• Vaccine Name: recombinant nontypeable H. influenzae protein P6 with AdDP
• Target Pathogen: Haemophilus influenzae
• Target Disease: Meningitis
• Vaccine Ontology ID: VO_0000558
• Type: Subunit vaccine
• Antigen: outer membrane protein P6
• Pal gene engineering:
  • Type: Recombinant protein preparation
  • Description:
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0000559
• Preparation: A DNA fragment encoding the mature P6 protein was amplified from the H. influenzae type a chromosomal DNA by use of PCR. The resulting PCR product was purified, digested with BamHI and SalI, ligated into the vector pCR2.1-TOPO (TOPO TA Cloning; Invitrogen), and then transformed into the E. coli strain XL-1 blue. The fragment encompassing the P6 gene was subsequently cloned into the expression vector pET23a+ (Novagen) and was transformed into E. coli BL21 (DE3) strain.The purity and identity of the purified rP6 protein was verified by SDS-PAGE and Western blot analysis, respectively, by use of mice antiserum raised against native P6 protein (Bertot et al., 2004).

Mouse Response

• Host Strain: BALB/c mice
• Vaccination Protocol: Groups of 5 mice were vaccinated by intranasal inoculation (5 μL/nostril) with either native P6 (10 μg/dose) or rP6 (10 μg/dose) plus AdDP (100 μg/dose) as a mucosal adjuvant diluted into sterile PBS on days 0, 7, and 14. No endotoxin was detected in either native or rP6 preparations (<1 ng/mg) by the limulus assay (Whittaker Bioproducts). Control groups received only AdDP (100 μg/dose) or PBS.
• Immune Response: High titers of P6-specific serum antibodies were elicited in mice vaccinated with either native P6 or rP6, which cross-recognized both antigens. However, rP6 stimulated stronger mucosal responses.
• Challenge Protocol: Bacteria were grown overnight on CBA plates at 37°C in an incubator containing 5% CO2 in air and then were harvested, washed 3 times, and resuspended in sterile PBS. The pulmonary challenge was performed 7 days after the last boost as follows: mice were anesthetized by ip injection with 0.1 mL of PBS containing 2 mg of ketamine and 0.2 mg of xylazine, and a bolus inoculum of 5 × 10^8 cfu of live bacteria in 50 μL of PBS was introduced into the lungs via an intratracheal cannula. Mice were killed 4 h after lung inoculation, and lung lavages were performed twice, using 200 μL of sterile PBS each time. The efficiency of the bacterial clearance was established by determining the number of viable bacteria present in pooled BAL samples and by plating serial dilutions of the washes on CBA plates at 37°C in 5% CO2 in air (Bertot et al., 2004).
• Efficacy: Mice vaccinated with rP6 were protected against both pulmonary and middle-ear infections (P < .01).