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The etiological agent of melioidosis, Burkholderia pseudomallei, utilises a type III secretion system cluster 3 (T3SS3) to deliver proteins termed type III effectors (T3SEs) into the host cytoplasm in order to establish an intracellular lifecycle in phagocytic and non-phagocytic cells, thus playing an important role in pathogenesis. To gain insight into possible functional roles for BapA, a putative T3SE with unknown function, in the intracellular lifecycle of B.pseudomallei, bapA gene knockout mutant was constructed. The effect of the knockout on virulence to the otherwise isogenic parental strain, K96243, was studied by cellular infection assays and Caenorhabditis elegans killing assay. The attachment and subsequent entry into A549 cells was significantly (P < 0.05) attenuated in the Delta bapA compared to K96243. However, intracellular replication was not affected. Furthermore, the cell-to-cell spreading capacity of Delta bapA was impaired although the mutant exhibited no evident defect in its actin tail formation. Additionally, phagocytosis and intracellular replication rates of Delta bapA in U937 macrophage cells were significantly reduced relative to K96243 without phagosomal escape being affected. Based on these observations, we conclude that the BapA T3SE could play an important role in B.pseudomallei intracellular lifecycle, especially, in the early stages of attachment and entry into the host cell.

Avian influenza virus A (H7N9), after circulating in avian hosts for decades, was identified as a human pathogen in 2013. Herein, amino acid substitutions possibly essential for human adaptation were identified by comparing the 4706 aligned overlapping nonamer position sequences (1-9, 2-10, etc.) of the reported 2014 and 2017 avian and human H7N9 datasets. The initial set of virus sequences (as of year 2014) exhibited a total of 109 avian-to-human (A2H) signature amino acid substitutions. Each represented the most prevalent substitution at a given avian virus nonamer position that was selectively adapted as the corresponding index (most prevalent sequence) of the human viruses. The majority of these avian substitutions were long-standing in the evolution of H7N9, and only 17 were first detected in 2013 as possibly essential for the initial human adaptation. Strikingly, continued evolution of the avian H7N9 virus has resulted in avian and human protein sequences that are almost identical. This rapid and continued adaptation of the avian H7N9 virus to the human host, with near identity of the avian and human viruses, is associated with increased human infection and a predicted greater risk of human-to-human transmission. Bezmiâlem Vakıf Üniversitesi Avian Influenza H7N9 Virus Adaptation to Human Hosts

Laboratory pedagogy is moving away from step-by-step instructions and toward inquiry-based learning (IBL), but only now developing methods for integrating inquiry-based writing (IBW) practices into the laboratory course. Based on an earlier proposal (Science 332:919 (2011)), we designed and implemented an IBW sequence in a university bioinformatics course.We automatically generated unique, double-blinded, biologically plausible DNA sequences for each student. After guided instruction, students investigated sequences independently and responded through IBW writing assignments. IBW assignments were structured as condensed versions of a scientific research paper, and because the sequences were double blinded, they were also assessed as authentic science and evaluated on clarity and persuasiveness.We piloted the approach in a seven-day workshop (35 students) at Perdana University Graduate School of Medicine in Kuala Lumpur. We observed dramatically improved student engagement and indirect evidence of improved learning outcomes over a similar workshop without IBW. Based on student feedback, initial discomfort with the writing component abated in favor of an overall positive response and increasing comfort with the high demands of student writing. Similarly encouraging results were found in a semester length undergraduate module at the National University of Singapore (155 students).

Severe dengue can be lethal caused by manifestations such as severe bleeding, fluid accumulation and organ impairment. This study aimed to investigate the role of dengue non-structural 1 (NS1) protein and host factors contributing to severe dengue. Electrical cell-substrate impedance sensing system was used to investigate the changes in barrier function of microvascular endothelial cells treated NS1 protein and serum samples from patients with different disease severity. Cytokines and metabolites profiles were assessed using a multiplex cytokine assay and liquid chromatography mass spectrometry respectively. The findings showed that NS1 was able to induce the loss of barrier function in microvascular endothelium in a dose dependent manner, however, the level of NS1 in serum samples did not correlate with the extent of vascular leakage induced. Further assessment of host factors revealed that cytokines such as CCL2, CCL5, CCL20 and CXCL1, as well as adhesion molecule ICAM-1, that are involved in leukocytes infiltration were expressed higher in dengue patients in comparison to healthy individuals. In addition, metabolomics study revealed the presence of deregulated metabolites involved in the phospholipid metabolism pathway in patients with severe manifestations. In conclusion, disease severity in dengue virus infection did not correlate directly with NS1 level, but instead with host factors that are involved in the regulation of junctional integrity and phospholipid metabolism. However, as the studied population was relatively small in this study, these exploratory findings should be confirmed by expanding the sample size using an independent cohort to further establish the significance of this study.

BackgroundInfluenza A (H5N1) virus is a global concern with potential as a pandemic threat. High sequence variability of influenza A viruses is a major challenge for effective vaccine design. A continuing goal towards this is a greater understanding of influenza A (H5N1) proteome sequence diversity in the context of the immune system (antigenic diversity), the dynamics of mutation, and effective strategies to overcome the diversity for vaccine design.MethodsHerein, we report a comprehensive study of the dynamics of H5N1 mutations by analysis of the aligned overlapping nonamer positions (1–9, 2–10, etc.) of more than 13,000 protein sequences of avian and human influenza A (H5N1) viruses, reported over at least 50 years. Entropy calculations were performed on 9,408 overlapping nonamer position of the proteome to study the diversity in the context of immune system. The nonamers represent the predominant length of the binding cores for peptides recognized by the cellular immune system. To further dissect the sequence diversity, each overlapping nonamer position was quantitatively analyzed for four patterns of sequence diversity motifs: index, major, minor and unique.ResultsAlmost all of the aligned overlapping nonamer positions of each viral proteome exhibited variants (major, minor, and unique) to the predominant index sequence. Each variant motif displayed a characteristic pattern of incidence change in relation to increased total variants. The major variant exhibited a restrictive pyramidal incidence pattern, with peak incidence at 50% total variants. Post this peak incidence, the minor variants became the predominant motif for majority of the positions. Unique variants, each sequence observed only once, were present at nearly all of the nonamer positions. The diversity motifs (index and variants) demonstrated complex inter-relationships, with motif switching being a common phenomenon. Additionally, 25 highly conserved sequences were identified to be shared across viruses of both hosts, with half conserved to several other influenza A subtypes.DiscussionThe presence of distinct sequences (nonatypes) at nearly all nonamer positions represents a large repertoire of reported viral variants in the proteome, which influence the variability dynamics of the viral population. This work elucidated and provided important insights on the components that make up the viral diversity, delineating inherent patterns in the organization of sequence changes that function in the viral fitness-selection. Additionally, it provides a catalogue of all the mutational changes involved in the dynamics of H5N1 viral diversity for both avian and human host populations. This work provides data relevant for the design of prophylactics and therapeutics that overcome the diversity of the virus, and can aid in the surveillance of existing and future strains of influenza viruses.

Plasma leakage is the main pathophysiological feature in severe dengue, resulting from altered vascular barrier function associated with an inappropriate immune response triggered upon infection. The present study investigated functional changes using an electric cell-substrate impedance sensing system in four (brain, dermal, pulmonary and retinal) human microvascular endothelial cell (MEC) lines infected with purified dengue virus, followed by assessment of cytokine profiles and the expression of inter-endothelial junctional proteins. Modelling of changes in electrical impedance suggests that vascular leakage in dengue-infected MECs is mostly due to the modulation of cell-to-cell interactions, while this loss of vascular barrier function observed in the infected MECs varied between cell lines and DENV serotypes. High levels of inflammatory cytokines (IL-6 and TNF-alpha), chemokines (CXCL1, CXCL5, CXCL11, CX3CL1, CCL2 and CCL20) and adhesion molecules (VCAM-1) were differentially produced in the four infected MECs. Further, the tight junctional protein, ZO-1, was down-regulated in both the DENV-1-infected brain and pulmonary MECs, while claudin-1, PECAM-1 and VE-cadherin were differentially expressed in these two MECs after infection. Non-purified virus stock was also studied to investigate the impact of virus stock purity on dengue-specific immune responses, and the results suggest that virus stock propagated through cell culture may include factors that mask or alter the DENV-specific immune responses of the MECs. The findings of the present study show that high DENV load differentially modulates human microvascular endothelial barrier function and disrupts the function of inter-endothelial junctional proteins during early infection with organ-specific cytokine production.

Background The sequence diversity of dengue virus (DENV) is one of the challenges in developing an effective vaccine against the virus. Highly conserved, serotype-specific (HCSS), immune-relevant DENV sequences are attractive candidates for vaccine design, and represent an alternative to the approach of selecting pan-DENV conserved sequences. The former aims to limit the number of possible cross-reactive epitope variants in the population, while the latter aims to limit the cross-reactivity between the serotypes to favour a serotype-specific response. Herein, we performed a large-scale systematic study to map and characterise HCSS sequences in the DENV proteome. Methods All reported DENV protein sequence data for each serotype was retrieved from the NCBI Entrez Protein (nr) Database (txid: 12637). The downloaded sequences were then separated according to the individual serotype proteins by use of BLASTp search, and subsequently removed for duplicates and co-aligned across the serotypes. Shannon’s entropy and mutual information (MI) analyses, by use of AVANA, were performed to measure the diversity within and between the serotype proteins to identify HCSS nonamers. The sequences were evaluated for the presence of promiscuous T-cell epitopes by use of NetCTLpan 1.1 and NetMHCIIpan 3.2 server for human leukocyte antigen (HLA) class I and class II supertypes, respectively. The predicted epitopes were matched to reported epitopes in the Immune Epitope Database. Results A total of 2321 nonamers met the HCSS selection criteria of entropy 0.8. Concatenating these resulted in a total of 337 HCSS sequences. DENV4 had the most number of HCSS nonamers; NS5, NS3 and E proteins had among the highest, with none in the C and only one in prM. The HCSS sequences were immune-relevant; 87 HCSS sequences were both reported T-cell epitopes/ligands in human and predicted epitopes, supporting the accuracy of the predictions. A number of the HCSS clustered as immunological hotspots and exhibited putative promiscuity beyond a single HLA supertype. The HCSS sequences represented, on average, ~ 40% of the proteome length for each serotype; more than double of pan-DENV sequences (conserved across the four serotypes), and thus offer a larger choice of sequences for vaccine target selection. HCSS sequences of a given serotype showed significant amino acid difference to all the variants of the other serotypes, supporting the notion of serotype-specificity. Conclusion This work provides a catalogue of HCSS sequences in the DENV proteome, as candidates for vaccine target selection. The methodology described herein provides a framework for similar application to other pathogens.

There is a growing attention toward personalized medicine. This is led by a fundamental shift from the 'one size fits all' paradigm for treatment of patients with conditions or predisposition to diseases, to one that embraces novel approaches, such as tailored target therapies, to achieve the best possible outcomes. Driven by these, several national and international genome projects have been initiated to reap the benefits of personalized medicine. Exome and targeted sequencing provide a balance between cost and benefit, in contrast to whole genome sequencing (WGS). Whole exome sequencing (WES) targets approximately 3% of the whole genome, which is the basis for protein-coding genes. Nonetheless, it has the characteristics of big data in large deployment. Herein, the application of WES and its relevance in advancing personalized medicine is reviewed. WES is mapped to Big Data "10 Vs" and the resulting challenges discussed. Application of existing biological databases and bioinformatics tools to address the bottleneck in data processing and analysis are presented, including the need for new generation big data analytics for the multi-omics challenges of personalized medicine. This includes the incorporation of artificial intelligence (AI) in the clinical utility landscape of genomic information, and future consideration to create a new frontier toward advancing the field of personalized medicine.

Background Ebolavirus (EBOV) is responsible for one of the most fatal diseases encountered by mankind. Cellular T-cell responses have been implicated to be important in providing protection against the virus. Antigenic variation can result in viral escape from immune recognition. Mapping targets of immune responses among the sequence of viral proteins is, thus, an important first step towards understanding the immune responses to viral variants and can aid in the identification of vaccine targets. Herein, we performed a large-scale, proteome-wide mapping and diversity analyses of putative HLA supertype-restricted T-cell epitopes of Zaire ebolavirus (ZEBOV), the most pathogenic species among the EBOV family. Methods All publicly available ZEBOV sequences (14,098) for each of the nine viral proteins were retrieved, removed of irrelevant and duplicate sequences, and aligned. The overall proteome diversity of the non-redundant sequences was studied by use of Shannon’s entropy. The sequences were predicted, by use of the NetCTLpan server, for HLA-A2, -A3, and -B7 supertype-restricted epitopes, which are relevant to African and other ethnicities and provide for large (~86%) population coverage. The predicted epitopes were mapped to the alignment of each protein for analyses of antigenic sequence diversity and relevance to structure and function. The putative epitopes were validated by comparison with experimentally confirmed epitopes. Results & discussion ZEBOV proteome was generally conserved, with an average entropy of 0.16. The 185 HLA supertype-restricted T-cell epitopes predicted (82 (A2), 37 (A3) and 66 (B7)) mapped to 125 alignment positions and covered ~24% of the proteome length. Many of the epitopes showed a propensity to co-localize at select positions of the alignment. Thirty (30) of the mapped positions were completely conserved and may be attractive for vaccine design. The remaining (95) positions had one or more epitopes, with or without non-epitope variants. A significant number (24) of the putative epitopes matched reported experimentally validated HLA ligands/T-cell epitopes of A2, A3 and/or B7 supertype representative allele restrictions. The epitopes generally corresponded to functional motifs/domains and there was no correlation to localization on the protein 3D structure. These data and the epitope map provide important insights into the interaction between EBOV and the host immune system. Electronic supplementary material The online version of this article (10.1186/s12864-017-4328-8) contains supplementary material, which is available to authorized users.

Background Viral vaccine target discovery requires understanding the diversity of both the virus and the human immune system. The readily available and rapidly growing pool of viral sequence data in the public domain enable the identification and characterization of immune targets relevant to adaptive immunity. A systematic bioinformatics approach is necessary to facilitate the analysis of such large datasets for selection of potential candidate vaccine targets. Results This work describes a computational methodology to achieve this analysis, with data of dengue, West Nile, hepatitis A, HIV-1, and influenza A viruses as examples. Our methodology has been implemented as an analytical pipeline that brings significant advancement to the field of reverse vaccinology, enabling systematic screening of known sequence data in nature for identification of vaccine targets. This includes key steps (i) comprehensive and extensive collection of sequence data of viral proteomes (the virome), (ii) data cleaning, (iii) large-scale sequence alignments, (iv) peptide entropy analysis, (v) intra- and inter-species variation analysis of conserved sequences, including human homology analysis, and (vi) functional and immunological relevance analysis. Conclusion These steps are combined into the pipeline ensuring that a more refined process, as compared to a simple evolutionary conservation analysis, will facilitate a better selection of vaccine targets and their prioritization for subsequent experimental validation. Electronic supplementary material The online version of this article (10.1186/s12920-017-0301-2) contains supplementary material, which is available to authorized users.

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design., Author Summary Although there is an increased recognition of the role of T-cells in both dengue pathogenesis and protection, comprehensive analysis of T-cell activation during dengue infection is hampered by the small repertoire of known human dengue T-cell epitopes. Although dengue serotype 3 (DENV3) is responsible for numerous outbreaks worldwide, most of the known epitopes are from studies of dengue 2 serotype (DENV2). In this study, we identified novel DENV3 T-cell epitopes in HLA transgenic mice that were confirmed by HLA binding assays. A subset of these epitopes activated memory T-cells from subjects who were dengue IgG positive and primed naïve T-cells from dengue IgG negative individuals. Notably, some of HLA class II epitopes bearing highly conserved regions common to all four dengue serotypes could bind to multiple HLAs. We postulate that these highly conserved and HLA promiscuous T-helper epitopes can be important components of a dengue tetravalent vaccine.

The rapid mutation of human immunodeficiency virus-type 1 (HIV-1) and the limited characterization of the composition and incidence of the variant population are major obstacles to the development of an effective HIV-1 vaccine. This issue was addressed by a comprehensive analysis of over 58,000 clade B HIV-1 protein sequences reported over at least 26 years. The sequences were aligned and the 2,874 overlapping nonamer amino acid positions of the viral proteome, each a possible core binding domain for human leukocyte antigen molecules and T-cell receptors, were quantitatively analyzed for four patterns of sequence motifs: (1) “index”, the most prevalent sequence; (2) “major” variant, the most common variant sequence; (3) “minor” variants, multiple different sequences, each with an incidence less than that of the major variant; and (4) “unique” variants, each observed only once in the alignment. The collective incidence of the major, minor, and unique variants at each nonamer position represented the total variant population for the position. Positions with more than 50% total variants contained correspondingly reduced incidences of index and major variant sequences and increased minor and unique variants. Highly diverse positions, with 80 to 98% variant nonamer sequences, were present in each protein, including 5% of Gag, and 27% of Env and Nef, each. The multitude of different variant nonamer sequences (i.e. nonatypes; up to 68%) at the highly diverse positions, represented by the major, multiple minor, and multiple unique variants likely supported variants function both in immune escape and as altered peptide ligands with deleterious T-cell responses. The patterns of mutational change were consistent with the sequences of individual HXB2 and C1P viruses and can be considered applicable to all HIV-1 viruses. This characterization of HIV-1 protein mutation provides a foundation for the design of peptide-based vaccines and therapeutics.

This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d) and HLA-DR4 (DRA1*0101, DRB1*0401) transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag) chimera antigen. Three immunization protocols were compared: 1) primary subcutaneous immunization with 1x10(5) immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2) primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3) immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-gamma ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a) the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b) the value of HLA transgenic mice as a model system for the identification and evaluation of epitope-based vaccine strategies, and c) the application of variability analysis across reported sequences in public databases for selection of historically conserved HIV epitopes as vaccine targets.

Background Effectors of Type III Secretion System (T3SS) play a pivotal role in establishing and maintaining pathogenicity in the host and therefore the identification of these effectors is important in understanding virulence. However, the effectors display high level of sequence diversity, therefore making the identification a difficult process. There is a need to collate and annotate existing effector sequences in public databases to enable systematic analyses of these sequences for development of models for screening and selection of putative novel effectors from bacterial genomes that can be validated by a smaller number of key experiments. Results Herein, we present T3SEdb http://effectors.bic.nus.edu.sg/T3SEdb, a specialized database of annotated T3SS effector (T3SE) sequences containing 1089 records from 46 bacterial species compiled from the literature and public protein databases. Procedures have been defined for i) comprehensive annotation of experimental status of effectors, ii) submission and curation review of records by users of the database, and iii) the regular update of T3SEdb existing and new records. Keyword fielded and sequence searches (BLAST, regular expression) are supported for both experimentally verified and hypothetical T3SEs. More than 171 clusters of T3SEs were detected based on sequence identity comparisons (intra-cluster difference up to ~60%). Owing to this high level of sequence diversity of T3SEs, the T3SEdb provides a large number of experimentally known effector sequences with wide species representation for creation of effector predictors. We created a reliable effector prediction tool, integrated into the database, to demonstrate the application of the database for such endeavours. Conclusions T3SEdb is the first specialised database reported for T3SS effectors, enriched with manual annotations that facilitated systematic construction of a reliable prediction model for identification of novel effectors. The T3SEdb represents a platform for inclusion of additional annotations of metadata for future developments of sophisticated effector prediction models for screening and selection of putative novel effectors from bacterial genomes/proteomes that can be validated by a small number of key experiments.