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.

Prosthetic joint infections are frequently associated with biofilm formation and the presence of viable but non-culturable (VBNC) bacteria. Conventional sample culturing remains the gold standard for microbiological diagnosis. However, VBNC bacteria lack the ability to grow on routine culture medium, leading to culture-negative results. Bacteriophages are viruses that specifically recognize and infect bacteria. In this study, we wanted to determine if bacteriophages could be used to detect VBNC bacteria. Four staphylococcal strains were cultured for biofilm formation and transferred to low-nutrient media with different gentamycin concentrations for VBNC state induction. VBNC bacteria were confirmed with the BacLight^TM viability kit staining. Suspensions of live, dead, and VBNC bacteria were incubated with bacteriophage K and assessed in a qPCR for their detection. The VBNC state was successfully induced 8 to 19 days after incubation under stressful conditions. In total, 6.1 to 23.9% of bacteria were confirmed alive while not growing on conventional culturing media. During the qPCR assay, live bacterial suspensions showed a substantial increase in phage DNA. No detection was observed in dead bacteria or phage non-susceptible E. coli suspensions. However, a reduction in phage DNA in VBNC bacterial suspensions was observed, which confirmed the detection was successful based on the adsorption of phages.

Our study introduces a novel bacteriophage-based vaccine strategy and evaluates its antitumor efficacy, both as a standalone therapy and in combination with gene electrotransfer (GET) of interleukin-12 (IL-12) plasmids. Using phage display technology, we produced engineered M13 bacteriophages expressing tumour peptides MAGE-A1, gp100, or MART-1/MELAN-A on the surface of the capsid. The therapeutic potential of bacteriophage vaccination alone or in combination with GET IL-12 was tested in vivo in a mouse malignant melanoma model. Response to treatment was further characterized by histological and immunohistochemical analyses of tumour tissue. No negative side effects were observed during treatment in mice. Engineered bacteriophage therapy significantly delayed tumour growth. GET IL-12 contributed to the therapeutic effect of engineered bacteriophages and increased tumour growth delay. Both therapies had a synergistic effect and led to complete responses in 30% of cases. Histological and immunohistochemical analyses have shown that both bacteriophage monotherapy and, especially in combination with GET IL-12, activate the immune system and increase the proportion of necrosis and the infiltration of macrophages, CD4 + and CD8 + T lymphocytes in tumours. For the first time, a cocktail of three engineered M13 bacteriophages displaying different melanoma-associated antigens with intratumoral IL-12 gene electrotransfer were applied, demonstrating a synergistic therapeutic effect in a highly aggressive melanoma model. Nanotechnological approaches, such as the use of genetically engineered bacteriophages, offer promising new avenues for the development of anti-tumour vaccines.