Otero Carrera, Jennifer

Universitat Autònoma de Barcelona. Facultat de Biociències

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Staphylococcus aureus vaccine. Use of ISD system components in the development of a S. aureus vaccine. Jennifer Otero Carrera. Microbiology, UAB. Tutor Jordi Barbé. 15 May 2013. Background Objectives Staphylococcus aureus is a gram positive ubiquitous pathogen that [1,2] may cause a variety of diseases ranging from moderate to severe skin and soft tissue infections to serious infections. [3,4]. Over the past decade different clonal types resistant to a wide range of antibiotics have emerged, as well as methicillin resistant Staphylococcus aureus (MRSA) [4,5], and becoming the predominant strain in S. aureus infections [2]. This increased underlines the need to develop new strategies to prevent infection by S. aureus [2]. Despite the need for a S. aureus vaccine and the continuing efforts of the scientific community, clinical trials have failed [1]. This emphasizes the need to develop new vaccine strategies that provide cellular memory, particularly Th17 [2,5]. §To develop a vaccine strategy against Staphylococcus aureus that involves multiple antigens that would trigger various immune mechanisms, especially Th17-mediated response and IL17, is essential for eliminating the infection. §Mass vaccination strategy for the general public, including sectors of the population with a high risk of infection. S. aureus immunologyEven though S. aureus stimulates thehumoral response, this may not play an important role in the removal of the pathogen [6]. The main immune mechanisms required for protection against staphylococcal infections include neutrophils and T cells, which regulate the phagocytic activity [3]. Various studies establish the connection between T cells and neutrophils through the IL-17 cytokine, which is important for the recruitment and activation of neutrophils at the site of infection, increases chemotaxis and their synergistic actions with the TLR2 (Figure 1) [2,6].  Isd system Iron is essential in the pathogenesiS [2,7]. S. aureus has developed a collection system called iron-regulated surface determinants (Isd) for heme acquisition [7]. Isd system is comprised of cell wall-anchored surface proteins (IsdA, IsdB, IsdC and IsdH), a membrane transporter (IsdD, IsdE and IsdF), a transpeptidase (SrtB) and two cytoplasmatic heme-degrading monooxygenases (IsdG and IsdH) (Figure 2) [7,8].  The Joshi et al. study using a murine sepsis model demonstrated that the protein IsdB provides cellular immunity, specifically Th17. They also found pre-existing titers of antibodies against this protein in serum of various mammals [6]. This project will make a subunit vaccine consisting of Isd system surface proteins, IsdA, IsdB, IsdC and IsdH, formulated with AAHSA (amorphous aluminum hydroxyphosphate sulfate adjuvant).  Expected results The vaccine formed by the Isd system surface proteins should provide protective memory against S. aureus. The immune response must be mediated by Th17 cells, so that it is expected to increase production of the cytokine IL-17 and th17 cell in the vaccinated individuals compared to the control ones. Likewise an increase of survival in mice immunized in the lethal challenge is expected. It should also increase the titer of antibodies against proteins of the vaccine as a consequence of enhancing the T response [6,9,10]. Material and MethodsThe project provides a solution to a major problem of public health, which is the increasing infections by S.aureus, particularly multi-resistant strains. The scientific community has attempted to solve this problem from different angles, but so far has failed.  This project offers an easily constructed vaccine that steers the immune response to a cellular one, more particularly to Th17 which is essential to bacterial clearance. Moreover, the vaccine is composed of various antigens, resulting in a multivalent approach. Project Benefits 1. Richard A. Proctor. Is there a future for a Staphylococcus aureus vaccine? 2012. Vaccine. 20: 2921-2927. 2. Brad Spellberg, Robert Daum. Development of a vaccine against Staphylococcus aureus. 2012. Semin Immmunophatol. 34: 335-348. 3. Robert Daum, Brad Spellberg. Progress toward a Staphylococcus aureus vaccine. 2012. Vaccines. CID. 54: 560-567. 4. Michael Otto, Ph. D. Novel targeted immunotherapy approaches for staphylococcal infection. NIH Author Manuscript. 2010. Expert Opin Biol Ther. 10(7):1049-1059. 5. Richard A. Proctor. Challenge for a universal Staphylococcus aureus vaccine. Clinical Practice. CID. 54: 1179-1186.6. Amita Joshi, Greg Pancari, Leslie Cope, Edward P. Bowman, Daniel Cua, Richard A. Proctor, Tessie McNeely. Immunization with Staphylococcus aureus iron regulated surface determinant B (IsdB) confers protection via Th17/IL17 pathway in a murine sepsis model. 2012. Human Vaccines & Immunotherapeutics. 8(3): 336–346. 7. Andrea DeDent, Hwan Keun Kim, Dominique Missiakas, Olaf Schneewind. Exploring Staphylococcus aureus pathways to disease for vaccine development. 2012. Semin Immunopathol. 34(2): 317–333. 8. Victor J. Torres, Gleb Pishchany, Munir Humayun, Olaf Schneewind, Eric P. Skaar. Staphylococcus aureus IsdB Is a Hemoglobin Receptor Required for Heme Iron Utilization. 2006. Journal of Bacteriology. 188: 8421–8429. 9. Yukiko K. Stranger-Jones, Taeok Bae, Olaf Schneewind. Vaccine assembly from surface proteins of Staphylococcus aureus. 2006. PNAS. 103(45): 16942–16947. 10. Nelly A. Kuklin, Desmond J. Clark, Susan Secore, James Cook, Leslie D. Cope, Tessie McNeely. A Novel Staphylococcus aureus Vaccine: Iron Surface Determinant B Induces Rapid Antibody Responses in Rhesus Macaques and Specific Increased Survival in a Murine S. aureus Sepsis Model. 2006. Infection and Immunity. 74: 2215–2223. 11. Ronald W. Ellis. Technoloies for the design, discovery, formulation and administration of vaccines. 2001. Vaccine. 19: 2681–2687. 12. Fleur Samantha Benghiat, Lous Marie Charbonnier, Benoit Vokaer, Virginie De Wild, Alain Le Moine.Interleukin 17–producing T helper cells in alloimmunity. 2009. Transplantation reviews. 23(1): 11-18.  13. Jessica R. Sheldon and David E. Heinrichs. The iron-regulated staphylococcal lipoproteins. 2012. Frontiers in Cellular and Infection Microbiology. 2(41): 1-13. 14. Lloyd S. Miller, John S. Cho. Immunity against Staphylococcus aureus cutaneous infections. 2011. Nature Reviews Immunology. 11: 505-518. Bibliography Figure 1: IL-17 activates production of antimicrobial peptides, chemokines and adhesion molecules that promote the recruitment of neutrophils from the circulation to the site of infection. Neutrophils help control infection spread and are required for bacterial clearance. IL-17-mediated signalling also promotes TH17 cells recruitment [14]. Figure 2: The different Isd system components involved in heme uptake (red star) of/from hemoglobin (Hb) [13]. PCR amplify genes isdA, isdB, isdC and isdH adding restriction enzyme targets BamHI and NdeI (Figure 3) (Table 2) Separete PCR product by agarose eletrophoresis Murine sepsis model and lethal challenge Cut the plasmid pET-15b and PCR products with BamHI and NdeI Ligation (Figure 3) Transform in E. coli BL21 (DE3) by electroporation  Selection the plasmids with the insert in LB + Amp Seed the colonies in LB liquid medium until an Abs600nm = 0.8 Induce expresion of T7 RNA polymerase with IPTG for 3 hours Lyse cells by sonication Recovering chimeric proteins (Figure 3) Purify proteins that are labelled with a histidine tag in an affinity chromatography Remove histidine tail with thrombin Purify proteins by gel filtration Formulate puryfy protein with adjuvant AAHSA Vaccine: 20mg/dose antigen + 450mg/dose AAHSA Control: 450mg/dose AAHSA Inject 50 µl/doses intramuscularly to BALB/c mice on days 0, 7 and 21 (Figure 4) Day 28: perform and ELISA to determine antibody titer and a flow citometry to determine Th17 levels (Figure 4) Day 35: infect mice with lethal dose of S. aureus. Perform a  survival study of 10 days (Figure 4) Perform a statistical  analysis Antigens production After the concession of the project, undertake the necessary research and experiments to obtain viable results and to perform a patent. Submit an application to the Oficina Española de Patentes y Marcas (OEPM http://www.oepm.es/es/index.html) with international scope by applying the Patents Cooperation Treaty (PCT). Once the patent is granted, publish one or more scientific papers with the obtained results so far in one of the following scientific journals:  •Vaccine (http://www.journals.elsevier.com/vaccine/)  •Journal of Bacteriology (http://jb.asm.org/). Project dissemination Table 2: Primers IsdAF 5’- CATCATG GCAGAAAATACAAATA -3’ IsdAR 5’- GGATCCTTTACATTTTAGATTGACT-3’ IsdBF 5’- CATCATGTTATTTAGATTCTTTTCT -3’ IsdBR 5’- GGATCCATGACAAAACATTAT -3’ IsdCF 5’- CATCATGTTAGTTTTTACGTTTTCT -3’ IsdCR 5’- GGATCCATGAACAAACAGC -3’ IsdHF 5’- CATCATGTTGAAAAATATTTTAAAAG -3’ IsdHR 5’- GGATCCTTATTCCACATTGC -3’ pET-15bF 5’- GATCTCGATCCCGCGAA-3’ pET-15bR 5’- CCTCAAATGGGCTCTTAAAACC-3’ Figure 3: Sketch of genic construction used in gene amplification for the production of antigens and chimeric protein obtained. Figure 4: Murine sepsis model and lethal challenge diagram to test  the S. aureus vaccine developed in this project.  

Staphylococcus aureus vaccine : use of ISD system components in the develpment of a S. aureus vaccine

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Staphylococcus aureus vaccine : use of ISD system components in the develpment of a S. aureus vaccine

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