Virus-like particles as powerful vaccination strategy against human viruses.

Nowadays, viruses are not only seen as causative agents of viral infectious diseases but also as valuable research materials for various biomedical purposes, including recombinant protein production. When expressed in living or cell-free expression systems, viral structural proteins self-assemble into virus-like particles (VLPs). Mimicking the native form and size of viruses and lacking the genetic material, VLPs are safe and highly immunogenic and thus can be exploited to develop antiviral vaccines. Some vaccines based on VLPs against various infectious pathogens have already been licenced for human use and are available in the commercial market, the latest of which is a VLP-based vaccine to protect against the novel Coronavirus. Despite the success and popularity of VLP subunit vaccines, many more VLPs are still in different stages of design, production, and approval. There are still many challenges that require to be addressed in the future before this surface display system can be widely used as an effective vaccine strategy in combating infectious diseases. In this review, we highlight the use of structural viral proteins to produce VLPs, emphasising their intrinsic properties, structural classification, and main expression host systems. We also compiled the recent scientific literature about VLP-based vaccines to underline the recent advances in their application as a vaccine strategy for preventing and fighting virulent human pathogens. Finally, we presented the key challenges and possible solutions for VLP-based vaccine production.

The introduction of mutations in the wild type coxsackievirus B3 (CVB3) IRES RNA leads to different levels of in vitro reduced replicative and translation efficiencies.

Coxsackievirus B3 (CVB3) is a principal causative agent of viral myocarditis, meningitis and pancreatitis. There is no vaccine available for clinical use. It has been demonstrated that the primary molecular determinant of virulence phenotype is located in the 5' UTR of the viral genome. Translation initiation of CVB3 RNA is directed by the IRES element situated in the 5'UTR. In the present study, we analyse the effects of single point mutations introduced in different positions in the domain V of the IRES RNA of CVB3 wild type. We characterize in vitro virus replicative capacitiy and translation efficiency and we test in vivo virulence of different CVB3 mutants produced by the introduction of different mutations in the domain V of IRES by site-directed mutagenesis to abolish its structure. Our results demonstrate that all RNA mutants display different levels of decreased replication and translation initiation efficiency in vitro. The translation defect was correlated with significant reduced viral titer of mutant particles in comparison with the wild type. When inoculated in mice, mutant viruses were checked for inflammation and necrosis.In vitro and in vivo Findings strongly suggest that the most attenuated mutant strain could be considered a candidate for live-attenuated CVB3 vaccine.

In-Vitro Screening and Biosynthesis of Secondary Metabolites from a New Streptomyces sp. SA1 from a Marine Environment.

BACKGROUND: Streptomyces sp. produces various antibiotic agents and the number of lead molecules from the genus Streptomyces increased rapidly in recent years. Drug resistance against various commercially available antibiotics is one of the important problems throughout the world. Streptomyces spp. produce various antimicrobials with potent activity against drug-resistant bacteria. METHODS: Streptomyces sp. SA1 was isolated from the marine environment for the biosynthesis of antibiotics. The important variables influencing secondary metabolite biosynthesis were optimized to increase the biosynthesis of antimicrobial agents using the traditional method and statistical approach. RESULTS: Streptomyces sp. SA1 produced novel antibiotics and the process variables were optimized by the traditional method (One-variable-at-a-time approach). Maltose showed maximum antimicrobial activity (220 U/mL). Analysis of the nitrogen, the effect of nitrogen sources revealed that beef extract incorporated culture medium showed rich antibacterial activity (188/mL). Among the ionic sources, KCl significantly influenced antibiotic production. Maltose, beef extract and KCl were considered as the most influencing medium components. Antimicrobial agent biosynthesis was achieved with maltose 1.22 g/L, beef extract 0.93 g/L and KCl 0.27 g/L in response surface methodology. CONCLUSION: Actinomycetes, especially Streptomyces, play an important role as a source for bioactive compounds that are used to treat infections, and many other diseases. The isolated Streptomyces sp. was a good producer of antibacterial agent, which required various nutritional supplements in the culture medium. The optimized medium components investigated in this study will be useful for future studies with the mass production of secondary metabolites.

Characterization of Coxsackievirus B4 virus-like particles VLP produced by the recombinant baculovirus-insect cell system expressing the major capsid protein.

Coxsackievirus B4 (CV-B4) is suspected to be an environmental factor that has the intrinsic capacity to damage the pancreatic beta cells and therefore causes insulitis and type 1 diabetes (T1D). Although vaccination against CV-B4 could reduce the incidence of this chronic auto-immune disease, there is currently no therapeutic reagent or vaccine in clinical use. By the employment of the Bac-to-Bac® vector system to express the major viral capsid protein, we contributed towards the development of a CV-B4 vaccine by producing CV-B4 virus-like particles (VLPs) from recombinant baculovirus in infected insect cells. In fact Western blot and Immunofluorescence analysis detected the viral protein 1 (VP1) in the cells resulting from the construction of a recombinant bacmid DNA carrying the key immunogenic protein then transfected in the insect cells. Sucrose gradient ultracentrifugation fractions of the infected cell lysates contained the recombinant protein and the electron microscopy demonstrated the presence of VLPs in these sucrose fractions. This study clearly shows for the first time the expression of CVB4 VP1 structure protein alone can form VLPs in the baculovirus-infected insect cell keeping conserved both characteristics and morphology.

Silver nanoparticles synthesis from Bacillus sp KFU36 and its anticancer effect in breast cancer MCF-7 cells via induction of apoptotic mechanism.

Biological synthesis of nanoparticles is a growing research trend because it has numerous applications in pharmaceutics and biomedicine. The aim of this study was to obtain silver nanoparticles (AgNPs) from Bacillus sp. KFU36, a marine strain, and to assess its anticancer activity. The supernatant of Bacillus sp. KFU36 was supplemented with silver nitrate and the nanoparticles obtained were characterized spectrophotometrically and microscopically. A band of surface plasmon resonance was appeared at 430 nm, as revealed by UV-vis spectrophotometry. X-ray diffraction spectrum and Energy Dispersive Spectroscopy confirmed the crystalline and metallic structure of the AgNPs, respectively. Scanning electron microscopy revealed that the shape of the synthesized AgNPs were spherical and the size extended between 5 and 15 nm. The AgNPs were investigated for their potential anticancer effects on the cell viability, migration and apoptosis using MTT and wound-healing assays, and flow cytometry, respectively. The cytotoxic effects of these nanoparticles were evidenced by the decreasing the cell viability (as 15% at 50 µg/ml), cell density, adhesion capacity and losing the normal shape and size, and inducing the apoptosis on MCF-7 by 61% at 50 µg/ml. These findings confirm that the synthesized AgNPs exhibited superior anticancer activities and therefore could be exploited as a promising, cost-effective, and environmentally benign strategy in treating this disease in future.

Isolation and characterization of polyketide drug molecule from Streptomyces species with antimicrobial activity against clinical pathogens.

BACKGROUND: Natural products derived from marine microbes have more potential toward to treatment of various diseases. Among the microbes, the secondary metabolites recovered from the marine actinomycetes were more added values. OBJECTIVE: A promising antimicrobial metabolite producing filamentous actinomycete SCA-7 recovered from Alkhobar marine region was investigated for its potential to inhibit Gram positive Enterococcus sp. In addition to the chemical characterization, the polyketide gene cluster of the actinomycete SCA-7 was sequenced. RESULTS: Among the 8 actinomycetes isolated from the marine sample, the isolate SCA-7 produced significant antimicrobial activity against Enterococcus sp. The biochemical, physiological and morphological characteristics and fermentation assimilation pattern confirmed that the isolate belonged to the genus Streptomyces. The 16S rRNA gene amplification and sequencing results showed 99% sequence similarity to Streptomyces felleus. The antimicrobial activity of the crude ethyl acetate extract was performed by disc diffusion method. The spectral characterization was done by 13C NMR and 1H NMR. The compound was polyketide in nature. The Minimum Inhibitory Concentration (MIC) of the polyketide compound against Enterococcus sp. was 25µg/mL. Among the agro-industrial waste materials, wheat bran showed increased secondary metabolite production. Antibacterial activity was found to be high when the isolate SCA-7 was grown in wheat bran substrate and maximum zone of inhibition (22mm) was recorded in it. Among the carbon and nitrogen sources, lactose enhanced the production of secondary metabolites and the zone of inhibition against Enterococcus sp. was 25mm. The amplification and sequencing of the ketoacyl synthase gene clearly indicated that it was type I polyketide synthase (PKS) gene of Streptomyces species. CONCLUSION: Overall, the therapeutic drug molecule isolated from the marine Streptomyces species might be used for the treatment of disease causing microbial clinical pathogen.

Silver nanopaticles synthesized using the seed extract of Trigonella foenum-graecum L. and their antimicrobial mechanism and anticancer properties.

BACKGROUND: Synthesis of silver nanoparticles (AgNPs) through biological route plays an important role in their applications in the medical field, especially in the prevention of disease causing microbial pathogens and arresting the propagation of cancer cells. The stable, green synthesis of AgNPs is very much welcomed in the medical field because of their low toxicity. Therefore, the demands of AgNPs synthesised biologically is on the rise. The present study aimed to investigate the antimicrobial mechanisms and anticancer properties of the AgNPs synthesized using the seed extract of Trigonella foenum-graecum L. The AgNPs were characterized by UV-vis, SEM, XRD, FTIR and EDAX analysis. The minimum inhibitory concentrations (MIC) of the AgNPs were determined by the broth micro dilution method. RESULTS: The formation of brownish red color indicated the formation NPs with the absorption maximum at 420 nm. The average size was found to be 33.93 nm and sphere shaped. The FTIR spectrum revealed the absorption bands at 3340 cm-1 and 1635 cm-1 indicated the presence of -OH or -COOH and amide group stretching in the AgNPs. The X-ray diffraction report confirmed the presence of strong peak values of 2θ within the angle of 37.1°. The lowest MIC of the AgNPs against Staphylococcus aureus was 62.5 µg mL-1. MIC values against Escherichia coli and Klebsiella pneumonia, were 125 and 250 µg mL-1 respectively. The MIC of the AgNPs against Aspergillus flavus, Trichophyton rubrum and Trichoderma viridiae were each 250 µg mL-1, respectively. The extracellular protein concentration, levels of lactate dehydrogenase and alkaline phosphtase enzyme in the AgNPs treated bacterial pathogens demonstrated greater antimicrobial mechanism. Additionally, the AgNPs exhibited significant anticancer activity against the MCF7 and Vero cell lines. CONCLUSION: The synthesized AgNPs could be further evaluated in large scale as a botanical antimicrobial agent.

Description of a methanotrophic strain BOH1, isolated from Al-Bohyriya well, Al-Ahsa City, Saudi Arabia.

Methanotrophic bacteria have a unique ability to utilize methane as their carbon and energy sources. Therefore, methanotrophs play a key role in suppressing methane emissions from different ecosystems and hence in alleviating the global climate change. Despite methanotrophs having many ecological, economical and biotechnological applications, little is known about this group of bacteria in Al-Ahsa. Therefore, the main objective of the current work was to expand our understanding of methane oxidizing bacteria in Al-Ahsa region. The specific aim was to describe a methanotrophic strain isolated from Al-Bohyriya well, Al-Ahsa using phenotypic, genotypic (such as 16S rRNA and pmoA gene sequencing) and phylogenetic characterization. The results indicated that the strain belongs to the genus Methylomonas that belongs to Gammaproteobacteria as revealed by the comparative sequence analysis of the 16S rRNA and pmoA genes. There is a general agreement in the profile of the phylogenetic trees based on the sequences of 16srRNA and pmoA genes of the strain BOH1 indicating that both genes are efficient taxonomic marker in methanotrophic phylogeny. The strain possesses the particulate but not the soluble methane monooxygenase as a key enzyme for methane metabolism. Further investigation such as DNA:DNA hybridization is needed to assign the strain as a novel species of the genus Methyomonas and this will open the door to explore the talents of the strain for its potential role in alleviating global warming and biotechnological applications in Saudi Arabia such as bioremediation of toxic by-products released in oil industry. In addition, the strain enhances our knowledge of methanotrophic bacteria and their adaptation to desert ecosystems.

Characterization of the plant growth promoting bacterium, Enterobacter cloacae MSR1, isolated from roots of non-nodulating Medicago sativa.

The aim of the present study was to characterize the endophytic bacterial strain designated MSR1 that was isolated from inside the non-nodulating roots of Medicago sativa after surface-sterilization. MSR1 was identified as Enterobacter cloacae using both 16S rDNA gene sequence analysis and API20E biochemical identification system (Biomerieux, France). Furthermore, this bacterium was characterized using API50CH kit (Biomerieux, France) and tested for antibacterial activities against some food borne pathogens. The results showed that E. cloacae consumed certain carbohydrates such as glycerol, d-xylose, d-maltose and esculin melibiose as a sole carbon source and certain amino acids such as arginine, tryptophan ornithine as nitrogen source. Furthermore, MSR1 possessed multiple plant-growth promoting characteristics; phosphate solubility, production of phytohormones acetoin and bioactive compounds. Inoculation of Pisum sativum with MSR1 significantly improved the growth parameters (the length and dry weight) of this economically important grain legume compared to the non-treated plants. To our knowledge, this is the first report addressing E. cloacae which exist in roots of alfalfa growing in Al-Ahsaa region. The results confirmed that E. cloacae exhibited traits for plant growth promoting and could be developed as an eco-friendly biofertilizer for P. sativum and probably for other important plant species in future.