Development of human monoclonal antibodies against conserved antigens from Streptococcus pneumoniae

Streptococcus pneumoniae ist ein weitverbreiteter Erreger verschiedenster lebensbedrohlicher Krankheiten wie Pneumonien, Bakteriämien oder Meningitis. Obgleich lizensierte Impfstoffe und Antibiotika zur Verfügung stehen, bleibt die Morbidität und Mortalität, nicht nur in Entwicklungsländern, sondern auch in Industrieländern auf einem hohen Niveau. Aufgrund zunehmender Antibiotikaresistenzen und der suboptimalen Wirksamkeit verfügbarer Impfstoffe in Risikogruppen, steigt der Bedarf für neue Therapien, wie beispielsweise einer passiven Immunisierung mit monoklonalen Antikörpern. In dieser Arbeit wurden drei konservierte Antigene von S. pneumoniae – Lipoteichonsäure (LTA), pneumococcal surface protein A (PspA) und Pneumolysin (PLY) – auf ihre Eignung als Zielmoleküle für die Entwicklung einer anti-infektiösen Antikörper-Therapie hin untersucht. Obwohl die Isolierung und Reinigung nativer LTA für den Erreger Streptococcus pyogenes erfolgreich optimiert wurde, konnte LTA nicht in ausreichender Menge und Reinheit aus S. pneumoniae gewonnen und somit keine weiterführenden Studien mit diesem Antigen durchgeführt werden. Verschiedenste Konstrukte von PspA und PLY wurden rekombinant exprimiert, gereinigt und nachfolgend deren immunogene Wirkung in naïven C3H/HeN Mäusen demonstriert. Die gewonnenen Antiseren wurden in Folge genauestens in vitro analysiert. Oberflächenfärbungen lebender Pneumokokken zeigten, dass PspA spezifische Antikörper nicht alle PspA-Varianten detektieren konnten, sondern nur solche des selben PspA-Typs. Polyklonale Antikörper gegen Typ 1 PspA reagierten auch mit anderen Prolin-reichen Oberflächenproteinen, vermutlich PspC, jedoch nicht mit PspA Typ 2. Des weiteren wurden die generierten Antiseren in zuvor etablierten Mausmodellen durch passiven Transfer und nachfolgeneder letaler Infektion mit verschiedenen S. pneumoniae Stämmen auf ihre Wirksamkeit getestet. Die Ergebnisse korrelierten mit den Daten der Oberflächenfärbung: Anti-PspA Hyperimmunseren induzierten eine Protektion gegen Pneumokokken mit homologen jedoch nicht heterologen PspA. Anti-PLY Seren hingegen konnten keine vollständig protektive Wirkung erzielen, bewirkten jedoch eine erhöhte Überlebensrate. Die beobachtete Protektion korrelierte mit der Stärke der durch die Infektion induzierte inflammatorischen IL-6 Reaktion in den Tieren. Im Hinblick auf die Wirkungsweise von PLY wurden zwei in vitro Analysen zur Antikörper-Charakterisierung entwickelt: ein Hämolyse-Inhibitions Assay und ein hTLR4-Reporter Assay. Pneumolysin spezifische murine poly- und monoklonale Antikörper bewirkten eine Reduktion der hämolytischen Aktivität von PLY auf Erythrozyten und interferierten auch mit der Aktivierung von TLR4 durch PLY. Humane monoklonale Antikörper sollen in weiterer Folge aus humanen B-Zellen basierend auf der „Sindbis Virus Based Mammalian Cell Surface Display“ Technologie generiert werden. Zu diesem Zweck wurden gesunde Spender aufgrund ihres Antigen-spezifischen Titers im ELISA identifiziert. Zudem wurden die für die B-Zell-Selektion notwendigen PBMC-Färbetechniken optimiert. Zusammenfassend demonstriert diese Arbeit einen vertiefenden Einblick in die Wirkungsweise von PspA und PLY-spezifischen Antikörpern sowie die Entwicklung von für die Selektion und Validierung humaner monoklonaler Antikörper notwendigen Analyseverfahren. Somit konnte die Grundlage für eine Antikörper-basierende Therapie zur Prävention und Behandlung von lebensbedrohlichen Pneumokokkeninfektionen geschaffen werden.Streptococcus pneumoniae is a common human pathogen that causes a variety of life-threatening invasive diseases such as pneumonia, bacteremia and meningitis. Despite the availability of licensed vaccines and antibiotic treatments, morbidity and mortality attributed to this bacterium remain significant in developing and developed countries. Due to increasing antibiotic resistance and limited efficacy of existing vaccines in at-risk populations, there is a need for new treatment strategies such as passive immunotherapy using human monoclonal antibodies (mAbs). In this study, three conserved antigens of S. pneumoniae – lipoteichoic acid (LTA), pneumococcal surface protein A (PspA) and pneumolysin (PLY) – were characterized for their suitability as targets for a mAb-based anti-infective therapy. Although isolation and purification procedures could be optimized for LTA from Streptococcus pyogenes, native LTA could not be extracted from S. pneumoniae in sufficient quantity and quality, thus limiting more in-depth studies of this antigen. Recombinant full-length PspA and PLY as well as domains thereof were expressed, purified and subsequently proven to be highly immunogenic in naïve C3H/HeN mice. These antisera were characterized in-depth in vitro: in surface staining and in ELISA, antibodies were shown to recognize PspA in a clade-specific manner. Polyclonal antibodies against Family 1 PspA also reacted with other Proline-rich cell-surface proteins – presumably PspC – but not with Family 2 PspA. Consequently these antisera were tested in vivo by passive transfer and subsequent lethal challenge with different S. pneumoniae strains in mice. The results correlated with surface staining data: anti-PspA hyperimmune sera were only effective against pneumococci expressing homologous PspA but not against those with a heterologous variant. Anti PLY sera were not fully protective although conferring prolonged survival. Interestingly the observed protection correlated with the level of inflammatory IL-6, induced in mice. Two in vitro assays exploiting the function of PLY were set up to allow a detailed characterization of selected antibodies: a Hemolysis-Inhibition Assay and an hTLR4-Reporter Assay. PLY-specific murine polyclonal and monoclonal antibodies reduced the hemolytic activity of PLY on erythrocytes and interfered with the activation of TLR4 through PLY. Since human mAbs against PLY will be generated from B-cells based on the “Sindbis Virus Based Mammalian Cell Surface Display” technology, healthy human donors were identified based on their antibody titers in ELISA. In addition PMBC staining conditions that are required for the selection of antigen-specific memory B cells were optimized. In conclusion, a deeper insight into the mode of action of PspA- and PLY-specific antibodies could be gained with this work and analytical methods that are required for the selection and validation of human mAbs were developed. This way a basis for the development of a mAb-based therapy for the prevention and treatment of life-threatening pneumococcal diseases was established

Improved Protection in a Rabbit Model of Community-Associated Methicillin-Resistant Staphylococcus aureus Necrotizing Pneumonia upon Neutralization of Leukocidins in Addition to Alpha-Hemolysin

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), especially the USA300 pulsotype, is a frequent cause of skin and soft tissue infections and severe pneumonia. Despite appropriate antibiotic treatment, complications are common and pneumonia is associated with high mortality. S. aureus strains express multiple cytotoxins, including alpha-hemolysin (Hla) and up to five bicomponent leukocidins that specifically target phagocytic cells for lysis. CA-MRSA USA300 strains carry the genes for all six cytotoxins. Species specificity of the leukocidins greatly contributes to the ambiguity regarding their role in S. aureus pathogenesis. We performed a comparative analysis of the leukocidin susceptibility of human, rabbit, and mouse polymorphonuclear leukocytes (PMNs) to assess the translational value of mouse and rabbit S. aureus models. We found that mouse PMNs were largely resistant to LukSF-PV, HlgAB, and HlgCB and susceptible only to LukED, whereas rabbit and human PMNs were highly sensitive to all these cytotoxins. In the rabbit pneumonia model with a USA300 CA-MRSA strain, passive immunization with a previously identified human monoclonal antibody (MAb), Hla-F#5, which cross-neutralizes Hla, LukSF-PV, HlgAB, HlgCB, and LukED, provided full protection, whereas an Hla-specific MAb was only partially protective. In the mouse USA300 CA-MRSA pneumonia model, both types of antibodies demonstrated full protection, suggesting that Hla, but not leukocidin(s), is the principal virulence determinant in mice. As the rabbit recapitulates the high susceptibility to leukocidins characteristic of humans, this species represents a valuable model for assessing novel, cytotoxin-targeting anti-S. aureus therapeutic approaches

Improved Protection in a Rabbit Model of Community-Associated Methicillin-Resistant Staphylococcus aureus Necrotizing Pneumonia upon Neutralization of Leukocidins in Addition to Alpha-Hemolysin

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), especially the USA300 pulsotype, is a frequent cause of skin and soft tissue infections and severe pneumonia. Despite appropriate antibiotic treatment, complications are common and pneumonia is associated with high mortality. S. aureus strains express multiple cytotoxins, including alpha-hemolysin (Hla) and up to five bicomponent leukocidins that specifically target phagocytic cells for lysis. CA-MRSA USA300 strains carry the genes for all six cytotoxins. Species specificity of the leukocidins greatly contributes to the ambiguity regarding their role in S. aureus pathogenesis. We performed a comparative analysis of the leukocidin susceptibility of human, rabbit, and mouse polymorphonuclear leukocytes (PMNs) to assess the translational value of mouse and rabbit S. aureus models. We found that mouse PMNs were largely resistant to LukSF-PV, HlgAB, and HlgCB and susceptible only to LukED, whereas rabbit and human PMNs were highly sensitive to all these cytotoxins. In the rabbit pneumonia model with a USA300 CA-MRSA strain, passive immunization with a previously identified human monoclonal antibody (MAb), Hla-F#5, which cross-neutralizes Hla, LukSF-PV, HlgAB, HlgCB, and LukED, provided full protection, whereas an Hla-specific MAb was only partially protective. In the mouse USA300 CA-MRSA pneumonia model, both types of antibodies demonstrated full protection, suggesting that Hla, but not leukocidin(s), is the principal virulence determinant in mice. As the rabbit recapitulates the high susceptibility to leukocidins characteristic of humans, this species represents a valuable model for assessing novel, cytotoxin-targeting anti-S. aureus therapeutic approaches

IgE Effector Mechanisms, in Concert with Mast Cells, Contribute to Acquired Host Defense against Staphylococcus aureus

International audienceIn our article entitled “IgE effector mechanisms, in concert with mast cells, contribute to acquired host defense against Staphylococcus aureus”, the following incorrect statement appears in the STAR Methods section under the subheading “Mass Spectrometry Data Analysis”: “Searches were performed with full tryptic digestion against the human SwissProt database v2017.06 (20456 sequences and appended known contaminants) with up to two miscleavage sites.” The correct sentence should read: “Searches were performed with full tryptic digestion against the mouse SwissProt database v2017.12 (25293 sequences and appended known contaminants) with up to two miscleavage sites.” The authors apologize for any confusion that may have arisen as a result of this error