• Vaccine Name: Nontypeable H. influenzae cell membrane (CM-Hi) vaccine
• Target Pathogen: Haemophilus influenzae
• Target Disease: Meningitis
• Vaccine Ontology ID: VO_0000479
• Type: Subunit vaccine
• Antigen: Nontypeable Haemophilus influenzae cell membrane
• Adjuvant:
  • Adjuvant name:
  • VO adjuvant ID: VO_0000143
• Preparation: NTHi (strain 76), which was isolated from the nasopharynx of a patient with OME at Oita Medical University, was stored at -80°C and used for the preparation of antigen and nasal inoculation. NTHi was cultured overnight on chocolate agar and harvested by being scraped from the plate, suspended in EDTA buffer (pH 7.4). The bacterial cells were then disrupted by sonication on ice, and the unbroken cells and debris were removed by centrifugation at 10,000 g for 20 min. The supernatants were pooled and centrifuged at 80,000 g for 2 h at 4°C. The clear, gel-like pellet was suspended in distilled water and lyophilized. The resulting powder, referred to as cell membrane preparation from NTHi (CM-Hi), was stored until used in the experiments (Kurono et al., 1999).

Mouse Response

• Host Strain: Male BALB/c mice
• Vaccination Protocol: Mice were immunized nasally, intratracheally, or intraperitoneally (ip) with 10 ug of CM-Hi together with 1 &mgr;g of cholera toxin (CT; List Biological Laboratories, Campbell, CA) as a mucosal adjuvant diluted in sterile PBS. The antigens, diluted in 10 uL of PBS, were inoculated into the nostrils (5 uL/nostril) by use of a pipette or into the trachea by intratracheal intubation connected to a microinjector under visualization with the aid of an electric otoscope. The procedures were performed under anesthesia with ip injection of 0.1 mL of a mixture containing 2 mg of ketamine and 0.2 mg of xylazine. For ip immunization, we diluted the antigens, including 10 ug of CM-Hi and 1 ug of CT, in 50 uL of PBS. Our previous studies demonstrated that oral immunization requires a higher dose of antigen and CT [13–15]; therefore, we orally immunized the other mouse group with 250 &mgr;g of CM-Hi and 10 ug of CT by gastric intubation without anesthesia. Prior to oral immunization, the mice were deprived of food for 2 h, and 30 min before immunization the mice were gavaged with 0.5 mL of a solution consisting of 8 parts Hanks' balanced salt solution and 2 parts 7.5% sodium bicarbonate by gastric intubation, to neutralize stomach acidity . The vaccine was administered 3 times, on days 0, 7, and 14 (Kurono et al., 1999).
• Immune Response: Antigen-specific IgA antibody titers in nasal washes and the numbers of antigen-specific IgA-producing cells in nasal passages showed the greatest increases in mice immunized nasally. Cytokine analysis showed that interferon-&ggr;, interleukin (IL)-2, IL-5, IL-6, and IL-10 were induced by nasal immunization, suggesting that Th2- and Th1-type cells were generated.
• Challenge Protocol: The same strain of NTHi used for the preparation of CM-Hi was cultured on chocolate agar plates overnight at 37°C in 5% CO2, removed by scraping, and resuspended in PBS (109 cfu/mL) for nasal challenge. For challenge, 10uL aliquots of the live NTHi suspension were administered into the nose 1 week after the 3d immunization. The same dose of live NTHi was also inoculated into nonimmunized mice; 12 h later, mice were sacrificed, and nasal washes were obtained by flushing the nasal cavity with 200 uL of PBS. Nasal washes were also obtained from nonimmunized mice that were not inoculated with NTHi, and the numbers of NTHi were counted, to determine whether contamination by H. influenzae other than the inoculated bacteria had occurred (Kurono et al., 1999).
• Efficacy: Bacterial clearance of a homologous strain of NTHi from the nasal tract was significantly enhanced in the nasal immunization group (Kurono et al., 1999).