Vaccine administration via the oral route is preferable to parenteral routes due to ease of administration. To date, most available oral vaccines comprises of live attenuated pathogens as oppose to peptide-based vaccines due to its low bioavailability within the gastrointestinal (GI) tract. Over the years, probiotic-based peptide delivery vehicles comprising of lactic acid bacteria such as Lactococcus lactis has emerged as an interesting alternative due to its generally recognized as safe (GRAS) status, a fully sequenced genome, transient gut colonization time, and is an efficient cellular factory for heterologous protein production. However, its survivability through the GI tract is low, thus better delivery approaches are being explored to improve its bioavailability. In this study, we employ the incorporation of a double coated mucoadhesive film consisting of sodium alginate and Lycoat RS 720 film as the inner coat. The formulated film exhibits good mechanical properties of tensile strength and percent elongation for manipulation and handling with an entrapment yield of 93.14±2.74%. The formulated mucoadhesive film is subsequently loaded into gelatin capsules with an outer enteric Eudragit L100-55 coating capable of a pH-dependent breakdown above pH 5.5 to protect against gastric digestion. The final product and unprotected controls were subjected to in vitro simulated gastrointestinal digestions to assess its survivability. The product demonstrated enhanced protection with an increase of 69.22±0.67% (gastric) and 40.61±8.23% (intestinal) survivability compared to unprotected controls after 6 hours of sequential digestion. This translates to a 3.5 fold increase in overall survivability. Owing to this, the proposed oral delivery system has shown promising potential as a live gastrointestinal vaccine delivery host. Further studies involving in vivo gastrointestinal survivability and mice immunization tests are currently being carried out to assess the efficacy of this novel oral delivery system in comparison to parenteral routes.
BackgroundSomatic point substitution mutations in the KRAS proto-oncogene primarily affect codons 12/13 where glycine is converted into other amino acids, and are highly prevalent in pancreatic, colorectal, and non-small cell lung cancers. These cohorts are non-responsive to anti-EGFR treatments, and are left with non-specific chemotherapy regimens as their sole treatment options. In the past, the development of peptide vaccines for cancer treatment was reported to have poor AT properties when inducing immune responses. Utilization of bioinformatics tools have since become an interesting approach in improving the design of peptide vaccines based on T- and B-cell epitope predictions.MethodsIn this study, the region spanning exon 2 from the 4th to 18th codon within the peptide sequence of wtKRAS was chosen for sequence manipulation. Mutated G12V and G13D K-ras controls were generated in silico, along with additional single amino acid substitutions flanking the original codon 12/13 mutations. IEDB was used for assessing human and mouse MHC class I/II epitope predictions, as well as linear B-cell epitopes predictions, while RNA secondary structure prediction was performed via CENTROIDFOLD. A scoring and ranking system was established in order to shortlist top mimotopes whereby normalized and reducing weighted scores were assigned to peptide sequences based on seven immunological parameters. Among the top 20 ranked peptide sequences, peptides of three mimotopes were synthesized and subjected to in vitro and in vivo immunoassays. Mice PBMCs were treated in vitro and subjected to cytokine assessment using CBA assay. Thereafter, mice were immunized and sera were subjected to IgG-based ELISA.ResultsIn silico immunogenicity prediction using IEDB tools shortlisted one G12V mimotope (68-V) and two G13D mimotopes (164-D, 224-D) from a total of 1,680 candidates. Shortlisted mimotopes were predicted to promote high MHC-II and -I affinities with optimized B-cell epitopes. CBA assay indicated that: 224-D induced secretions of IL-4, IL-5, IL-10, IL-12p70, and IL-21; 164-D triggered IL-10 and TNF-α; while 68-V showed no immunological responses. Specific-IgG sera titers against mutated K-ras antigens from 164-D immunized Balb/c mice were also elevated post first and second boosters compared to wild-type and G12/G13 controls.DiscussionIn silico-guided predictions of mutated K-ras T- and B-cell epitopes were successful in identifying two immunogens with high predictive scores, Th-bias cytokine induction and IgG-specific stimulation. Developments of such immunogens are potentially useful for future immunotherapeutic and diagnostic applications against KRAS(+) malignancies, monoclonal antibody production, and various other research and development initiatives.
α-Mangostin, one of the major constituents of Garcinia mangostana, has been reported to possess several biological activities, including antioxidant, anti-inflammatory, antibacterial, and cytotoxic activities associated with the inhibition of cell proliferation and activation of apoptosis. However, the cellular signaling pathway mediated by α-mangostin has not been firmly established. To investigate the cellular activities of α-mangostin, human cancer cells, MCF-7 and MCF-7-CR cells, were treated with α-mangostin to measure the cellular responses, including cytotoxicity, protein-protein interaction, and protein expression. Cancer cells stably expressed Myc-BCL-XL and HA-MOAP-1 were also included in the studies to delineate the cell signaling events mediated by α-mangostin. Our results showed that the apoptosis signaling mediated by α-mangostin involves the upregulation of endogenous MOAP-1, which interacts with α-mangostin activated BAX (act-BAX) while downregulating the expression of BCL-XL. Moreover, α-mangostin was found to induce BAX oligomerization, the release of mitochondrial cytochrome C, and activation of caspase in MCF-7 cells. In overexpression studies, MCF-7 cells and spheroids stably expressed HA-MOAP-1 and Myc-BCL-XL exhibited differential chemosensitivity toward α-mangostin in which the stable clones expressing HA-MOAP-1 and MYC-BCL-XL were chemosensitive and chemoresistant to the apoptosis signaling events mediated by α-mangostin, respectively, when compared to untreated cells. Together, the data suggest that the cytotoxicity of α-mangostin involves the activation of MOAP-1 tumor suppressor and its interaction with act-BAX, leading to mitochondria dysfunction and cell death.
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