Sazaly Abu Bakar

DNA vaccine constructs against enterovirus 71 elicit immune response in mice

BackgroundEnterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and construct a DNA vaccine against Enterovirus 71 using the viral capsid protein (VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo.MethodsThe VP1 gene of EV71 from two local outbreak isolates were amplified using PCR and then inserted into a eukaryotic expression vector, pVAX1. The 3.9 kb recombinant constructs were transformed into competent E. coli cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in Vero cells. Subsequently, in the in vivo studies, female Balb/c mice were immunized with the DNA vaccine constructs. Enzyme Linked Immunosorbent Assay (ELISA) and virus neutralizing assay were performed to detect the presence of anti-VP1 IgG in mice and its neutralizing effect against the EV71.ResultsThe pVAX1 vector was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame. The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting. The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster. The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71.ConclusionThe promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccine.

DengueTools: innovative tools and strategies for the surveillance and control of dengue

Dengue fever is a mosquito-borne viral disease estimated to cause about 230 million infections worldwide every year, of which 25,000 are fatal. Global incidence has risen rapidly in recent decades with some 3.6 billion people, over half of the worlds population, now at risk, mainly in urban centres of the tropics and subtropics. Demographic and societal changes, in particular urbanization, globalization, and increased international travel, are major contributors to the rise in incidence and geographic expansion of dengue infections. Major research gaps continue to hamper the control of dengue. The European Commission launched a call under the 7th Framework Programme with the title of ‘Comprehensive control of Dengue fever under changing climatic conditions’. Fourteen partners from several countries in Europe, Asia, and South America formed a consortium named ‘DengueTools’ to respond to the call to achieve better diagnosis, surveillance, prevention, and predictive models and improve our understanding of the spread of dengue to previously uninfected regions (including Europe) in the context of globalization and climate change. The consortium comprises 12 work packages to address a set of research questions in three areas: Research area 1 Develop a comprehensive early warning and surveillance system that has predictive capability for epidemic dengue and benefits from novel tools for laboratory diagnosis and vector monitoring. Research area 2 Develop novel strategies to prevent dengue in children. Research area 3 Understand and predict the risk of global spread of dengue, in particular the risk of introduction and establishment in Europe, within the context of parameters of vectorial capacity, global mobility, and climate change. In this paper, we report on the rationale and specific study objectives of ‘DengueTools’. DengueTools is funded under the Health theme of the Seventh Framework Programme of the European Community, Grant Agreement Number: 282589 Dengue Tools.

IL10 and IL12B polymorphisms each influence IL-12p70 secretion by dendritic cells in response to LPS

Dendritic cells (DC) are the main producers of the cytokine IL‐12p70, through which they play a direct role in the development of IFN‐γ‐secreting Th1 cells, costimulation of CTL differentiation and NK‐cell activation. In contrast, IL‐10, which is also produced by DC, negatively regulates IL‐12 production. IL‐12p70 production varies widely between individuals, and several polymorphisms in the gene encoding IL‐12p40 (IL12B) have been identified that influence susceptibility and severity of infectious, autoimmune and neoplastic disease. Here we show that polymorphisms not only of IL12B, but also in the IL10 promoter, influence IL‐12p70 secretion by monocyte‐derived DC in response to LPS. Although IL12B promoter homozygotes were prone to making more IL‐12p70, presence of the IL10 high genotype restricted IL‐12p70 production in these individuals. These observations provide a further genetic control of IL‐12p70 regulation and emphasize the complexity of production of this cytokine. They also suggest genotypes that might influence the outcome of DC immunotherapy.

C-phycocyanin confers protection against oxalate-mediated oxidative stress and mitochondrial dysfunctions in MDCK cells

Oxalate toxicity is mediated through generation of reactive oxygen species (ROS) via a process that is partly dependent on mitochondrial dysfunction. Here, we investigated whether C-phycocyanin (CP) could protect against oxidative stress-mediated intracellular damage triggered by oxalate in MDCK cells. DCFDA, a fluorescence-based probe and hexanoyl-lysine adduct (HEL), an oxidative stress marker were used to investigate the effect of CP on oxalate-induced ROS production and membrane lipid peroxidation (LPO). The role of CP against oxalate-induced oxidative stress was studied by the evaluation of mitochondrial membrane potential by JC1 fluorescein staining, quantification of ATP synthesis and stress-induced MAP kinases (JNK/SAPK and ERK1/2). Our results revealed that oxalate-induced cells show markedly increased ROS levels and HEL protein expression that were significantly decreased following pre-treatment with CP. Further, JC1 staining showed that CP pre-treatment conferred significant protection from mitochondrial membrane permeability and increased ATP production in CP-treated cells than oxalate-alone-treated cells. In addition, CP treated cells significantly decreased the expression of phosphorylated JNK/SAPK and ERK1/2 as compared to oxalate-alone-treated cells. We concluded that CP could be used as a potential free radical-scavenging therapeutic strategy against oxidative stress-associated diseases including urolithiasis.

Antiviral activity of silymarin against chikungunya virus

The mosquito-borne chikungunya virus (CHIKV) causes chikungunya fever, with clinical presentations such as severe back and small joint pain, and debilitating arthritis associated with crippling pains that persist for weeks and even years. Although there are several studies to evaluate the efficacy of drugs against CHIKV, the treatment for chikungunya fever is mainly symptom-based and no effective licensed vaccine or antiviral are available. Here, we investigated the antiviral activity of three types of flavonoids against CHIKV in vitro replication. Three compounds: silymarin, quercetin and kaempferol were evaluated for their in vitro antiviral activities against CHIKV using a CHIKV replicon cell line and clinical isolate of CHIKV of Central/East African genotype. A cytopathic effect inhibition assay was used to determine their activities on CHIKV viral replication and quantitative reverse transcription PCR was used to calculate virus yield. Antiviral activity of effective compound was further investigated by evaluation of CHIKV protein expression using western blotting for CHIKV nsP1, nsP3, and E2E1 proteins. Briefly, silymarin exhibited significant antiviral activity against CHIKV, reducing both CHIKV replication efficiency and down-regulating production of viral proteins involved in replication. This study may have important consequence for broaden the chance of getting the effective antiviral for CHIKV infection.

Blockade of CXCR2 signalling: A potential therapeutic target for preventing neutrophil-mediated inflammatory diseases

Polymorphonuclear neutrophils (PMN) play a key role in host innate immune responses by migrating to the sites of inflammation. Furthermore, PMN recruitment also plays a significant role in the pathophysiology of a plethora of inflammatory disorders such as chronic obstructive pulmonary disease (COPD), gram negative sepsis, inflammatory bowel disease (IBD), lung injury, and arthritis. Of note, chemokine-dependent signalling is implicated in the amplification of immune responses by virtue of its role in PMN chemotaxis in most of the inflammatory diseases. It has been clinically established that impediment of PMN recruitment ameliorates disease severity and provides relief in majority of other immune-associated disorders. This review focuses on different novel approaches clinically proven to be effective in blocking chemokine signalling associated with PMN recruitment that includes CXCR2 antagonists, chemokine analogs, anti-CXCR2 monoclonal antibodies, and CXCR2 knock-out models. It also highlights the significance of the utility of nanoparticles in drugs used for blocking migration of PMN to the sites of inflammation.

Chlamydia muridarum infection associated host microRNAs in the murine genital tract and contribution to generation of host immune response

Chlamydia trachomatis (CT) is the leading sexually transmitted bacterial infection in humans and is associated with reproductive tract damage. However, little is known about the involvement and regulation of microRNAs (miRs) in genital CT.

Inhibition of chikungunya virus replication by hesperetin and naringenin

Chikungunya virus (CHIKV) is an emerging arbovirus, which has recently become globally important. In recent years, it has posed a progressive threat to humankind, causing the development of possibly life-threatening and incapacitating arthritis. Currently, there is no available effective antiviral drug for chikungunya infection. Thus, finding and developing lead compounds with anti-CHIKV activity that could be further developed into a practical treatment is urgently required. Several studies have reported the wide-ranging antiviral activities of flavanones; however, an inhibitory effect of selected compounds has yet to be shown against CHIKV. In this study, we investigated the antiviral properties of two types of flavanones, namely naringenin and hesperetin, against CHIKV in vitro replication. Our data have shown dose dependent inhibitory effects for naringenin and hesperetin against CHIKV intracellular replication using different assays, including the CHIKV replicon cell line, time-of-addition and virus yield assays. The antiviral activity of the compounds was further investigated by the evaluation of CHIKV protein expression using a quantitative immunofluorescence assay and western blotting. In brief, these compounds presented significant antiviral activity against CHIKV, reducing both the CHIKV replication efficiency and down-regulating the production of viral proteins involved in replication. Naringenin with IC50 = 6.818 μM (SI = 80.27) and hesperetin with IC50 = 8.500 μM (SI = 23.34) inhibited the post entry stages of CHIKV replication activity. In conclusion, the obtained data from the current study suggest that naringenin and hesperetin could be potential candidates to be developed further as anti-CHIKV therapeutic agents.

The high-affinity human IgG receptor Fc gamma receptor I (FcγRI) is not associated with vascular leakage of dengue

BackgroundDengue is a major public health problem in many tropical and sub-tropical countries. Vascular leakage and shock are identified as the major causes of deaths in patients with severe dengue. Studies have suggested the potential role of Fc gamma receptors I (FcγRI) in the pathogenesis of dengue. We hypothesized that the circulating level of Fcγ receptor I could potentially be used as an indicator in assisting early diagnosis of severe dengue.ResultsA selected cohort of 66 dengue patients including 42 dengue with signs of vascular leakage, and 24 dengue without signs of vascular leakage were identified and were afterwards referred to as ‘cases’ and ‘controls’ respectively. Thirty seven normal healthy controls were also recruited in this study. The circulating level of FcγRI was quantified from the serum using enzyme-link immunosorbent assay (ELISA). The levels of FcγRI in both groups of patients with and without vascular leakage were found to be significantly higher than the normal healthy controls (P < 0.001). However, there was no significant difference found between patients with vascular leakage and those without vascular leakage (p = 0.777).ConclusionWe suggest that FcγRI is not associated with the vascular leakage in dengue. However, further studies are necessary to delineate the role of FcγRI in antibody-dependent enhancement (ADE) mechanism.

Deciphering the potential of baicalin as an antiviral agent for Chikungunya virus infection

Abstract The past decade has seen the re‐emergence of Chikungunya virus (CHIKV) as a major global health threat, affecting millions around the world. Although fatal infections are rare among infected patients, the occurrence of long‐lasting polyarthralgia has a significant impact on patients’ quality of lives and ability to work. These issues were the stimuli for this study to determine the potential of baicalin, a bioflavonoid, as the novel antiviral compound against CHIKV. It was found that baicalin was well tolerated by Vero, BHK‐21 and HEK 293T cells with maximal nontoxic doses >600 &mgr;M, ≈ 350 &mgr;M and ≈110 &mgr;M, respectively. Antiviral assays indicated that baicalin was the most effective inhibitor when tested for its direct virucidal activity with EC50 ≈ 7 &mgr;M, followed by inhibition of virus entry into the host cell, attachment of virus particle to cellular receptors and finally intracellular replication of viral RNA genome. In silico analysis using molecular docking demonstrated close interactions between baicalin and CHIKV envelope protein with considerably strong binding affinity of −9.7 kcal/mol. qRT‐PCR analysis revealed that baicalin had the greatest effect on the synthesis of viral negative stand RNA with EC50 ≈ 0.4 &mgr;M followed by the inhibition of synthesis of positive‐strand genomic (EC50 ≈ 13 &mgr;M) and subgenomic RNAs (EC50 ≈ 14 &mgr;M). These readings indicate that the compound efficiently inhibits replicase complexes formation but is a less potent inhibitor of existing replicase complexes. Coherent with this hypothesis, the use of recombinant CHIKV replicons harboring Renilla luciferase marker showed that replication of corresponding replicon RNAs was only slightly downregulated at higher doses of baicalin, with EC50 > 100 &mgr;M. Immunofluorescence and western blotting experiments demonstrated dose‐dependent inhibition of expression of different viral proteins. It was also observed that levels of important protein markers for cellular autophagy (LC3) and apoptosis (Bax) were reduced in baicalin treatment groups as compared with untreated virus infected controls. In summary, given its low toxicity and high efficacy against CHIKV, baicalin has great potential to be developed as the novel antiviral compound for CHIKV. In vivo studies to evaluate its activity in a more complexed system represent a necessary step for future analysis. HighlightsBaicalin inhibits different stages of CHIKV replication cycle.CHIKV proteins production and expression were effectively suppressed by baicalin treatment.Cellular‐death mechanisms induced by CHIKV infections were downregulated in baicalin‐treated cells.