Bordetella pertussis Professor Alison Weiss

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1 Department of Molecular Genetics University of Cincinnati 1 : the causative agent of Whooping Cough This disease represents a medical success story...and underscores the limits of medical success 2 Whooping cough (or Pertussis) Estimated 3, deaths annually worldwide Highly contagious respiratory infection caused by the Gram negative bacterium, Transmission respiratory droplets Only humans are susceptible 3 1

2 Whooping cough case presentation This is an 18-month old girl; she did not receive her childhood immunizations because her parents were afraid of adverse effects ; her teenage brother, who was vaccinated, had a slight cough for the last three weeks; she developed what her parents described as a mild cold, but five days ago she began to experience periodic choking, progressing to turning red and gasping for breath; she vomited thick mucus in association with the choking spells; her white cell count was elevated with a predominance of lymphocytes The parents were informed that it was unlikely that the organism could be successfully cultured at this late stage in the disease and antibiotic therapy would not alleviate her symptoms; they decided not to allow her to be cultured, saying she had been through enough; the older brother agreed to be cultured; a nasopharyngeal exudate was collected by swap and Bordetella pertussis was recovered 4 Pertussis the a medical limits of success medical success story 25, Pertussis vaccine introduced s Cases 2, 15, 1, Vaccination lead to a 95.7% reduction in cases 5, ear 5 Pertussis United States, Cas ses Increase in incidence from low in 198s ear 6 2

3 Pertussis is cyclic (like influenza), and epidemics occur every 3 5 years 3 Influenza Case es Deaths ear 7 Pertussis vaccine Pertussis vaccine protects from severe disease better than carriage 8 1 Pertussis vaccine efficacy following household exposure* Pe ercent symp ptomatic Unvaccinated Vaccinated Days of coughing *Adapted from: Storsaeter et al., Vaccine 16:197 (1998) 9 3

4 Pertussis vaccine Pertussis vaccine protects from severe disease better than carriage Promotes little herd immunity, and little protection from transmission 1 Cases Pertussis United States Incidence Haemophilus influenzae United States Pertussis vaccine prevents disease, not carriage Incidence of disease is increasing, even with high vaccination rates Hib vaccine prevents carriage and disease Due to herd immunity, the incidence of invasive disease has declined dramatically 11 Pertussis vaccine Pertussis vaccine protects from severe disease better than carriage Promotes little herd immunity, and little protection from transmission Immunization does not yield life-long immunity, which may account for more frequent occurrence in teenagers and adults today 12 4

5 Pertussis by age group Accounts for up to 7% of cough illnesses per year Disease often milder than in infants and children Adults often source of infection for children No. of reported cases 2 Wide age distribution 19 yrs 15 Pediatric disease yrs 5 < 11 yrs ear 13 Whooping cough Human to human transmission by respiratory droplets Bacteria remain localized to upper respiratory tract attached to ciliated cells 14 Classical whooping cough Catarrhal Lasts 1-3 weeks Localized Symptoms: Fever, cough, runny nose Disease Process: Growth of bacteria in respiratory tract Paroxysmal Lasts 2-6 weeks Systematic Symptoms: Paroxysmal cough, lymphocytosis, hyperinsulinemia, elevated IgE, histamine, cold Toxin release into body (bacteria non-culturable) Convalescent Lasts 2-3 weeks Lingering Symptoms: Increased susceptibility to all respiratory infections Compromised immune function 15 5

6 Chronic coughing disease Catarrhal Lasts 1-3 weeks Localized Symptoms: Fever, cough, runny nose Paroxysmal Lasts 2-6 weeks Persistent cough especially at night Convalescent Lasts 2-3 weeks Disease Process: Growth of bacteria in respiratory tract 16 Pertussis is two diseases One disease is classical whooping cough Non-immune individuals experience whooping cough The other disease is a milder, chronic coughing disease Previously infected or vaccinated individuals get chronic coughing disease 17 B. pertussis is a strict human pathogen Needs mechanism for long-term maintenance or the ability to re-infect previously infected hosts Strategies for host-restricted pathogens include: Long-term maintenance latency in tuberculosis Antigenic variation Neisseria gonorrhoeae Antigenic elimination Syphilis Immune manipulation B. pertussis 18 6

7 Infection (and vaccination) with B. pertussis diverts the immune system to favor the development of suboptimal immune responses that will make humans susceptible to re-infection every 5 to 1 years 19 Virulence factors of Toxins: Pertussis toxin Adenylate cyclase toxin Dermonecrotic toxin Tracheal cytotoxin Fimbriae Adenylate cyclase toxin FHA Pertussis toxin TCT Pertactin Dermonecrotic toxinn BrkA TCF 2 Pertussis toxin Structure: AB5, six subunits, five different Mode of action: ADP-ribosylation and inactivation of GTP-binding proteins that regulate mammalian cells Activation of signaling by receptor cross-linking (T cell receptor) Role in disease: Inhibition of chemotaxis Inhibition of cellular migration (lymphocytosis) Immunosuppression Lethal factor 21 7

8 Adenylate cyclase toxin Structure: Single polypeptide, 2 kda Mode of action: Elevation of camp, Hemolysis Role in disease: Inhibition of immune response Aids colonization 22 Adenylate cyclase toxin inhibits neutrophil phagocytosis? camp 23 Dermonecrotic toxin Structure: Single polypeptide, 16 kda Mode of action: Modifies GTP-binding protein, Rho Alters cytoskeletal functions, and growth Role in disease: Unknown 24 8

9 Tracheal cytotoxin Structure: Peptidoglycan Mode of action: Promotes release of IL-1 (toxicity resides in host response) Role in disease: Ciliastasis and destruction Promotes transmission (coughing) 25 Virulence factors of Adhesins: Filamentous hemagglutinin Fimbriae (or pili) Pertactin BkA BrkA Vag8 TCF (tracheal colonization factor) Others Fimbriae Adenylate cyclase toxin Pertactin FHA Pertussis toxin TCT Dermonecrotic toxinn BrkA TCF 26 Adhesins of Filamentous hemagglutinin (FHA) 2, Daltons Fimbriae FHA Pertussis toxin Cell surface-associated and released Adenylate cyclase toxin TCT Pertactin Dermonecrotic toxinn BrkA TCF 27 9

10 Adhesins not just sticky proteins Modulate the immune response All bind to cell-surface signaling molecules Activate signaling pathways Induce cytokine production Example, FHA has immuno-modulatory activity FHA suppresses IL-12 production by macrophages, shifting the balance from a Th1 to a Th2 response Eur J Immunol 3: (2) 28 Composition of acellular pertussis vaccine Pertussis toxin (toxoid) Filamentous Hemagglutinin Pertactin ti Fimbriae (two antigens) Fimbriae Adenylate cyclase toxin FHA Pertussis toxin TCT Pertactin Dermonecrotic toxinn BrkA TCF 29 Mechanisms of vaccine-induced antibody-mediated immunity to pertussis Anti-toxin antibodies neutralize the action of toxins Anti-adherence antibodies inhibit attachment to the ciliated cells Bactericidal antibodies kill the bacteria 3 1

11 Anti-toxin antibodies neutralize the action of toxins Effective against pertussis toxin Adenylate cyclase toxin Dermonecrotic toxin Tracheal cytotoxin Not in the vaccine 31 Anti-adherence antibodies inhibit attachment to the ciliated cells Filamentous hemagglutinin Fimbriae Pertactin Included in the pertussis vaccine BrkA Vag8 Tracheal colonization factor Etc. Not in the pertussis vaccine 32 Bactericidal antibodies kill the bacteria Opsonizing antibodies Promote phagocytosis and intracellular killing by neutrophils or macrophages Complement fixing antibodies Promote bacterial lysis through the membrane attack complex 33 11

12 Opsono-phagocytosis: killing by neutrophils or macrophages Countered by adenylate cyclase toxin, FHA and pertussis toxin 34 Phagocytosis assay GFP expressing B. pertussis +/- Opsonizing antibody Incubate with human neutrophils or macrophages Counterstain with ethidium bromide Adherent = orange Inside = green 35 Neutrophil phagocytosis B. pertussis attach to neutrophils, but are not efficiently internalized and remain viable Human Neutrophil 8 - extracellular adherent 1 - intracellular phagocytosed Phagocytosed B. pertussis are killed 36 12

13 *Neutralizing antibodies to adenylate cyclase toxin promote phagocytosis of opsonized bacteria Phagoc cytosed per 1 ne eutrophils Opsonized: Monoclonal: none none 3D1 6E1 Neutralize toxin: es No * Neutralizing antibodies to ACT promote phagocytosis 37 Opsono-phagocytosis activity after vaccination 38 Phagocytosis of B. pertussis opsonized with serum from 2-component acellular pertussis vaccine recipients agocytosd r 1 PMNs Ph per No AB Internalized Bacteria Pre Post Pre and post-vaccine activity is the same Vaccination with acellular pertussis vaccine does not enhance phagocytosis by neutrophils 39 13

14 Opsono-phagocytosis phagocytosis activity after infection 4 Phagocytosis of B. pertussis opsonized with convalescent serum Phagoc cytosd Per 1 PMNs Phagocytosis with serum depleted for antibodies to adenylate Internalized cyclase toxinbacteria No AB + AB ACT depleted Only one sample had improved uptake compared to unopsonized control 41 Was the lack of phagocytosis due to a lack of opsonizing antibodies? gocytosd 1 PMNs Pha per * * Wild type ACT mutant Internalized bacteria * * * # * * * * No Ab Mutants lacking ACT were efficiently internalized when opsonized with the convalescent sera Therefore, opsonizing antibodies were present, but phagocytosis of the wild type strain was blocked by ACT 42 14

15 Phagocytosis with serum depleted for antibodies to FHA Adherent Per 1 PMNs Adherent bacteria No ab intact t intact t intact t -FHA -FHA -FHA Phagocytosd Per 1 PMNs Phagocytosed bacteria No ab intact -FHA intact -FHA intact -FHA FHA is both bound to the bacteria and secreted ACT can bind to FHA Heparin Binding RGD CRD FHA ACT Antibody binding sites FHA possesses a few immuno-dominant regions, none of which map to regions necessary for binding to host cells 44 Antibody to FHA delivers the FHA-adenylate cyclase complex to the Fc receptors on neutrophils FHA ACT camp 45 15

16 Role of Complement in immunity to B. pertussis 46 Complement killing assay Rela ative survival 1% 1% 1%.1% WT BrkA.1% Time (min) BrkA confers resistance complement, but a sufficiently potent immune response can overcome BrkA-resistance 47 Complement cascade Classical Alternative Activated by: Antigen/Antibody Repeating Structures C1 D Opsonization C4 P and Recruitment C2 C3 B BrkA C5 Recruitment Membrane C6 Attack C7 Complex C8 C9 Lysis 48 16

17 Complement killing activity after vaccination 49 Complement activity after vaccination Significant loss of killing activity survival Relative 1% 1% 1% Control Placebo Vaccinees Pre and post-vaccine activity No enhancement of complement killing Pre-immune Post-immune 5 Complement killing activity after infection survival Relative 1% 1% 1%.1%.1% Good activity survival 1% 1%.1%.1% Antibody concentration Relative Blocking activity 1% 1%.1%.1% Antibody concentration IgA antibodies do not fix complement; Blocking activity may be due to IgA 51 17

18 Blocking response decays with time Relative survival Convalescent One year later 1% 1% 1%.1%.1%.1% 1% 1%.1% Antibody dilution Serum half life: IgG1 = 23 days IgA = 6 days Increased activity may be due to accelerated loss of IgA Increased killing was observed one year later 52 IgA responses of 15 adults in APERT trial 3-component acellular pertussis vaccine Some vaccinees developed extremely high IgA titers to either FHA or Pertactin (not PT) These vaccinees were more likely to display complement-blocking activity ELISA Units/ml FHA PRN Pre immune Post immune 53 Antibody-dependent complement killing C C C C C C C Lysis Good killing activity Complement Fixing Antibodies (IgG) Promote lysis Lysis variable Blocking serum Blocking Antibodies (IgA) & Complement Fixing Antibodies (IgG) Lysis depends on the relative concentration 54 18

19 Summary Antibodies to ACT block phagocytosis but it is difficult to generate neutralizing antibodies to ACT, and ACT is not included in the current pertussis vaccine FHA is highly immunogenic, but antibodies to FHA block phagocytosis Vaccination can generate a robust IgA response which is poorly bactericidal Neutralizing antibodies to pertussis toxin and pertactin are protective, but circulating strains now express antigenic variants of these proteins which allow for escape from vaccine control 55 I gratefully acknowledge contributions of the following people Phagocytosis studies: Chris Weingart Lyndsay Schaeffer Derrick Lenz Paula Mobberley-Schuman Simon Newman Complement studies: Rachel Fernandez (UBC) Mike Barnes Angie Patton Scott Millen Adenylate Cyclase Toxin Erik Hewlett - U. VA. Mary C. Gray Human Serum Samples Wendy Keitel - Baylor Kathryn Edwards - Vanderbilt David Bernstein - Children s Hospital Joel Ward - UCLA Beverly Connelly - Children s Hospital Mineo Watanabe (Kitasato) Support from NIH/National Institute of Allergy and Infectious Diseases 56 Thank you for your attention! 57 19

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