Characterization of Staphylococcus aureus antigen-specific antibodies and application of their epitopes as active vaccine against MRSA
Characterization of Staphylococcus aureus antigen-specific antibodies and application of their epitopes as active vaccine against MRSA
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DissertationPrüfungsdatum
31.05.2017Datum der Veröffentlichung
21.09.2017Erstgutachter
Krönke, MartinZweitgutachter
Sahl, Hans-GeorgMetadaten
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Inhalt
Patients infected with the life-threatening, methicillin-resistant, human pathogen Staphylococcus aureus (MRSA) can be treated only with antibiotics of last resort. The development of a vaccine as an alternative to antibiotics is therefore of great clinical importance to fight MRSA.
Based on a S. aureus proteome screen with S. aureus specific human antiserum, novel surface-associated proteins were identified as vaccine candidates. Proto-porphyrinogen oxidase (pOxi), Triosephosphate isomerase (Triiso) and the hypo-thetical protein 2160 (hp2160) provided protection in a murine sepsis model upon active immunization. Monoclonal antibodies (moAbs) against these target proteins were generated of which three demonstrated significant, repetitive, epitope-specific protection against S. aureus infection in mice.
The present work focused on the characterization of these protective moAbs and their epitopes. Binding of these moAbs to a preparation of cell wall-associated proteins as well as to the S. aureus surface depended on in vitro growth conditions. For binding of anti-Triiso moAb H8 and anti-hp2160 moAb 16-2 to the bacterial surface, biofilm growth of S. aureus was essential. Anti-pOxi moAb D3 showed a surface binding also after planktonic growth. In addition to biofilm conditions it was detected that the pH of the surrounding medium influences the occurrence of Triiso and the epitope accessibility for moAb H8 on the cell surface whereas it does not influence the pOxi presence. Triiso was also identified as exoenzyme in the medium supernatant and it was shown that the surface of S. aureus can be spiked with exogenous Triiso and pOxi.
The moAbs’ mode of action was investigated first with regard to in vitro opsonophagocytosis but we did not observe this. An enzymatic activity of recombinant Triiso was measured, as well as an enzymatic activity of native Triiso in the cell wall-associated protein fraction and cell lysate. Furthermore, an interaction between Triiso and human plasminogen was demonstrated. These measurements were not influenced by moAb H8. Additional experiments with other blood compounds have to be performed to investigate this further.
Using deletion mutants, an impaired in vitro growth of ΔpOxi, but not Δhp2160, compared to wildtype was demonstrated. In the mouse infection model the virulence of ΔpOxi was also impaired, highlighting pOxi as a potential virulence factor for S. aureus. Moreover, we succeeded to show that the protein pOxi contributes to host cell adherence. This unexpected moonlighting function was identified using epithelial and endothelial cells and demonstrated that the addition of moAb D3 leads to a detachment of pOxi from these cells. Furthermore, it was detected that exogenous pOxi increases the binding of S. aureus to HeLa cells and that this increased binding was inhibited by our moAb D3.
The identified epitopes of the three moAbs were further characterized and used in different approaches for active immunization of mice. Using the pOxi- and hp2160-epitope-peptides for immunization, an antigen-specific IgG response and a significant higher bacterial clearance of organs after S. aureus challenge was measured. It was also demonstrated that the protection is epitope-specific because another epitope of Triiso, derived from a non-protective anti-Triiso antibody, triggered an IgG-response in immunized mice but failed to show protective efficacy after S. aureus challenge. Out of several mono- and multivalent epitope fusion peptides the pOxi epitope peptide and a pOxi and Triiso diepitope fusion peptide demonstrated to be the best candidates, providing significant protection upon lethal MRSA challenge. Additionally, the T-cell response was analyzed upon immunization with the fusion epitope peptides. The exposure of isolated splenocytes to unconjugated fusion peptides led to an increased cell proliferation and to a strong IFN-γ (Th1 response) and IL-17 release (Th17), described to be important to combat S. aureus infection.
The triepitope construct was also used as part of MVA and AAV vaccine vectors. Vaccination with rMVA-HA-Triepitope resulted in a strong humoral immune response. In addition to the viral genome encoding the triepitope peptide, the triepitope was also fused to a viral protein, which supports the integration into the membrane of the host cell. Vaccination with rAAV encoding the triepitope peptide in addition to a triepitope fusion with a viral capsid protein induced just a moderate IgG titer.
The achieved results gave new insights in the moonlighting functions and surface occurrence of our vaccine candidates and in the mode of action of the generated, protective antibodies. The successful peptide vaccination underlines the feasibility of such a “reverse immunology”-approach and therefore the importance of differentiating between protective and non-protective epitopes.
Based on a S. aureus proteome screen with S. aureus specific human antiserum, novel surface-associated proteins were identified as vaccine candidates. Proto-porphyrinogen oxidase (pOxi), Triosephosphate isomerase (Triiso) and the hypo-thetical protein 2160 (hp2160) provided protection in a murine sepsis model upon active immunization. Monoclonal antibodies (moAbs) against these target proteins were generated of which three demonstrated significant, repetitive, epitope-specific protection against S. aureus infection in mice.
The present work focused on the characterization of these protective moAbs and their epitopes. Binding of these moAbs to a preparation of cell wall-associated proteins as well as to the S. aureus surface depended on in vitro growth conditions. For binding of anti-Triiso moAb H8 and anti-hp2160 moAb 16-2 to the bacterial surface, biofilm growth of S. aureus was essential. Anti-pOxi moAb D3 showed a surface binding also after planktonic growth. In addition to biofilm conditions it was detected that the pH of the surrounding medium influences the occurrence of Triiso and the epitope accessibility for moAb H8 on the cell surface whereas it does not influence the pOxi presence. Triiso was also identified as exoenzyme in the medium supernatant and it was shown that the surface of S. aureus can be spiked with exogenous Triiso and pOxi.
The moAbs’ mode of action was investigated first with regard to in vitro opsonophagocytosis but we did not observe this. An enzymatic activity of recombinant Triiso was measured, as well as an enzymatic activity of native Triiso in the cell wall-associated protein fraction and cell lysate. Furthermore, an interaction between Triiso and human plasminogen was demonstrated. These measurements were not influenced by moAb H8. Additional experiments with other blood compounds have to be performed to investigate this further.
Using deletion mutants, an impaired in vitro growth of ΔpOxi, but not Δhp2160, compared to wildtype was demonstrated. In the mouse infection model the virulence of ΔpOxi was also impaired, highlighting pOxi as a potential virulence factor for S. aureus. Moreover, we succeeded to show that the protein pOxi contributes to host cell adherence. This unexpected moonlighting function was identified using epithelial and endothelial cells and demonstrated that the addition of moAb D3 leads to a detachment of pOxi from these cells. Furthermore, it was detected that exogenous pOxi increases the binding of S. aureus to HeLa cells and that this increased binding was inhibited by our moAb D3.
The identified epitopes of the three moAbs were further characterized and used in different approaches for active immunization of mice. Using the pOxi- and hp2160-epitope-peptides for immunization, an antigen-specific IgG response and a significant higher bacterial clearance of organs after S. aureus challenge was measured. It was also demonstrated that the protection is epitope-specific because another epitope of Triiso, derived from a non-protective anti-Triiso antibody, triggered an IgG-response in immunized mice but failed to show protective efficacy after S. aureus challenge. Out of several mono- and multivalent epitope fusion peptides the pOxi epitope peptide and a pOxi and Triiso diepitope fusion peptide demonstrated to be the best candidates, providing significant protection upon lethal MRSA challenge. Additionally, the T-cell response was analyzed upon immunization with the fusion epitope peptides. The exposure of isolated splenocytes to unconjugated fusion peptides led to an increased cell proliferation and to a strong IFN-γ (Th1 response) and IL-17 release (Th17), described to be important to combat S. aureus infection.
The triepitope construct was also used as part of MVA and AAV vaccine vectors. Vaccination with rMVA-HA-Triepitope resulted in a strong humoral immune response. In addition to the viral genome encoding the triepitope peptide, the triepitope was also fused to a viral protein, which supports the integration into the membrane of the host cell. Vaccination with rAAV encoding the triepitope peptide in addition to a triepitope fusion with a viral capsid protein induced just a moderate IgG titer.
The achieved results gave new insights in the moonlighting functions and surface occurrence of our vaccine candidates and in the mode of action of the generated, protective antibodies. The successful peptide vaccination underlines the feasibility of such a “reverse immunology”-approach and therefore the importance of differentiating between protective and non-protective epitopes.
Schlagwörter
MRSA, S. aureus, Vaccine, Monoclonal Antibodies, Epitopes, Immunization
Klassifikation (DDC)
570 Biowissenschaften, Biologie 610 Medizin, GesundheitNicolai, Anne Kristin: Characterization of Staphylococcus aureus antigen-specific antibodies and application of their epitopes as active vaccine against MRSA. - Bonn, 2017. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-48506
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-48506
@phdthesis{handle:20.500.11811/7271,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-48506,
author = {{Anne Kristin Nicolai}},
title = {Characterization of Staphylococcus aureus antigen-specific antibodies and application of their epitopes as active vaccine against MRSA},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2017,
month = sep,
note = {Patients infected with the life-threatening, methicillin-resistant, human pathogen Staphylococcus aureus (MRSA) can be treated only with antibiotics of last resort. The development of a vaccine as an alternative to antibiotics is therefore of great clinical importance to fight MRSA.
Based on a S. aureus proteome screen with S. aureus specific human antiserum, novel surface-associated proteins were identified as vaccine candidates. Proto-porphyrinogen oxidase (pOxi), Triosephosphate isomerase (Triiso) and the hypo-thetical protein 2160 (hp2160) provided protection in a murine sepsis model upon active immunization. Monoclonal antibodies (moAbs) against these target proteins were generated of which three demonstrated significant, repetitive, epitope-specific protection against S. aureus infection in mice.
The present work focused on the characterization of these protective moAbs and their epitopes. Binding of these moAbs to a preparation of cell wall-associated proteins as well as to the S. aureus surface depended on in vitro growth conditions. For binding of anti-Triiso moAb H8 and anti-hp2160 moAb 16-2 to the bacterial surface, biofilm growth of S. aureus was essential. Anti-pOxi moAb D3 showed a surface binding also after planktonic growth. In addition to biofilm conditions it was detected that the pH of the surrounding medium influences the occurrence of Triiso and the epitope accessibility for moAb H8 on the cell surface whereas it does not influence the pOxi presence. Triiso was also identified as exoenzyme in the medium supernatant and it was shown that the surface of S. aureus can be spiked with exogenous Triiso and pOxi.
The moAbs’ mode of action was investigated first with regard to in vitro opsonophagocytosis but we did not observe this. An enzymatic activity of recombinant Triiso was measured, as well as an enzymatic activity of native Triiso in the cell wall-associated protein fraction and cell lysate. Furthermore, an interaction between Triiso and human plasminogen was demonstrated. These measurements were not influenced by moAb H8. Additional experiments with other blood compounds have to be performed to investigate this further.
Using deletion mutants, an impaired in vitro growth of ΔpOxi, but not Δhp2160, compared to wildtype was demonstrated. In the mouse infection model the virulence of ΔpOxi was also impaired, highlighting pOxi as a potential virulence factor for S. aureus. Moreover, we succeeded to show that the protein pOxi contributes to host cell adherence. This unexpected moonlighting function was identified using epithelial and endothelial cells and demonstrated that the addition of moAb D3 leads to a detachment of pOxi from these cells. Furthermore, it was detected that exogenous pOxi increases the binding of S. aureus to HeLa cells and that this increased binding was inhibited by our moAb D3.
The identified epitopes of the three moAbs were further characterized and used in different approaches for active immunization of mice. Using the pOxi- and hp2160-epitope-peptides for immunization, an antigen-specific IgG response and a significant higher bacterial clearance of organs after S. aureus challenge was measured. It was also demonstrated that the protection is epitope-specific because another epitope of Triiso, derived from a non-protective anti-Triiso antibody, triggered an IgG-response in immunized mice but failed to show protective efficacy after S. aureus challenge. Out of several mono- and multivalent epitope fusion peptides the pOxi epitope peptide and a pOxi and Triiso diepitope fusion peptide demonstrated to be the best candidates, providing significant protection upon lethal MRSA challenge. Additionally, the T-cell response was analyzed upon immunization with the fusion epitope peptides. The exposure of isolated splenocytes to unconjugated fusion peptides led to an increased cell proliferation and to a strong IFN-γ (Th1 response) and IL-17 release (Th17), described to be important to combat S. aureus infection.
The triepitope construct was also used as part of MVA and AAV vaccine vectors. Vaccination with rMVA-HA-Triepitope resulted in a strong humoral immune response. In addition to the viral genome encoding the triepitope peptide, the triepitope was also fused to a viral protein, which supports the integration into the membrane of the host cell. Vaccination with rAAV encoding the triepitope peptide in addition to a triepitope fusion with a viral capsid protein induced just a moderate IgG titer.
The achieved results gave new insights in the moonlighting functions and surface occurrence of our vaccine candidates and in the mode of action of the generated, protective antibodies. The successful peptide vaccination underlines the feasibility of such a “reverse immunology”-approach and therefore the importance of differentiating between protective and non-protective epitopes.},
url = {https://hdl.handle.net/20.500.11811/7271}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-48506,
author = {{Anne Kristin Nicolai}},
title = {Characterization of Staphylococcus aureus antigen-specific antibodies and application of their epitopes as active vaccine against MRSA},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2017,
month = sep,
note = {Patients infected with the life-threatening, methicillin-resistant, human pathogen Staphylococcus aureus (MRSA) can be treated only with antibiotics of last resort. The development of a vaccine as an alternative to antibiotics is therefore of great clinical importance to fight MRSA.
Based on a S. aureus proteome screen with S. aureus specific human antiserum, novel surface-associated proteins were identified as vaccine candidates. Proto-porphyrinogen oxidase (pOxi), Triosephosphate isomerase (Triiso) and the hypo-thetical protein 2160 (hp2160) provided protection in a murine sepsis model upon active immunization. Monoclonal antibodies (moAbs) against these target proteins were generated of which three demonstrated significant, repetitive, epitope-specific protection against S. aureus infection in mice.
The present work focused on the characterization of these protective moAbs and their epitopes. Binding of these moAbs to a preparation of cell wall-associated proteins as well as to the S. aureus surface depended on in vitro growth conditions. For binding of anti-Triiso moAb H8 and anti-hp2160 moAb 16-2 to the bacterial surface, biofilm growth of S. aureus was essential. Anti-pOxi moAb D3 showed a surface binding also after planktonic growth. In addition to biofilm conditions it was detected that the pH of the surrounding medium influences the occurrence of Triiso and the epitope accessibility for moAb H8 on the cell surface whereas it does not influence the pOxi presence. Triiso was also identified as exoenzyme in the medium supernatant and it was shown that the surface of S. aureus can be spiked with exogenous Triiso and pOxi.
The moAbs’ mode of action was investigated first with regard to in vitro opsonophagocytosis but we did not observe this. An enzymatic activity of recombinant Triiso was measured, as well as an enzymatic activity of native Triiso in the cell wall-associated protein fraction and cell lysate. Furthermore, an interaction between Triiso and human plasminogen was demonstrated. These measurements were not influenced by moAb H8. Additional experiments with other blood compounds have to be performed to investigate this further.
Using deletion mutants, an impaired in vitro growth of ΔpOxi, but not Δhp2160, compared to wildtype was demonstrated. In the mouse infection model the virulence of ΔpOxi was also impaired, highlighting pOxi as a potential virulence factor for S. aureus. Moreover, we succeeded to show that the protein pOxi contributes to host cell adherence. This unexpected moonlighting function was identified using epithelial and endothelial cells and demonstrated that the addition of moAb D3 leads to a detachment of pOxi from these cells. Furthermore, it was detected that exogenous pOxi increases the binding of S. aureus to HeLa cells and that this increased binding was inhibited by our moAb D3.
The identified epitopes of the three moAbs were further characterized and used in different approaches for active immunization of mice. Using the pOxi- and hp2160-epitope-peptides for immunization, an antigen-specific IgG response and a significant higher bacterial clearance of organs after S. aureus challenge was measured. It was also demonstrated that the protection is epitope-specific because another epitope of Triiso, derived from a non-protective anti-Triiso antibody, triggered an IgG-response in immunized mice but failed to show protective efficacy after S. aureus challenge. Out of several mono- and multivalent epitope fusion peptides the pOxi epitope peptide and a pOxi and Triiso diepitope fusion peptide demonstrated to be the best candidates, providing significant protection upon lethal MRSA challenge. Additionally, the T-cell response was analyzed upon immunization with the fusion epitope peptides. The exposure of isolated splenocytes to unconjugated fusion peptides led to an increased cell proliferation and to a strong IFN-γ (Th1 response) and IL-17 release (Th17), described to be important to combat S. aureus infection.
The triepitope construct was also used as part of MVA and AAV vaccine vectors. Vaccination with rMVA-HA-Triepitope resulted in a strong humoral immune response. In addition to the viral genome encoding the triepitope peptide, the triepitope was also fused to a viral protein, which supports the integration into the membrane of the host cell. Vaccination with rAAV encoding the triepitope peptide in addition to a triepitope fusion with a viral capsid protein induced just a moderate IgG titer.
The achieved results gave new insights in the moonlighting functions and surface occurrence of our vaccine candidates and in the mode of action of the generated, protective antibodies. The successful peptide vaccination underlines the feasibility of such a “reverse immunology”-approach and therefore the importance of differentiating between protective and non-protective epitopes.},
url = {https://hdl.handle.net/20.500.11811/7271}
}
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