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Vaccine Comparison

H. influenzae D15 protein vaccine H. influenzae Omp26 protein vaccine Nontypeable H. influenzae NucA Protein vaccine nontypeable H. influenzae P5 peptide MVF/H3 vaccine Nontypeable H. influenzae protein P6 with cholera toxin Nontypeable H. influenzae rTbpB vaccine
Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information Vaccine Information
  • Vaccine Ontology ID: VO_0011551
  • Type: Subunit vaccine
  • Status: Research
  • Antigen: H. influenzae protective surface antigen D15
  • D15 gene engineering:
    • Type: Recombinant protein preparation
    • Description: Plasmid DNA for sequencing clones DS-712-2-1, DS-691-1-5, JB-1042-5-1, and JB-1042-9-4 was prepared from 50-ml overnight cultures with the Qiagen Plasmid Midi kit. DNA samples were sequenced on an ABI model 373A DNA sequencer with dye terminator chemistry. Plasmid JB-1042-5-1 contains the entire SB33 d15 gene (see Fig. 4). The ;2.9-kb HindIII-EcoRI d15 gene fragment was subcloned into pUC8-BgXb to generate plasmid pRY-60-1. Digestion of pRY- 60-1 with BsrFI and EcoRI excised a ;2.7-kb fragment containing most of the coding sequence of d15. The remainder of the DNA sequences coding for the amino terminus of D15 were recreated from ;90-bp NdeI-BsrFI oligonucleotides (Loosmore et al., 1997).
    • Detailed Gene Information: Click Here.
  • Adjuvant: Freunds emulsified oil adjuvant
  • Immunization Route: Intramuscular injection (i.m.)
  • Vaccine Ontology ID: VO_0011562
  • Type: Subunit vaccine
  • Status: Research
  • Antigen: H. influenzae outer membrane protein 26
  • skp gene engineering:
    • Type: Recombinant protein preparation
    • Description: A crude outer membrane preparation was obtained from bacteria grown overnight on agar plates, and OMP26 was purified by preparative polyacrylamide gel electrophoresis (PAGE) as previously described. Preparative SDS-PAGE to purify OMP26 was performed with a Bio-Rad model 491 Prep Cell, using a 60-ml 14% T-1.42% C acrylamide-BIS (N,N′-methylenebisacrylamide) separating gel with a 10-ml 4% T-0.36% C acrylamide-BIS stacking gel polymerized in a 37-mm (internal diameter) column. Fractions were concentrated by lyophilization and analyzed for protein content by analytical SDS-PAGE. OMP26 isolated under these conditions contained SDS, which was subsequently removed. Fractions containing OMP26 were pooled and dialyzed prior to determination of protein concentration. The presence of lipooligosaccharide (LOS) was assessed by both silver staining of SDS-PAGE minigels and assaying with the E-TOXATE Limulus lysate test (Sigma, Castle Hill, New South Wales, Australia) (Kyd and Cripps, 1998).
    • Detailed Gene Information: Click Here.
  • Adjuvant: incomplete Freunds adjuvant
  • Immunization Route: Subserosal injection
  • 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: MPL vaccine 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_0000509
  • Type: Subunit vaccine
  • Antigen: Peptides representing conserved regions of the NTHi P5 outer membrane protein which have been fused to a promiscuous measles virus F protein T-cell eptitope (MVF) (Webb et al., 2000).
  • Adjuvant: incomplete Freunds adjuvant
  • Preparation: The sequences of the peptides were based on the sequence of P5 from NTHi strain UC19. Peptide L1A encompasses the amino acid motif GINNNGAIK, which is found in loop one in a subset of NTHi strains, including UC19; L4 encompasses the highly conserved central region of loop four; and H3 encompasses a region in P5 that is homologous to OprF peptide 10 from P. aeruginosa .The peptides also contained the measles virus F protein promiscuous T-cell epitope (MVF) and a linker region composed of a 4-residue (LSPG) beta -turn. Peptides were synthesized and purified and by the Biomolecular Resource Facility (Webb et al., 2000) .
  • Vaccine Ontology ID: VO_0000614
  • Type: Subunit vaccine
  • Antigen: outer membrane protein P6 (Sabirov et al., 2004)
  • Adjuvant: cholera toxin
  • Preparation: NTHI was grown on chocolate agar plates and suspended in phosphate-buffered saline (PBS). The suspension was sonicated and centrifuged at 21,000 × g for 30 min at room temperature. The pellet was resuspended in 1% sodium dodecyl sulfate with 0.1 M Tris, 0.5 M NaCl, and 0.1% 2-mercaptoethanol (buffer B, pH 8.0) with RNase (10 mg/ml), sonicated, incubated, and centrifuged. This procedure was repeated twice. The pellet was then suspended in buffer B without RNase, sonicated, incubated, and centrifuged again. The pellet was finally suspended in buffer A (0.01 M Tris, 0.15 M NaCl; pH 7.4) and incubated at 65°C for 30 min. The insoluble material was removed by centrifugation at 100,000 × g for 60 min at 30°C. The protein contained in the supernatant was thought to be pure P6 (Sabirov et al., 2004).
  • Vaccine Ontology ID: VO_0000510
  • Type: Subunit vaccine
  • Antigen: Nontypeable H. influenzae vaccine recombinant transferrin binding protein B
  • Adjuvant: incomplete Freunds adjuvant
  • Preparation: The NTHI strain UC19 (289-I) was originally derived from the sputum of a patient with chronic bronchitis and has been routinely used in this laboratory as the challenge strain when assessing the efficacy of immunization with NTHI antigens. The gene encoding the mature form of UC19 TbpB was then amplified and cloned into the BamHI restriction sites in plasmid pGEX2T (Pharmacia Biotech, Uppsala, Sweden) to produce plasmid pCU17. This plasmid is engineered to express recombinant TbpB as a glutathione S-transferase (GST) fusion protein with a thrombin cleavage recognition site between the two proteins (Webb et al., 1999).
  • Description: The transferrin receptor is composed of two subunits. The interaction of the receptor with transferrin is probably initiated by transferrin binding protein B (TbpB), a peripheral lipoprotein that forms a complex with TbpA, a TonB-dependent integral outer membrane protein that is thought to form a gated pore to facilitate the transport of transferrin-derived iron across the outer membrane (Webb et al., 1999).
Host Response Host Response Host Response Host Response Host Response Host Response

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.

Rat Response

  • Host Strain: Sprague- Dawley
  • Vaccination Protocol: In the bacteremia models, Sprague-Dawley infant rats (Harlan Sprague Dawley, Indianapolis, Ind.) from three mothers were mixed and then randomly divided into three groups. In the type b model, groups of 12 to 13 5-day-old infant rats were inoculated s.c. on the dorsum close to the neck with 0.1 ml of rabbit anti-rD15 or anti-MinnA antiserum. The animals in the control group were injected with rabbit prebleed serum (Loosmore et al., 1997).
  • Challenge Protocol: 24 hours after immunization, the animals were challenged intraperitoneally (i.p.) with 100 CFU of freshly grown Hib MinnA (0.1 ml) (Loosmore et al., 1997).
  • Efficacy: Purified rD15 was found to be highly immunogenic in mice, guinea pigs, and rabbits, and passive transfer of anti-rD15 antibodies protected infant rats from challenge with H. influenzae type b or type a in infant rat models of bacteremia (Loosmore et al., 1997).

Rat Response

  • Host Strain: DA
  • Vaccination Protocol: The immunization protein was prepared by emulsifying 200 or 800 μg of protein per ml in a 1:1 ratio of incomplete Freund’s adjuvant (IFA; Difco Laboratories, Detroit, Mich.), and phosphate-buffered saline (PBS), and a total inoculum of 10 or 40 μg of protein, respectively, was administered to each animal via subserosal injection of intestinal Peyer’s patches (IPP) (Kyd and Cripps, 1998).
  • Challenge Protocol: Pulmonary challenge with live bacteria was performed on day 21 post-IPP immunization. The animals were sedated with halothane, and a bolus inoculum of 5 × 108 CFU of live H. influenzae in 50 μl of PBS was introduced into the lungs via an i.t. cannula and dispersed with two 5-ml volumes of air (Kyd and Cripps, 1998).
  • Efficacy: OMP26 was used to immunize rats via intestinal Peyer's patches, followed by an intratracheal boost. Immunization was found to significantly enhance bacterial clearance following pulmonary challenge with both the homologous NTHI strain and a different NTHI strain (Kyd and Cripps, 1998).
  • Host IgA response
    • Description: High OMP26-specific antibody titers for IgG, IgA, and IgM were found in the serum. The results were significantly higher than non-immunized mice for groups receiving 10 micrograms and 40 micrograms of the vaccine (Kyd and Cripps, 1998).
    • Detailed Gene Information: Click Here.
  • Host IgG (partial) response
    • Description: High OMP26-specific antibody titers for IgG, IgA, and IgM were found in the serum. The results were significantly higher than non-immunized mice for groups receiving 10 micrograms and 40 micrograms of the vaccine (Kyd and Cripps, 1998).
    • Detailed Gene Information: Click Here.
  • Host IgM response
    • Description: High OMP26-specific antibody titers for IgG, IgA, and IgM were found in the serum. The results were significantly higher than non-immunized mice for groups receiving 10 micrograms and 40 micrograms of the vaccine (Kyd and Cripps, 1998).
    • Detailed Gene Information: Click Here.

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).

Rat Response

  • Host Strain: Wistar rat
  • Vaccination Protocol: MVF/L1A, MVF/L4, and MVF/H3 were solubilized at a concentration of 6.5 mg/ml in 6 M guanidine-HCl and then diluted to 800 µg/ml with phosphate-buffered saline (PBS) and emulsified with an equal volume of incomplete Freund's adjuvant. Peyer's patch immunization was performed in Wistar rats. Each animal receiving 20 µg of peptide. Control rats were unimmunized or immunized with the same concentration of incomplete Freund's adjuvant-PBS-guanidine-HCl as that used for the peptide-treated group (Webb et al., 2000).
  • Efficacy: Immunization of rats with MVF/H3 was the most efficacious in significantly reducing the number of viable NTHi in both the broncho-alveolar lavage fluid (74%) and lung homogenates (70%), compared to control rats. Importantly, despite significantly increased rates of clearance, immunization with MVF/H3 elicited poor antibody responses (Webb et al., 2000).

Rat Response

  • Host Strain: White Wistar male rats
  • Vaccination Protocol: White Wistar rats were randomly assigned to four experimental groups: intranasal immunization with P6 plus CT or CT alone, intraperitoneal immunization with P6 plus CT or CT alone. Rats were immunized intranasally 5 times, on days 0, 7, 14, 21 and 28 with 60 μl of antigen solution containing 50 μg of P6 and 10 μg of cholera toxin (CT; Sigma, St. Louis, MO) as a mucosal adjuvant or 60 μl of PBS containing 10 μg of CT alone into the left nasal cavity. Control rats were given PBS without antigen.In the experiments on systemic immunization, animals were intraperitoneally injected with 60 μl of the same vaccine on days 0, 7, 14, 21 and 28 (Sabirov et al., 2004).
  • Immune Response: Intranasal immunization induced P6-specific sinus mucosal and systemic immunological responses, mainly of the IgA and IgG isotype.
  • Challenge Protocol: At day 35, 50 μl (109 CFU) of the live NTHi suspension was injected slowly through exposed fibrous tissue into left or right maxillary sinus using a microsyringe. Six rats from each group were sacrificed at 12 and 24 h after the inoculation. Thereafter, the nose was lengthwise split in the equal halves and challenged sinuses were washed with PBS. The sinus wash samples were serially diluted 10-fold, and 10 μl aliquots of the diluted samples were spread on chocolate agar plates to determine the concentration of live bacteria (Sabirov et al., 2004).
  • Efficacy: The protective effect of intranasal immunization was demonstrated by enhancement of sinus clearance of NTHi. Therefore, unilateral intranasal immunization has a capacity to induce protective immunity against NTHi in the bilateral maxillary sinuses. Systemic administration of the vaccine did not affect sinus clearance of NTHi (Sabirov et al., 2004).

Rat Response

  • Host Strain: Wistar rat
  • Vaccination Protocol: Lyophilized rTbpB was resuspended in PBS and emulsified in an equal volume of incomplete Freund’s adjuvant (IFA) to give a final protein concentration of either 400 or 800 μg/ml. Peyer’s patches in male, 8-week-old Wistar rats were each injected with 2 to 5 μl of antigen, with each rat receiving a total of either 20 or 40 μg of rTbpB. A control group of animals was either sham immunized with PBS-IFA or left unimmunized. The animals were boosted intratracheally 14 days later with the same antigen dose as in the primary immunization in 50 μl of PBS (Webb et al., 1999).
  • Challenge Protocol: 7 days after the booster dose, the rats were lightly sedated with halothane and 5 × 108 CFU of UC19 in 50 μl was instilled into the lungs via an intratracheal cannula. After 4 h, the animals were killed and bronchoaveolar lavage (BAL) fluid, serum, and homogenized lung samples were obtained. The numbers of viable bacteria in BAL fluid and lung homogenates were estimated by plating serial dilutions onto chocolate blood agar (Webb et al., 1999).
  • Efficacy: The efficacy of immunization with a recombinant form of TbpB (rTbpB) was determined by assessing the pulmonary clearance of viable bacteria 4 h after a live challenge with NTHI. There was a significant reduction in the number of viable bacteria in both the bronchoalveolar lavage fluid (34% for the 20-μg dose and 58% for the 40-μg dose) and lung homogenates (26% for the 20-μg dose and 60% for the 40-μg dose) of rats immunized with rTbpB compared to the control animals. While rTbpB-specific antibodies from immunized rats were nonspecific in the recognition of TbpB from six heterologous NTHI strains on Western blots, these antibodies differed in their ability to block transferrin binding to heterologous strains and to cross-react in bactericidal assays (Webb et al., 1999).
References References References References References References
Loosmore et al., 1997: Loosmore SM, Yang YP, Coleman DC, Shortreed JM, England DM, Klein MH. Outer membrane protein D15 is conserved among Haemophilus influenzae species and may represent a universal protective antigen against invasive disease. Infection and immunity. 1997; 65(9); 3701-3707. [PubMed: 9284140].
Kyd and Cripps, 1998: Kyd JM, Cripps AW. Potential of a novel protein, OMP26, from nontypeable Haemophilus influenzae to enhance pulmonary clearance in a rat model. Infection and immunity. 1998; 66(5); 2272-2278. [PubMed: 9573117].
Zagursky et al., 2000: Zagursky RJ, Ooi P, Jones KF, Fiske MJ, Smith RP, Green BA. Identification of a Haemophilus influenzae 5'-nucleotidase protein: cloning of the nucA gene and immunogenicity and characterization of the NucA protein. Infection and immunity. 2000 May; 68(5); 2525-34. [PubMed: 10768940].
Webb et al., 2000: Webb DC, Cripps AW. A P5 peptide that is homologous to peptide 10 of OprF from Pseudomonas aeruginosa enhances clearance of nontypeable Haemophilus influenzae from acutely infected rat lung in the absence of detectable peptide-specific antibody. Infection and immunity. 2000 Jan; 68(1); 377-81. [PubMed: 10603411].
Sabirov et al., 2004: Sabirov A, Kodama S, Sabirova N, Mogi G, Suzuki M. Intranasal immunization with outer membrane protein P6 and cholera toxin induces specific sinus mucosal immunity and enhances sinus clearance of nontypeable Haemophilus influenzae. Vaccine. 2004 Aug 13; 22(23-24); 3112-21. [PubMed: 15297063].
Webb et al., 1999: Webb DC, Cripps AW. Immunization with recombinant transferrin binding protein B enhances clearance of nontypeable Haemophilus influenzae from the rat lung. Infection and immunity. 1999 May; 67(5); 2138-44. [PubMed: 10225866].