Contributions
Abstract: PB2059
Type: Publication Only
Background
The complement cascade, responsible for the detection and clearance of pathogens, is activated by the classical (CP), lectin (LP) or alternative (AP) pathways, each of which can be independently activated by pathway-specific pattern recognition receptors. However, aberrant complement activation is observed in numerous diseases. While therapeutic complement inhibition at the level of C5 has proven to be a successful approach for treating various diseases, it is associated with an increased risk of infection, in particular, invasive meningococcal disease, even when vaccinating prophylactically. Targeting pathway-specific components provides the theoretical advantage of selectively inhibiting the pathway that triggers disease pathogenesis, while leaving the other pathways intact for immune surveillance.
Aims
Here we aimed to address the potential increased infection risk associated with complement pathway specific inhibition using a C1s inhibitor (TNT005; CP inhibitor), and a factor Bb inhibitor (anti-fBb; AP inhibitor). We performed in vitro bactericidal (complement-mediated) and whole blood killing assays (complement- and immune cell-mediated) to assess the relative contribution of the CP and AP in killing N. meningitidis and S. pneumoniae.
Methods
Experiments were performed using S. pneumoniae strain TIGR4 and group C N. meningitidis strain 4243. Flow cytometry was used to measure deposition of C3 and C4; pathway specific inhibitors were used to determine the relative contribution of the different pathways in depositing opsonizing complement fragments on bacteria. Bactericidal experiments in normal human plasma and whole blood killing assays containing both intact complement and phagocytes, were performed in the presence of CP and AP inhibitors (either alone or in combination). Experiments were performed in the presence or absence of capsular antibody to mimic vaccinated and non-vaccinated states, respectively.
Results
Inhibiting the CP alone using saturating concentrations of TNT005 prevented C4 deposition and killing of N. meningitidis in both normal human plasma and whole blood. However, in whole blood killing assays that contained specific anti-meningococcal antibodies, simultaneous inhibition of both the CP and AP was required to prevent killing of antibody-coated N. meningitidis. For antibody-coated S. pneumoniae, anti-fBb alone completely blocked C3 deposition, whereas TNT005 only partially inhibited (~40% decrease in fluorescence) C3 deposition. As expected, killing of S. pneumoniae was observed only in whole blood in the presence of phagocytes; blocking either the CP or AP alone did not impair killing of pneumococci in the presence of specific antibody (>90% killing at 3 h), but blocking both pathways resulted in >50% bacterial survival at 3 h.
Conclusion
The data presented here suggest that antibody-coated N. meningitidis can activate both the CP and AP of complement, and that inhibition of either pathway alone would not significantly affect N. meningitidis killing in the presence of anti-N. meningitidis antibodies as membrane attack complex mediated killing could still occur via the unblocked pathway. Antibody-mediated killing of S. pneumoniae, which requires phagocytes, also proceeded in an unimpeded manner when the CP or AP were blocked individually. These data suggest that vaccination against N. meningitidis and S. pneumoniae is critical and likely to be effective when administering a therapeutic CP or AP inhibitor.
Session topic: 31. Infectious diseases, supportive care
Keyword(s): Complement, Infection, Monoclonal antibody, Vaccination
Abstract: PB2059
Type: Publication Only
Background
The complement cascade, responsible for the detection and clearance of pathogens, is activated by the classical (CP), lectin (LP) or alternative (AP) pathways, each of which can be independently activated by pathway-specific pattern recognition receptors. However, aberrant complement activation is observed in numerous diseases. While therapeutic complement inhibition at the level of C5 has proven to be a successful approach for treating various diseases, it is associated with an increased risk of infection, in particular, invasive meningococcal disease, even when vaccinating prophylactically. Targeting pathway-specific components provides the theoretical advantage of selectively inhibiting the pathway that triggers disease pathogenesis, while leaving the other pathways intact for immune surveillance.
Aims
Here we aimed to address the potential increased infection risk associated with complement pathway specific inhibition using a C1s inhibitor (TNT005; CP inhibitor), and a factor Bb inhibitor (anti-fBb; AP inhibitor). We performed in vitro bactericidal (complement-mediated) and whole blood killing assays (complement- and immune cell-mediated) to assess the relative contribution of the CP and AP in killing N. meningitidis and S. pneumoniae.
Methods
Experiments were performed using S. pneumoniae strain TIGR4 and group C N. meningitidis strain 4243. Flow cytometry was used to measure deposition of C3 and C4; pathway specific inhibitors were used to determine the relative contribution of the different pathways in depositing opsonizing complement fragments on bacteria. Bactericidal experiments in normal human plasma and whole blood killing assays containing both intact complement and phagocytes, were performed in the presence of CP and AP inhibitors (either alone or in combination). Experiments were performed in the presence or absence of capsular antibody to mimic vaccinated and non-vaccinated states, respectively.
Results
Inhibiting the CP alone using saturating concentrations of TNT005 prevented C4 deposition and killing of N. meningitidis in both normal human plasma and whole blood. However, in whole blood killing assays that contained specific anti-meningococcal antibodies, simultaneous inhibition of both the CP and AP was required to prevent killing of antibody-coated N. meningitidis. For antibody-coated S. pneumoniae, anti-fBb alone completely blocked C3 deposition, whereas TNT005 only partially inhibited (~40% decrease in fluorescence) C3 deposition. As expected, killing of S. pneumoniae was observed only in whole blood in the presence of phagocytes; blocking either the CP or AP alone did not impair killing of pneumococci in the presence of specific antibody (>90% killing at 3 h), but blocking both pathways resulted in >50% bacterial survival at 3 h.
Conclusion
The data presented here suggest that antibody-coated N. meningitidis can activate both the CP and AP of complement, and that inhibition of either pathway alone would not significantly affect N. meningitidis killing in the presence of anti-N. meningitidis antibodies as membrane attack complex mediated killing could still occur via the unblocked pathway. Antibody-mediated killing of S. pneumoniae, which requires phagocytes, also proceeded in an unimpeded manner when the CP or AP were blocked individually. These data suggest that vaccination against N. meningitidis and S. pneumoniae is critical and likely to be effective when administering a therapeutic CP or AP inhibitor.
Session topic: 31. Infectious diseases, supportive care
Keyword(s): Complement, Infection, Monoclonal antibody, Vaccination