Periodic Reporting for period 1 - EpIg Men (The Role of Antibodies (Ig) and the Respiratory Epithelium in the Prevention of Invasive Meningococcal Infections in Different Age Groups)
Reporting period: 2019-07-01 to 2021-06-30
Meningococci causing invasive meningococcal disease (IMD) have distinct genetic signatures, referred to as clonal complexes or sequence types, compared to non-invasive meningococci found in carriage studies. A well-known clonal complex associated with IMD is cc11. In this project we aim to elucidate how distinct genetic lineages differ in their ability to infect the cells that form the wall of the upper respiratory tract, the epithelial cells. Upon infection and crossing the epithelial barrier the complement system is the main line of defense against meningococci. Therefore we also aimed to investigate whether genetic lineages vary in their resistant to complement-mediated killing. Antibodies enhance the ability of complement to kill meningococci. Therefore antibody levels and the ability of vaccine-induced antibodies to promote complement-mediated killing is studied.
Genetic analyses of invasive and carriage isolates
Clinical (n=56) and carriage (n=20) meningococcal isolates were selected based on diverse clinical presentation and genetic composition (serogroup and clonal complex). Whole genome sequences of these isolates were obtained and isolates were analysed in a spanning tree to identify genetic distances. Some clusters with only invasive isolates were observed indicating clonal relatedness. Also clusters consisting of invasive and carriage isolates appeared in the tree, which indicates that limited numbers of genes may be responsible for invasiveness. Several branches consisted of the same clonal complex only. Also branches consisting of more than one clonal complex appeared which indicates that the clonal complexes have additional underlying heterogeneity.
Interaction of invasive and carriage isolates with respiratory epithelium.
Meningococcal isolates were allowed to bind to two different epithelial cell lines (cells that form the wall of the respiratory tract): Calu-3 and RPMI2650 to reveal differences in binding ability of the various isolates. Binding to these cells varied within both serogroup and clonal complex. However, cell lines failed to reliably predict binding to primary epithelial cells. Therefore, the more advanced and time-consuming primary cell cultures need to be used in future studies. Different meningococcal isolates varied in their ability to bind to the primary nasal epithelial cells. The un-encapsulated meningococcus showed the strongest binding after 2 hours. After 24 hours bacterial numbers still varied between the isolates, with varying growth rates of cc11 isolates.
Meningococci colonize the upper respiratory tract, for which binding to the epithelial cells that from the wall of the respiratory tract is needed. Pilot experiments using cell lines to study infection of epithelial cells with meningococci showed no responses for RPMI2560 cells. And since binding data using epithelial cell lines did not properly predict primary epithelial cell responses, cytokine and chemokine production was studied using primary nasal epithelial cells obtained from 7 different donors. Both invasive and non-invasive isolates induced similar levels of production of cytokines and chemokines. In addition to studying cytokine and chemokine production, binding to the cells and barrier changes were studied. The strongest binding was observed for an un-encapsulated (lacking the polysaccharide capsule) meningococcus, but faster growth rates of invasive bacteria. Infection of epithelial cells resulted in a partial loss of the mechanical epithelial barrier.
Meningococcus protein-specific antibody levels
Young children had low levels of IgG antibodies to unencapsulated meningococci with different genetic makeup. These antibodies are directed against proteins of the bacterium, in contrast to polysaccharide-capsule-specific antibodies as induced by vaccination with MenACWY vaccine. Levels peaked in adolescents and then declined again with increasing age. Interestingly, the proportion of individuals with IgM antibodies declined with increasing age, which was not observed for IgG.
Agglutinate of bacteria by antibodies
Antibodies are capable of binding to the meningococci and agglutinating the bacteria, a mechanisms postulated to hinder interaction of the bacteria with host cells. Mainly IgG followed by IgM seemed implicated in agglutinating the bacteria. The proportion of agglutinated un-encapsulated meningococci did not vary with age.
The addition of serum together with bacteria only reduced the number of bacteria attached to epithelial cells when low (bacteria per cells, multiplicity of infection [MOI] of 1) numbers of bacteria were used. With higher numbers of bacteria added (MOI 10) increased binding of meningococci to the cells was observed in the presence of suboptimal concentrations of serum. This suggests that high degrees of opsonization is needed to prevent adherence of the meningococci to epithelial cells. The number of meningococci that entered the cells was reduced when serum was added.
Resistance of invasive clinical isolates to complement mediated killing
Sera from 11 non-vaccinated adults were pooled and used to test all invasive (n=56) and carriage (n=20) isolates. Various carriage isolates were very sensitive to killing whereas some carriage isolates showed resistance to killing. All invasive isolates showed moderate to high resistance to complement-mediated killing. This resistance was partly reflected in the spanning tree mentioned above. All carriage and invasive isolates were killed when sera of vaccinated persons was used.
Dissemination and Communication
- Throughout the project findings were shared within the cross-disciplinary ream of the national Institute for Public Health and the Environment. This meeting is attended by immunologist, microbiologist, epidemiologist and infectious disease control staff.
- Results have been reported in the annual Dutch national Immunization Report 2021.
- A press release shared on social media (twitter and LinkedIn) and the RIVM webpage was published (2022) describing complement resistance by clinically invasive isolates and the effectiveness of vaccine-induced antibodies to overcome this resistance.
- Data are presented on scientific conferences of basic scientist (ICMI) and translational scientists (ISPPD, EMGM, IPNC). Attendance: DSI (250), ICMI (350 attendants), ISPPD (1000 delegates), IPNC (450)
- A preprint is published in Medrxiv (https://doi.org/10.1101/2022.05.20.22275303(opens in new window)) and submitted to a journal and a second manuscript is being prepared.
Clinical (n=56) and carriage (n=20) meningococcal isolates were selected based on diverse clinical presentation and genetic composition (serogroup and clonal complex). Whole genome sequences of these isolates were obtained and isolates were analysed in a spanning tree to identify genetic distances. Some clusters with only invasive isolates were observed indicating clonal relatedness. Also clusters consisting of invasive and carriage isolates appeared in the tree, which indicates that limited numbers of genes may be responsible for invasiveness. Several branches consisted of the same clonal complex only. Also branches consisting of more than one clonal complex appeared which indicates that the clonal complexes have additional underlying heterogeneity.
Interaction of invasive and carriage isolates with respiratory epithelium.
Meningococcal isolates were allowed to bind to two different epithelial cell lines (cells that form the wall of the respiratory tract): Calu-3 and RPMI2650 to reveal differences in binding ability of the various isolates. Binding to these cells varied within both serogroup and clonal complex. However, cell lines failed to reliably predict binding to primary epithelial cells. Therefore, the more advanced and time-consuming primary cell cultures need to be used in future studies. Different meningococcal isolates varied in their ability to bind to the primary nasal epithelial cells. The un-encapsulated meningococcus showed the strongest binding after 2 hours. After 24 hours bacterial numbers still varied between the isolates, with varying growth rates of cc11 isolates.
Meningococci colonize the upper respiratory tract, for which binding to the epithelial cells that from the wall of the respiratory tract is needed. Pilot experiments using cell lines to study infection of epithelial cells with meningococci showed no responses for RPMI2560 cells. And since binding data using epithelial cell lines did not properly predict primary epithelial cell responses, cytokine and chemokine production was studied using primary nasal epithelial cells obtained from 7 different donors. Both invasive and non-invasive isolates induced similar levels of production of cytokines and chemokines. In addition to studying cytokine and chemokine production, binding to the cells and barrier changes were studied. The strongest binding was observed for an un-encapsulated (lacking the polysaccharide capsule) meningococcus, but faster growth rates of invasive bacteria. Infection of epithelial cells resulted in a partial loss of the mechanical epithelial barrier.
Meningococcus protein-specific antibody levels
Young children had low levels of IgG antibodies to unencapsulated meningococci with different genetic makeup. These antibodies are directed against proteins of the bacterium, in contrast to polysaccharide-capsule-specific antibodies as induced by vaccination with MenACWY vaccine. Levels peaked in adolescents and then declined again with increasing age. Interestingly, the proportion of individuals with IgM antibodies declined with increasing age, which was not observed for IgG.
Agglutinate of bacteria by antibodies
Antibodies are capable of binding to the meningococci and agglutinating the bacteria, a mechanisms postulated to hinder interaction of the bacteria with host cells. Mainly IgG followed by IgM seemed implicated in agglutinating the bacteria. The proportion of agglutinated un-encapsulated meningococci did not vary with age.
The addition of serum together with bacteria only reduced the number of bacteria attached to epithelial cells when low (bacteria per cells, multiplicity of infection [MOI] of 1) numbers of bacteria were used. With higher numbers of bacteria added (MOI 10) increased binding of meningococci to the cells was observed in the presence of suboptimal concentrations of serum. This suggests that high degrees of opsonization is needed to prevent adherence of the meningococci to epithelial cells. The number of meningococci that entered the cells was reduced when serum was added.
Resistance of invasive clinical isolates to complement mediated killing
Sera from 11 non-vaccinated adults were pooled and used to test all invasive (n=56) and carriage (n=20) isolates. Various carriage isolates were very sensitive to killing whereas some carriage isolates showed resistance to killing. All invasive isolates showed moderate to high resistance to complement-mediated killing. This resistance was partly reflected in the spanning tree mentioned above. All carriage and invasive isolates were killed when sera of vaccinated persons was used.
Dissemination and Communication
- Throughout the project findings were shared within the cross-disciplinary ream of the national Institute for Public Health and the Environment. This meeting is attended by immunologist, microbiologist, epidemiologist and infectious disease control staff.
- Results have been reported in the annual Dutch national Immunization Report 2021.
- A press release shared on social media (twitter and LinkedIn) and the RIVM webpage was published (2022) describing complement resistance by clinically invasive isolates and the effectiveness of vaccine-induced antibodies to overcome this resistance.
- Data are presented on scientific conferences of basic scientist (ICMI) and translational scientists (ISPPD, EMGM, IPNC). Attendance: DSI (250), ICMI (350 attendants), ISPPD (1000 delegates), IPNC (450)
- A preprint is published in Medrxiv (https://doi.org/10.1101/2022.05.20.22275303(opens in new window)) and submitted to a journal and a second manuscript is being prepared.
Data studying the interaction of meningococci wit primary human epithelial cells are scarce. This project shows distinct interaction of meningococci and primary cells compared to epithelial cell lines, indicating the importance of using these more advanced models.
Since multiple recent clinical isolates with distinct genetic characteristics were, data are relevant to currently circulating isolates. The epithelial cells from all donors were able to sense the presence of meningococci and initiated an immune response. However, the response varied between donors and isolates, implicating an interaction between bacterial and host characteristics for mucosal infections.
This is one of few studies that compared multiple clinically invasive isolates for resistance to complement on how these very same bacteria are killed through complement activation by vaccine-induced antibodies.
Since multiple recent clinical isolates with distinct genetic characteristics were, data are relevant to currently circulating isolates. The epithelial cells from all donors were able to sense the presence of meningococci and initiated an immune response. However, the response varied between donors and isolates, implicating an interaction between bacterial and host characteristics for mucosal infections.
This is one of few studies that compared multiple clinically invasive isolates for resistance to complement on how these very same bacteria are killed through complement activation by vaccine-induced antibodies.