• Vaccine Name: Nontypeable H. influenzae outer membrane recombinant P4 vaccine
• Target Pathogen: Haemophilus influenzae
• Target Disease: Meningitis
• Vaccine Ontology ID: VO_0004114
• Type: Subunit vaccine
• Antigen: Recombinant lipidated P4 and the non-fatty acylated recombinant P6 protein that contains a 7-amino acid peptide genetically fused to the N-terminus (rP6) were used as vaccine candidates (Hotomi et al., 2005).
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0000143
• Preparation: The lipidated form of recombinant P4 protein (rP4) was expressed in E. coli strain BLR (Novagen, Madison, WI) transformed with plasmid pLP339. Plasmid pLP339 contains the wild type P4 gene cloned into the multiple cloning sites of pBAD18 cm (Invitrogen Corp., Carlsbad, CA) under control of the arabinose promoter. The non-fatty acylated recombinant P6 protein (rP6) was expressed in plasmid pRSM1007 in E coli strain BL21(DE3). Plasmid pRSM1007 contain A P6 DNA fragment encoding the mature protein devoid of lipidation signal sequence was amplified from Hib strain MinnA chromosomal DNA (Hotomi et al., 2005).

Mouse Response

• Host Strain: BALB/c mice
• Vaccination Protocol: BALB/c mice (n = 60) were randomly assigned into four groups (A–D) and immunized intranasally with 30 μg of recombinant proteins with or without 2 μg of CT (Sigma Chemical Co., St. Louis, MO) as mucosal adjuvant. Mice were immunized every 2 days for 2 weeks (on days 0, 2, 4, 6, 8, 10, 12 and 14). Group A, B, and C mice were immunized with rP4 + CT, rP4 and rP6 without CT, and mixture of rP4 and rP6 + CT, respectively. Group D mice were sham immunized controls and intranasally received CT alone (Hotomi et al., 2005).
• Challenge Protocol: Nontypeable H. influenzae strain SR7332, biotype II, originated from the nasopharynx of an 8-year-old male patient with acute sinusitis (Shionogi Ph. Co., Osaka, Japan). The strain was grown at 37 °C in BHI-XV to mid-log phase. Mid-log phase cells (1 × 108 cfu) were diluted in PBS to a concentration of 5 × 108 cfu ml−1 to prepare the inoculums. One week after the final immunization, mice (n = 15) were intranasally inoculated with 5 × 106 cfu (10 μl) of live NTHi SR7332. The reduction in nasopharyngeal colonization with NTHi was determined by plate count of viable NTHi in nasal washes from immunized mice versus sham-immunized mice. Mice were sacrificed on day 0 (12 h), and day 3 after challenge and NPWs were collected by meticulous washing of the nasopharynx with 100 μl sterile PBS. Ten microlitres of NPWs were plated on chocolate agar (BBL Microbiology System, Cockeysville, MD) and incubated at 37 °C in 5% CO2 for 24 h. After the incubation, colonies were counted. The nasopharyngeal clearance was expressed as the percentage of cfu at day 3 to the cfu present on day 0 (12 h). Thus, the percent clearance is measured for each group at day 3 compared to its day 0 time point, with a maximum percent colonization at day 3 of 100% of the cfu's present at day 0 (Hotomi et al., 2005).
• Efficacy: Intranasal immunization with either rP4 + CT, a mixture of rP4 and rP6 + CT, or rP4 and rP6 without CT elicited anti-rP4 specific IgG antibody in serum of mice. Intranasal immunization with either rP4 + CT or a mixture of rP4, rP6 + CT elicited anti-rP4 specific IgA antibody in nasopharyngeal washing (NPW), while intranasal immunization with rP4 and rP6 without CT did not induced anti-rP4 specific IgA antibody responses in NPWs. Sera from mice intranasally immunized with rP4 + CT and a mixture of rP4, rP6 + CT also showed bactericidal activity. Significant clearance of NTHi in nasopharynx was seen 3 days after the inoculation of live NTHi in mice intranasally immunized with rP4 + CT (Hotomi et al., 2005).