K-ras peptide mimotope induces a humoral immune response against G12V K-ras antigen in BALB/c mice

Abstract

<jats:p>Background. KRAS mutations are highly prevalent in pancreatic, lung, and colorectal carcinomas with G12V point substitution being one of the most prevalent mutations. While developments of peptide vaccines for KRAS(+) cancers are usually associated with poor immunogenicity, coupling mutant K-ras vaccines with universal CD4+ carrier molecules may enhance its outcome. Additionally, recent immunotherapeutic advances also suggest the possibility of inducing mucosal immunity against cancers using Lactococcus lactis as a live gastrointestinal delivery vehicle. Methods. A region of wild-type K-ras peptide was previously modified with a V7D substitution flanking the G12V mutation, generating a K-ras peptide (termed 68-V) with improved predicted antigenicity. This peptide was fused with a diphtheria toxoid sequence, and cloned into pNZ8048 vector within Lactococcus lactis NZ9000. BALB/c mice were then immunized orally, and then subjected to T/B cells immunophenotyping, as well as IgG and IgA detection. Results. Modified 68-V K-ras peptide and controls were successfully cloned and detection of His-tagged proteins expressed following induction by nisin was observed. Populations of CD3-CD19+ immune cells increased following immunization of 68-V, while K-ras specific-IgG and -IgA sera titers were elevated compared to wild-type and G12V K-ras controls. Conclusion. 68-V K-ras mimotope was shown to induce humoral-mediated immunity, highlighting the ability of an additional mutation flanking the G12V KRAS mutation to induce B cell activation and production of K-ras specific antibodies, while diphtheria toxoid was unable to stimulate an enhanced response when fused to 68-V. Nevertheless, these findings showed that further assessments are required to understand the role of K-ras specific antibodies within a KRAS(+) environment.</jats:p>

K-Ras Peptide Mimotope Induces Antigen Specific Th1 and B-Cell Immune Responses against G12A-Mutated K-Ras Antigen in Balb/c Mice

“…Therefore, minute elevation of NK cells (Figure 3d) could probably be elicited by the vaccine delivery host L. lactis. This is further supported by studies reporting that ingestion of probiotics such as L. lactis could stimulate NK cell production which is important in early lytic defence against foreign microbial, infected or malignant T-cells [31,[38][39][40][41]. Collectively, significant elevations in B-cell population suggests the potential of 139A-K-ras mimotopes in triggering a humoral immune response.…”

“…Recombinant pIDT:SMART plasmid was transformed via heat-shock into the X-10 GOLD E. coli. The cDNA of wtKRAS which was obtained from a previous study [31] was used as the template for PCR-based site direct mutagenesis to generate the first 50 codon of KRAS sequence with G12A mutation, incorporating restriction sites at both ends, and a 6× histidine tag at the C-terminal. PCR amplification was carried out using 0.2-1.0 µg template and the PCR mix contained 2.0 mM MgCl 2 , 0.2 mM dNTP mix, 0.05 mM primers and 0.02 U/µL Q5 Hi-Fidelity DNA polymerase (New England Biolabs, Boston, MA, USA).…”

Effect of Secretion Efficiency of Mutant KRAS Neoantigen by Lactococcus lactis on the Immune Response of a Mucosal Vaccine Delivery Vehicle Targeting Colorectal Cancer

“…Two unique RE sites, NcoI and KpnI, were included in the primers at N-and C-terminal ends of the SPs, respectively, for directional cloning. Meanwhile, the gene-coding sequences of the 68V-DT and wtKRAS were amplified from the previously constructed plasmids pNZ-USP45-68V-DTD and pNZ-USP45-wtKRAS, respectively (Hoo et al, 2020) [51]. Primers 4 and 5 were used to amplify the KRAS 68V-DT gene, whereas primers 6 and 7 were used to amplify the wtKRAS gene.…”