It is an important virulence factor of Streptococcus pneumoniae and a key vaccine target against pneumococcal disease. Pneumolysin (PLY) is a cholesterol-binding, pore-forming protein toxin. , Infecções Pneumocócicas/prevenção & controle These results are important to understand human PlyD1 T cell immunogenicity, to guide cell mediated immunity studies in the context of vaccine development.Īssuntos Epitopos de Linfócito T/imunologia In conclusion, PlyD1 evokes T cell responses in mice directed against multiple epitope regions, that is dependent on Major Histocompatibility Complex (MHC) background. Cytokine responses show mostly a Th2 profile, with low levels of Th1 cytokines, in both mouse strains. H-2d restricted T cell epitopes were localized in pneumolysin domains 2 and 3, whereas H-2b epitopes were scattered over the protein. Overlap between H-2d and H-2b restricted T cell epitopes was limited, yet similarities were found between epitopes processed in mice and predicted to be immunogenic in humans. For both mouse strains, one immunodominant and three subdominant natural epitopes were identified. In vitro restimulation of splenocytes with PlyD1 and 18-mer synthetic peptides derived from pneumolysin revealed specific proliferative and cytokine responses.
Adjuvantation of PlyD1 enhanced the serological responses in both strains. Vaccination with adjuvanted and non-adjuvanted PlyD1 induced anti-pneumolysin IgG antibodies with neutralizing capacity in both mouse strains. BALB/c (H-2d) and C57BL/6 (H-2b) mice were vaccinated with Al(OH)3-adjuvanted or non-adjuvanted PlyD1, or placebo, on day 0, 21 and 42 and were sacrificed at day 56 for collection of sera and spleens. Here we interrogated epitope breadth and functional characteristics of the T cell response to PlyD1 in two mouse strains. Despite extensive knowledge on antibody responses against PlyD1, limited information is available about PlyD1 induced T cell recognition. Application of the FEP approach in conjunction with experimental assessment techniques offers opportunities to lower the time and expense of product development and reduce the risk of costly late-stage failures.ĭetoxified pneumolysin, PlyD1, is a protein vaccine candidate that induces protection against infections with Streptococcus pneumoniae in mouse models. On the entire validation dataset, consisting of 328 single point mutations spread across 14 distinct protein structures, our results show good overall correlation with experiment with an R2 of 0.65 and a low mean unsigned error of 0.95 kcal/mol.
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In contrast to many of the commonly available computational methods for stability prediction, the Free Energy Perturbation approach applied in this paper explicitly accounts for solvent effects and samples conformational dynamics using a rigorous molecular dynamics simulation process.
Although experimental techniques for measuring the impact of amino acid residue mutation on the stability of proteins exist, they tend to be time consuming and costly, hence the need for accurate prediction methods. Physical stability is a key determinant of the clinical and commercial success of biological therapeutics, vaccines, diagnostics, enzymes and other protein-based products.
This work describes the application of a physics-based computational approach to predict the relative thermodynamic stability of protein variants, and evaluates the quantitative accuracy of those predictions compared to experimental data obtained from a diverse set of protein systems assayed at variable pH conditions.
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