An Engineered M13 Filamentous Nanoparticle as an Antigen Carrier for a Malignant Melanoma Immunotherapeutic Strategy

Abstract

Bacteriophages, prokaryotic viruses, hold great potential in genetic engineering to open up new avenues for vaccine development. Our study aimed to establish engineered M13 bacteriophages expressing MAGE-A1 tumor peptides as a vaccine for melanoma treatment. Through in vivo experiments, we sought to assess their ability to induce robust immune responses. Using phage display technology, we engineered two M13 bacteriophages expressing MAGE-A1 peptides as fusion proteins with either pVIII or pIIII coat proteins. Mice were intraperitoneally vaccinated three times, two weeks apart, using two different engineered bacteriophages; control groups received a wild-type bacteriophage. Serum samples taken seven days after each vaccination were analyzed by ELISA assay, while splenocytes harvested seven days following the second boost were evaluated by ex vivo cytotoxicity assay. Fusion proteins were confirmed by Western blot and nano-LC-MS/MS. The application of bacteriophages was safe, with no adverse effects on mice. Engineered bacteriophages effectively triggered immune responses, leading to increased levels of anti-MAGE-A1 antibodies in proportion to the administered bacteriophage dosage. Anti-MAGE-A1 antibodies also exhibited a binding capability to B16F10 tumor cells in vitro, as opposed to control samples. Splenocytes demonstrated enhanced CTL cytotoxicity against B16F10 cells. We have demonstrated the immunogenic capabilities of engineered M13 bacteriophages, emphasizing their potential for melanoma immunotherapy.

Keywords: bacteriophage-based vaccine; filamentous bacteriophages; malignant melanoma immunotherapy; melanoma-associated antigen; nanoparticles; phage display technology.

Figure 1

Schematic representation of the strategy used to construct recombinant phagemid (a) pComb8-MAGE- and (b) pComb3XSS-MAGE-expressing tumor peptide EADPTGHSY.

Figure 2

Schematic representation of in vivo experiments.

Figure 3

Ex vivo spleen cytotoxicity assay. The specific survival rate (%) of B16F10 malignant melanoma cells after incubation with splenocytes isolated from C57BL/6NCrl mice immunized with genetically engineered M13 bacteriophages (pVIII::MAGE-A1), genetically engineered M13 bacteriophages (pIII::MAGE-A1), wild-type M13 bacteriophages, and from the CTRL group. Legend: *, p < 0.05; ns, not statistically significant. The values are presented as the AM ± SEM. Repeated measurements are represented by black dots.

Figure 4

ELISA assay for the detection of (a) anti-M13 bacteriophage and (b) anti-MAGE antibodies in mouse sera. The antibody response was assessed in C57BL/6NCrl mice immunized with genetically engineered M13 bacteriophages (pVIII::MAGE-A1), genetically engineered M13 bacteriophages (pIII::MAGE-A1), wild-type M13 bacteriophages, and in the CTRL group. Legend: *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001, values were considered statistically significant for the comparison of immune responses across different vaccine doses; #: p ≤ 0.05 and ####: p ≤ 0.0001 were considered statistically significant for the comparison of immune responses between the genetically engineered M13 bacteriophages (pVIII::MAGE-A1) and genetically engineered M13 bacteriophages (pIII::MAGE-A1) groups within one vaccine dose; ns: not statistically significant. The values are presented as the AM ± SEM. Repeated measurements are represented by black dots.

Figure 5

Immunocytochemical analyses of B16F10 melanoma cells expressing MAGE-1_161–169 tumor epitopes after incubation with sera from (a) genetically engineered M13 bacteriophages (pIII::MAGE-A1), (b) genetically engineered M13 bacteriophages (pVIII::MAGE-A1), (c) wild-type M13 bacteriophages, and (d) in the CTRL group. Red staining represents the expression of MAGE-1_161–169 tumor epitopes.