Diphtheria, Tetanus, Pertussis

1. Diphtheria, Tetanus, Pertussis MedCh 401 Lecture 3 KL Vadheim Lecture 3

2. Adjuvant • Substances that enhance the immune response • Two categories: • vehicles • immunomodulators KL Vadheim Lecture 3

3. Adjuvants functioning as vehicles I • Human use: • Alum compounds • Aluminum hydroxide and phosphate • the only licensed adjuvants in U.S. • MF59 • Oil and water emulsion • Marketed in Europe KL Vadheim Lecture 3

4. Adjuvants functioning as vehicles II • Animal use: • Freund’s Complete Adjuvant (CFA) • dessicated Mycobacterium butyricum, mineral oil and an emulsifying agent, mannide monooleate • causes potentially secere local inflammatory lesions, chronic granulomas, abscesses, and tissue sloughs. Injected into the murine footpad, it can cause chronic lameness and arthritis; injected intraperitoneally, it can cause peritonitis • Freund’s Incomplete Adjuvant • Mineral oil and Mannide monooleate • Fewer side effects, adequate for boosting KL Vadheim Lecture 3

5. Immunomodulatory Adjuvants • Purified Protein Derivative (PPD) • Lipopolysaccharide (LPS; bacterial endotoxin) • Lipid A - lipid portion of LPS • Cholera toxin B subunit • CpG KL Vadheim Lecture 3

6. Diphtheria vaccine • Detoxified bacterial, protein toxin • Injectable, IM administration • Toxigenic Corynebacterium diphtheriae (infected withbphage) • Primarily a childhood disease KL Vadheim Lecture 3

7. Diphtheria Transmission • Person-to-person by respiratory droplet • No animal reservoir KL Vadheim Lecture 3

8. Manufacturing Process • Toxigenic strain of C. diphtheriae grown in Fenton medium with a bovine extract • After suitable growth, toxin purified from cells by centrifugation • Toxoided by incubation with formaldehyde for several weeks • Concentrated with ultrafiltration • Purified by precipitation, dialysis and sterile filtered • Adsorbed onto aluminum hydroxide, Al(OH)3 KL Vadheim Lecture 3

9. Excellent Vaccine Efficacy • Mortality • 1860-1897 46-196/100,000 people • Case fatality rates >50% during outbreaks • 1920 15/100,000 people • Cases • 1971 - 1981 - 1,288 in U.S. • 1980 - 1995 - 41 • occasional in developing countries and Native populations in U.S. and Canada KL Vadheim Lecture 3

10. Tetanus Vaccine • Detoxified bacterial, protein toxin • Clostridium tetani • Injectable dosage form • IM KL Vadheim Lecture 3

11. Tetanus Transmission • Not a communicable disease • The only vaccine-preventable infection that is not communicable • Disease acquired through exposure to bacterial spores in the environment • inoculation of bacterial spores into body by puncture or deep cut KL Vadheim Lecture 3

12. Transmission II • Neonatal tetanus most common worldwide • Case fatality increases with age (~50% for 80+ years of age) • Disease reservoirs • Soil • Animal feces KL Vadheim Lecture 3

13. Tetanus toxins • Tetanolysin - possible role in establishing infection at inoculation site • Tetanospasm • accumulates intracellularly during log-phase growth • released into medium upon autolysis • Minimum human lethal dose ~ 2.5 ng/kg KL Vadheim Lecture 3

14. Tetanus disease • Tetanospasms • localized - spasm of muscles close to site of injection; weeks to months duration; rare but may precede generalized symptoms • generalized - 80% of cases • Complications of the spasms: • fractures of the long bones and vertebrae • asphyxia from glottic obstruction KL Vadheim Lecture 3

15. Nervous system effects • Toxin travels up nerve endings by intra-axonal transport • Gains entry to neuromuscular junctions by binding to gangliosides • Interferes with release of neurotransmitters from presynaptic inhibitory fibers • Excitatory reflexes multiply unchecked, causing spasms KL Vadheim Lecture 3

16. Tetanus • Toxin-mediated disease • Symptoms may progress clinically despite use of parenteral antibiotics • Treatment: • hyperimmune serum • supportive care • Recovery may depend on development of new functional nerve connections KL Vadheim Lecture 3

17. Manufacturing Process • Growth of C. tetani in modified Latham broth in fermenters • Harvest extracellular toxin by filtration • Purify • Detoxify with formaldehyde for ~3 weeks • Adsorb with Alum adjuvant • Diafiltration KL Vadheim Lecture 3

18. Pertussis (Whooping Couth) • Bordetella pertussis • Whole cell and acellular vaccines • traditional and recombinant • Injectable dosage form • IM KL Vadheim Lecture 3

19. Pertussis Transmission • Person-to-person • Respiratory droplets KL Vadheim Lecture 3

20. Pertussis (whooping cough) • Killed Whole cell - • old, not licensed in U.S. or Europe • still used in developing countries • relatively cheap • Acellular (aP) - • currently licensed in U.S., Japan and Europe • some are recombinant • expensive KL Vadheim Lecture 3

21. B. pertussis virulence factors • Pertussis toxin (PT) • Filamentous hemagglutinin (FHA) • Fimbriae (fimbrial agglutinogens) (Fim) • Pertactin (PRN) • Adenylate cyclase • Tracheal cytotoxin • Dermonecrotic or heat-labile toxin • BrkA • Endotoxin (LPS) KL Vadheim Lecture 3

22. Vaccine Efficacy • 1914 (Pre-vaccine) • ~270,000 cases, 10,000 deaths annually • killed >5 of every 1000 children born in U.S. • 1976 - 1,010 cases • 1996 - 7,796 • 2002 • 8,296 cases • 3.01/100,000 pop. KL Vadheim Lecture 3

23. Vaccine History • 1914 First whole-cell vaccine (P) • 1948 DTP • 1991 - 2002 DTaP for children • 2005 Tdap for adults 19-64 KL Vadheim Lecture 3

24. Manufacturing • B. pertussis cultures grown in modified Stainer-Scholte broth • Acellular antigens: • purified from culture medium (PT and FHA) or • extracted from cells (PRN, FIM) • Formaldehyde and/or glutaraldehyde detoxification of PT • Aluminum adjuvants KL Vadheim Lecture 3

25. Manufacturing II • Novartis-Behring (Chiron) - Recombinant • Genetically detoxified PT • Two other components • Not available in U.S. yet KL Vadheim Lecture 3

26. DTaP Vaccine Formulations KL Vadheim Lecture 3

27. Licensed DTaP Vaccines KL Vadheim Lecture 3