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Staphylococcus aureus has been recognized as a highly versatile gram-positive bacterium that has a long history of interconnections with humans as both commensal organisms and pathogens. Staphylococcal infection management with antibiotic intervention has become increasingly difficult with the pressing matter of global health crisis caused by Methicillin-resistant S. aureus (MRSA). As a promising alternative, immunological approaches have been earning the reputation to effectively and successfully reduce the incidence of infectious diseases. But due to the pleomorphic characteristics of S. aureus and lacking detection of one or more well-characterized protective antigenic targets there is no successful immunotherapy has been developed against this organism. Epitope based immunological strategies have promising attributes to induce optimal immune specificities targeting protective surface antigens and if conserved in all clinical isolates, a novel broadly protective “universal” immunotherapy can be achieved for staphylococcal infections. Staphylococcus aureusApproximately 30% of the population is colonized by S. aureus on the skin, throat, anterior nares and gastrointestinal tract 1. S. aureus was first reported in 1882 by a Scotish surgeon Sir Alexander Ogston who detected S. aureus cells in the pus from surgical abscesses as micrococci growing in clusters and in chains intermittently 2. S. aureus is known for its capacity to cause a broad range of serious infections in humans. Infectious diseases like boils, impetigo, cellulitis, bacteremia, sepsis, food poisoning and toxic shock syndrome may appear due to the infection itself or due to the production of toxins by S. aureus. Hospitalized patients with injury, chronic illness and suppressed immunity are susceptible to serious infections. More than 50% of hospital acquired infections in intensive care unit patients are caused by MRSA 3. Due to the ability to affect wide range of species, S. aureus can be transmitted from one species to another including transmission between humans and animals. Humans possess highly diverse immune response towards S. aureus that can be predisposed by history of exposure, differences in the serotype of S. aureus that have been encountered, host genetic variation, and environmental factors. Cell Surface AntigenS. aureus has capacity to express an array of virulence factors that participate in pathogenesis of infection, allowing this bacterium to adhere to surfaces/tissues, avoid or invade the immune system, and cause harmful toxic effects to the host. These virulence determinants can be divided into cell-surface-associated (adherence) and secreted (exotoxins) factors. The colonization process of S. aureus to the host cell surface is manifested by several adhesins of the cell surface factors. The microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) contain proteins that are covalently anchored to cell peptidoglycans having a conserved LPXTG motif at their C-terminus. The MSCRAMM family typically includes staphylococcal protein A, fibronectin-binding proteins A and B, collagen-binding protein, and clumping factor A and B proteins 4. There are also non-covalently linked, cell wall-associated proteins that possess neither a conserved signal peptide nor an LPXTG motif. Members of this group are tissue adhesins, toxins and immune evasion factors. There is evidence of high immune responses against some members of this group including IsaA, Efb and Atl 5.Evading Host Immune ResponseThere are staphylococcal virulence factors that are specific for human immune responses and have minimal activity in other organisms. These include the Panton-Valentine leukocidin (PVL), ?-Hemolysin CB (HlgCB), Enterotoxin A (SEA), the staphylococcal complement inhibitor (SCIN), the chemotaxis inhibitor proteins (CHIPS) and the staphylokinase (Sak) 6. The first line immune response against S. aureus infection is comprised of professional phagocytes like neutrophils and macrophages that ingest and eliminate the bacteria. However, phagocytosis of S. aureus relies on the opsonization of bacteria by antibodies and complement. The killing of phagocytosed bacteria by oxidative burst as well as induction of a long-term immune response require recognition of opsonizing antibodies bound to S. aureus surface via neutrophil Fc? receptors 7. On the other hand, S. aureus has the ability to derange the effectiveness of neutrophils and macrophages by expressing proteins that inhibit complement activation and neutrophil chemotaxis or that lyse neutrophils, neutralizes antimicrobial defensin peptides as well as can modify its cell surface to reduce their effectiveness. The organism can survive in phagosomes and its cell wall is resistant to lysozyme. Moreover, S. aureus can cause anergy and immunosuppression by expressing several types of superantigen that can exploit the normal humoral immune response 8.                                                      Antibiotic Therapy IncompetencyHistorically, ?-lactam antibiotics have been the drug of choice to treat staphylococcal infections owing to its good safety profiles and potent activity against S. aureus. By the late 1960s, MRSA emerged as a public health concern where more than 80% of both community and hospital-acquired staphylococcal isolates found to be resistant to all ?-lactam antibiotics. This pattern of resistance now persists with each new wave of antimicrobial resistance, initially emerging in the hospitals and then spreading to the community. Over the past decade, the development of new antimicrobial agents has been undermined by the propensity of resistance pattern in S. aureus. Despite the availability of effective antimicrobials, S. aureus bacteremia mortality rate still remains about 20–40%. There have been developments of intermediate or complete resistance to vancomycin by S. aureus isolates that is indicative of an era in which effective antimicrobials against this organism may no longer exist 9. Consequently, the need to develop novel prophylactic or therapeutic treatments against S. aureus infections as alternatives to antibiotic therapy has become inevitable.ImmunotherapyRecently researchers have focused the search for alternatives to antibiotic therapy particularly in the field of immunological treatment. Active and passive immunization for combating virulent strains are often targeted at molecules involved in pathogenesis. Encountering staphylococcal infection, the bacteria induced immune response does not provide significant protection against reinfection. However, a continuous exposure to staphylococcal antigens may improve the effectiveness of the immune response. Older patients with blistering disease epidermolysis bullosa (EB) have high S. aureus colonization rates in their chronic wounds. Interestingly S. aureus bacteremia is rarely observed in this kind of patients signifying high antistaphylococcal IgG levels due to continuous exposure to multiple strains, may be protective against invasive S. aureus infections 10. Therefore, this could support the indication of active immunization of healthy individuals with appropriate antigens that can similarly produce an immune response providing effective protection against S. aureus infections. In order to develop S. aureus vaccines against variety of different antigens, several active immunization approaches have been undertaken not precisely selecting the best antigen, rather based on information obtained from the literature on surface exposition, involvement in pathogenesis, or distribution in virulent strains 11. Conversely, lack of successful active immunization strategies against acute staphylococcal infections has shifted S. aureus immunotherapy research toward passive immunization. Passive immunization is able to conduct a major protective role in specific immunity against MRSA and with antigen specific antibody can provide partial protection against staphylococcal infections 12. However, antibodies against certain staphylococcal antigens can react to the targeted antigens more efficiently than other antibodies in order to induce phagocytosis and subsequently eliminate S. aureus. Therefore, identification of protective antigens is a crucial step for immunotherapy. Epitope-based ImmunotherapyEpitope-based vaccines could effectively elicit protective immune responses against diverse pathogens. Epitopes are known as the antigenic determinant, which represents the minimal immunogenic region of a protein antigen and precisely elicit specific immune responses 13. The basic premise of the epitope-based approach to vaccine development is that, in certain cases, the responses induced by the natural immunogen are not optimal, and can be improved upon by isolation or optimization of specific components of the response 14. In some cases, immunization with the entire pathogen or even infection by it may not provide sufficient protection, since the immune response they elicit is not towards protective epitopes. The use of epitope-based vaccine offers practical advantages, such as inclusion of specific protective epitopes and their exposure to the immune system, exclusion of suppressive epitopes, relative ease of construction and production, chemical stability and an avoidance of any infectious or autoimmune potential hazard 15. Vaccine design might be attempted to elicit protective antibody responses only against carefully selected pathogen epitopes which may be broadly conceptualized in terms of high-level functional immunization regimens. LITERATURE REVIEWSeveral studies worked on well-characterized determinants derived from infectious disease agents as well as showed protective immune responses of epitope-based vaccines against diverse pathogens. A multi-scale scaffolding strategy was utilized in epitope vaccine design for hepatitis C virus which exhibited promising HCV immunogens with increased avidity of antibody binding 16. A multivalent vaccine candidate against hepatitis B virus (HBV) and HCV infections was constructed carrying HBV and HCV epitopes which induced a specific cytotoxic-T-lymphocyte (CTL) reaction high-titer antibody response 17. An active area of research on epitope-based influenza vaccines aims to develop broadly protective “universal” vaccines based on conserved protein regions or peptides that are shared by all strains. An advanced-stage influenza vaccine embodying this concept is Multimeric-001®, which is a “universal” vaccine containing 9 conserved linear epitopes (both B- and T-cell epitopes) from HA, nucleoprotein and matrix protein-1 18. A novel Severe acute respiratory syndrome (SARS)-associated coronavirus-specific CTL epitope was derived from the S protein that could be a potential target for characterization and evaluation of candidate SARS vaccines 19. There were two monoclonal antibodies (mAbs) were generated against the nucleocapsid (N) protein of infectious bronchitis virus (IBV) and using phage display peptide library screening and peptide scanning two linear B-cell epitopes were recognized by the mAbs that can be useful for developing diagnostic assays for IBV infections 20. The epitope-focused recombinant protein-based malaria vaccine is a next-generation approach that successfully reached phase-III trials, and will potentially become the first commercial vaccine against a human parasitic disease 18. In an epitope-based vaccine setting, the use of conserved epitopes would be expected to provide broader protection across multiple strains, or even species, than epitopes derived from highly variable genome regions. An epitope conservancy analysis tool was developed to analyze the variability or conservation of epitopes. The tool is user friendly, and is expected to aid in the design of epitope-based vaccines and diagnostics 21. Studies used molecular docking approach to design epitope-based meningitis vaccine in silico by using polysaccharide capsule protein of S. pneumoniae, N. meningitidis serogroup A, N. meningitidis serogroup W, and H. influenzae type b and analyzed the complex stability between predicted epitopes and HLA molecules. The designed epitopes may serve as promising candidates for the development of epitope-based vaccine against the meningitis-inducing bacteria 22. Mycobacterium tuberculosis antigen epitope based recombinant BCG vaccines showed greater antigen specific proliferation, characterized with higher IFN-? response and reduced IL-4 secretion compared to the conventional BCG immunized animals 23. Immunization with a single T cell epitope derived from Salmonella secreted effector I could provide protection against lethal Salmonella infection in mice via the induction of a potent effector T cell response specific for that epitope 24. A novel cell epitope-based polypeptide, OmpC-EP, for E. coli was designed and confirmed to possess a significant immune protective function that can be useful for epitope-based vaccine development against E. coli infection 25. A potential vaccine antigen OMP26 for nontypeable Haemophilus influenzae (NTHi) has newly mapped antigenic epitopes for lymphocyte recognition where antibodies against the protein consistently yielded the greatest reactivity 26. Coccidioides challenged mice immunized with the epitope-based vaccine admixed with a synthetic oligodeoxynucleotide adjuvant or loaded into yeast glucan particles, showed early lung infiltration of activated T helper cells, elevated gamma interferon (IFN-?) and interleukin-17 production, significant reduction of fungal burden, and prolongation of survival compared to nonvaccinated mice 27. Analyses of antibody levels against defined epitopes within S. aureus antigens showed comparable immune reactivity with sera from healthy adults and infected patients, suggesting an overlapping pattern of expression of the corresponding antigens during invasive disease and during colonization and interaction without infection. The majority of the identified epitopes and almost all of the most frequently selected epitopes belonged to surface located or secreted proteins of S. aureus 28. S. aureus iron-regulated surface determinant B (IsdB) antigenic epitopes are recognized by the IsdB-specific MAbs in mice but not all of the epitopes induce protective antibodies 29. A particular mAbs among a panel of mAbs against S. aureus cell wall had weaker reactivity than that of most of the other mAbs, but had protective activity against S. aureus in mouse infection models. These indicate that the epitopes that trigger production of high-yield and/or high-affinity antibodies may not be the most suitable epitopes for developing anti-infective antibodies 30. The S. aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S. aureus vaccine and identification of protective epitopes have been reported that can be useful for the characterization of potential protective antibody responses induced by vaccines that are in clinical development 31. A protective mAb with strong specificity for GapC protein showed ability to induce macrophages to phagocytose S. aureus where the identified epitope peptide induced a protective humoral immune response against S. aureus infection in immunized mice 32. The immunodominant staphylococcal antigen A (IsaA) is a potential target for active or passive immunization against S aureus where the mapped epitope exhibited different preference of domain selection by mouse and human sera. IgGs against the C-terminal region of IsaA do not effectively protect against S. aureus infection. On the other hand, it cannot be claimed that the high titers of anti-IsaA antibodies in EB patients, which bind to the N-terminal domain of IsaA, are protective 33. HYPOTHESISDetecting protective epitope derived from cell surface antigen of S. aureus could provide the potential to design immunotherapy that could provide protection against serious staphylococcal infection via eliciting a relevant immune response specific for that epitope. An understanding of the protective epitopes of surface proteins will inform decisions on the type of antibody response necessary for protection from S. aureus challenge. Epitope-specific and protective mAbs are also important as reagents to ensure the maintenance of appropriate structural integrity of cell surface antigen during vaccine formulation.EXPERIMENTAL DESIGNThe conventional method for T cell epitopes identification involves the experimental screening of overlapping peptides in the protein of interest. Nevertheless, the development of various epitope predicting programs has facilitated the prediction of most promising epitope candidates and reduces the number of peptides selected for experimental validation. Peptide displaying phage libraries against a pool of cell surface antigen specific affinity-purified polyclonal human IgG can be panned. The selected peptides could be divided into groups of sequences depending on the represented dominant sequence clones. Binding to human affinity-purified IgG could be verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide could be chemically synthesized and antibodies affinity-purified on this peptide would bind to the cell surface molecule which can be studied by ELISA and Surface Plasmon Resonance. Furthermore, potential conformational epitopes responsible for antibody recognition could be identified by mapping phage selected peptide sequences on the surface protein using the NMR structure of the recombinant cell surface protein. Mapped epitopes can be verified by in vitro mutational analysis of the cell surface molecule. Single mutations introduced in the proposed antibody epitopes altering antibody binding to cell surface antigen can be analysed. The biological function in terms of signalling could be studied by flow cytometry. A few mutations could be carried out in order to affect the biological function as well as the antibody binding. Regarding vaccine design, even moderate successes might lead to significant progress on epitope-based vaccine efficacy against S. aureus.