants who were previously diagnosed with COVID-19 suggesting the unpredictability of after-infection immunity. To sum up, this study demonstrated an initial lack of evidence on the association between iron deficiency and the effectiveness of COVID-19-vaccine-induced neutralizing humoral immunity. Similar studies with larger sample size remain necessary to obtain comprehensive conclusions about the effect or lack of effect of iron on COVID-19-vaccine effectiveness.

Genomic profiling and network-level understanding uncover the potential genes and the pathways in hepatocellular carcinoma.

Data integration with phenotypes such as gene expression, pathways or function, and protein-protein interactions data has proven to be a highly promising technique for improving human complex diseases, particularly cancer patient outcome prediction. Hepatocellular carcinoma is one of the most prevalent cancers, and the most common cause is chronic HBV and HCV infection, which is linked to the majority of cases, and HBV and HCV play a role in multistep carcinogenesis progression. We examined the list of known hepatocellular carcinoma biomarkers with the publicly available expression profile dataset of hepatocellular carcinoma infected with HCV from day 1 to day 10 in this study. The study covers an overexpression pattern for the selected biomarkers in clinical hepatocellular carcinoma patients, a combined investigation of these biomarkers with the gathered temporal dataset, temporal expression profiling changes, and temporal pathway enrichment following HCV infection. Following a temporal analysis, it was discovered that the early stages of HCV infection tend to be more harmful in terms of expression shifting patterns, and that there is no significant change after that, followed by a set of genes that are consistently altered. PI3K, cAMP, TGF, TNF, Rap1, NF-kB, Apoptosis, Longevity regulating pathway, signaling pathways regulating pluripotency of stem cells, Cytokine-cytokine receptor interaction, p53 signaling, Wnt signaling, Toll-like receptor signaling, and Hippo signaling pathways are just a few of the most commonly enriched pathways. The majority of these pathways are well-known for their roles in the immune system, infection and inflammation, and human illnesses like cancer. We also find that ADCY8, MYC, PTK2, CTNNB1, TP53, RB1, PRKCA, TCF7L2, PAK1, ITPR2, CYP3A4, UGT1A6, GCK, and FGFR2/3 appear to be among the prominent genes based on the networks of genes and pathways based on the copy number alterations, mutations, and structural variants study.

Re-infection with a different SARS-CoV-2 clade and prolonged viral shedding in a hematopoietic stem cell transplantation patient.

Immunocompromised patients who have a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection pose many clinical and public health challenges. We describe the case of a hematopoietic stem cell transplantation patient with lymphoma who had a protracted illness requiring three consecutive hospital admissions. Whole genome sequencing confirmed two different SARS-CoV-2 clades. Clinical management issues and the unanswered questions arising from this case are discussed.

Test-based de-isolation in COVID-19 immunocompromised patients: Cycle threshold value versus SARS-CoV-2 viral culture.

BACKGROUND: Immunocompromised patients with coronavirus disease 2019 (COVID-19) have prolonged infectious viral shedding for more than 20 days. A test-based approach is suggested for de-isolation of these patients. METHODS: The strategy was evaluated by comparing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load (cycle threshold (Ct) values) and viral culture at the time of hospital discharge in a series of 13 COVID-19 patients: six immunocompetent and seven immunocompromised (five solid organ transplant patients, one lymphoma patient, and one hepatocellular carcinoma patient). RESULTS: Three of the 13 (23%) patients had positive viral cultures: one patient with lymphoma (on day 16) and two immunocompetent patients (on day 7 and day 11). Eighty percent of the patients had negative viral cultures and had a mean Ct value of 20.5. None of the solid organ transplant recipients had positive viral cultures. CONCLUSIONS: The mean Ct value for negative viral cultures was 20.5 in this case series of immunocompromised patients. Unlike those with hematological malignancies, none of the solid organ transplant patients had positive viral cultures. Adopting the test-based approach for all immunocompromised patients may lead to prolonged quarantine. Large-scale studies in disease-specific populations are needed to determine whether a test-based approach versus a symptom-based approach or a combination is applicable for the de-isolation of various immunocompromised patients.

Amotosalen and ultraviolet A light treatment efficiently inactivates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human plasma.

BACKGROUND AND OBJECTIVES: During the ongoing pandemic of COVID-19, SARS-CoV-2 RNA was detected in plasma and platelet products from asymptomatic blood donors, raising concerns about potential risk of transfusion transmission, also in the context of the current therapeutic approach utilizing plasma from convalescent donors. The objective of this study was to assess the efficacy of amotosalen/UVA light treatment to inactivate SARS-CoV-2 in human plasma to reduce the risk of potential transmission through blood transfusion. METHODS: Pools of three whole-blood-derived human plasma units (630-650 ml) were inoculated with a clinical SARS-CoV-2 isolate. Spiked units were treated with amotosalen/UVA light (INTERCEPT Blood System™) to inactivate SARS-CoV-2. Infectious titres and genomic viral load were assessed by plaque assay and real-time quantitative PCR. Inactivated samples were subject to three successive passages on permissive tissue culture to exclude the presence of replication-competent viral particles. RESULTS: Inactivation of infectious viral particles in spiked plasma units below the limit of detection was achieved by amotosalen/UVA light treatment with a mean log reduction of >3·32 ± 0·2. Passaging of inactivated samples on permissive tissue showed no viral replication even after 9 days of incubation and three passages, confirming complete inactivation. The treatment also inhibited NAT detection by nucleic acid modification with a mean log reduction of 2·92 ± 0·87 PFU genomic equivalents. CONCLUSION: Amotosalen/UVA light treatment of SARS-CoV-2 spiked human plasma units efficiently and completely inactivated >3·32 ± 0·2 log of SARS-CoV-2 infectivity, showing that such treatment could minimize the risk of transfusion-related SARS-CoV-2 transmission.

Reducing mortality and morbidity in patients with severe COVID-19 disease by advancing ongoing trials of Mesenchymal Stromal (stem) Cell (MSC) therapy - Achieving global consensus and visibility for cellular host-directed therapies.

As of May 17th 2020, the novel coronavirus disease 2019 (COVID-19) pandemic has caused 307,395 deaths worldwide, out of 3,917,366 cases reported to the World Health Organization. No specific treatments for reducing mortality or morbidity are yet available. Deaths from COVID-19 will continue to rise globally until effective and appropriate treatments and/or vaccines are found. In search of effective treatments, the global medical, scientific, pharma and funding communities have rapidly initiated over 500 COVID-19 clinical trials on a range of antiviral drug regimens and repurposed drugs in various combinations. A paradigm shift is underway from the current focus of drug development targeting the pathogen, to advancing cellular Host-Directed Therapies (HDTs) for tackling the aberrant host immune and inflammatory responses which underlie the pathogenesis of SARS-CoV-2 and high COVID-19 mortality rates. We focus this editorial specifically on the background to, and the rationale for, the use and evaluation of mesenchymal stromal (Stem) cells (MSCs) in treatment trials of patients with severe COVID-19 disease. Currently, the ClinicalTrials.gov and the WHO Clinical Trials Registry Platform (WHO ICTRP) report a combined 28 trials exploring the potential of MSCs or their products for treatment of COVID-19. MSCs should also be trialed for treatment of other circulating WHO priority Blueprint pathogens such as MERS-CoV which causes upto 34% mortality rates. It's about time funding agencies invested more into development MSCs per se, and also for a range of other HDTs, in combination with other therapeutic interventions. MSC therapy could turn out to be an important contribution to bringing an end to the high COVID-19 death rates and preventing long-term functional disability in those who survive disease.

Anti-hepatitis B virus activity of new 1,2,4-triazol-2-yl- and 1,3,4-oxadiazol-2-yl-2-pyridinone derivatives.

A number of 1,3,4-oxadiazole, 3-9, and 1,2,4-triazole derivatives, 13-15, were synthesized starting form the acid hydrazide 1. The 1,3,4-thiadiazole derivative 12 was prepared from the substituted phenylthiosemicarbazide derivative 11 by treatment with sulfuric acid. The aryl hydrazone derivatives 10a-c were synthesized by reaction of the hydrazide 1 with the corresponding ketones. The thioalkyl derivatives 16a-e were prepared by akylation of the thiol derivatives 3 and 13 with different alkylating agents. The newly synthesized compounds were tested for their anti-HBV activity and some of these compounds showed high antiviral activity.

P53 mutations in hepatocellular carcinoma patients in Egypt.

The p53 gene plays a major role in hepatocellular carcinoma (HCC). Acquired mutations may provide clues to etiology, as some carcinogenic agents are associated with specific genetic changes in p53. Our aim was to analyze the spectrum of p53 mutations in tumor tissues from subjects with HCC in Egypt, where there is a rising incidence of HCC due to hepatitis C virus (HCV). We collected tumor tissues from 41 subjects with HCC diagnosed at the National Cancer Institute of Cairo University during 2000-2003. Sequence mutations were analyzed by the Affymetrix GeneChip technique. HCV RNA was detected in the sera of 37 subjects (90%). Only one patient had a current HBV infection. A total of 17 of the 41 subjects (41%) had p53 mutations. Thirteen of these were in exon 7, of which 10 were in codon 249, but only 8 of the 10 were the R249S mutation, previously reported to be associated with aflatoxin exposure. The other three exon 7 mutations were found in codons 232, 242 and 248. A total of three mutations were detected in exon 5 codons 133, 144 and 176. One mutation was detected in exon 8 codon 275. Unlike previous studies, this population is characterized by a high prevalence of chronic HCV infection. The presence of the R249S mutation in exon 7 may indicate that these subjects with HCC have been exposed to aflatoxin (AFB1), and further investigation is in progress to measure AFB1-albumin adducts in the sera of these subjects.