• Vaccine Ontology ID: VO_0011499
• Type: Subunit vaccine
• Status: Research
• Antigen: H. influenzae Hap
• hap gene engineering:
  • Type: Recombinant protein preparation
  • Description: Bacteria were grown in 10 liters of BHI broth for 18 h at 35°C with aeration. All the following steps were performed at 4°C. Bacterial cells were removed by centrifugation at 10,000 × g. The culture supernatant was concentrated 20-fold by using an Amicon stir cell and was fractionated overnight with ammonium sulfate at 60% saturation. After centrifugation at 17,000 × g for 1 h, the precipitate was dissolved in 20 mM Tris buffer at pH 7.4 containing 50 mM NaCl and 1 mM EDTA, dialyzed against the same buffer, and then centrifuged at 100,000 × g for 1 h to remove insoluble material. The resulting supernatant was loaded at a flow rate of 2 ml/min onto a 20-ml SP Sepharose column (Amersham Pharmacia Biotech) equilibrated with the same buffer. The column was washed until the OD280 reached the baseline, and nHapS-P860295 was eluted at a flow rate of 3 ml/min with a linear gradient of NaCl (from 55 to 500 mM) in 20 mM Tris at pH 7.5 with 1 mM EDTA. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, fractions containing nHapS-P860295 were pooled (Liu et al., 2004).
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000143
  • Description: Mutant cholera toxin CT-E29H
• Immunization Route: Subcutaneous injection

• Vaccine Ontology ID: VO_0004115
• Type: Conjugate vaccine
• Antigen: NTHi detoxified lipooligosaccharide (dLOS) conjugated with outer membrane protein P6
• Adjuvant:
  • VO ID: VO_0001238
• Preparation: LOS was purified from a clinical isolate of NTHi strain 9274 and five major LOS prototype strainsby a modified phenol–water extraction method. A hydrazinolysis was used to produce the detoxified LOS (dLOS). An adipic acid dihydrazide (ADH, Aldrich Chemical Co., Milwaukee, WI) was then bound covalently to the dLOS to form an adipic hydrazide derivative, AH-dLOS. P6 was isolated from strain 9274 (Wu et al., 2005). P6 was covalently conjugated to dLOS of strain 9274 through adipic acid dihydrazide with different ratios of dLOS to P6. It resulted in two conjugate formulations with weight ratios of dLOS to P6 of 3.7 for dLOS–P6 (I) and 1.6 for dLOS–P6 (II). Binding activity of the conjugates was examined by ELISA with mouse monoclonal antibodies specific to LOS and P6 and a rabbit anti-P6 serum. The results showed that the conjugates bound not only to the LOS antibody but also to both P6 antibodies. It suggested that the conjugates retained epitopes of both LOS and P6 antigens (Wu et al., 2005).
• Virulence: detoxified

• Vaccine Ontology ID: VO_0000479
• Type: Subunit vaccine
• Antigen: Nontypeable Haemophilus influenzae cell membrane
• Adjuvant:
  • VO 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).

• Vaccine Ontology ID: VO_0000432
• Type: Subunit vaccine
• Antigen: Recombinant proteins corresponding to the C-terminal region of HapS from H. influenzae strains N187, P860295, and TN106.
• hap gene engineering:
  • Type: recombinant
  • Detailed Gene Information: Click Here.
• hap gene engineering:
  • Type: recombinant
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0000143
  • Description: CT-E29H (a mutant cholera toxin)
• Preparation: Recombinant proteins corresponding to the C-terminal region of HapS from H. influenzae strains N187, P860295, and TN106 were used. To prepare the protein antigens for vaccination, 15 µg of rCBD was diluted in Dulbecco's PBS (D-PBS) to a final volume of 20 to 40 µl with or without 0.1 µg of CT-E29H (a mutant cholera toxin) as an adjuvant (Liu et al., 2004).
• Description: Nontypeable Haemophilus influenzae (NTHi), a nonencapsulated gram-negative bacterium, is the cause of a number of human respiratory tract diseases, such as otitis media, sinusitis, bronchitis, and pneumonia. Hap adhesin, promotes bacterial interaction with human respiratory epithelial cells and extracellular matrix proteins as well as mediates bacterial aggregation and microcolony formation. Hap belongs to the autotransporter family of proteins common among gram-negative pathogens . It is synthesized as a 155-kDa precursor protein, which consists of an N-terminal 25-amino-acid signal peptide, an internal 110-kDa passenger domain called HapS, and a C-terminal 45-kDa outer membrane domain called Hapß. Domain in Hap responsible for promoting adherence to epithelial cells resides in the C-terminal 311 amino acids the (cell binding domain [CBD]) of HapS (Liu et al., 2004).

• Vaccine Ontology ID: VO_0004038
• Type: Subunit vaccine
• Status: Research
• Antigen: NucA Protein
• nucA gene engineering:
  • Type: Recombinant protein preparation
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO ID: VO_0001250
  • Description: MPL (3-O-deacylated monophosphoryl lipid A.
• Preparation: The native protein was extracted from NTHi strain P860295 with KSCN and purified. The recombinant protein was cloned, sequenced, and expressed in Escherichia coli. The recombinant protein is localized in the periplasm of E. coli and has been purified to homogeneity. Both the recombinant and native proteins possess 5'-nucleotidase activity (Zagursky et al., 2000).
• Immunization Route: Intraperitoneal injection (i.p.)
• Description: A surface-exposed, highly conserved, immunogenic NTHi protein was identified, which elicits cross-reactive bactericidal antibodies against NTHi (Zagursky et al., 2000). This protein, called NucA, has been identified as a 5' nucleotidase and has been cloned, sequenced, and expressed recombinantly. It elicits broadly cross-reactive antibody against NTHi strains and has vaccine potential.

• 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:
  • VO 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).

• Vaccine Ontology ID: VO_0000558
• Type: Subunit vaccine
• Antigen: outer membrane protein P6
• Pal gene engineering:
  • Type: Recombinant protein preparation
  • Detailed Gene Information: Click Here.
• Adjuvant:
  • VO 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
• Vaccination Protocol: Groups of 10 female, 6- to 8-week-old Swiss Webster or BALB/c mice (Taconic Farms, Germantown, N.Y.) were immunized subcutaneously at weeks 0 and 4 with protein antigens. To prepare the protein antigens for vaccination, 10 μg of rCBD or 2 μg of an induced E. coli BL21(DE23)/pLysS/pGEX-6P-1 cell lysate was absorbed onto 100 μg of aluminum phosphate at 37° for 1 h, and then 50 μg of 3-O-deacylated monophosphoryl lipid A (MPL; Corixa Corp., Hamilton, Mont.) was added. Sera collected at weeks 0, 4, and 6 were pooled for analysis (Liu et al., 2004).
• Challenge Protocol: Three weeks after the final immunization, mice were challenged intranasally with approximately ×10^6 CFU of strain TN106.P2 (Liu et al., 2004).
• Efficacy: When mice immunized intranasally with recombinant protein corresponding to the C-terminal region of Hap(S) from H. influenzae strains N187, P860295, and TN106 plus mutant cholera toxin CT-E29H were challenged with strain TN106, they were protected against nasopharyngeal colonization (Liu et al., 2004).

Mouse Response

• Vaccination Protocol: Ten mice per group were given a total of three subcutaneous injections at 2-week intervals with conjugates, conjugates with Ribi adjuvant, P6, or a mixture of P6 and dLOS. Blood samples were collected 14 days after the first injection and 7 days after the second and third injections (Wu et al., 2005).
• Immune Response: In mice, the mixture of P6 and dLOS or P6 alone did not elicit anti-LOS IgG responses. In contrast, both conjugates were able to elicit a significant rise of anti-LOS IgG, whereas dLOS–P6 (II) was a better immunogen for eliciting mouse IgG as compared to dLOS–P6 (I) ( P < 0.01). There was a 648-fold rise of anti-LOS IgG elicited by dLOS–P6 (II) and a 14-fold rise by dLOS–P6 (I) after three injections when compared to their pre-immune sera that contained 1 ELISA unit (p < 0.01). Formulation of the conjugates with Ribi adjuvant enhanced the antibody responses in both conjugates, especially for dLOS–P6 (I). In addition, dLOS–P6 (I) alone elicited better responses of anti-LOS IgM (Wu et al., 2005).

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).

Mouse Response

• Host Strain: BALB/c
• Vaccination Protocol: Groups of 10 female, 6-week-old BALB/c mice were immunized intranasally with protein antigens as described previously at weeks 0, 1, 3, and 5. To prepare the protein antigens for vaccination, 15 µg of rCBD was diluted in Dulbecco's PBS (D-PBS) to a final volume of 20 to 40 µl with or without 0.1 µg of CT-E29H (a mutant cholera toxin) as an adjuvant . Control mice received D-PBS alone or D-PBS with 0.1 µg of CT-E29H. Sera collected at weeks 0, 3, 5, and 8 were pooled for analysis. Prior to immunization, mice were anesthetized with a mixture of ketamine (80 mg per kg of body weight) and xylazine (7 mg per kg of body weight), a dosage that maintains a state of anesthesia for 15 to 20 min. Vaccines were delivered by pipette in a volume of 20 µl per nostril. The pipette was positioned so that the tip touched the opening of the nostril, allowing the liquid to be drawn into the nasopharynx with breathing. Immediately following immunization, mice were placed in a supine position for 3 to 5 min (Liu et al., 2004).
• Challenge Protocol: Three weeks after the final immunization, mice were challenged intranasally with approximately 10^6 CFU of strain TN106.P2 . Three days after challenge, mice were sacrificed and the nasal tissue was harvested, weighed, homogenized, and plated on BHI-XV plates containing 100 µg of streptomycin/ml. The plates were incubated overnight, and the colonies were counted (Liu et al., 2004).
• Efficacy: Antisera against the recombinant proteins from all three strains not only recognized native HapS purified from strain P860295 but also inhibited H. influenzae Hap-mediated adherence to Chang epithelial cells. Furthermore, when mice immunized intranasally with recombinant protein plus mutant cholera toxin CT-E29H were challenged with strain TN106, they were protected against nasopharyngeal colonization (Liu et al., 2004).

Mouse Response

• Host Strain: Swiss-Webster mice
• Vaccination Protocol: Swiss-Webster mice were immunized subcutaneously with 5 µg of NucA protein and 50 µg of MPL (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, Mont.) as adjuvant per dose at weeks 0, 4, and 6. Blood samples were collected at weeks 0 and 10 for analyses of antibody titers to protein, whole cells, and bactericidal activity (Zagursky et al., 2000).
• Efficacy: Mouse antiserum generated against the purified protein was reactive on whole-cell enzyme-linked immunosorbent assay (ELISA) with seven NTHi strains and type b Eagan and Whittier strains and exhibited bactericidal activity to homologous and heterologous NTHi strains.

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).

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).

Rat Response

• Vaccination Protocol: Four-day-old Sprague-Dawley rats were randomized into 10 groups with a mother for each group of 10 infants. The infants were immunized i.p. with 0.1 ml of the appropriate dilutions of mouse rNucA antiserum from week 10. Preimmune serum and PCM buffer were used as negative controls. The positive control group received a monoclonal antibody raised against Hib capsular polysaccharide (MAbE117.5). All dilutions of sera and cells were done in PCM buffer (Zagursky et al., 2000).
• Challenge Protocol: Approximately 24 h after immunization, the infant rats were challenged i.p. with approximately 50 CFU (0.1 ml) of virulent Hib Eagan. Approximately 20 to 24 h postchallenge, 10 µl of blood was taken from the tail and viable Hib CFU were determined from duplicate dilutions of blood (Zagursky et al., 2000).
• Efficacy: The group receiving a 1:2 dilution of mouse anti-rNucA pooled sera showed about a 10-fold reduction in the level of bacteremia compared to the group vaccinated with week 0 (preimmune) pooled mouse sera (Zagursky et al., 2000).

Rabbit Response

• Vaccination Protocol: Two or three rabbits per group were immunized subcutaneously and intramuscularly twice on days 0 and 28 with conjugates, conjugates with Ribi adjuvant, P6, or a mixture of P6 and dLOS. Blood samples were collected on days 0, 14 and 38 (Wu et al., 2005).
• Efficacy: Both conjugates were further tested for their immunogenicity in rabbits. The mixture of P6 and dLOS or P6 alone did not elicit significant rises of anti-LOS IgG. In contrast, both conjugates elicited high levels of anti-LOS IgG after the second injection as compared to those in their pre-immune sera (122–243-fold rise). Unlike with mice, formulation of both conjugates with Ribi adjuvant did not further enhance the immunogenicity of the conjugates (Wu et al., 2005).