A Seeded Genetic Algorithm for RNA Secondary Structural Prediction with Pseudoknots

This work explores a new approach in using genetic algorithm to predict RNA secondary structures with pseudoknots. Since only a small portion of most RNA structures is comprised of pseudoknots, the majority of structural elements from an optimal pseudoknot-free structure are likely to be part of the true structure. Thus seeding the genetic algorithm with optimal pseudoknot-free structures will more likely lead it to the true structure than a randomly generated population. The genetic algorithm uses the known energy models with an additional augmentation to allow complex pseudoknots. The nearest-neighbor energy model is used in conjunction with Turner’s thermodynamic parameters for pseudoknot-free structures, and the H-type pseudoknot energy estimation for simple pseudoknots. Testing with known pseudoknot sequences from PseudoBase shows that it out performs some of the current popular algorithms

Investigating cross-clade immune responses in HIV-1 subtype C-infected individuals from South Africa: implications for HIV vaccine design

Background An effective HIV vaccine remains the main hope for controlling the HIV epidemic and is a global health priority. The genetic diversity of the virus across the globe is a major impediment to developing an effective vaccine. Whether a universal vaccine is possible still remain elusive. Therefore, there is need to fully characterise clusters of commonly targeted regions across the different HIV-1 clades. Centralized sequences have been suggested as vaccine immunogens and peptide reagents for assessing vaccine-induced responses, but their cross-reactivity has not been fully assessed in larger cohorts of subtype C-infection and in regions of differing HIV epidemics. In addition, the functional profile of HIV-specific T-cells recognizing variant epitopes has not been fully characterized. Whether cross-reactivity observed by IFN-γ production in an ELISpot assay can be observed at physiological concentrations of the peptides and for other functions of HIV-specific T-cells is an important question that remains to be answered. Methods The cross-reactivity of HIV-specific T-cells was assessed using clade-specific peptide reagents forming part of current candidate vaccine inserts based on the HIV-1 Gag protein from clades CDu422, CCH, A, B and D in 40 clade C-infected study participants using the IFN-γ ELISpot assay. To test the reactivity of group M consensus peptide reagents, 66 individuals, 44 of whom were ARV naïve, were assessed for HIV-specific T cell responses to group M Gag and Nef peptides. A selection of these individuals was screened for HIVspecific T-cell responses to clade CDu422 Gag peptides. Cross-reactivity of peptide variants was assessed at physiologically relevant peptide concentrations by functional avidity studies using peptide dilution IFN-γ ELISpot assays. Additionally, the cytokine profile, cytotoxic potential and proliferative capacity of cross-reactive peptide variants was characterised using multiparameter flow cytometry. Results The magnitude and breadth of HIV-specific T-cell responses were similar between the two clade-C peptide reagents in a clade C-infected population. However, the magnitude and breadth of responses to peptides based on clades A, B and D were significantly lower compared to the clade C peptides. Clusters of commonly targeted regions cross-reactive across the four clades investigated resided predominantly in conserved regions. Interestingly, there were Gag regions that were exclusively recognized in the different clades that had significantly lower entropy scores for the reactive variants than their nonreactive counterparts, suggesting that the variability in targeted regions could have been shaped by host immune pressure. For consensus group M peptides, the magnitude and breadth of Gag responses were significantly higher than that of Nef in clade C-infected individuals. In addition, consensus group M Gag peptides had significantly lower magnitude and breadth of HIV-specific T-cell responses compared to clade C peptide reagents, suggesting loss of responses by centralised reagents despite their central nature to group M viruses. On the contrary, the magnitude and breadth of responses to consensus group M Gag peptides were comparable to that of clade-mismatched peptides, namely clades A, B and D, suggesting that these reagents can be used interchangeably. Peptide dilution assays showed that amino acid mismatches have discordant effects on functional avidity and that some peptides are cross-reactive at physiological concentrations. Similarly, discordant effects (differences in functional avidity, cytokine and cytotoxic profiles and proliferative capacity) of amino acid mismatches on cytokine and cytotoxic potential profiles as well as proliferative capacity were observed. Conclusion People infected by a particular HIV clade can recognize HIV peptides based on other clades. However, the magnitude and breadth of responses are greater for the matched clades compared to mismatched clades, suggesting that there may be an advantage of using vaccines based on matched over unmatched clades. Group M based consensus sequences can be recognized in HIV-infected individuals, but with a lower frequency, magnitude and breadth of responses compared to clade-matched peptides, suggesting a limitation of these peptides both as reagents and vaccine immunogens. However, the frequency, magnitude and breadth T-cell reponses to consensus group M peptides were comparable to clademismatched responses, suggesting that these reagents may be used interchangeably. Furthermore, amino acid variations across corresponding viral regions have discordant effects on the functional avidity, cytokine profile, cytotoxic potential and proliferative capacity; implying that qualitative measures of cross-reactivity beyond IFN-γ frequencies need to be considered. These data may aid in the development of reagents for the assessment of vaccine-induced responses as well as in HIV vaccine immunogen design

A New Rodent Model to Understand Host-Virus Interactions at the Early Stages of Retroviral Endogenization

Unlike other viruses, retroviruses replicate by copying their RNA genomes to DNA. They therefore become a largely inseparable part of the cell's genome upon infection. Retroviruses can be transmitted horizontally and vertically by infection and often have wide cellular tropism. Exogenous retroviral infection (XRV) occurs in somatic cells, but when infection is in the germline, the resulting provirus is known as an endogenous retrovirus (ERVs). Accumulation of these retroviral sequences over evolutionary time has granted them ~ 8% occupancy of the human genome and, along with other transposable elements (TEs), makes them a major determinant of DNA sequence diversity and driver of species evolution. Millions of years of evolution have obscured the history of mutation, indel, rearrangement and distribution events that ERVs have experienced since they integrated. Understanding how ERVs establish themselves in a host genome is crucial to infer vertebrate adaptive immunity and the generated memory of these genome invaders. Koala retrovirus (KoRV), as the only known mammalian retrovirus currently undergoing genome colonization, is generally used as a model system for mechanism of endogenization. However the precursor vector species that gave rise to KoRV and the closely related pathogenic Gibbon Ape Leukemia Virus (GALV) remains obscure. In an attempt to identify the reservoir of GALV-KoRV, we have identified a novel infectious GALV virus in a specific population of a native rodent of Papua New Guinea, Melomys leucogaster. We named this virus, complete melomys woolly monkey virus (cMWMV). Using cell culture methods, fluorescence, and electron microscopy, we have characterized this gammaretrovirus. The significance of cMWMV is that like KoRV, it is currently invading the genome of a new host species. As KoRV is restricted to koalas, cMWMV could provide an additional rodent model to further study the evolutionary processes that contribute to the germline invasion and adaptation to a new host. This recent retroviral invasion can help us elucidate the general principles of antiretroviral gene evolution within Melomys and between rodent species that are known to be under diversifying selection in the primate orthologs. PacBio sequencing was used to sequence the whole genome of Melomys. Guided sequence alignment was performed and exons corresponding to genes of interest were extracted. Coding sequences (CDS) were de novo assembled and manually curated. We then used various substitution models to quantify the selection pressure in these immune genes. Our data suggest that, similar to primates, these genes may have experienced positive selection at some sites (codons) in Mus musculus and Rattus norvegicus lineages. However the excess of synonymous sites asserts a long-term trend of purifying selection. A weak intensified diversifying selection pattern in Melomys lineage of ZAP (zinc-finger CCCH-type antiviral protein 1) gene could indicate an effort to inhibit viral mRNA translation of the endogenizing cMWMV.Im Gegensatz zu anderen Viren replizieren sich Retroviren, indem sie ihr RNS-Genom in DNS kopieren. Daher werden sie nach der Infektion zu einem weitgehend untrennbaren Teil des Zellgenoms. Retroviren können durch Infektion horizontal und vertikal übertragen werden und haben oft einen breiten Zelltropismus. Eine exogene retrovirale Infektion (XRV) findet in somatischen Zellen statt. Erfolgt jedoch die Infektion in der Keimbahn, wird das resultierende Provirus als endogenes Retrovirus (ERV) bezeichnet. Die Anhäufung dieser retroviralen Sequenzen im Laufe der Evolution hat dazu geführt, dass sie ca. 8 % des menschlichen Genoms einnehmen und zusammen mit anderen transponierbaren Elementen (TEs) eine wichtige Determinante der DNS-Sequenzvielfalt sowie eine treibende Kraft für die Evolution der Arten darstellen. Jahrmillionen der Evolution haben den Verlauf von Mutation, Indel, Umlagerung und Verbreitung, die ERVs seit ihrer Integration erfahren haben, verschleiert. Die Art und Weise, wie sich ERVs in einem Wirtsgenom etablieren, ist entscheidend, um Rückschlüsse auf die adaptive Immunität von Wirbeltieren und das erzeugte Gedächtnis dieser Genom-Invasoren zu ziehen. Das Koala-Retrovirus (KoRV), das einzige bekannte Säugetier-Retrovirus, das derzeit eine Genomkolonisierung durchläuft, wird im Allgemeinen als Modellsystem für den Mechanismus der Endogenisierung verwendet. Die Vorläufer-Vektorspezies, die KoRV und das eng verwandte pathogene Gibbon-Affen-Leukämievirus (GALV) hervorgebracht hat, ist jedoch nach wie vor unbekannt. In einem Versuch, das Reservoir von GALV-KoRV zu identifizieren, haben wir ein neuartiges infektiöses GALV-Virus in einer bestimmten Population eines in Papua-Neuguinea heimischen Nagetiers, Melomys leucogaster, nachgewiesen. Das Virus wurde complete Melomys Woolly Monkey Virus (cMWMV) genannt. Mit Hilfe von Zellkulturmethoden, Fluoreszenz- und Elektronenmikroskopie haben wir dieses Gammaretrovirus charakterisiert. Die Besonderheit von cMWMV besteht darin, dass es, wie KoRV, derzeit in das Genom einer neuen Wirtsart eindringt. Da KoRV nur bei Koalas vorkommt, könnte cMWMV ein zusätzliches Nagetiermodell sein, um die evolutionären Prozesse zu untersuchen, die zur Keimbahninvasion und Anpassung an einen neuen Wirt beitragen. Diese jüngste retrovirale Invasion kann uns helfen, die allgemeinen Prinzipien der antiretroviralen Genevolution innerhalb von Melomys und zwischen Nagetierarten zu verdeutlichen, die bekanntermaßen der diversifizierenden Selektion der Primatenorthologen unterliegen. Mittels PacBio-Sequenzierung wurde das gesamte Genom von Melomys sequenziert. Ein geführter Sequenzabgleich wurde vorgenommen und die den relevanten Genen entsprechenden Exone extrahiert. Die kodierenden Sequenzen (CDS) wurden de novo assembliert und manuell kuratiert. Anschließend haben wir verschiedene Substitutionsmodelle angewandt, um den Selektionsdruck in diesen Immungenen zu quantifizieren. Unsere Daten deuten darauf hin, dass diese Gene, ähnlich wie bei den Primaten, in den Abstammungslinien von Mus musculus und Rattus norvegicus an einigen Stellen (Codons) einer positiven Selektion unterlegen haben könnten. Der Überschuss an synonymen Stellen deutet jedoch auf einen langfristigen Trend der reinigenden Selektion hin. Ein schwaches, verstärkt diversifizierendes Selektionsmuster in der Melomys-Abstammungslinie des ZAP-Gens (Zink-Finger-CCCH-Typ antivirales Protein 1) könnte auf einen Versuch hindeuten, die virale mRNA-Translation des endogenisierenden cMWMV zu inhibieren

Evaluation of vaccine candidates for HIV prevention in Tanzania

Background: This thesis describes the capability of an HIV-1 DNA and HIV-1 modified vaccinia virus Ankara (MVA) prime boost vaccination strategy to elicit immune responses known to correlate with reduced risk of HIV infection. Additionally, it describes the confounding effect of vaccine-induced HIV antibodies on the performance of HIV testing algorithms in sub-Saharan Africa, as well as the recruitment and preparation of a cohort of female sex workers (FSW) for potential participation in an HIV vaccine efficacy trial. Methods: Study I evaluated the capability of the HIV-DNA/MVA vaccine regimen to elicit potent and durable binding antibody responses to the V1V2 domain of HIV-1 gp120. Plasma samples collected at peak immunogenicity and three years later, were analyzed for frequency, magnitude and persistence of antibodies to gp70V1V2 proteins. Study II investigated whether addition of an envelope protein (CN54rgp140/GLA-AF) boost would increase anti- V1V2 responses elicited by the HIV-DNA/HIV-MVA vaccine. Study III assessed the impact of vaccine-induced seroreactivity on the performance of rapid test-based HIV diagnostic algorithms. The HIV diagnostic strategies of Mozambique and Tanzania were evaluated using samples collected from vaccine recipients in the HIVIS and TaMoVac clinical trials. Study IV assessed the suitability of FSW in Dar es Salaam for participation in a phase IIb HIV vaccine efficacy trial (PrEPVacc). HIV incidence, retention rate and risk behaviours were determined in a cohort of 700 FSW after one year of follow up. Results: Frequent and durable anti-V1V2 responses were elicited in the majority of the vaccine recipients. At peak immunogenicity, 97% of the vaccinees had binding IgG antibodies to the V1V2 loop of CRF01_AE A244. The anti-A244 V1V2 IgG persisted for at least three years in 75% of vaccinees and the response rate was improved by a late HIVMVA boost given three years after the second boost (study I). The CN54rgp140/GLA-AF boost did not enhance the V1V2 response (study II). The HIV diagnostic algorithms in sub- Saharan Africa misdiagnosed a substantial proportion of HIV vaccine recipients. More than half (54%) of the vaccinees would have been incorrectly identified as HIV infected in Tanzania, while, 26.3% would have been misclassified in Mozambique (study III). A high HIV incidence of 3.45 per 100 person years at risk was observed among FSW in the PrEPVacc preparedness study. The rate of HIV acquisition was higher among the young (18-24 years), drug using FSW, and those with syphilis or hepatitis infections (study IV). Conclusion: Priming with HIV-DNA followed by boosting with HIV-MVA elicited robust and durable anti-V1V2 responses in a majority of vaccinees. Boosting with a combination of CN54rgp140/GLA-AF and HIV-MVA did not augment anti-V1V2 responses elicited by HIV-DNA priming and HIV-MVA boosting. The current HIV testing algorithms in sub- Saharan Africa cannot sufficiently discriminate vaccine-induced seroreactivity from true HIV infection. Young FSW in Dar es Saalam are a suitable target population for HIV vaccine efficacy trials

DNA microarray integromics analysis platform

Background: The study of interactions between molecules belonging to different biochemical families (such as lipids and nucleic acids) requires specialized data analysis methods. This article describes the DNA Microarray Integromics Analysis Platform, a unique web application that focuses on computational integration and analysis of "multi-omics" data. Our tool supports a range of complex analyses, including - among others - low- and high-level analyses of DNA microarray data, integrated analysis of transcriptomics and lipidomics data and the ability to infer miRNA-mRNA interactions. Results: We demonstrate the characteristics and benefits of the DNA Microarray Integromics Analysis Platform using two different test cases. The first test case involves the analysis of the nutrimouse dataset, which contains measurements of the expression of genes involved in nutritional problems and the concentrations of hepatic fatty acids. The second test case involves the analysis of miRNA-mRNA interactions in polysaccharide-stimulated human dermal fibroblasts infected with porcine endogenous retroviruses. Conclusions: The DNA Microarray Integromics Analysis Platform is a web-based graphical user interface for "multi-omics" data management and analysis. Its intuitive nature and wide range of available workflows make it an effective tool for molecular biology research. The platform is hosted at https://lifescience.plgrid.pl

Using signal processing, evolutionary computation, and machine learning to identify transposable elements in genomes

About half of the human genome consists of transposable elements (TE's), sequences that have many copies of themselves distributed throughout the genome. All genomes, from bacterial to human, contain TE's. TE's affect genome function by either creating proteins directly or affecting genome regulation. They serve as molecular fossils, giving clues to the evolutionary history of the organism. TE's are often challenging to identify because they are fragmentary or heavily mutated. In this thesis, novel features for the detection and study of TE's are developed. These features are of two types. The first type are statistical features based on the Fourier transform used to assess reading frame use. These features measure how different the reading frame use is from that of a random sequence, which reading frames the sequence is using, and the proportion of use of the active reading frames. The second type of feature, called side effect machine (SEM) features, are generated by finite state machines augmented with counters that track the number of times the state is visited. These counters then become features of the sequence. The number of possible SEM features is super-exponential in the number of states. New methods for selecting useful feature subsets that incorporate a genetic algorithm and a novel clustering method are introduced. The features produced reveal structural characteristics of the sequences of potential interest to biologists. A detailed analysis of the genetic algorithm, its fitness functions, and its fitness landscapes is performed. The features are used, together with features used in existing exon finding algorithms, to build classifiers that distinguish TE's from other genomic sequences in humans, fruit flies, and ciliates. The classifiers achieve high accuracy (> 85%) on a variety of TE classification problems. The classifiers are used to scan large genomes for TE's. In addition, the features are used to describe the TE's in the newly sequenced ciliate, Tetrahymena thermophile to provide information for biologists useful to them in forming hypotheses to test experimentally concerning the role of these TE's and the mechanisms that govern them

Review on Approaches to Reverse Vaccinology Against Dangerous Pathogens in Animals

Vaccines have been recognized as major and effective tools For controlling disease impact, vaccinology is a field with great opportunity. Contribution of vaccines towards societal development by improvement of health status and increasing life-expectancy has been paramount. The conventional way of vaccine development includes culturing of pathogens in laboratory but this is not possible in case of highly infectious pathogens that are hazardous to culture in laboratory. The concept of reverse vaccinology is based on selecting specific epitope of interest that are capable of provoking cellular as well as humoral immune response which  is the heart of reverse vaccinology. Some approaches against viruses have also been done by reverse vaccinology. Applying genomic approaches to study both the pathogen and host will ultimately increase our fundamental understanding of pathogen biology, mechanisms responsible for the development of protective immunity, and guide next-generation vaccine design. This review paper show development of reverse vaccinology, their relevance, and limitations in the timely development of useful  against the most dangerous pathogens. Keywords: Conventional Vaccinology, Epitope prediction, Reverse Vaccinology, Vaccines DOI: 10.7176/JBAH/13-11-01 Publication date:July 31st 2023

Review on Approaches to Reverse Vaccinology Against Dangerous Pathogens in Animals

Vaccines have been recognized as major and effective tools For controlling disease impact, vaccinology is a field with great opportunity. Contribution of vaccines towards societal development by improvement of health status and increasing life-expectancy has been paramount. The conventional way of vaccine development includes culturing of pathogens in laboratory but this is not possible in case of highly infectious pathogens that are hazardous to culture in laboratory. The concept of reverse vaccinology is based on selecting specific epitope of interest that are capable of provoking cellular as well as humoral immune response which  is the heart of reverse vaccinology. Some approaches against viruses have also been done by reverse vaccinology. Applying genomic approaches to study both the pathogen and host will ultimately increase our fundamental understanding of pathogen biology, mechanisms responsible for the development of protective immunity, and guide next-generation vaccine design. This review paper show development of reverse vaccinology, their relevance, and limitations in the timely development of useful  against the most dangerous pathogens. Keywords: Conventional Vaccinology, Epitope prediction, Reverse Vaccinology, Vaccines DOI: 10.7176/JHMN/109-01 Publication date:June 30th 202