This study produced recombinant versions of the Bordetella pertussis proteins pertactin (Prn), fimbriae 2 (Fim2), and fimbriae 3 (Fim3) and characterized their immune responses. Prn elicited both antibody production and a cellular immune response, and provided partial protection against lethal B. pertussis infection at multiple doses. Fim2 and Fim3 stimulated antibody production but did not impact cytokine levels or survival. The results suggest Prn warrants further investigation for whooping cough vaccine development.

1. Production and characterization of recombinant pertactin, fimbriae 2 and fimbriae 3 from Bordetellapertussis Xu, Wang, Tan, Zhang, Wu, Wang, Hou, and Zhang 3/25/2010 Presented by Krista DeLuca

2. Whooping Cough Top ten causes of Childhood mortality worldwide. There are nearly 300,000 deaths annually. 39 million cases a year

3. B. Pertussis vaccine history Whole cell pertussis vaccines (1960’s) Side effects: swelling, redness, fever Need booster shot every 10 or so years Acellularpertussis vaccine (1981) Pertussis toxin (PT), FHA, Prn, Fim2, or Fim3 2 or more have greater effect Problems: Low yield of Prn, Fim2 and Fim 3 are difficult to isolate.

4. Pertactin (Prn) Outermembrane protein of B. pertussis used to adhere to epithelial cells. Elicits cellular and humoral immune responses. Virulence factor

5. Fimbriae (Fim) 2 and 3 Bacterial adhesins Fim 2 and Fim 3 have similar molecular weight. Serologically, they are different. No isolation before.

6. Hypothesis Prn, Fim2, and Fim3 can be isolated and produced in large amounts in vitro by new recombinant technology.

7. Figure 1 Q: Were the correct proteins purified from the recombinant vectors? SDS-PAGE Lanes 1- Molecular Mass Marker 2- rPrn (10 μg) 3-Fim 2 (10 μg) 4- Fim 3 (10 μg) A: Yes, rPrn, rFim2, and rFim3 all migrated as a single band to the correct size. B: Yes, all three protein specificities were confirmed by using specific antibodies against naïve forms of Prn, Fim2, and Fim3. Western Blot Lanes 1- Molecular Mass Marker 2-rFim2. 3-rFim3 4- Molecular Mass Marker 5- rPrn 6-Molecular Mass Marker 10% SDS-PAGE gel, reducing conditions, stained with Coomaisse blue Incubated in 5% skim milk in phosphate-buffered solution containing 0.05% Tween 20 for 1 hr. signals captured with DAG kit.

8. Figure 2 Q: Do the proteins elicite an IgG immune repsonse? A: Yes A: At both high and low dosages, there is no significant difference between Ab titer. Both are successful. Q: How does dosage affect IgG antibody titer? Conditions: ELISA used Taken 2 weeks after immunization

9. Figure 3 Q: How does rPrn, rFim2, and rFim3 affect levels of cytokines that elicite a cellular immune response? A: IL-2 was increased for all three immunizations. TNF-a only increased in rPrn IL-4 showed no significant response to any of the immunizations. Conditions: 2 weeks after immunization, 5 mice each, determined by ELISA

10. Figure 4 Q: Does immunization with rPrn, rFim2, or rFim3 decrease CFU counts? A: rPrn shows a significant decrease in CFU count compared to the conrtrol. rFim2 and rFim3 do not. Conditions: 2 weeks after immunization, injected intranasally with B. pertussis 18323, from BG agar with 20% sheep blood, 50 μL of bacterial suspension at approximately 1 × 106 CFU was injected into the nostril, after 7 days lungs were removed and homogenized in 1 ml of PBS, viable counts were plated on BG agar medium

11. Figure 5 A- 100 μg B- 20 μg C- 4 μg Q: Can any of the proteins at 4 μg, 20 μg, or 100 μg protect against a lethal dose of B. pertussis? A: rPrn alone can confer partial protection at all 3 dose levels. rFim2 and rFim3 do not show any survival. Conditions: 3 wks after immunization, used B. pertussis 18232, Bacterial suspension (30 μL) with approximately 8 × 104 CFU suspended in PBS containing 1% casamino acids were injected into the mouse brain using a 0.25-mL glass syringe, survival number counted 14 days after infection.

12. Conclusion B. pertussis proteins Prn, Fim2, and Fim3 can be genetically manipulated to produce large amounts of protein in vitro. The proteins proved to be successful in eliciting both a humoral and cellular immune response. Prn has very strong effects on the immune system and should be further researched in vaccine development studies.

14. Only supported statistically significant findings.