In the search for universal vaccine candidates for the prevention of avian influenza, the non-structural (NS)-1 protein of avian influenza virus (AIV) H5N1 has shown promising potential for its ability to effectively stimulate the host immunity. This study was aimed to produce a bacterial expression plasmid using pRSET B vector to harbour the NS1 gene of AIV H5N1 (A/Chicken/Malaysia/5858/2004 (H5N1)) for protein expression in Escherichia coli (E. coli). The NS1 gene (687 bp) was initially amplified by polymerase chain reaction (PCR) and then cloned into a pGEM-T Easy TA vector. The NS1 gene was released from pGEM-T-NS1 using EcoRI and XhoI restriction enzymes (RE). The pRSET B vector was also linearized using the same RE. The digested NS1 gene and linearized pRSET B were ligated using T4 DNA ligase to form the expression plasmid, pRSET B-NS1. The NS1 gene sequence in pRSET B-NS1 was confirmed by DNA sequencing. To prepare recombinant bacterial cells for protein expression in the future, pRSET B-NS1 was transformed into E. coli strain BL21 (DE3) by heat-shock. Colonies bearing the recombinant plasmid were screened using PCR. The DNA sequencing analysis revealed that the NS1 gene sequence was 97% homologous to that of AIV H5N1 A/Chicken/Malaysia/5858/2004 (H5N1). These results indicated that the NS1 gene of influenza A/Chicken/Malaysia/5858/2004 (H5N1) was successfully amplified and cloned into a pRSET B vector. Bacterial colonies carrying pRSET B-NS1 can be used for the synthesis of NS1-based influenza vaccine in the future and thereby aid in the prevention of avian influenza.
There has been a significant increase in research on probiotics-associated health benefits in the last 20 years. Many studies carried out in vitro and clinically show that consumption of probiotics inhibits the growth of pathogenic microorganisms. Furthermore, the consumption of probiotics also enhances the host immune response and decreases the levels of carcinogenesis-inducing enzymes. These positive outcomes have led to the use of probiotics in prevention and treatment of infectious diseases like bacterial or antibiotic associated diarrhea, chronic inflammatory bowel diseases and colon cancer. This review summarises literature pertaining to mechanistic actions of probiotics in improving the well-being of hosts.
Many studies have shown that probiotic strains added to a number of probiotic products are not compatible to that of
claimed. It is thus of note to validate probiotic strains added to probiotic products. In this study, three probiotic drinks,
A, B and C, were cultured on MRS agar and the number of bacterial colonies was enumerated. The bacterial counts
recovered from A (9.3 ± 6.9 log CFU/ml) and C (9.0 ± 6.9 log CFU/ml) were signifi cantly higher than B (5.2 ± 3.5 log
CFU/ml) and achieved the minimal amount recommended for probiotic bacteria. All of the isolates appeared as gram
positive rods microscopically and were proven to be catalase negative. However, there were only A1, A2, B4 and C1 that
were highly tolerant to the gastrointestinal pH 3 to 6. The four isolates produced and secreted antimicrobial substances
which inhibited the growth of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). C1 showed the greatest
growth inhibition by forming 17.50-mm and 17.85-mm inhibition zones against E. coli and S. aureus, respectively. The
16s rDNA sequencing and phylogenetic analysis were performed to further identify the twelve isolates. The twelve isolates
were found to be Lactobacillus (L.), particularly L. casei and L. paracasei. However, the bacteria isolated from drink B
were incompatible to the labelled ones. In conclusion, probiotic drinks are possible to contain different bacterial counts
and probiotic strains from the labelled ones. These differences might affect health benefi ts rendered by probiotic strains
to consumers.
The occasional influenza pandemics and the seasonal influenza epidemics have destroyed millions of lives since
the last century. It is therefore necessary to understand the virus replication patterns as this provides essential
information on the virus infectivity, pathogenicity and spread patterns. This study aimed to investigate the replication
of avian influenza A virus H5N1 (A/Chicken/Malaysia/5858/2004) in MDCK cells. In this study, the TCID50 (50% tissue
culture infectious dose) of AIV H5N1 was first determined. The MDCK cells were then infected with AIV H5N1 at TCID50
for 0-48 h. The CPE (cytopathic effect) was observed and cell death was determined hourly. The virus-infected cells
and media were subsequently collected for gene analysis. The results showed that the TCID50 of AIV H5N1 was 10-9
dilution. The CPE percentage showed a strong and positive correlation with the infection period (r = 1.0, n = 9, p <
0.01). The amount of a highly conserved influenza viral gene, M2 gene amplified from infected media (r = 0.471, n =
9, p= > 0.05) and infected cell (r = 0.73, n = 9, p < 0.05) were also positively correlated with the infection period. In
conclusion, although CPE started to be observed in the early time points of infection, however, the M2 gene was only
amplified from the infected media and cells after 48 h and 24 h, respectively. This signifies that AIV H5N1 used in this
study is pathogenic and it is able to cause severe cytopathology to host cells even at low virus load.
Breast cancer is one of the commonest cancers among women. Conventional therapies cause adverse side effects in patients. Cytokine immunotherapy such as interleukin-27 (IL-27) has been sought as an alternative cancer treatment in recent years. IL-27 has been shown to improve anticancer immunity and anti-angiogenesis in cancers, however, its effect on apoptotic and anti-apoptotic gene expression especially in breast cancers is yet to be explored. Cytotoxicity of IL-27 in non-cancerous (184b5) and cancerous (MCF-7 and MDA-MB-231) breast cell lines was first determined for 24-72 h in this study. The results indicated that IL-27 treatment did not retard 184b5 cell growth, however, did inhibit MCF-7 (48 h) and MDA-MB-231 (72 h) cell growth with IC50 at 442 and 457 ng/ml, respectively. Apoptotic (TRAIL, FADD, FAS, caspase-3 and caspase-8) and anti-apoptotic (BCL-2, AKT, and COX-2) genes were then amplified from untreated (control) and treated breast cancer cells and studied. TRAIL, caspase-3, caspase-8 gene expression was significantly (p < 0.05) upregulated in treated MCF-7 (442 ng/ml) and MDA-MB-231 (457 ng/ml) cells. Expression of FADD and FAS genes was not detected in both control and treated MCF-7 and MDA-MB-231 cells. COX-2 gene was also not expressed by MCF-7 cells, but reduced significantly (p < 0.05) in treated MDA-MB-231 cells. In MDA-MB-231 cells, IL-27 treatment seemed to slightly enhance the expression of AKT and BCL-2 genes which, on the other hand, was downregulated in treated MCF-7 cells. Conclusively, IL-27 is able to inhibit breast cancer cell growth and regulate apoptotic and anti-apoptotic gene expression in breast cancer cells.
Mutagenic and antimutagenic activities of lactic acid bacteria (LAB) Lactobacillus plantarum isolated from the local fermented durian (tempoyak) was determined by Ames test (Salmonella/microsome mutagenicity assay). Our study also involved pre-incubation assay against Salmonella typhimurium TA 98 and TA 100 bacterial strain in the presence and absence of metabolic activator S9 system. It was found that the L. plantarum showed no mutagenic activity on both S. typhimurium strain TA 98 and TA 100 in the presence and absence of metabolic activator. Significant antimutagenic activity (p < 0.05) was observed in both cell-free supernatant and bacterial cell suspension of L. plantarum as compared to the mutagenicity induced by 2-Aminoanthracene in the presence of metabolic activator. Meanwhile, in the absence of metabolic activator, only the bacterial cells of L. plantarum showed antimutagenicity acitivity against Sodium Azide and 2-Nitrofluorene. In conclusion, L. plantarum could play a vital role as chemopreventive agent by binding to mutagens and suppressing mutagenesis. Thus, L. plantarum could be consider as a good candidate for functional food development as a supplement product to prevent development of colon cancer.
Each year, influenza A infections have caused tremendous death rate as high as 300,000-500,000 globally. Although
there are effective anti-influenza agents and vaccines, high mutational rate among influenza A viruses renders dramatic
decline in the effectiveness of anti-influenza agents or vaccines in certain individuals. The situation is further complicated
by limitations in influenza vaccine production, for instance, long production period, limited vaccine capacity and lack
of cross-protection against various influenza A virus strains. To solve these issues, development of universal influenza
vaccine based on conserved antigens such as non-stuctural protein 1 (NS1) has been endeavoured. NS1 protein is highly
conserved in all influenza A virus strains known by far, produced abundantly on infected cell surfaces and responsible for
maintaining virulence. Furthermore, cytotoxic T-lymphocytes that are active against NS1 were also reported to be able
to avoid shedding of influenza in hosts. To better inhibit influenza infections, oral immunization has long been proposed
due to feasibility of this method to be implemented and safer for recipients while able to target influenza A viruses from
the entry point. Lactobacillus has been vastly studied for its roles as bacterial carrier in oral vaccine development due
to its significant probiotic properties. For examples, stimulation of immune responses in oral and airway mucosal layers,
high colonization in oral and airway mucosal layers and great natural adjuvant effects. In this light, influenza universal
oral vaccine developed using NS1 dan Lactobacillus should be further studied in influenza oral vaccine design.
In light of the limited protection conferred by current influenza vaccines, immunisation using universal influenza vaccines has been proposed for protection against all or most influenza sub-types. The fundamental principle of universal influenza vaccines is based on conserved antigens found in most influenza strains, such as matrix 2, nucleocapsid, matrix 1 and stem of hemagglutinin proteins. These antigens trigger cross-protective immunity against different influenza strains. Many researchers have attempted to produce the conserved epitopes of these antigens in the form of peptides in the hope of generating universal influenza vaccine candidates that can broadly induce cross-reactive protection against influenza viral infections. However, peptide vaccines are poorly immunogenic when applied individually owing to their small molecular sizes. Hence, strategies, such as combining peptides as multi-epitope vaccines or presenting peptides on vaccinia virus particles, are employed. This review discusses the clinical and laboratory findings of several multi-epitope peptide vaccine candidates and vaccinia-based peptide vaccines. The majority of these vaccine candidates have reached the clinical trial phase. The findings in this study will indeed shed light on the applicability of universal influenza vaccines to prevent seasonal and pandemic influenza outbreaks in the near future.
The C-terminal domain of Nipah virus (NiV) nucleocapsid protein (NP₄₀₁₋₅₃₂) was inserted at the N-terminus and the immunodominant loop of hepatitis B core antigen (HBc). The stability of NP₄₀₁₋₅₃₂ increased tremendously when displayed on the HBc particles. These particles reacted specifically with the swine anti-NiV and the human anti-HBc antisera.
Hepatitis B core antigen (HBcAg) is used as a diagnostic reagent for the detection of hepatitis B virus infection. In this study, immobilized metal affinity-expanded bed adsorption chromatography (IMA-EBAC) was employed to purify N-terminally His-tagged HBcAg from unclarified bacterial homogenate. Streamline Chelating was used as the adsorbent and the batch adsorption experiment showed that the optimal binding pH of His-tagged HBcAg was 8.0 with a binding capacity of 1.8 mg per ml of adsorbent. The optimal elution condition for the elution of His-tagged HBcAg from the adsorbent was at pH 7 in the presence of 500 mM imidazole and 1.5 M NaCl. The IMA-EBAC has successfully recovered 56% of His-tagged HBcAg from the unclarified E. coli homogenate with a purification factor of 3.64. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of the recovered His-tagged HBcAg was not affected throughout the IMA-EBAC purification process and electron microscopy revealed that the protein assembled into virus-like particles (VLP).
The current influenza vaccines only confer protection against the circulating influenza subtypes, therefore universal vaccines are needed to prevent upcoming influenza outbreaks caused by emerging influenza subtypes. The extracellular domain of influenza A M2 protein (M2e) is highly conserved among different subtypes of influenza A viruses, and it is able to elicit protective immunity against the viruses. The influenza nucleoprotein (NP) was used to display the M2e in this study due to its promising T-cell response and adjuvanticity. The M2e gene was fused to the 5'-end of the NP gene and then cloned into pRSET B vector. The DNA sequencing analysis revealed six point mutations in the M2e-NP fusion gene, including one mutation in the M2e peptide and five mutations in the NP. The mutations were reverted using PCR site-directed mutagenesis. The recombinant plasmids (pRSET B-M2e-NP and pRSET B-mM2e-NP) were introduced into Escherichia coli (E. coli) BL21 (DE3) for protein expression. The mutated and non-mutated proteins were subsequently expressed and named mM2e-NP and M2e-NP, respectively. The expression of mM2e-NP and M2e-NP was not affected by the mutations. The binding of anti-M2e antibody to the purified native mM2e-NP and M2e-NP also remained active. However, when the anti-NP antibody was tested, the signal produced by mM2e-NP was very weak. The results implied that the amino acid changes in the NP had adversely impacted on the conformation of mM2e-NP and subsequently affected the antibody binding. In light of the remarkable antibody binding to the M2e-NP fusion protein, this study highly recommends the potential of M2e-NP as a universal influenza vaccine candidate.
Lactic acid bacterium isolated from fermented durian (tempoyak) was investigated for its potentials as a probiotic strain. Bacterial tolerance toward gastrointestinal environment, adhesion, and cytotoxic activity in human colon adenocarcinoma cell line HT-29 was evaluated. 16S rRNA sequencing identified the lactic acid bacterium as Lactobacillus plantarum. The bacterium demonstrated good tolerance toward gastrointestinal pH 2.0 and 0.3% bile salts. It showed strong adhesive capacity in human intestinal cell line, HT-29, with an adhesion index of 159 ± 10. Cytotoxicity of L. plantarum was investigated using both live bacterial cells (BC) and cell-free supernatant (CFS). Findings showed that both BC and CFS of L. plantarum reduced proliferation of HT-29 colon adenocarcinoma cells using MTT assay. The results imply potential probiotic properties of L. plantarum isolated from tempoyak.
Discovery of conserved antigens for universal influenza vaccines warrants solutions to a number of concerns pertinent to the currently licensed influenza vaccines, such as annual reformulation and mismatching with the circulating subtypes. The latter causes low vaccine efficacies, and hence leads to severe disease complications and high hospitalization rates among susceptible and immunocompromised individuals. A universal influenza vaccine ensures cross-protection against all influenza subtypes due to the presence of conserved epitopes that are found in the majority of, if not all, influenza types and subtypes, e.g., influenza matrix protein 2 ectodomain (M2e) and nucleoprotein (NP). Despite its relatively low immunogenicity, influenza M2e has been proven to induce humoral responses in human recipients. Influenza NP, on the other hand, promotes remarkable anti-influenza T-cell responses. Additionally, NP subunits are able to assemble into particles which can be further exploited as an adjuvant carrier for M2e peptide. Practically, the T-cell immunodominance of NP can be transferred to M2e when it is fused and expressed as a chimeric protein in heterologous hosts such as Escherichia coli without compromising the antigenicity. Given the ability of NP-M2e fusion protein in inducing cross-protective anti-influenza cell-mediated and humoral immunity, its potential as a universal influenza vaccine is therefore worth further exploration.
Hypertension can be caused by various factors while the predominant causes include increase in body fluid volume and resistance in the circulatory system that elevate the blood pressure. Consumption of probiotics has been proven to attenuate hypertension; however, the effect is much strain-dependent. In this study, a newly isolated Lactobacillus casei (Lb. casei) strain C1 was investigated for its antihypertensive properties in spontaneously hypertensive rats (SHR). Lactic acid bacteria (LAB) suspension of 11 log colony-forming unit (CFU) was given to SHR (SHR+LAB, n=8), and phosphate buffer saline (PBS) was given as a control in SHR (SHR, n=8) and in Wistar rats as sham (WIS, n=8). The treatment was given via oral gavage for 8 weeks. The results showed that the weekly systolic blood pressure (SBP), mean arterial pressure (MAP), diastolic blood pressure (DBP) and aortic reactivity function were remarkably improved after 8 weeks of bacterial administration in SHR+LAB. These effects were mostly attributed by restoration of wall tension and tensile stress following the bacterial treatment. Although not statistically significant, the level of malondialdehye (MDA) in SHR+LAB serum was found declining. Increased levels of glutathione (GSH) and nitric oxide (NO) in SHR+LAB serum suggested that the bacterium exerted vascular protection through antioxidative functions and relatively high NO level that induced vasodilation. Collectively, Lb. casei strain C1 is a promising alternative for hypertension improvement.
Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM.
The coronavirus disease 2019 (COVID-19) became a worldwide concern at the beginning of 2020 and has affected millions. Several previous studies revealed the impact of the imbalanced innate immune response on the progression of COVID-19 and its disease outcomes. High levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukins are produced readily by innate immune cells to fight Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infections. Nonetheless, cytokine-mediated inflammatory events are also linked to detrimental lung injury and respiratory failure, which can result in deaths among COVID-19 patients. TNF-α is amongst the early cytokines produced to mediate proinflammatory responses and enhance immune cell infiltration in response to SARS-CoV-2 infections. In COVID-19, TNF-α-mediated inflammation can cause detrimental tissue damage and gradually promotes lung fibrosis, which later results in pneumonia, pulmonary edema, and acute respiratory distress syndrome. This review, therefore, aims to deliberate the immunomodulatory roles of TNF-α in promoting inflammation and its relation with COVID-19 morbidity and mortality. In addition, this review also proposes the potential of TNF-α as a biomarker for the prognosis of severe COVID-19 and its related complications and as a molecular target for anti-TNF-α therapy.
The core antigen of the hepatitis B virus (HBcAg) has been used widely as a diagnostic reagent for the identification of the viral infection. However, purification using the conventional sucrose density gradient ultracentrifugation is time consuming and costly. To overcome this, HBcAg particles displaying His-tag on their surface were constructed and produced in Escherichia coli. The recombinant His-tagged HBcAgs were purified using immobilized metal affinity chromatography. Transmission electron microscopy and enzyme-linked immunosorbent assay (ELISA) revealed that the displayed His-tag did not impair the formation of the core particles and the antigenicity of HBcAg.