Designing an efficient multi-epitope peptide vaccine against Vibrio cholerae via combined immunoinformatics and protein interaction based approaches

Highlights

• Designing epitope peptide vaccines is a novel strategy for protection against Vibrio cholerae.

• The designed cholera vaccine consists of two main sections: four protective antigens (OmpW, OmpU, TcpA and TcpF) and adjuvant (CTB).

• The selected epitopes and adjuvant were fused together by proper linkers in order to enhance the immunogenicity of vaccine.

• In silico analysis indicates that the epitope vaccine is able to incite long-lasting protective immunity against V. cholerae.

Abstract

Cholera continues to be a major global health concern. Among different Vibrio cholerae strains, only O1 and O139 cause acute diarrheal diseases that are related to epidemic and pandemic outbreaks. The currently available cholera vaccines are mainly lived and attenuated vaccines consisting of V. cholerae virulence factors such as toxin-coregulated pili (TCP), outer membrane proteins (Omps), and nontoxic cholera toxin B subunit (CTB). Nowadays, there is a great interest in designing an efficient epitope vaccine against cholera. Epitope vaccines consisting of immunodominant epitopes and adjuvant molecules enhance the possibility of inciting potent protective immunity. In this study, V. cholerae protective antigens (OmpW, OmpU, TcpA and TcpF) and the CTB, which is broadly used as an immunostimulatory adjuvant, were analyzed using different bioinformatics and immunoinformatics tools. The common regions between promiscuous epitopes, binding to various HLA-II supertype alleles, and B-cell epitopes were defined based upon the aforementioned protective antigens. The ultimately selected epitopes and CTB adjuvant were fused together using proper GPGPG linkers to enhance vaccine immunogenicity. A three-dimensional model of the thus constructed vaccine was generated using I-TASSER. The model was structurally validated using the ProSA-web error-detection software and the Ramachandran plot. The validation results indicated that the initial 3D model needed refinement. Subsequently, a high-quality model obtained after various refinement cycles was used for defining conformational B-cell epitopes. Several linear and conformational B-cell epitopes were determined within the epitope vaccine, suggesting likely antibody triggering features of our designed vaccine. Next, molecular docking was performed between the 3D vaccine model and the tertiary structure of the toll like receptor 2 (TLR2). To gain further insight into the interaction between vaccine and TLR2, molecular dynamics simulation was performed, corroborating stable vaccine-TLR2 binding. In sum, the results suggest that our designed epitope vaccine could incite robust long-term protective immunity against V. cholera.

Introduction

Cholera is an acute diarrheal disease caused by gram-negative bacterium Vibrio cholerae leading to 100 000–120 000 worldwide deaths annually (Organization, 2010). The first report of cholera goes back to 1854, when Filippo Pacini, professor of anatomy observed curved bacterium form in stool of individuals who had been infected by cholera and named it V. cholera (Harris et al., 2012). About 30 years later, Robert Koch first isolated deadly V. cholerae in pure culture (Thompson et al., 2004). Koch found that many of V. cholerae strains, which living in aquatic environments were non-pathogenic. He also suggested that filtrating drinking water is useful to remove the bacteria. In the context of cholera treatment, According to WHO reports, More than 80% cholera patient can be treated successfully via oral rehydration therapy (World, 1991).

V. cholerae species is categorized into more than 200 serogroups according to the O antigen of the lipopolysaccharide (Chatterjee and Chaudhuri, 2003); among them, only two toxigenic serogroups of O1 and O139 were determined to cause epidemic and pandemic outbreaks (Morris and Acheson, 2003). Moreover, V. cholerae O1 variant is subcategorized into two biotypes, El Tor and Classical. In 1992, the O139 serogroup was first appeared in Bangladesh, southern and eastern of India as a cause of epidemic cholera (John Albert et al., 1993, Ramamurthy et al., 1993). On the other hand, the Classical O1 serogroup caused the fifth and sixth pandemics, but it seems that the seventh pandemic is associated with the El Tor biotype.

Immunization with cholera vaccines is an important strategy to control and prevent cholera epidemic or pandemic. V. cholerae virulence factors ranging from cholera toxin (CTX) (Sánchez and Holmgren, 2011), toxin-coregulated pili (TCP) (Taylor et al., 1987), Lipopolysaccharide (LPS) (Waldor et al., 1994) to outer membrane proteins (Omps) (Nandi et al., 2005, Sengupta et al., 1992, Sperandio et al., 1995) are ideal candidates for devising the cholerae vaccine.

V. cholerae CT belongs to the AB class of bacterial toxins. The heterodimeric A subunit (CTA) is responsible for toxicity of bacteria, while the homopentameric B subunit (CTB) is necessary for host cell binding (Merritt et al., 1995). Recently, CTB subunit is widely used as a potent oral-mucosal adjuvant for stimulation of mucosal antibody responses. Moreover, it has been shown that linking of CTB to antigens increases immunogenicity of antigens through induction of B and T cells responses; hence this strategy exploited in different kinds of peptide and DNA vaccines (Pinkhasov et al., 2010).

TCP has a crucial role in intestinal colonization and virulence of V. cholerae. The TCP filament is a member of type IVb pilus subclass consisting of 1000 copies of the pilin subunit, TcpA, forming interwoven filaments at the bacterial surface (Li et al., 2008). TcpA DNA sequence is 100% identical between El Tor O1 and O139 strains; consequently, serum against TcpA generates immune responses against both aforementioned strains (Rhine and Taylor, 1994). Toxin-coregulated pili A incites robust systemic and mucosal immune responses via increasing the level of TcpA-specific antibody in patients infected with V. cholerae El Tor O139 and O1 (Asaduzzaman et al., 2004). Due to the principal role of TcpA in V. cholerae pathogenesis, it has attracted more attentions as an ideal target in designing the vaccine. Besides TcpA, TcpF is another soluble colonization factor that is secreted by V. cholerae O1 and O139, acting as a protective antigen in the suckling mouse cholera model (Kirn and Taylor, 2005).

Omps are essential proteins in outer membrane (OM) of gram-negative bacteria playing crucial roles in bacterial pathogenesis as well as interaction between organism and its niche (Lin et al., 2002). Due to high immunogenicity of Omps especially in a variety of pathogenic strains, they have been considered as prominent candidates for designing antimicrobial drugs and vaccines (Das et al., 1998, Galdiero et al., 2003). It has been reported that about six main Omps present on V. cholerae surface (Chakrabarti et al., 1996). OmpU of some Vibrio species such as V. alginolyticus and V. harveyi have been applied as effective vaccine candidates in Lutjanus erythropterus and Scophthalmus maximus, respectively (Cai et al., 2013, Wang et al., 2011). In addition, the V. cholerae OmpU is able to incite pro-inflammatory responses, in this regard act similar to other gram-negative bacterial Omps (Sakharwade et al., 2013). V. cholerae OmpW, a 22 kDa surface protein, appears to be conserved and highly immunogenic in different V. cholerae species, hence it could be considered as a potential vaccine target to incite protective immunity against pathogenic serogroups O1 and O139 (Jalajakumari and Manning, 1990, Nandi et al., 2005).

Omps and CTB along with other highly conserved microbial components are known as pathogen associated molecular patterns (PAMPs); they are recognized by pattern-recognition receptors (PRRs), which are located on most surfaces of immune and non-immune cells (Kang and Lee, 2011). Toll-like receptors (TLR) are a subset of PRRs, which interact with toll-like ligands (member of PAMPs) and consequently stimulating innate immune response in the host. Each TLR ligands is recognized with its cognate TLR, for instance, lipids and lipoprotein, like lipopolysaccharides (LPS) (recognized by TLR4, TLR2/6 and TLR1/2), nucleic acids such as: dsRNA, ssRNA, CpG motif, viral DNA (recognized by TLR3, 7, 8 and TLR9) and proteins, which include flagellin, profilin (recognized by TLR5, TLR11) (Kawai and Akira, 2010). Heat-labile enterotoxins (B subunit) of Escherichia coli and V. cholerae, monophosphoryl lipid A (MPL) and some bacterial Omps, which belongs to TLR2 ligands, indicate robust immune adjuvanticity when they are applied with various antigens; hence they are utilized in the constituent of vaccines as immunostimulatory adjuvants (Connell, 2007, Moyle and Toth, 2008, Wetzler, 2010).

Due to some drawbacks of live attenuated or killed bacterial vaccines such as reactogenicity, limited long-term protection and instability in different storage conditions, attempts have been begun to use epitope-based vaccines (EV) (Pastor et al., 2013). EVs are safe, cost-effective, stable in different conditions and highly specific due to defined epitopes (De Groot et al., 2009). More potent protective immunity is incited by multi-epitope vaccine than single epitope; consequently, more effective immunoprotection could be generated against V. cholerae infection by such vaccines. The identification of ideal epitopes (B and T cell epitopes), which is bound efficiently to major histocompatibility complex (MHC) molecules class I or II, B cell receptors (BCRs) and T-cell receptors (TCRs) are laborious and time-consuming. Recently, bioinformatic approaches can considerably reduce time and costs for vaccine advance (Six et al., 2012). In this study, we intend to apply bioinformatics methods to design a novel epitope peptide vaccine against cholera infection. In our designed epitope vaccine, OmpW, OmpU and TcpA are applied as protective antigens and CTB playing a role as adjuvant. Moreover, to enhance vaccine efficacy, each part of vaccine is fused together by proper GPGPG and EAAAK linkers. Different features of chimeric protein vaccine are evaluated by in silico approach. Finally, due to the important role of the molecular dynamics simulation (MD simulation) for a better perception of biological processes (Boehr et al., 2009, Hansson et al., 2002), this method is applied to monitor the vaccine behavior and its stability toward a receptor. MD simulation allows to the both protein and vaccine to get into their best conformations and form the optimum modes of interactions against each other.