Development of a potent recombinant scFv antibody against the SARS-CoV-2 by in-depth bioinformatics study: Paving the way for vaccine/diagnostics development.

BACKGROUND: The SARS-CoV-2 has led to a worldwide disaster. Thus, developing prophylactics/therapeutics is required to overcome this public health issue. Among these, producing the anti-SARS-CoV-2 single-chain variable fragment (scFv) antibodies has attracted a significant attention. Accordingly, this study aims to address this question: Is it possible to bioinformatics-based design of a potent anti-SARS-CoV-2 scFv as an alternative to current production approaches? METHOD: Using the complexed SARS-CoV-2 spike-antibodies, two sets analyses were performed: (1) B-cell epitopes (BCEs) prediction in the spike receptor-binding domain (RBD) region as a parameter for antibody screening; (2) the computational analysis of antibodies variable domains (VH/VL). Based on these primary screenings, and docking/binding affinity rating, one antibody was selected. The protein-protein interactions (PPIs) among the selected antibody-epitope complex were predicted and its epitope conservancy was also evaluated. Thereafter, some elements were added to the final scFv: (1) the PelB signal peptide; (2) a GSGGGGS linker to connect the VH-VL. Finally, this scFv was analyzed/optimized using various web servers. RESULTS: Among the antibody library, only one met the various criteria for being an efficient scFv candidate. Moreover, no interaction was predicted between its paratope and RBD hot-spot residues of SARS-CoV-2 variants-of-Concern (VOCs). CONCLUSIONS: Herein, a step-by-step bioinformatics platform has been introduced to bypass some barriers of traditional antibody production approaches. Based on existing literature, the current study is one of the pioneer works in the field of bioinformatics-based scFv production. This scFv may be a good candidate for diagnostics/therapeutics design against the SARS-CoV-2 as an emerging aggressive pathogen.

Preparation, Purification and Performance Evaluation of Polyclonal Antibody Against SARS-CoV-2 Produced in Rat.

Purpose: Among all known human coronaviruses, some viruses (e.g., SARS-CoV-2) cause severe pneumonia or even death. With the regard to its spread and the importance of its rapid identification/treatment, and because pAbs are relatively cheap, able to bind to more sites on antigens and even neutralize them, this study was done for the production and purification of anti-SARS-CoV-2 polyclonal antibodies (pAb) in rats. Methods: Viral antigen purification was performed by PEG/NaCl precipitation. The efficiency of the sucrose cushion method was also investigated to produce a purer antigen. Immunization was done and antibody purification was performed by ammonium sulfate precipitation (33%), dialysis, and ion-exchange chromatography. Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to verify the antibody specificity. All data were analyzed by SPSS software. Results: The results showed that the amount of concentrated virus increased with the increase of PEG concentration. Moreover, the sucrose cushion method increased its purity. Besides, induction of immune response in rats for pAb production with high titers was reached via these antigens and ELISA/western blot results indicated a suitable antibody-antigen interaction. Additionally, it was shown that ion-exchange chromatography could be a suitable technique for IgG purification. Conclusion: Herein, we presented a simple and cheap method for the purification of whole viral particles with relatively high quality. The results verified that these antigens could elicit a good immune response in the rat. The obtained pAbs showed a good specificity toward SARS-CoV-2 antigens. Accordingly, this study proposes a promising method for viral vaccine development.

Virulence characterization and clonal analysis of uropathogenic Escherichia coli metallo-beta-lactamase-producing isolates.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infection (UTI); however, treatment of UTI has been challenging due to increased antimicrobial resistance (AMR). One of the most important types of AMR is carbapenem resistance (CR). CR bacteria are known as an important threat to global public health today. Class B metallo-beta-lactamases (MBLs) are one of the major factors for resistance against carbapenems. We aimed to investigate the characteristics of UPEC isolates producing MBL. METHODS: A cross-sectional study was conducted from October 2018 to December 2019 in Ahvaz; Iran. UPEC isolates were identified by biochemical and molecular methods. Metallo-beta-lactamase-producing isolates were detected using modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) tests. MBL genes, phylogenetic group, and virulence genes profile of carbapenem resistant isolates were determined. Conjugation assay and plasmid profiling were conducted to evaluate the ability of transferring of CR to other E. coli isolates. Clonal similarity of isolates were assessed using Enterobacterial intergenic repetitive element sequence (ERIC)-PCR. RESULTS: Among 406 UPEC isolates, 12 (2.95%) carbapenem-resistant were detected of which 11 were phenotypically MBL-producing strains. Four isolates were resistant to all investigated antimicrobial agents and were considered possible pandrug-resistant (PDR). blaNDM, blaOXA-48, blaIMP-1, and blaIMP-2 genes were found in 9, 5, 1, and 1 isolates, respectively. Among 30 virulence genes investigated, the traT, fyuA followed by fimH, and iutA with the frequency of 8 (66.7%), 8 (66.7%), 7 (58.3%), and 7 (58.3%) were the most identified genes, respectively. Siderophore production was the main virulence trait among carbapenem-resistant UPEC isolates. Except for two, all other isolates showed weak to moderate virulence index. In all recovered isolates, CR was readily transmitted via plasmids to other isolates during conjugation experiments. CONCLUSION: MBL and carbapenemase genes, especially blaNDM and blaOXA-48 are spreading rapidly among bacteria, which can be a threat to global public health. Therefore monitoring the emergence and dissemination of new AMR is necessary to continuously refine guidelines for empiric antimicrobial therapy. Understanding the mechanisms of resistance and virulence in this group of bacteria can play an effective role in providing new therapeutic methods.

Genotypic and phenotypic characterization of enteroaggregative Escherichia coli (EAEC) isolates from diarrheic children: An unresolved diagnostic paradigm exists.

OBJECTIVES: The enteroaggregative Escherichia coli (EAEC) has been one of the most intriguing emerging bacterial pathogens in children that occur both in developing countries and the industrial world. Although various phenotypic and genotypic based protocols have been suggested for diagnosis of EAEC, they are not conclusive or practical to be used in most clinical laboratories. MATERIALS AND METHODS: In this study, we analyzed and compared 36 typical EAEC strains (aggR-positive) by various genotypic and phenotypic methods. RESULTS: Briefly, pCVD432 was detected in all of isolates along with aggR, then it was followed by other virulence genes including app, astA, aggA, and pet genes in 32 (88.8%), 21 (58.3%), 9 (25%), and 2 (5.5%) isolates, respectively. Biofilm was formed by 34 (94.4%) isolates, while only 26 (72.2%) isolates showed an aggregative adherence pattern to HEp-2 cells. CONCLUSION: The genetic and phenotypic features of EAEC were highly inconsistent, which may have considerable diagnostic implications. The variations in the virulence genes, phenotypic characteristics, and genetic profiles among the EAEC isolates again emphasized the genetic heterogeneity of this emerging pathotype. Biofilm formation may be an important phenotypic virulence property of this pathotype, especially in strains with the aggR-pCVD432-aap-astA profile.

Characterization of diarrheagenic Escherichia coli strains associated with diarrhea in children, Khouzestan, Iran.

INTRODUCTION: Diarrheagenic Escherichia coli (DEC) is a major etiologic agent among the pathogens that cause diarrhea in children. METHODOLOGY: To investigate the presence and pathotypes of DEC in children under five years of age, living in the province of Khouzestan, Iran. 208 diarrhea stool samples were screened by multiplex-PCR. The isolated DEC isolates were investigated for resistance to various antimicrobials including the production of extended-spectrum beta-lactamases (ESBLs) and phylogenetic groups were determined. RESULTS: DEC isolates were identified in 54 (26%) diarrhea samples, and 4 (7%) cases contained two DEC pathotypes. DEC isolated included 35 (16.8%) enteroaggregative E. coli (EAEC), ten (4.8%) enteropathogenic E. coli (EPEC), six (2.9%) enteroinvasive E. coli (EIEC), six (2.9%) enterotoxigenic E. coli (ETEC) and one (0.48%) LEE-positive EAEC. Shiga-toxin producing E. coli (STEC) was not identified in any diarrheal samples. The most prevalent resistance was observed with ceftazidime (88%), followed by ceftizoxime (83%) and ceftriaxone (71%). The majority of isolates (> 75%) were sensitive to Imipenem, ciprofloxacin, and amikacin. More than 65% of the pathogenic isolates showed a multidrug-resistant phenotype. ESBL-producing strains was observed in 79.3% of all DEC isolates. Phylogenetic group B2 was the most predominant group with a frequency of 44.8%. A significant association was observed between the B2 phylogenetic group and the DEC isolates (P < 0.05). CONCLUSIONS: Overall, our findings highlight the importance of the role of DEC isolates in the etiology of diarrhea in children in Iran. The progressive increase in antimicrobial resistance among DEC isolates makes it imperative to implement policies to control the spread of resistant bacteria.

Gold Nanoparticles Impair Foot-and-Mouth Disease Virus Replication.

In this study, we evaluated the antiviral activity of gold nanoparticles (AuNPs) against the foot-and-mouth disease virus (FMDV), that causes a contagious disease in cloven-hoofed animals. The anti-FMDV activity of AuNPs was assessed using plaque reduction assay. MTT assay was used for quantitatively measuring the cytopathic effect caused by the viral infection. The 50% cytotoxicity concentration of nanoparticles was measured and found to be 10.4 µg/ml. The virus yield reduction assay showed that AuNP have an approximately 4-fold virus titer reduction compared with controls. Plaque reduction assay showed that at non-cytotoxic concentrations, AuNPs do not show extracellular virucidal activity and inhibition of FMDV growth at the early stages of infection including attachment and penetration. Time-of-addition experiments revealed that AuNPs inhibited post-entry stages of viral replication concomitant with the onset of intracellular viral RNA synthesis; however, the mechanism of AuNPs against FMDV was unclear.

Isolation of a potent antibiotic producer bacterium, especially against MRSA, from northern region of the Persian Gulf.

Nowadays, emergence and prevalence of MRSA (Methicillin Resistant Staphylococcus aureus) strain have become a great global concern in 21st century, so, it is necessary to discover new antibiotics against this pathogen. The aim of this study was isolation and evaluation marine bacteria from the Persian Gulf in order to finding antibiotic compounds against some pathogenic bacteria. For this purpose, water and sediment samples were collected from the Persian Gulf during March to October 2009. The antibacterial activity of the isolated bacteria was assessed using disc diffusion method. The Growth Curve Interference (GCI) parameter against MRSA was determined for the high potential antibiotic producing strain. The most important factors affecting fermentation conditions in antibiotic production were also optimized. Definite identification of intended isolate was confirmed by 16S rRNA sequencing. Altogether, 51 bacterial colony was isolated and among them only 3 bacterium showed antibacterial activity. Pseudoalteromonas piscicida PG-01 isolated from a sediment sample was chosen as the best antibiotic producing strain. This strain was effective against all tested Gram-positive bacteria, had good anti-MRSA activity and also GCI value against MRSA was two times lower than MIC value. Among the optimized fermentation parameters, carbon and nitrogen sources play major role in efficacy of optimized antibiotic production. Ultrastructural study on the effect of intended antibiotic compounds on MRSA using TEM revealed that the target site for this compound is cell wall. Considering the antibacterial effect of PG-01 strain especially against MRSA, intended antibiotic compounds can gives hope for treatment of diseases caused by multi-drug resistant bacteria.

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates.

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209-216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.