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.

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Individual studies have assessed the association between TNF-α-308G>A and TNF-α-238 G>A polymorphisms and severity of dengue infection. However, the results are inconclusive and most studies had small sample sizes. The objective of this study was to summarize the evidence of association between TNF-α-308 G>A and TNF-α-238 G>A and severity of dengue infection.This study follows the preferred reporting items for systematic reviews and meta- analyses of genetic association studies, recommended by PLOS One. We calculated pooled odds ratio and its 95% confidence interval (CI) to estimate the association between TNF-α-308 G>A or TNF-α-238 G>A and the risk of severe dengue infections. To determine the information size required for this meta-analysis study, a trial sequential analysis (TSA) was done. Eight studies (640 cases and 1275 controls), which assessed the association of TNF-α-308 G>A or TNF-α-238 G>A and the risk of DHF were included. Overall, we found no significant association between TNF-α-308 G>A and the DHF risk in the allelic model (OR, 0.91; 95% CI, 0.51–1.63), the recessive model (OR,1.32;95%CI,0.73–2.37), the dominant model (OR,0.93;95%CI:0.59–1.47) or the additive model (OR,1.43,95;95%CI:0.79–2.59). There was also no significant association between TNF-α-238 G>A and DHF risk under the allele contrast model (OR:1.51;95%CI:0.88–2.58), the recessive model (OR,1.48,95% CI:0.33–6.58), the dominant model (OR,1.48;95%CI:0.56–3.92), or the additive model (OR:1.5;95%CI:0.34–6.69). On subgroup analysis, neither the Asian population nor the non-Asian population showed significant association between TNF-α-308 G>A/TNF-α-238 G>A and the DHF risk under any genetic models. Leave-one-out meta-analysis showed stability of the results. TSA plots suggested that the sample size in this meta-analysis study was below the required information size.The findings suggest an inclusive evidence of the association between TNF-α-308/ TNF-α-238 G>A and the risk of developing severe dengue infection. Large studies with evidence of Hardy-Weinberg equilibrium, assessing gene-gene interactions are recommended.

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Background: Gastric cancer is globally the fifth most common cancer. Several studies have assessed the relationship between tumour necrosis factor-alpha (TNF-a- 308) and the risk of gastric cancer. These individual genetic association studies showed inconclusive results. The objective of the present study was to synthesis evidence on the association between TNF-a-308 polymorphisms and gastric cancer risk by meta-analysis of data from eligible studies.Methods: We performed a meta-analysis of genetic association studies, according to the PLOS One checklist. We searched relevant case-control studies in health-related electronic databases. The methodological quality of included studies was assessed by the Newcastle-Ottawa quality assessment scale. The strength of association was calculated as odds ratios (ORs) with 95% confidence intervals (CIs). Pooled ORs and 95 % CIs were estimated using random-effects model or fixed effect model, based on between-study heterogeneity. We analysed the strength of association under four genetic models (allele, dominant, recessive and additive models). Subgroup analyses on ethnic groups, Hardy-Weinberg equilibrium (HWE) status, status of Helicobacter pylori infection and study quality were done for robustness of the estimates. Publication bias was detected by inspection of funnel plot asymmetry. To estimate the required information size, we performed trial sequential analysis (TSA) that classified the effect estimates as ‘firm evidence of effect’ or ‘potentially spurious evidence of effect’.Results: A total of 35 studies, comprising 11353 cases and 12827 controls were identified. Based on 28 studies that met HWE, there was overall significant association between TNF-α-308 polymorphisms and gastric cancer risk under the dominant model (OR 1.19, 95%CI 1.1-1.29, I2:37%), as well as Asians (OR 1.2, 95%CI 1.05-1.38, I2:53%) and Cassian subgroups (OR 1.19, 95%CI 1.07-1.31, I2:28%). Based on 13 high quality studies under the dominant model, overall significant association was also found (OR 1.38, 95%CI 1.07, 1.77). The TSA plot indicated the analyses was with the required information size. There was no publication bias. In the subgroup analysis by ethnic groups, the quality of studies impacted on the estimates. Conclusions: The findings suggest that TNF-α-308 gene polymorphism plays an important predisposing role for gastric carcinogenesis, and can serve as a useful screening marker.