Cholera is a sudden illness characterized by diarrhea, and it is caused by a gram-negative bacillus bacterium known as Vibrio cholerae. Humans are the primary hosts for most of the serogroups O1 and O139 that cause cholera. The development of a highly effective and affordable vaccine is crucial due to the prevalence of cholera in underprivileged areas with inadequate water sanitation management [1], [2], [3], [4].

Vaccine manufacturers have made advancements in two areas: creating vaccines from killed bacteria and developing vaccines from live attenuated recombinant strains [5], [6], [7]. The World Health Organization [8] has established a set of criteria for an optimal vaccine, including the vaccine's ability to be administered with minimal frequency, provide long-lasting immunity, have no disease-causing effects, and be suitable for immunodeficiency patients along with all age groups [9]. Vaccines from killed bacteria do not produce long-term immunity and only have 66 % immunogenicity, necessitating multiple doses. Live attenuated vaccines may cause moderate diarrhea and associated symptoms; they should not be administered to immunodeficient individuals or children under two years old. Based on previous studies virulence factors of V. cholerae such as cholera toxin (CTX), toxin-coregulated pili (TCP), and outer membrane proteins (Omps) specially OmpU and OmpW are excellent options for developing a cholera vaccine [10], [11], [12], [13], [14]. Vibrio cholerae's virulence component, cholera toxin, has two subunits, A and B. Both subunits are essential for cholera's acute watery diarrhea [15]. The toxin also manipulates dendritic cell activation to generate regulatory T lymphocytes that target bystander antigens. TCP, another virulence factor, helps Vibrio cholera colonize bacteria. By raising TcpA-specific antibody levels, TCPA boosts systemic and mucosal immune responses in V. cholera patients. Furthermore, gram-negative bacteria need their outer membrane proteins (OMPs) for pathogenicity and environmental interactions [16]. OmpU from Vibrio species as V. alginolyticus [17], V. mimicus [18], and V. harveyi [19] may be vaccine candidates for Lutjanus erythropterus, yellow catfish, and Scophthalmus maximus. Like other Gram-negative bacteria Omps, V. cholerae OmpU causes pro-inflammatory reactions [20]. Additionally, Vibrio cholerae's highly conserved extracellular protein OmpW is an important virulence component with heightened immunogenicity. Researchers found that vaccine candidates with the chimeric gene OTC (OmpW, TcpA, and CtxB) induced specific IgGs and protected against V. cholera [21], [22], [23].

Reverse vaccination is a new technique that has replaced conventional methods due to their limitations. Conventional vaccinations have challenges like the need for special cultivation conditions for dangerous microorganisms, the risk of disease transmission, and difficulty in growing certain microorganisms in the lab [24], [25]. Additionally, it is impossible to check all microorganism proteins which increases manufacturing costs. To overcome these obstacles, reverse vaccinology is used a part of immunoinformatics that uses computational data to identify potential vaccine antigens [26]. This approach aims to target specific antigen regions or immune system-recognized epitopes, offering several advantages such as stability in various conditions and excellent specificity [27]. Additionally, it enables a faster and more cost-effective completion of the primary research phase of vaccine development by combining multiple antigens' epitopes to stimulate the immune system strongly. Multiepitope vaccines have been forecasted for various bacterial diseases like Brucella melitensis [28], Staphylococcus aureus [29], and Vibrio cholera [20], [30]. However, additional research is required to assess their effectiveness in animal studies. The multi-epitope produced through this in silico investigation can be tested on animals but requires adjuvants due to low immunogenicity caused by rapid destruction from proteinases and difficulty recognizing them by the body's immune receptors [31], [32], [33], [34].

Bacteriophages are a crucial component in vaccine development. These viruses infect and replicate within bacteria, but they do not cause harm to humans or other animals. The most widely used vectors for displaying antigenic determinants for vaccine production are filamentous non-lytic bacteriophages, such as M13, which belong to the genus Inovirus in the Inoviridae family [35], [36]. Phage particles can be modified genetically or chemically to transport the necessary antigenic domains, making them suitable for use as vaccines [37], [38].

We previously identified phage clones expressing the multi-epitope of V. cholerae proteins using an immunoinformatics approach. In this study, we used M13KO7ΔpIII, derived from M13 with an origin of replication from P15A and a kanamycin resistance gene, together with a selective phagemid. Our hypothesis was that M13KO7ΔpIII might integrate five copies of the poly epitope-pIII fusion protein during assembly to avoid heterogeneity. Typically, all rescued phages express the recombinant protein because the helper phage genome does not encode the pIII proteins [39].