- Identification of polysaccharides and protein as vaccine targets
- Development of monoclonal antibodies against bacterial pathogens
- Role of bacterial surface components in virulence
Prof. Dr. med. Johannes Hübner
Nosocomial infections caused by multi-resistant bacteria are among the biggest concerns in modern medicine. Many bacterial pathogens have developed or acquired resistance against most if not all clinically available antibiotics. Therefore, we focus our research on alternative treatment therapies and prevention strategies to fight nosocomial (i.e. hospital-acquired) pathogens.
Capsular polysaccharides and extracellular proteins are the bacterial first line of defense against complement and bacterial phagocytes being therefore, good antibody targets that are used for vaccine development. Several cell-surface polysaccharides and protein structures have been proposed as potential vaccine candidates to fight bacterial pathogens. Our group has identified and characterized various surface antigens in Enterococcus faecalis and Enterococcus faecium. These bacteria are among the most important pathogens of nosocomial infections. The antigens we have found are potential vaccine candidates for the prevention and/or treatment of infections caused by these opportunistic pathogens.
We have identified several polysaccharides from E. faecalis and E. faecium that could be used as potential vaccine antigens against these pathogens and other Gram-positive antigens [1–5]. Also, we have identified several enterococcal proteins that could serve as potential vaccine candidates against enterococcal infections by the implementation of different approaches, e.g. transcriptomic and proteomic analysis [6–9].
Since polysaccharides are poorly immunogenic, chemical conjugation with a carrier protein is necessary to induce an effective immune humoral response. Following this rational, we had evaluated the potential use of proteins from E. faecium as carrier proteins conjugated with polysaccharides from E. faecalis in order to develop a cross-species vaccine with broad coverage against different enterococcal species . Our developed glycoconjugates induced specific, opsonic and protective antibodies against E. faecalis and E. faecium strains.
Synthetic glycoconjugate vaccines are currently an attractive concept due to micro-heterogeneous mixtures obtained after purification of capsular polysaccharides from bacterial cultures. In our group, we have studied synthetic structures that mimic the antigenic properties of natural polysaccharides from E. faecalis [11,12].
In addition we have described how these and other bacterial structures contribute to enterococcal pathogenesis and to virulence [13–20].
1. Huebner J, Wang Y, Krueger WA, et al. Isolation and Chemical Characterization of a Capsular Polysaccharide Antigen Shared by Clinical Isolates of Enterococcus faecalis and Vancomycin-Resistant Enterococcus faecium. Infect Immun. 1999; 67(3):1213–1219.
2. Wang Y, Huebner J, Tzianabos AO, Martirosian G, Kasper DL, Pier GB. Structure of an antigenic teichoic acid shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium. Carbohydrate Research. 1999; 316(1–4):155–160.
3. Bychowska A, Theilacker C, Czerwicka M, et al. Chemical structure of wall teichoic acid isolated from Enterococcus faecium strain U0317. Carbohydrate Research. 2011; 346:2816–2819.
4. Theilacker C, Kaczyński Z, Kropec A, et al. Serodiversity of Opsonic Antibodies against Enterococcus faecalis —Glycans of the Cell Wall Revisited. Lu S, editor. PLoS ONE. 2011; 6(3):e17839.
5. Theilacker C, Kropec A, Hammer F, et al. Protection against Staphylococcus aureus by antibody to the polyglycerolphosphate backbone of heterologous lipoteichoic acid. Journal of Infectious Diseases. 2012; 205(7):1076–1085.
6. Romero-Saavedra F, Laverde D, Wobser D, et al. Identification of peptidoglycan-associated proteins as vaccine candidates for enterococcal infections. PloS one. 2014; 9(11):e111880.
7. Romero-Saavedra F, Laverde D, Budin-Verneuil A, et al. Characterization of Two Metal Binding Lipoproteins as Vaccine Candidates for Enterococcal Infections. PloS one. Public Library of Science; 2015; 10(8):e0136625.
8. Kropec A, Sava IG, Vonend C, et al. Identification of SagA as a novel vaccine target for the prevention of Enterococcus faecium infections. Microbiology (Reading, England). 2011; 157(Pt 12):3429–34.
9. Laverde D, Probst I, Romero-Saavedra F, et al. Targeting Type IV Secretion System Proteins to Combat Multidrug-Resistant Gram-positive Pathogens. The Journal of Infectious Diseases. Springer Berlin Heidelberg, Berlin, Heidelberg; 2017; 215(12):1836–1845.
10. Romero-Saavedra F, Laverde D, Kalfopoulou E, et al. Conjugation of different immunogenic enterococcal vaccine target antigens leads to extended strain coverage. The Journal of Infectious Diseases. 2019; .
11. Hogendorf WFJ, Meeuwenoord N, Overkleeft HS, et al. Automated solid phase synthesis of teichoic acids. Chemical communications (Cambridge, England) [Internet]. 2011 [cited 2014 Nov 23]; 47(31):8961–8963. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21734985
12. Laverde D, Wobser D, Romero-Saavedra F, et al. Synthetic Teichoic Acid Conjugate Vaccine against Nosocomial Gram-Positive Bacteria. PloS one. 2014; 9(10):e110953.
13. Theilacker C, Sava IG, Sanchez-Carballo P, et al. Deletion of the glycosyltransferase bgsB of Enterococcus faecalis leads to a complete loss of glycolipids from the cell membran and to impaired biofilm formation. BMC Microbiology. 2011; 11:67.
14. Bao Y, Sakinc T, Laverde D, et al. Role of mprF1 and mprF2 in the pathogenicity of enterococcus faecalis. PLoS ONE. 2012; 7(6):e38458–e38458.
15. Theilacker C, Sanchez-Carballo P, Toma I, et al. Glycolipids are involved in biofilm accumulation and prolonged bacteraemia in Enterococcus faecalis. Molecular Microbiology. 2009; 71(4):1055–1069.
16. Fabretti F, Theilacker C, Baldassarri L, et al. Alanine esters of enterococcal lipoteichoic acid play a role in biofilm formation and resistance to antimicrobial peptides. Infection and immunity. 2006; 74(7):4164–71.
17. Diederich A-K, Wobser D, Spiess M, Sava IG, Huebner J, Sakιnç T. Role of glycolipids in the pathogenesis of Enterococcus faecalis urinary tract infection. Schaik W van, editor. PloS one. Public Library of Science; 2014; 9(5):e96295.
18. Sava IG, Zhang F, Toma I, et al. Novel interactions of glycosaminoglycans and bacterial glycolipids mediate binding of enterococci to human cells. The Journal of biological chemistry. 2009; 284(27):18194–201.
19. Sava IG, Heikens E, Kropec A, Theilacker C, Willems R, Huebner J. Enterococcal surface protein contributes to persistence in the host but is not a target of opsonic and protective antibodies in Enterococcus faecium infection. Journal of medical microbiology. 2010; 59(Pt 9):1001–4.
20. Theilacker C, Kaczyński Z, Kropec A, et al. Serodiversity of opsonic antibodies against Enterococcus faecalis--glycans of the cell wall revisited. PloS one. 2011; 6(3):e17839.
Monoclonal antibodies (mAbs) could be used as a prophylactic therapy against enterococci either as monotherapy or as a supplementary therapy with antibiotics having a synergistic effect on the eradication of enterococcal infections. We have established a pipeline (see Figure) for the discovery and development of opsonic and protective mAbs that target E. faecalis and/or E. faecium. First, we used well-characterized antigens to raise antibodies in mice. After immunization, we use the hybridoma technology and a combination of ELISA and opsonophagocytic assays for the selection process of the cells producing the desire antibodies . After the screening, we fully sequence the variable domains of the selected mAbs. To confirm the observed activity, we recombinantly produce the mAbs in eukaryotic cells and test them on the same immunological assays. Finally, we proceed with the humanization of the murine mAbs and test them in different in vitro and in vivo assays to complete their preclinical development.
21. Kalfopoulou E, Laverde D, Miklic K, et al. Development of opsonic mouse monoclonal antibodies against multidrug resistant enterococci. Infection and immunity. American Society for Microbiology Journals; 2019; :IAI.00276-19.
Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital,
Klinikum der Universität München, LMU München
Postal Address: Lindwurmstr. 4
Visiting Address: Research Building, Lindwurmstraße 2a
Room: K 0.14
Phone: +49 (0)89 - 4400 - 57712
Fax: +49 (0)89 - 4400 - 57702
Lab Manager: Diana Laverde, Ph.D.
Postal Address: Lindwurmstr. 2a
Visiting Address: Research Building, Lindwurmstraße 2a.
Room: K 0.14
Phone: +49 (0)89 - 4400 - 57712
Fax: +49 (0)89 - 4400 - 57614