Epitope Presentation of Dengue Viral Envelope Glycoprotein Domain III on Hepatitis B Core Protein Virus-Like Particles Produced in Nicotiana benthamiana.

Dengue fever is currently ranked as the top emerging tropical disease, driven by increased global travel, urbanization, and poor hygiene conditions as well as global warming effects which facilitate the spread of Aedes mosquitoes beyond their current distribution. Today, more than 100 countries are affected most of which are tropical Asian and Latin American nations with limited access to medical care. Hence, the development of a dengue vaccine that is dually cost-effective and able to confer a comprehensive protection is ultimately needed. In this study, a consensus sequence of the antigenic dengue viral glycoprotein domain III (cEDIII) was used aiming to provide comprehensive coverage against all four circulating dengue viral serotypes and potential clade replacement event. Utilizing hepatitis B tandem core technology, the cEDIII sequence was inserted into the immunodominant c/e1 loop region so that it could be displayed on the spike structures of assembled particles. The tandem core particles displaying cEDIII epitopes (tHBcAg-cEDIII) were successfully produced in Nicotiana benthamiana via Agrobacterium-mediated transient expression strategy to give a protein of ∼54 kDa, detected in both soluble and insoluble fractions of plant extracts. The assembled tHBcAg-cEDIII virus-like particles (VLPs) were also visualized from transmission electron microscopy. These VLPs had diameters that range from 32 to 35 nm, presenting an apparent size increment as compared to tHBcAg control particles without cEDIII display (namely tEL). Mice immunized with tHBcAg-cEDIII VLPs showed a positive seroconversion to cEDIII antigen, thereby signifying that the assembled tHBcAg-cEDIII VLPs have successfully displayed cEDIII antigen to the immune system. If it is proven to be successful, tHBcAg-cEDIII has the potential to be developed as a cost-effective vaccine candidate that confers a simultaneous protection against all four infecting dengue viral serotypes.

Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA) by combination of ampicillin and a bioactive fraction from Duabanga grandiflora.

BACKGROUND: The inhibition of penicillin-binding protein 2a (PBP2a) is a promising solution in overcoming resistance of methicillin resistance Staphylococcus aureus (MRSA). A potential approach in achieving this is by combining natural product with currently available antibiotics to restore the activity as well as to amplify the therapeutic ability of the drugs. We studied inhibition effects of a bioactive fraction, F-10 (isolated from the leaves of Duabanga grandiflora) alone and in combination with a beta-lactam drug, ampicillin on MRSA growth and expression of PBP2a. Additionally, phytochemical analysis was conducted on F-10 to identify the classes of phytochemicals present. METHODS: Fractionation of the ethyl acetate leaf extract was achieved by successive column chromatography which eventually led to isolation of an active fraction, F-10. Both extract and F-10 were analyzed for the presence of major classes of phytochemicals in addition to obtaining a high performance liquid chromatography (HPLC) profile to reveal the complexity of the fraction F-10. Broth microdilution method was employed to determine minimum inhibitory concentration (MIC) of the extract and fractions against MRSA. Evaluation of synergistic activity of the active fraction with ampicillin was determined using checkerboard methodand kinetic growth experiments. Effect of combination treatments on expression of PBP2a, a protein that confers resistance to beta-lactam antibiotics, was elucidated with the Western blot assay. RESULTS: MIC of F-10 against MRSA was 750 mg/L which showed an improved activity by 4-fold compared to its crude extract (MIC = 3000 mg/L). Phytochemical analysis revealed occurrence of tannins, saponin, flavonoids, sterols, and glycosides in F10 fraction. In FIC index interpretation, the most synergistic activity was achieved for combinations of 1/64 × MIC ampicillin + 1/4 × MIC F-10. The combination also evidently inhibited MRSA growth in kinetic growth curve assay. As a result of this synergistic interaction, MIC of ampicillin against MRSA was reduced to 0.78 mg/L (64-fold) from initial value of 50 mg/L. Western blot analysis suggested inhibition of PBP2a in MRSA cultures grown in synergistic combination treatment in which no PBP2a band was expressed. CONCLUSIONS: The results demonstrated synergism between fraction F-10 of D. grandiflora with ampicillin in suppressing MRSA growth via PBP2a inhibition.

Reversal of ampicillin resistance in MRSA via inhibition of penicillin-binding protein 2a by Acalypha wilkesiana.

The inhibitory activity of a semipure fraction from the plant, Acalypha wilkesiana assigned as 9EA-FC-B, alone and in combination with ampicillin, was studied against methicillin-resistant Staphylococcus aureus (MRSA). In addition, effects of the combination treatment on PBP2a expression were investigated. Microdilution assay was used to determine the minimal inhibitory concentrations (MIC). Synergistic effects of 9EA-FC-B with ampicillin were determined using the fractional inhibitory concentration (FIC) index and kinetic growth curve assay. Western blot experiments were carried out to study the PBP2a expression in treated MRSA cultures. The results showed a synergistic effect between ampicillin and 9EA-FC-B treatment with the lowest FIC index of 0.19 (synergism ≤ 0.5). The presence of 9EA-FC-B reduced the MIC of ampicillin from 50 to 1.56 µg mL(-1). When ampicillin and 9EA-FC-B were combined at subinhibitory level, the kinetic growth curves were suppressed. The antibacterial effect of 9EA-FC-B and ampicillin was shown to be synergistic. The synergism is due the ability of 9EA-FC-B to suppress the activity of PBP2a, thus restoring the susceptibility of MRSA to ampicillin. Corilagin was postulated to be the constituent responsible for the synergistic activity showed by 9EA-FC-B.