In silico Analysis of Immunologic Regions of Surface Antigens (Sags) of Toxoplasma gondii

Background: Surface antigens (SAGs) of Toxoplasma gondii are known candidates for diagnostic tests and vaccines. The present study argues about the main necessary properties for determination and prediction of T-cell agretopes and B-cell epitopes of surface antigens of Toxoplasma gondii.Materials and Methods: Primary, secondary and tertiary structures of the proteins were analyzed by different methods. The three-dimensional structures were determined by use of ab initio method for prediction of discontinues epitopes. The agretopes and epitopes were predicted via several various web servers with different methods employed.Results: The results of in silico analyses showed that the regions 129-GAPAGRNNDGSSAPT-143 for protein p22, 234-SENPWQGNASSD-245 for protein p30 and 348-PGTEGESQAGT-358 for protein p43, have the highest immunogenic potential.Conclusion: We reached to three antigenic epitopes for cloning and protein expression. In following the purified polypeptide will be applied for diagnosis of Toxoplasma gondii

A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis

Background: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. Results: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. Conclusions: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far

Evaluation of tumour-associated antigens to optically label cutaneous basal cell carcinoma for surgical excision

Basal cell carcinoma (BCC) is the most common skin cancer worldwide, with South Africa having the highest incidence rate only after Australia. The most effective treatment modality for BCC is tumor excision via Mohs surgery (pioneered by Dr. Frederic Mohs of the University of Wisconsin in 1930), a microscopically controlled surgery that removes a tumor piecemeal in layers until each layer is free of any neoplastic tissue. The major drawback of Mohs excision is that the surgeon might miss any neoplastic tissue as the tumor margin is not always well defined, and the tumor often could extend beyond the superficial layers of skin. Moreover, it's a time-consuming, expensive procedure that takes generally 3-4 h, at times even more, if several rounds of excisions are warranted. In South Africa, at the time of writing, therapy using the surgery cost around R45,000. The status quo thus necessitates identifying BCC cells both in the superficial layers and beyond the layers of the skin in individual patients. Our aim was to identify BCC-specific cell surface proteins and design, engineer, and test a range of SNAPtag–based antibody fusion proteins that would specifically bind to and detect such BCC cell surface receptors. The SNAP-tag antibody technology is based on the genetic fusion of a disease-specific ligand to a protein tag derived from O6-alkylguanine-DNA alkyltransferase, which would allow for covalent auto-labeling of the corresponding antibody based fusion proteins with benzylguanine-modified (BG) substrates (e.g., fluorophores) under physiological conditions with high efficiency at 1:1 stoichiometry. This would allow to develop a unique immunological screening modality which should allow to visually label BCC lesions for a more precise surgical excision. The best-performing SNAP-tag–based diagnostic antibodies resulting from these studies would be further evaluated in the future in suitable mouse models, thus aiming to reduce the time needed for surgical removal of BCC lesions and complete removal of the tumor from both superficial and deep layers of the skin by a single-excision procedure. We used an integrated computational tool to re-analyze publicly available cDNA microarray data in combination with theoretical search to identify BCC-associated antigens. Accordingly, six different antigens were selected and single-chain variable fragments (scFv) targeting these antigens were cloned in fusion with SNAP-tag encoding gene into a custom expression vector for production in a secretory mammalian system. scFv-SNAP-tag protein was isolated from the cell culture supernatant by immobilized metal affinity chromatography and eluted protein samples were analyzed by gel electrophoresis and immunoblotting. The absolute amount of the full-length protein was quantified by densitometry. Purified scFv-SNAP-tag proteins were validated for specific binding to corresponding antigen-positive cells by flow cytometry and confocal microscopy. Of the six different scFv-SNAP-tag fusion proteins cloned, four were successfully expressed in HEK293T cells. The specific binding to EpCAM, EMA, CSPG4, and CD138 antigenexpressing cell lines was observed on incubation with scFvUBS54-SNAP-tag, scFvID405- SNAP-tag, 9.2.27scFv-SNAP-tag, and scFvh-STL002-SNAP-tag, respectively. In addition, we showed the selective cell killing effect of scFvUBS54-SNAP after conjugating it with the cytotoxic drug BG-modified auristatin-F (BG-AF). In conclusion, we identified various cell surface antigens along with one possibly novel antigen for BCC detection and therapy. Further, we successfully designed and synthesized SNAP tag based antibody fusion proteins and showed their functional activity by selective binding to the corresponding antigens on the surface of tumor cells. Based on these findings, we presume that these antibodies can effectively bind to BCC and can confirm EpCAM as one of the target antigens, which has already been reported to be a standard immunophenotypic marker for differential BCC diagnosis

LIPOSOMAL TECHNOLOGIES TO IMPROVE GENE DELIVERY

Lipid based nanoparticles (LBNs) are used in myriad applications in medicine from small molecule drug delivery to mRNA vaccines. A major contributing factor to the development of the field has been the ongoing development of novel compounds that retain the functionality of natural lipids but expand upon them through inclusion of functional moieties that can be applied to specific scientific and biomedical questions. In the body of this dissertation, an extensive overview of LBNs is provided, focusing primarily on their use in immune modulation. The research presented herein begins with the synthesis of a novel class of lipids based on the triazine (TZ) cyanuric chloride. Twelve compounds were synthesized and assessed for their biophysical behavior and ability to form LBNs. Of the 12 compounds, 10 were able to form nanoparticles and these were assessed for in vitro toxicity. The toxicity of the nanoparticles differs based on the nanoparticle charge and approximate that observed for similarly charged compounds. The cationic TZ lipids were then tested in vitro for their ability to deliver plasmid DNA into cells where they showed improved efficacy compared with the cationic lipid DOTMA, and similar toxicity. Finally, TZ lipids were used to lipidate peptides in a liposomal peptide vaccine where they induced similar anti-peptide titers as a CHEMS conjugate. Following these experiments, the in vivo toxicity and potential for plasmid delivery was evaluated for the cationic TZ lipids. TZ lipids led to toxicity similar to other cationic lipids. Of note, the PEG length in the nanoparticles was studied for its effect on transfection efficiency as was the effect of the helper lipid in the formulation. These experiments showed improved transfection efficiency with DOPE and with shorter length PEG chains on the nanoparticle surface. Evaluation of immune responses toward the transgene studied showed a similar titer response as the free protein. However, when the protein was delivered with a cationic lipid as control, titers increased significantly, particularly for the TZ lipid used, which increased titers 1000-fold. These data provide evidence for continued evaluation of TZ lipids as gene delivery vectors and as potential vaccine adjuvants. Finally, in continuing the evaluation of LBNs to improve gene therapy, an LBN based system was evaluated to deplete anti-AAV8 antibodies. As one of the most promising strategies to deliver transgenes since AAV provides an excellent platform that is unfortunately affected by the presence of anti-viral antibodies. This system, using doxorubicin liposomes coated with recombinant VP1 protein bound to DGS-NTA-Ni lipid or DSPE-PEG2000-Maleimide, failed to deplete circulating antibodies to AAV. However, the results of the experiments carried out shed light on how this strategy might be improved upon at a later time. Finally, in an attempt to better understand the immune targets on AAV, the antibody response toward AAV8 was tested in human samples from deidentified blood donors and compared with that of mice and monkeys treated with the virus. Serum from these species was scrutinized for its ability to neutralize the virus in vitro and evaluated using a peptide array for targets against the viral capsid protein VP1. Collectively, the studies presented in the body of this dissertation demonstrate the utility of LBNs in gene delivery, both as vectors and as aids for viral delivery

Anatomical heterogeneity of tendon: Fascicular and interfascicular tendon compartments have distinct proteomic composition

This study was funded by the BBSRC (BB/K008412/1)

Group A Streptococcal Peptides expressed in HBsAg-S VLPs as a vaccine candidate

Streptococcus pyogenes, or Group A Streptococcus (GAS) is responsible for significant patient morbidity and mortality in the developing world and within the Australian Indigenous population. GAS is responsible for a variety of diseases such as invasive necrotizing fasciitis and toxic shock syndrome, as well as non-invasive diseases, such as pharyngitis, impetigo, scarlet fever and otitis media. However, GAS sequelae such as rheumatic fever and rheumatic heart disease are responsible for the highest morbidity. The 30-valent vaccine candidate currently in trials is inappropriately specialised to serotypes present in areas with low GAS incidence, such as the United States. The difficulty in creation of a suitable vaccine lies in part with the variety of GAS virulence factors. The M protein is a highly abundant, multifunctional immunogenic surface protein which confers resistance to phagocytes and complement mediated protection. As sections of the M protein is highly conserved, it has been the focus of vaccination research. Furthermore, protein fragments J8 and J14 within the M protein have given encouraging results within a mouse model. Virus-like particle (VLP) technology offers a promising alternative to existing vaccination delivery systems. VLPs are able to induce both cell mediated and humoral immune responses. In this study, the use of a chimeric hepatitis B surface antigen VLP expressing M protein epitopes p145, J8 and J14 for use as a dual vaccine against Hepatitis B virus (HBV) and GAS is investigated. Specifically, PCR generated DNA sequences of J8, J14 and p145 from the M protein of GAS have been cloned into the highly immunogenic ‘a’ determinant region of the HBsAg-S VLP and transformed into human embryonic kidney (HEK293T) cells. Expressed recombinant HBsAg-S-GAS-m protein constructs were assayed by ELISA to confirm presentation of GAS epitopes. ELISA results showing high titres were obtained for VLP:p145 but low titres were obtained for VLP:J8 and VLP:J14. Further sequencing of plasmid constructs, protein expression and antigenic screening of proteins is required before the study can progress to proof-of-concept murine challenge models

Comparative and functional genomics provide insights into the pathogenicity of dermatophytic fungi

ABSTRACT: BACKGROUND: Millions of humans and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which exclusively infect keratinized host structures. To provide broad insights into the molecular basis of the pathogenicity-associated traits, we report the first genome sequences of two closely phylogenetically related dermatophytes, Arthroderma benhamiae and Trichophyton verrucosum, both of which induce highly inflammatory infections in humans. RESULTS: 97% of the 22.5 megabase genome sequences of A. benhamiae and T. verrucosum are unambiguously alignable and collinear. To unravel dermatophyte-specific virulence-associated traits, we compared sets of potentially pathogenicity-associated proteins, such as secreted proteases and enzymes involved in secondary metabolite production, with those of closely related onygenales (Coccidioides species) and the mould Aspergillus fumigatus. The comparisons revealed expansion of several gene families in dermatophytes and disclosed the peculiarities of the dermatophyte secondary metabolite gene sets. Secretion of proteases and other hydrolytic enzymes by A. benhamiae was proven experimentally by a global secretome analysis during keratin degradation. Molecular insights into the interaction of A. benhamiae with human keratinocytes were obtained for the first time by global transcriptome profiling. Given that A. benhamiae is able to undergo mating, a detailed comparison of the genomes further unraveled the genetic basis of sexual reproduction in this species. CONCLUSIONS: Our results enlighten the genetic basis of fundamental and putatively virulence-related traits of dermatophytes, advancing future research on these medically important pathogens