Shigellosis is an acute inflammatory bowel disease caused by a group of Gram-negative intracellular enterobacteria known as Shigella. Food and water sources that contaminated with human or animal waste contribute primarily towards the transmission of Shigella [1]. Based on the structure of their lipopolysaccharide O-antigen, the bacteria are divided into four species namely Shigella flexneri, S. sonnei, S. dysenteriae, and S. boydii [2]. At present, shigellosis has led to 169 million morbidities in developing countries and is responsible for 1.1 million deaths per year in young age children who are less than 5 years of age, attributed to Shigella-associated diarrhoea cases [3]. Apart from young children, Shigella also targets elderly and immunocompromised patients [4]. Its mode of transmission is by fecal-oral route and requires a minimum infectious dose of <10 to 100 bacilli to cause the infection, due to its ability to survive gastric acidity better than other enterobacteria [5]. In the context of pathogenesis, Shigella sp. invades and colonizes the colonic mucosa leading to its disruption. The bacilli invade the villi of the large intestine, multiply and spread laterally to adjacent epithelial cells and also penetrate into the lamina propria [5]. Since the pathogen is intracellular it becomes even more difficult to treat the disease. The symptoms associated with shigellosis range from mild self-limited diarrhea to severe dysentery with frequent passage of blood and mucus, high fever, abdominal cramps, malaise, chills, nausea, vomiting and in rare cases known to cause bacteremia.

The uncontrolled use of antimicrobial agents Shigella sp. has inadvertently led to the development of resistant strains, which hinder appropriate selection of effective antibiotics, causing it poses a threat to public health [6]. Shigella strains have been reported to develop resistance to sulphonamide followed by with an increasing rate of resistance to tetracycline, chloramphenicol, ampicillin, nalidixic acid, and fluoroquinolones, making therapeutic treatment against Shigella infection a challenge [6]. In Malaysia, literature study strongly suggests that Shigella spp. exhibits frequencies of resistance against tetracycline and trimethoprim-sulfamethoxazole. The existing antibiotics can no longer be used to treat cases of severe diarrhoea and dysentery [5]. Therefore, there is an urgent need for vaccine development for the long-term control of Shigella transmission in affected tropical regions.

Until now, no practical vaccine available for shigellosis has yet been licensed. Current existing vaccine candidates are either not sufficiently attenuated or less immunogenic to the host [7]. Further identification of immunogenic protective antigens is essentially required to develop effective subunit vaccines for shigellosis. Recent literatures reveal that the outer membrane proteins (OMPs) of Shigella are ideal targets for vaccine development [8]. It has been well established that the OMPs of Shigella are capable of induction of immunological response in animal models given these proteins play crucial role as dynamic interface during host-pathogen interaction [9]. In most of the bacteria, OMPs involve in cell structure maintenance, providing cell's integrity, maintaining selective permeable of bacterial membrane, an adaptation of bacteria in the host niches, and bacterial pathogenesis through enhancing the bacterial adaptability in various type of environment. Most of the OMPs are multifaceted surface exposed antigens which play an important role in outer membrane structure integrity and represent a virulence factor among various Shigella strains [10].

Development of vaccine through conventional techniques takes decades to develop. With the availability of whole genome sequences, the genomic data could be used in silico to help identify and screen for potential vaccine candidates. This vaccine development technique using genome-based in silico method is known as “reverse vaccinology” [11]. To select a potential vaccine candidate, it is essential to identify the virulent protein that is capable to evoke an immune response within the host organism. Some features required for an effective vaccine candidate protein include: (1) sub-cellular localization; (2) presence of a signal peptide; (3) transmembrane domain; and (4) antigenic epitopes. The main strategy behind identification of potential vaccine candidates is recognizing the antigenic and virulence factor as well as predicting those sequences which are likely to bind to major histocompatibility complex (human leukocyte antigen [HLA] in human) molecules on the antigen presenting cells within the host. Reverse vaccinology approach was firstly adopted by Rappuoli and his colleagues to develop a safe and protective vaccine against Group B meningococcus [12].

This study is based on the use of reverse vaccinology approach for the identification and search of putative vaccine targets against S. flexneri 2a. The most conserved and immunogenic OMPs of S. flexneri 2a which can be potent vaccine candidates were initially identified. The shortlisted proteins were then predicted for their antigenicity as potential epitopic sequences using validated online immunoinformatics software. A schematic diagram showing computational pipeline corresponds to reverse vaccinology used in this study is represented in Fig. 1.

Diarrhoeal disease caused by shigellosis continues to be a leading cause of morbidity and mortality worldwide. Without a proper control, the infection could potentially lead to major human life-threatening disease. Increased resistance level in Shigella to a wide range of antibiotics has led the disease to a global health issue. Until now, efforts to develop safe and efficacious vaccines against Shigella are still unsuccessful. Attenuated live vaccines developed for human were found not to be safe partly due to their possibility in occasional back mutations. Later, lipopolysaccharide-based vaccines containing the sugar moiety were also reported to have low immunogenicity when evaluated in infants and young children [27]. The emergence of multiple drug resistance in Shigella and lack of vaccine against Shigella have necessitated a search for alternative strategies to further discover potential vaccine targets against Shigella infections.

The advent of genome sequencing and advancement in bioinformatics is now possible to identify potential vaccine candidates computationally without the need for living culture. This approach termed as “reverse vaccinology” reduces the time and cost of designing and identifying vaccine candidates, it also provide us with new solutions for those diseases for whom conventional approach have been unsuccessful [28]. The first vaccine developed using this approach was against the serogroup B Neisseria meningitidis [12]. In this study, we adopted “reverse vaccinology” method to identify potential OMPs from S. flexneri 2a that conserved among S. dysentery and S. sonnei for vaccine development. It is interesting to note that bacterial cell surface and secreted antigens could be important targets for developing vaccine given that they are responsible for the initial host-pathogen interaction [9]. Based on the proteins sequence conservedness, immunogenicity, subcellular localization, and unrelatedness with human proteome, five OMPs were identified as potential vaccine candidates against S. flexneri. The selected proteins were outer membrane receptor (fepA), outer membrane porin protein C (ompC), lipoprotein NlpD (nlpD_1), outer membrane channel protein (tolC), and lipoprotein (nlpD_2).

Surface proteins of many pathogens are mostly antigenic and are responsible for host pathogenesis [29]. During colonization, the attachment of pathogens to host cells surfaces is usually mediated by adhesins primarily built up of OMPs. OMPs, which are characterized as a large group of β-barrel proteins found in the outer membrane of Gram-negative bacteria have been reported to possess multiple roles during bacteria pathogenesis including mediating of the initial adhesion of bacteria to host cells, modulation of host-pathogen interactions as well as propagation of virulence factors [30]. It is well documented that OMPs of Gram-negative bacteria consist of components which are easily recognized as foreign antigens by immunological defense systems of the hosts [31]. This strongly suggests that OMPs of Gram-negative bacteria are essentially to play crucial roles both in the process of bacterial pathogenesis and stimulation of the host immune response. In view of this, OMPs of Shigella have been reported to confer a significant protective immune response [32]. ompA has been identified as a major protective antigen and antigenically conserved among the Shigella spp. Immunization of mice with ompA was found to give protection against a lethal dose of virulent S. flexneri 2a [10]. Meanwhile, previous study also showed that mice immunized with ompC survived after being challenged with live S. flexneri 3a [33], suggesting the OMPs could be one of the ideal components for developing subunit vaccine against shigellosis.

Despite having essential role in pathogenesis, the bacterial membranes also known to possess functions as defensive barriers to protect the cells from harmful and toxic compounds [34]. In order to understand the functional and cellular processes at system-level, protein-protein interacting network within the outer membrane of the bacteria is of importance. In this study, protein-protein interaction analysis revealed that five selected S. flexneri 2a OMPs were involved primarily in beta-lactam resistance pathway (pathway ID no., 00312). Beta-lactamases are enzymes produced by bacteria that provide multi-resistance to beta-lactam antibiotics. Bacterial resistance to antibiotics can be associated with the degradation of the drug by these enzymes [35]. It has been reported that resistance toward beta-lactam antibiotic is associated with alteration in outer membrane porins ompC and ompF and cytosolic proteins of ompR in Shigella [36]. Hence, active efflux pump is another major factor responsible for the antibiotic resistance phenotype in Gram-negative bacteria [37]. Previous studies have shown that AcrAB-TolC pump is involved in multidrug resistance in Gram-negative bacteria including Escherichia coli, Enterobacter aerogenes, Salmonella enterica, and S. flexneri [38]. The drug metabolism can be associated with overexpression of acrA gene which leads to multiple-antibiotic resistance in clinical isolates of S. flexneri [38]. In this study, two OMPs (fepA and tolC) identified from the selection pipeline are among the OMPs reported to be involved in facilitating the internalization of the external irons into the bacterial cell for bacterial colonization and survival [39]. The data presented here indicates that five OMPs of S. flexneria 2a selected in this study may possess essential role in facilitating drug resistance for bacterial survival, supporting these proteins could serve as potential vaccine targets against multidrug resistant Shigella spp.

Shigella spp. infection involves both intracellular and extracellular phases. During invasion, the bacteria target macrophages and gut colonic epithelial cells. To evade host immune system, Shigella uses its T3SS-dependent mechanism to avoid rapid clearance by promoting in vivo apoptosis of immune cells in mucosa-associated lymphoid tissues of the gut, resulting in innate immune response is insufficient to control Shigella spp. infection [40]. Following entering the epithelial cells, Shigella spp. can elicit both the humoral and cell-mediated immune responses which are responsible to facilitate the clearance of Shigella spp. infection from the host [41]. Given the crucial role of adaptive immunity in protection against Shigella spp. infection, the selection of antigens containing both B- and T-cell epitopes may provide an efficient way for vaccine development against Shigella spp. [41]. In this study, five OMPs selected by immunoinformatics method were found to possess numbers of antigenic epitopes which could potentially induce both humoral (B-) and cellular (T-cell) mediated immune responses [42]. The immunogenic epitopes predicted from each of these OMPs are potentially to be used for designing multi-epitope vaccines against this bacterium. Therefore, computationally or better known as reverse vaccinology approach is both cost effective and less time consuming for screening of potential vaccine candidates and further providing a framework for future designing of an effective vaccine against Shigellosis. The selected vaccine candidates need to be further validated for their immunogenicity and protective efficacy experimentally for their future use as vaccine targets against various strains of Shigella spp.

In summary, five target OMPs of S. flexneri 2a selected through our bioinformatics pipeline have been verified computationally as potential vaccine candidates against S. flexneri 2a. Our data shown that these OMPs are conserved among the Shigella strains with particular S. boydii, S. dysentery, and S. sonnei and therefore suggested they are potentially effective against all the strains of Shigella spp. Reverse vaccinology based on the data derived from genomics, proteomics, and immunoinformatics is a promising strategy for the screening and identification of antigenic antigens which could have potential capacity to evoke cellular and humoral immune responses simultaneously against Shigella spp. infection. Further in vitro and in vivo experiment studies are required to validate the immunogenicity and protective efficacy of these antigens against experimentally Shigella infection in animal model. The current user-friendly vaccine identification pipeline can be extended to other target pathogens to pave a better way for the control of transmission of persistent infections.