"Immunoinformatic Identification of T-Cell and B-Cell Epitopes From Giardia lamblia Immunogenic Proteins as Candidates to Develop Peptide-Based Vaccines Against Giardiasis".

Giardiasis is one of the most common gastrointestinal infections worldwide, mainly in developing countries. The etiological agent is the Giardia lamblia parasite. Giardiasis mainly affects children and immunocompromised people, causing symptoms such as diarrhea, dehydration, abdominal cramps, nausea, and malnutrition. In order to develop an effective vaccine against giardiasis, it is necessary to understand the host-Giardia interactions, the immunological mechanisms involved in protection against infection, and to characterize the parasite antigens that activate the host immune system. In this study, we identify and characterize potential T-cell and B-cell epitopes of Giardia immunogenic proteins by immunoinformatic approaches, and we discuss the potential role of those epitopes to stimulate the host´s immune system. We selected the main immunogenic and protective proteins of Giardia experimentally investigated. We predicted T-cell and B-cell epitopes using immunoinformatic tools (NetMHCII and BCPREDS). Variable surface proteins (VSPs), structural (giardins), metabolic, and cyst wall proteins were identified as the more relevant immunogens of G. lamblia. We described the protein sequences with the highest affinity to bind MHC class II molecules from mouse (I-Ak and I-Ad) and human (DRB1*03:01 and DRB1*13:01) alleles, as well as we selected promiscuous epitopes, which bind to the most common range of MHC class II molecules in human population. In addition, we identified the presence of conserved epitopes within the main protein families (giardins, VSP, CWP) of Giardia. To our knowledge, this is the first in silico study that analyze immunogenic proteins of G. lamblia by combining bioinformatics strategies to identify potential T-cell and B-cell epitopes, which can be potential candidates in the development of peptide-based vaccines. The bioinformatics analysis demonstrated in this study provides a deeper understanding of the Giardia immunogens that bind to critical molecules of the host immune system, such as MHC class II and antibodies, as well as strategies to rational design of peptide-based vaccine against giardiasis.

Characterization of BIP protein of G. lamblia as a potential immunogen in a mouse infection model.

Giardia lamblia is a protozoan parasite that causes one of the most common gastrointestinal diseases worldwide. To eliminate the parasite from the host intestine, it is necessary the activation of B-cell and T-cell dependent mechanisms. The knowledge about Giardia antigens that can stimulate the host immune response is limited. Recently, it has been described the Binding Immunoglobulin Protein (BIP) of G. lamblia (71kDa) as a potential immunogen. Additionally, our group has identified a highly immunogenic antigen (5G8 protein) of G. lamblia with a relative molecular mass of approximately 70kDa. There is some evidence suggesting that the 5G8 protein may activate both humoral and cellular immune responses. Based on these observations and preliminary mass spectrometry analyses, we hypothesized that the antigen 5G8 could be the BIP protein. In the present study, we characterize immunochemically the BIP protein of Giardia. Flow cytometric assays and western blotting were used to determine the expression profile of BIP and 5G8 antigens in Giardia trophozoites. The differences in expression profile indicated that BIP and 5G8 are not the same molecule. ELISA and Western blotting assays revealed that BIP protein was recognized by antibodies produced during G. lamblia infection in C3H/HeN mice. MTT assays did not reveal the activation of cellular immune response induced by BIP protein in vitro. In addition, we identified the potential B-cell and T-cell epitopes of G. lamblia BIP protein. This molecule is a conserved protein among Giardia strains and other pathogens. The complete immunological characterization of this antigen will contribute to a better understanding of the host-parasite interactions in Giardia infection.

Isolation and partial characterization of an immunogenic antigen of Giardia lamblia.

Humoral and cellular immune responses play an important role during Giardia lamblia infection. Several Giardia proteins have been identified as immunogenic antigens based on their elicited humoral immune response. Poorly is known about Giardia antigens that stimulate a cellular immune response. The main purpose of this study was to isolate and partial characterize an immunogenic antigen (5G8) of G. lamblia. The 5G8 protein was isolated from G. lamblia trophozoite lysates by affinity chromatography using moAb 5G8-coupled CNBr-Sepharose. The isolated protein was analysed by electrospray tandem mass spectrometry (ESI-MS/MS), and by diverse bioinformatics tools (GiardiaDB, BLASTn, BLASTp and ExPASy). Additionally, several biochemical and immunological characteristics of the isolated protein were analysed. By ESI-MS/MS the amino acidic 5G8 sequence was deduced. The 5G8 antigen belongs to the VSP family proteins of G. lamblia. This protein is composed by one polypeptide chain (±71kDa). Using the algorithm SYFPHEITI, we identified candidate CD4+ T-cell epitopes from the 5G8 antigen, which can elicit cell-mediated immune responses. In this study, we have identified a G. lamblia protein that induces a strong immune response in infected mice. The biochemical and immunological characterization of the immunogenic 5G8 antigen may contribute to the rational design of a Giardia vaccine.

Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries.

Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.