Proof of concept in utilizing <Emphasis Type="Italic">in-trans</Emphasis> surface display system of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> as mucosal tuberculosis vaccine via oral administration in mice

Background

Tuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world’s population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0–80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed.

Results

A novel approach of utilizing an in-trans protein surface display system of Lactobacillus plantarum carrying and displaying combination of Mycobacterium tuberculosis subunit epitope antigens (Ag85B, CFP-10, ESAT-6, Rv0475 and Rv2031c) fused with LysM anchor motif designated as ACERL was constructed, cloned and expressed in Esherichia coli Rossetta expression host. Subsequently the binding capability of ACERL to the cell wall of L. plantarum was examined via the immunofluorescence microscopy and whole cell ELISA where successful attachment and consistent stability of cell wall binding up to 4 days was determined. The immunization of the developed vaccine of L. plantarum surface displaying ACERL (Lp ACERL) via the oral route was studied in mice for its immunogenicity effects. Lp ACERL immunization was able to invoke significant immune responses that favor the Th1 type cytokine response of IFN-γ, IL-12 and IL-2 as indicated by the outcome from the cytokine profiling of spleen, lung, gastrointestinal tract (GIT), and the re-stimulation of the splenocytes from the immunized mice. Co-administration of an adjuvant consisting of Lactococcus lactis secreting mouse IL-12 (LcIL-12) with Lp ACERL was also investigated. It was shown that the addition of LcIL-12 was able to further generate significant Th1 type cytokines immune responses, similar or better than that of Lp ACERL alone which can be observed from the cytokine profiling of the immunized mice’s spleen, lung and GIT.

Conclusions

This study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development.

     

Evaluation of sticky traps for adult Aedes mosquitoes in Malaysia: a potential monitoring and surveillance tool for the efficacy of control strategies

The present study compared the performance of sticky traps in order to identify the most effective and practical trap for capturing Aedes aegypti and Aedes albopictus mosquitoes. Three phases were conducted in the study, with Phase 1 evaluating the five prototypes (Models A, B, C, D, and E) of sticky trap release‐and‐recapture using two groups of mosquito release numbers (five and 50) that were released in each replicate. Similarly, Phase 2 compared the performance between Model E and the classical ovitrap that had been modified (sticky ovitrap), using five and 50 mosquito release numbers. Further assessment of both traps was carried out in Phase 3, in which both traps were installed in nine sampling grids. Results from Phase 1 showed that Model E was the trap that recaptured higher numbers of mosquitoes when compared to Models A, B, C, and D. Further assessment between Model E and the modified sticky ovitrap (known as Model F) found that Model F outperformed Model E in both Phases 2 and 3. Thus, Model F was selected as the most effective and practical sticky trap, which could serve as an alternative tool for monitoring and controlling dengue vectors in Malaysia.     

Floral and pollinator behaviour of flexistylous Bornean ginger, Alpinia nieuwenhuizii (Zingiberaceae)

Flexistyly is a unique floral mechanism involving extreme curving of the style. It was first described in Chinese ginger (Amomum, Zingiberaceae). This is a pioneer report on flexistylous gingers of Malesia, where most species of this family grow. We observed the floral behaviour and flower visitors in Alpinia nieuwenhuizii Val., a Bornean endemic. Although the floral behaviour and effective pollinators (carpenter bees, Xylocopa) were similar between the Bornean species and the previously reported flexistylous Alpinia, the pollinator behaviour between them strikingly differed with regard to the visit frequency of the pollinators showing a bimodal pattern during the day. This was a better match for the floral behaviour of the flexistylous Alpinia. Some gender differentiation observed between the two types of morphs is also discussed in the present study.     

Biochemical fractionation and characterization of proteins from Golgi-enriched membranes.

Fractions enriched in Golgi membranes were prepared from rat liver by sucrose gradient ultracentrifugation. These enriched membranes were further subfractionated on the basis of their solubilities in EGTA, 150 mM sodium carbonate, pH 11.5, sodium deoxycholate, Triton X-100, or sodium dodecyl sulfate. This led to isolation of peripheral, luminal, and integral membrane proteins of the Golgi-enriched membranes. Luminal and membrane proteins were further purified by wheat germ agglutinin and concanavalin A lectin affinity chromatographies. Some proteins from these lectin columns were resolved by preparative gel electrophoresis and microsequenced. Subsequently, antibodies were produced for two proteins by immunization of either mice or rabbits. Immunofluorescence microscopy suggests that these proteins are confined to Golgi apparatus-like structures. The protocol described is well suited for the study of organelle structure and function.     

Live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 and D4 for protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus)

Aims: To evaluate a live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 (Lac‐D1ae) and/or D4 (Lac‐D4ae) in protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus). Methods and Results: The polymerase chain reaction (PCR)‐amplified 250‐ and 750‐bp sequences coding for domains D1 and D4 of aerolysin were individually cloned into pNZ8048 and electrotransformed into L. lactis. The recombinant vaccine candidates were then either orally fed or injected intraperitoneally into tilapia. The development of antibodies in sampled fish compared to control groups implied that the recombinant epitopes expressed in L. lactis were able to elicit an immunogenic response in tilapia. Interestingly, the lower doses of both Lac‐D1ae and Lac‐D4ae gave higher antibody levels over the study period. Fish immunized with Lac‐D1ae and Lac‐D4ae together showed the highest level of protection, and the mortality was reduced significantly compared to control strains in both modes of vaccination. Conclusions: The recombinant L. lactis strain expressing D1 and D4 produced aerolysin‐specific serum IgM in tilapia. Both D1 and D4 promoted 55–82% relative per cent survival (RPS) against Aeromonas infection through intraperitoneal injection, whereas the RPS following oral feeding of the vaccine was 70–100%. Significance and Impact of the Study: The D1 and D4 regions of the aerolysin protein have been successfully identified as immunogenic regions that can elicit antibody production in tilapia and protect against challenge with Aer. hydrophila. A promising oral vaccine using L. lactis harbouring the D1 and D4 regions has been developed to control Aer. hydrophila.     

Utilization of palm kernel cake for production of β-mannanase by Aspergillus niger FTCC 5003 in solid substrate fermentation using an aerated column bioreactor

The production of β-mannanase from palm kernel cake (PKC) as a substrate in solid substrate fermentation (SSF) was studied using a laboratory column bioreactor. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and airflow rate, on β-mannanase production were evaluated by response surface methodology (RSM) on the basis of a central composite face-centered (CCF) design. Eighteen trials were conducted in which Aspergillus niger FTCC 5003 was cultivated on PKC in an aerated column bioreactor for seven days under SSF process. The highest level of β-mannanase (2117.89 U/g) was obtained when SSF process was performed at incubation temperature, initial moisture level and aeration rate of 32.5°C, 60% and 0.5 l/min, respectively. Statistical analysis revealed that the quadratic terms of incubation temperature and initial moisture content had significant effects on the production of β-mannanase (P < 0.01). A similar analysis also demonstrated that the linear effect of initial moisture level and an interaction effect between the initial moisture content and aeration rate significantly influenced the production of β-mannanase (P < 0.01). The statistical model suggested that the optimal conditions for attaining the highest level of β-mannanase were incubation temperature of 32°C, initial moisture level of 59% and aeration rate of 0.5 l/min. A β-mannanase yield of 2231.26 U/g was obtained when SSF process was carried out under the optimal conditions described above.     

A novel peptide inhibits the influenza virus replication by preventing the viral attachment to the host cells

Avian influenza viruses (AIV), the causative agent of avian flu or bird flu, cause widespread morbidity and mortality in poultry. The symptoms of the disease range from mild flu like symptoms to death. These viruses possess two important surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA) against which neutralizing antibodies are produced. Due to the highly mutative nature of the genes which encode these proteins, the viruses often confer resistance to the current anti-viral drugs making the prevention and treatment of infection challenging. In our laboratory, we have recently identified a novel anti-viral peptide (P1) against the AIV H9N2 from a phage displayed peptide library. This peptide inhibits the replication of the virus in ovo and in vitro by its binding to the HA glycoprotein. In the current study, we demonstrate that the peptide inhibits the virus replication by preventing the attachment to the host cell but it does not have any effect on the viral fusion. The reduction in the viral nucleoprotein (NP) expression inside the host cell has also been observed during the peptide (P1) treatment. This novel peptide may have the potential to be developed as a therapeutic agent for the treatment and control of avian influenza virus H9N2 infections.     

Response surface modeling of 1-stearoyl-3(2)-oleoyl glycerol production in a pilot packed-bed immobilized Rhizomucor miehei lipase reactor

A dual response approach using diacylglycerol (DAG) and triacylglycerol (TAG) as responses for optimization of 1-stearoyl-3(2)-oleoyl glycerol-enriched DAG synthesis using response surface methodology (RSM) was investigated. Four variables from a lipase-catalyzed esterification reaction were optimized using a central composite rotatable design. The following optimized conditions yielded 51 wt.% DAG and 22 wt.% TAG: reaction temperature of 55 °C, enzyme dosage of 9.5 wt.%, fatty acid/glycerol molar ratio of 2.1 and reaction time of 3 h. Results were repeatable at 10 kg production scale in a pilot packed-bed enzyme reactor. No significant losses in enzyme activity or changes in fatty acid selectivity on DAG synthesis were observed during the five pilot productions. Lipozyme RM IM showed selectivity towards the production of stearic acid enriched DAG. The purity of DAG oil after purification was 90 wt.%.     

Structural analysis of peptides that interact with Newcastle disease virus

A peptide with the sequence CTLTTKLYC has previously been identified to inhibit the propagation of Newcastle disease virus (NDV) in embryonated chicken eggs and tissue culture. NDV has been classified into two main groups: the velogenic group, and mesogenic with lentogenic strains as the other group based on its dissociation constants. In this study the peptide, CTLTTKLYC, displayed on the pIII protein of a filamentous M13 phage was synthesized and mutated in order to identify the amino acid residues involved in the interactions with NDV. Mutations of C1 and K6 to A1 and A6 did not affect the binding significantly, but substitution of Y8 with A8 dramatically reduced the interaction. This suggests that Y8 plays an important role in the peptide-virus interaction. The three-dimensional structure of the peptide was determined using circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular modeling. The peptide exhibited two possible conformers. One that consists of consecutive beta-turns around T2-L3-T4-T5 and K6-L7-Y8-C9. The other conformer exhibited a beta-hairpin bend type of structure with a bend around L3-T4-T5-K6.