Methods: In 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.
Results: In 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.
Discussion: In 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.
Materials and Methods: Samples were collected from the Tuberculosis Laboratory, Clinical Microbiology of Dr. Soetomo Hospital Surabaya Indonesia. DNA extraction used boiling extraction method and continued nucleic acid amplification using PCR techniques. Primer pairs used eccB5 SK.. The positivity of DNA specific revealed amplicon in 1592 bp. PCR product was sequenced by 1st Base (First BASE Laboratories Sdn Bhd, Selangor, Malaysia). The sequence analysis used Genetyx-Win version 10.0 (Genetyx Corporation, Tokyo, Japan).
Results: Total isolates of Mycobacterium spp. were 28 and those that showed positive MTBC were 24 isolates and 4 nontuberculosis mycobacteria (NTM) using immunochromatographic test (ICT). The amount of homology from MTBC using blast NCBI was 99%-100%. Two SNPs were found in position c.1277 which revealed replacement of amino acid in 426 of codon position.
Conclusion: The sequence of eccB5 gene of MTBC showed high significant homology, while proposed non-synoymous single nucleotide polymorphisms (nsSNP) may associated with clinical outcomes.