Bioinformatics Resources and Tools for Conformational B-Cell Epitope Prediction

Identification of epitopes which invoke strong humoral responses is an essential issue in the field of immunology. Localizing epitopes by experimental methods is expensive in terms of time, cost, and effort; therefore, computational methods feature for its low cost and high speed was employed to predict B-cell epitopes. In this paper, we review the recent advance of bioinformatics resources and tools in conformational B-cell epitope prediction, including databases, algorithms, web servers, and their applications in solving problems in related areas. To stimulate the development of better tools, some promising directions are also extensively discussed

An overview of bioinformatics tools for epitope prediction: Implications on vaccine development

AbstractExploitation of recombinant DNA and sequencing technologies has led to a new concept in vaccination in which isolated epitopes, capable of stimulating a specific immune response, have been identified and used to achieve advanced vaccine formulations; replacing those constituted by whole pathogen-formulations. In this context, bioinformatics approaches play a critical role on analyzing multiple genomes to select the protective epitopes in silico. It is conceived that cocktails of defined epitopes or chimeric protein arrangements, including the target epitopes, may provide a rationale design capable to elicit convenient humoral or cellular immune responses. This review presents a comprehensive compilation of the most advantageous online immunological software and searchable, in order to facilitate the design and development of vaccines. An outlook on how these tools are supporting vaccine development is presented. HIV and influenza have been taken as examples of promising developments on vaccination against hypervariable viruses. Perspectives in this field are also envisioned

Peptídeos solúveis de Leishmania spp. conjugados em nanopartículas superparamagnéticas como alternativa para ensaios de imunoadsorção enzimática

Orientador: Prof º. Drº. Ricardo Andrez Machado de ÁvilaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Defesa : Curitiba, 19/12/2022Inclui referênciasResumo: As leishmanioses são um espectro de doenças, causadas por protozoários do gênero Leishmania, que se apresentam nas formas de Leishmaniose Tegumentar cutânea, muco cutânea ou disseminada; e Leishmaniose Visceral dependendo da espécie do parasito. Em diversos países do mundo, incluindo o Brasil, é um problema de saúde pública. Assim é importante o controle epidemiológico da doença e a necessidade de um diagnóstico eficaz como medidas profiláticas. No entanto, os diagnósticos existentes apresentam deficiências, principalmente no que se diz respeito a reatividade cruzada com proteínas de outros parasitas. Para aprimorar o sorodiagnóstico, as nanopartículas magnéticas têm sido aplicadas no diagnóstico imunológico principalmente devido às suas propriedades de se desprender rapidamente de um campo magnético externo, o que reduz o tempo de diagnóstico. A funcionalização desses nanomateriais também permite o isolamento específico de analitos alvo, melhorando a sensibilidade e a eficiência de imunoensaios, como o ensaio ELISA. O presente estudo teve como objetivo conjugar um peptídeo antigênico de Leishmania spp. preditos a partir de ferramentas de bioinformática em nanopartículas superparamagnéticas (NPSPMs) funcionalizadas com poli(etilenoglicol) (PEG) sintetizadas pelo método de coprecipitação como alternativa para melhorar o sorodiagnóstico de leishmaniose. Por meio de ferramentas de bioinformática, foi selecionada uma das 17 proteínas hipotéticas de L. infantum: LiHyT (XP_001465138.1). Essa proteína demonstrou ser conservada entre as principais espécies de Leishmania e pouco conservada em outras espécies de Trypanosoma, Plasmodium, Mycobacterium, Paracoccidioides, Histoplasma e Toxoplasma. A partir desta proteína, foram preditos dois epítopos de células B através do programa ABCPred. Em seguida, o epítopo de célula B que apresentou boa solubilidade em água e continha pelo menos um aminoácido cisteína em sua sequência foi selecionado e nomeado de pept1. Este antígeno peptídico foi então associado as NPSPMs/PEG através do ancoramento pela cisteína e avaliado sua capacidade de diagnóstico em um imunoensaio enzimático otimizado. Os resultados apresentaram acurácia diagnóstica de LTH de 0,8398 com sensibilidade de 75% (95 IC% 50,50 - 89,82) e especificidade de 87,50% (95 IC% 71,93 - 95,03) e acurácia de LVH de 0,9258 com sensibilidade de 87,50% (95 IC% 63,98 - 97,78) e especificidade 87,50% (95 IC% 71,93 - 95,03).Os resultados obtidos podem levar ao uso do antígeno proposto para fins comerciais e levar a novos métodos de nanodiagnóstico nos locais de atendimento.Abstract: Leishmaniases are a spectrum of diseases, caused by protozoa of the genus Leishmania, which are presented in the forms of Cutaneous Tegumentary Leishmaniasis, mucocutaneous or disseminated Leishmaniasis; and Visceral Leishmaniasis depending on the parasite species. In several countries around the world, including Brazil, it is a public health problem. Thus, the epidemiological control of the disease is important and the need for an effective diagnosis as prophylactic measures. However, existing diagnostics have shortcomings, especially with regard to cross-reactivity with proteins from other parasites. To improve serodiagnosis, magnetic nanoparticles have been applied in immunological diagnosis mainly due to their properties of quickly detaching themselves from an external magnetic field, which reduces diagnostic time. The functionalization of these nanomaterials also allows the specific isolation of target analytes, improving the sensitivity and efficiency of immunoassays, such as the ELISA assay. The present study aimed to conjugate an antigenic peptide from Leishmania spp. predicted from bioinformatics tools in superparamagnetic nanoparticles (SPMSPs) functionalized with poly(ethylene glycol) (PEG) synthesized by the coprecipitation method as an alternative to improve the serodiagnosis of leishmaniasis. Using bioinformatics tools, one of the 17 hypothetical L. infantum proteins was selected: LiHyT (XP_001465138.1). This protein proved to be conserved among the main species of Leishmania and poorly conserved in other species of Trypanosoma, Plasmodium, Mycobacterium, Paracoccidioides, Histoplasma and Toxoplasma. From this protein, two B-cell epitopes were predicted using the ABCPred program. Then, the B cell epitope that showed good solubility in water and contained at least one cysteine amino acid in its sequence was selected and named pept1. This peptide antigen was then associated with SPMNPs/PEG through cysteine anchorage and evaluated for its diagnostic capacity in an optimized enzyme immunoassay. The results showed an TL diagnostic accuracy of 0.8398 with a sensitivity of 75% (95 CI% 50.50 - 89.82) and specificity of 87.50% (95 CI% 71.93 - 95.03) and accuracy of VL of 0.9258 with sensitivity of 87.50% (95 CI% 63.98 - 97.78) and specificity 87.50% (95 CI% 71.93 - 95.03). The results obtained may lead to the use of the proposed antigen for commercial purposes and lead to new nanodiagnostic methods at the point of care

The in silico prediction of foot-and-mouth disease virus (FMDV) epitopes on the South African territories (SAT)1, SAT2 and SAT3 serotypes

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease that affects even-toed hoofed mammals. The FMD virus (FMDV) is the causative agent of FMD, of which there are seven clinically indistinguishable serotypes. Three serotypes, namely, South African Territories (SAT)1, SAT2 and SAT3 are endemic to southern Africa and are the most antigenically diverse among the FMDV serotypes. A negative consequence of this antigenic variation is that infection or vaccination with one virus may not provide immune protection from other strains or it may only confer partial protection. The identification of B-cell epitopes is therefore key to rationally designing cross-reactive vaccines that recognize the immunologically distinct serotypes present within the population. Computational epitope prediction methods that exploit the inherent physicochemical properties of epitopes in their algorithms have been proposed as a cost and time-effective alternative to the classical experimental methods. The aim of this project is to employ in silico epitope prediction programmes to predict B-cell epitopes on the capsids of the SAT serotypes. Sequence data for 18 immunologically distinct SAT1, SAT2 and SAT3 strains from across southern Africa were collated. Since, only one SAT1 virus has had its structure elucidated by X-ray crystallography (PDB ID: 2WZR), homology models of the 18 virus capsids were built computationally using Modeller v9.12. They were then subjected to energy minimizations using the AMBER force field. The quality of the models was evaluated and validated stereochemically and energetically using the PROMOTIF and ANOLEA servers respectively. The homology models were subsequently used as input to two different epitope prediction servers, namely Discotope1.0 and Ellipro. Only those epitopes predicted by both programmes were defined as epitopes. Both previously characterised and novel epitopes were predicted on the SAT strains. Some of the novel epitopes are located on the same loops as experimentally derived epitopes, while others are located on a putative novel antigenic site, which is located close to the five-fold axis of symmetry. A consensus set of 11 epitopes that are common on at least 15 out of 18 SAT strains was collated. In future work, the epitopes predicted in this study will be experimentally validated using mutagenesis studies. Those found to be true epitopes may be used in the rational design of broadly reactive SAT vaccinesLife and Consumer SciencesM. Sc. (Life Sciences

Development of bioinformatic tools to identify and characterize linear protein epitopes

The adaptive immune system produces antibodies to specifically target antigens. Identifying and characterizing epitopes on target antigens enables numerous medical applications such as vaccine design, diagnostic discovery, and therapeutic antibody development. We have developed computational tools that characterize epitopes using large sets of antibody-binding peptides. To identify epitopes in target proteins, we used an approach termed K-mer Tiling of Protein Epitopes (K-TOPE). In this approach, we divided protein sequences into short overlapping subsequences of length k (k-mers). Then, we defined and scored epitopes using each k-mer’s enrichment in the sets of antibody-binding peptides. Using K-TOPE, we accurately identified epitopes for monoclonal and polyclonal antibodies. Next, using 250 specimens, we identified commonly targeted epitopes in nearly 3,000 viral proteins as well as two bacterial proteomes. Importantly, these epitopes agreed with previously reported results. To map antibody binding in epitopes, we developed Multiplexed Epitope Substitution Analysis (MESA). In this approach, target epitopes were divided into short overlapping k-mers. Then, each k-mer was exhaustively substituted with all amino acids. The effects of these substitutions were used to identify amino acid preferences at important binding positions in the epitopes. By applying this method to monoclonal antibodies and multiple sets of specimens, we identified binding motifs which agreed with an alternative computational approach. Finally, K-TOPE and MESA were used to characterize epitopes and antigens in age-related macular degeneration (AMD), herpes simplex virus (HSV), and Chagas disease. We identified 42 AMD-specific epitopes, 30 HSV2-specific epitopes, and 222 Chagas-specific epitopes. Several epitopes were in validated antigens while many were novel. MESA demonstrated that generally only 4-5 positions in these epitopes were important for binding. Future application of these approaches could enhance our understanding of the role that antibodies play in disease progression

Nuevos alergenos de Anisakis simplex: estudios in silico e in vitro

Anisakis simplex es un nematodo parásito cuyas larvas de tercer estadio pueden encontrarse en productos de la pesca de captura marina y ocasionar síntomas alérgicos tras ingerirlas vivas en pescados crudos o poco cocinados que no hayan sido congelados. Estos síntomas alérgicos pueden ser locales o generalizados según la respuesta inmunitaria del hospedador, distinguiéndose tres fenotipos clínicos: anisakiosis gástrica, anisakiosis gastroalérgica y urticaria crónica asociada a sensibilización por Anisakis. La anisakiosis gástrica (AG) se caracteriza por una reacción inflamatoria aguda, de tipo alérgico y local debida a la penetración gástrica o intestinal del nematodo. Por otro lado, la anisakiosis gastroalérgica (AGA) puede presentar igualmente síntomas digestivos, pero éstos son sobrepasados por síntomas alérgicos agudos generalizados que van desde urticaria o angioedema, hasta anafilaxia. Finalmente, la urticaria crónica asociada a sensibilización por Anisakis (UC+) se produce en pacientes que tras un episodio de parasitación previa por Anisakis desarrollan urticaria crónica. La urticaria crónica se caracteriza por la aparición de habones espontáneamente al menos dos veces por semana en un periodo de, como mínimo, seis semanas. La urticaria crónica es una patología sin una causa definida pero se conoce bien su mecanismo efector: la degranulación de los mastocitos. Debido a que el conocimiento de los mecanismos inmunológicos previos a la degranulación de los mastocitos es escaso para esta patología, intentamos descubrir nuevas posibles causas de urticaria crónica a través del estudio serológico de pacientes sensibilizados y no sensibilizados a A. simplex. Los síntomas alérgicos de la anisakiosis se deben a que el ser humano no es un hospedador natural de este parásito y además se trata de un parasitismo agudo o intermitente por lo que no parece posible que Anisakis module la respuesta inmunológica como sucede en otras helmintosis humanas. En el caso de Anisakis es preciso que la larva viva penetre desde la luz intestinal para que la reacción alérgica se produzca, por lo tanto los “alergenos” de Anisakis no se comportan como los alergenos alimentarios. Sin embargo, debido a la existencia de un área en el campo de la alergología que estudia la detección y caracterización de alergenos, se han estudiado estos antígenos parasitarios inductores de IgE presentes en las larvas de Anisakis de la misma manera que el resto de alergenos alimentarios..

The Development and application of pseudovirus based cell entry assays for emerging bat viruses

Recent years have seen the emergence of two serious coronavirus pathogens with the emergence of the MERS-CoV still an ongoing concern. In addition, the recent unprecedented Ebola outbreak has claimed more than 10,000 lives and affected the lives of countless more. All three of these viruses have been linked through differing strands of research to the second largest mammalian family, Chiroptera, the bats. Bats are among the most diverse a widespread of all mammalian species and have become subject of intensive research in recent years as various bats species have been linked to a number of severe viral outbreaks. In the studies described in this thesis attempts were made to develop, pseudotyped viruses (PV) bearing the glycoprotein of a number of highly pathogenic viruses including MERS-CoV, Ebolavirus, Marburgvirus and SARS-CoV coupled with envelope-defective Human Immunodeficiency Virus and envelope-defective Murine Leukemia Virus. These tools were then used to examine the potential for cross reactivity among related coronaviruses and a number of computational tools were employed to investigate the phenomenon and attempt to develop a better understanding of the antigenic regions that are responsible for the observed cross reactivity. The next stage in the thesis involved attempts to develop a novel form of multiplex assay that made use of PV to attempt to make serological screening of bat specimens more feasible and efficient. The novel bat-borne influenza A haemagglutinin H17 was then successfully incorporated into the PV system and screening of this PV against a number of cell lines led to improved understanding of the viral tropism and the role protease plays in this tropism. The final set of experiments carried out in this thesis involved a combination of computational biology and PV based protocols to both predict patterns of viral evolution through selection analysis and to then test these predictions in the PV framework. This study lead to the generation of a number of mutant MERS-CoV spike proteins and Ebolavirus glycoproteins. These were then incorporated into the PV system and the effects of these mutations of PV production and serum neutralization were investigated