Haemophilus influenzae 727 The most important virulence factor defining pathogenic H. influenzae strains is capsule, of which six serotypes, a-f, have been described. Among encapsulated strains, those possessing the type b capsule (Hib strains), which is composed of polyribosylribitol phosphate (PRP), are the most virulent, capable of causing sustained bacteremia and subsequent focal infection in non-immune, normal hosts. Like the capsules of other gram negative organisms, H. influenzae capsules enhance the pathogenicity of the bacteria by protecting them from phagocytosis. The strong negative charge of the polysaccharide capsules may provide electrostatic repulsion to phagocytic cells and the capsular material itself may sterically interfere with the binding of opsonizing antibodies and complement to the bacterial surface (Marrs et al., 2001). H. influenzae pili (sometimes called fimbriae) was the first well studied adherence factors of H. influenzae. These filamentous organelles are seen by electron microscopy distributed in a peritrichous manner on the H. influenzae bacterial cell surface of isolates that hemagglutinate and adhere to buccal epithelial cells, but are absent from non-hemagglutinating and non-adherent bacteria. Generally strains isolated from invasive infections do not express pili, while those colonizing the nasopharynx more commonly do. piliated H. influenzae binds to erythrocytes possessing the Anton (AnWj) antigen. Additional studies have shown that sialic acid-containing lactosylceramides, the gangliosides GM1, GM2, GM3 and GD1a, inhibit pilus- mediated H. influenzae binding to both buccal epithelial cells and erythrocytes (Marrs et al., 2001). Highly similar high-molecular-weight proteins (HMW1, HMW2) were first identified as H. influenzae-associated antigens by their ability to induce robust antibody responses in individuals with acute otitis media . Subsequent studies have revealed that these HMW proteins function as adherence factors and can facilitate the colonization of cultured human epithelial cells . HMW1 is a 160 kDa product of the hmw1A gene and HMW2 is a 155 kDa protein encoded by the hmw2A gene; hmw1A and hmw2A are 71% identical and 80% similar . To date the hmw genes have only been detected in nontypable strains, and a majority of HMW-positive strains retain both chromosomal loci (Marrs et al., 2001). Secretory IgA fulfills many protective functions on the mucosal surface; these include the neutralization of toxins, inhibition of pathogen attachment to the epithelium, and agglutination of organisms within mucus . To surmount this immunological obstacle, H. influenzae constitutively secrete IgA1 proteases which are enzymes that inactivate the predominant IgA species, IgA1, by cleaving the immunoglobulin molecule at the heavy chain hinge region, resulting in the production of two fragments (Fab and Fc). (Reinholdt et al., 1997) H. influenzae produces a lipopolysaccharide structure that lacks an O-specific antigen and, instead, incorporates short oligosaccharide side chains onto three conserved heptoses and is thus termed lipooligosaccharide (LOS). A wide variety of glyco-modifications to the core LOS molecule have been detected and include the addition of glucose, galactose, lactose, phosphatidylcholine (ChoP), and sialic acid. This repertoire of sugars allows the organism to mimic common eukaryotic glycolipid structures, and is presumably an adaptive strategy for subverting immune defenses (Marrs et al., 2001). Meningitis Newborns are protected by transplacental maternal type b antibodies and older children and adults are protected by "natural antibodies", which are H. influenzae specific antibodies in a host who has not had a known H. influenzae infection. Most likely these antibodies result from subclinical infection or asymptomatic colonization (Talan et al., 1999). H. influenzae live exclusively in humans, and although they may cause disease in a variety of body sites (such as the central nervous system, joints, skin, lungs, bronchi and genito-urinary tract), they are most commonly isolated from the nasopharynx, where they are carried asymptomatically . Throat or nasopharyngeal culture surveys of both healthy and ill individuals reveal the carriage rate to be between 20 and 85%, and the majority of colonizing strains lack a capsule. Person to person transmission of H. influenzae is assumed to occur by contact with infected respiratory droplets and has resulted in clusters of H. influenzae type b (Hib) invasive infections among children in households and day care centers (Marrs et al., 2001). The infant rat model of invasive Hib disease played a substantial role in the identification of Hib virulence factors. The model allows for respiratory tract colonization in rats less than three weeks of age by inhalation of only a few organisms. Colonization is followed closely by bacteremia and meningitis. Therefore, this model mimics the invasive disease seen in children . Older rats (up to three months of age) can also develop meningitis through intraperitoneal injection of Hib and subsequent bacteremia. Rabbits have also been used for experimental Hib infections and have contributed to understanding the pathophysiology of meningitis and in the design of treatment regiments. Rabbits require direct inoculation into the CNS or the perturbation of the CNS prior to i.v. injection (Marrs et al., 2001). Experimental infection studies for otitis media most often employ the chinchilla but the gerbil and rat have also been used . In the chinchilla, NTHi are often inoculated directly into the bulla and disease is monitored over a two- to eight-week period for fluid pressure changes using otoscopy and tympanometry. Tympanocentesis and nasopharyngeal lavage are performed to assess colonization and immune responses present in the middle ear . Direct inoculation of the middle ear results in 100% infectivity but bacterial and host factors involved in initial nasopharyngeal colonization cannot be studied through this route. While NTHi can colonize the nasopharynx of chinchillas after experimental inoculation, colonization does not consistently lead to otitis media. An otitis media model has, however, been developed that allows for initial colonization with NTHi. Chinchillas are first infected with adenovirus, and then NTHi, administered nasally, will colonize the nasopharynx and sometimes result in otitis media. This scenario mimics development of otitis media in children. This method has been used to assess the role of bacterial components in colonization and their potential as vaccine candidates . Pulmonary infection models used for vaccine efficacy tests have utilized both mice and rats. In these models, animals are immunized intravenously or at the mucosal surface and then challenged by direct administration of NTHi to the lungs. Bacterial clearance is then assessed a few hours after challenge by culturing homogenized lung tissue or lavage fluid. These pulmonary clearance models, however, do not allow for the study of persistent colonization as is seen with chronic infections in the lungs of adults with COPD. Attempts have been made to reflect this scenario in rats by administering NTHi encapsulated within agarose beads. This method can lead to infections that persist for weeks resulting in pathology and immune responses that parallel those seen in humans . Haemophilus influenzae are Gram negative, pleomorphic rods first described by Pfeiffer in 1892. Haemophilus influenzae includes six encapsulated types, a through f, as well as nonencapsulated or untypeable strains, and these are found as both respiratory tract commensals and respiratory and invasive pathogens (Kilian et al, 1991) (Tristram et al., 2007). Invasive H. influenzae infections, such as bacteremia, meningitis, pneumonia in children, epiglottis, and septic arthritis, are usually caused by H. influenzae strains possessing the type b capsule. Furthermore, these infections occur much more frequently in non-immune children between ages 2 months and 5 years, and this age-related susceptibility is inversely correlated with the bactericidal capacity of the blood , which is mediated by antibodies directed against the type b capsule.Although non-typable H. influenzae rarely cause invasive infections in normal hosts, they are important causes of respiratory infections, such as bronchitis and pneumonia in adults with underlying pulmonary disease and otitis media and sinusitis in healthy individuals. Recent studies show that H. influenzae cause 23-27% of acute otitis media in children, the same incidence as S. pneumoniae (Marrs et al., 2001). Prior to introduction of Hib vaccines, Hib was estimated to be responsible worldwide for some three million serious illnesses and an estimated 386,000 deaths per year, chiefly through meningitis and pneumonia, with 95% of cases and 98% of deaths occurring in patients in developing countries. Almost all victims are children under the age of 5 years, with those between 4 and 18 months of age especially vulnerable . Large, population-based disease burden studies showed annual rates of Hib meningitis at 10 to 60 cases per 100,000 children under 5 years of age in countries where Hib vaccine is not used . In developing countries, where the vast majority of Hib deaths occur, pneumonia accounts for a larger number of deaths than meningitis. However, Hib meningitis is also a serious problem in such countries with mortality rates several times higher than seen in developed countries, and 15 to 35% of survivors have permanent disabilities such as mental retardation or deafness. Systematic vaccination has virtually eliminated Hib disease in most industrialized nations, with 89 countries offering infant immunization against Hib by the end of 2004 compared with 38 in 1999. However, Hib vaccine is not available in Japan. Ninety-two percent of the populations of developed countries were vaccinated against Hib as of 2003, while vaccination coverage was 42% for developing countries and 8% for least-developed countries (Tristram et al., 2007). Baboon Papio cynocephalus 9556 Bank vole Clethrionomys glareolus 447135 Bear Ursus americanus 9643 Birds Passeroidea 175121 Brown Trout Salmo trutta 8032 Buffalo Bison bison 9901 Carnivores Vulpes 9625 Cat Felis catus 9685 Catfishes Siluriformes 7995 Cattle Bos taurus 9913 Chicken Gallus gallus 9031 Chimpanzee Pan troglodytes 9598 chinchillas Chinchillidae 10150 Copper Pheasant Syrmaticus soemmerringii 9067 Deer Cervus elaphus 9860 Deer mouse Peromyscus maniculatus 10042 Dog Canis familiaris 9615 Ducks Anas 8835 Ferret Mustela putorius furo 9669 Fish Hyperotreti 117565 Gerbil Gerbillina 10045 Goat Capra hircus 9925 Gray wolf Canis lupus 9612 Guinea pig Cavia porcellus 10141 Hamster Mesocricetus auratus 10036 Horse Equus caballus 9796 Human Homo sapiens 9606 Macaque Macaca fascicularis 9541 Mongolian Gerbil Meriones unguiculatus 10047 Monkey Platyrrhini 9479 Mouse Mus musculus 10090 None None Parrot Psittacidae 9224 Pig Sus scrofa 9823 Rabbit Oryctolagus cuniculus 9986 Rainbow trout Oncorhynchus mykiss 8022 Rat Rattus 10114 Raven Corvus corax 56781 sei whale Balaenoptera borealis 9768 Sheep Ovis aries 9940 Squirrel Spermophilus richardsonii 37591 Tree shrew Tupaiidae 9393 Trouts, salmons & chars Salmoninae 504568 Turkey Meleagris gallopavo 9103 Vole Microtus ochrogaster 79684 Water buffalo Bubalus bubalis 391902 Actacel Actacel Sanofi Pasteur Ltd VO_0010705 Subunit vaccine Licensed Intramuscular injection (i.m.) Canada Products: Proteins + Conjugate. Aluminum phosphate Intramuscular injection (i.m.) ActHIB Haemophilus b Conjugate Vaccine (Tetanus Toxoid Conjugate) ActHIB Sanofi Pasteur, SA VO_0000004 Conjugate vaccine Licensed Intramuscular injection (i.m.) USA (License #1724) ActHIB vaccine was among the first conjugated Hib vaccines developed at the National Institutes of Health (NIH). It is now manufactured by Sanofi Pasteur SA in Lyon, France. The conjugation process changes the polysaccharide from a “T-cell–independent antigen” to a “T-cell–dependent antigen”, which greatly improves immunogenicity, particularly in young children. In addition, repeated doses elicit a substantial booster response in children who have been previously vaccinated (ActHIB 2005). 2° to 8°C (35° to 46°F). DO NOT FREEZE. Not virulent The vaccine consists of the Haemophilus b capsular polysaccharide (polyribosyl-ribitol-phosphate, PRP), a high molecular weight polymer prepared from the Haemophilus influenzae type b (HiB) strain 1482 grown in a semi-synthetic medium, covalently bound to tetanus toxoid. The tetanus toxoid is prepared by extraction, ammonium sulfate purification, and formalin inactivation of the toxin from cultures of Clostridium tetani (Harvard strain) grown in a modified Mueller and Miller medium. The toxoid is filter sterilized prior to the conjugation process. When ActHIB vaccine is reconstituted with saline diluent, each single dose of 0.5 mL is formulated to contain 10 μg of purified capsular polysaccharide conjugated to 24 μg of inactivated tetanus toxoid, and 8.5% of sucrose (ActHIB 2005). Intramuscular injection (i.m.) Haemophilus b capsular polyribosyl-ribitol-phosphate(PRP)- tetanus toxoid ActHIB vaccine is indicated for active immunization of infants and children 2 through 18 months of age for the prevention of invasive disease caused by H influenzae type b. The number of doses of ActHIB vaccine indicated depends on the age at which immunization is begun. For previously unvaccinated children, the first, second, third and fourth dose were given at 2, 4, 6, and 12 to 18 months. A child 7 to 11 months of age should receive 2 doses at 8-week intervals and a booster dose at 15 to 18 months. A child 12 to 24 months of age should receive 1 dose and a booster dose 2 months later. Preterm infants should be vaccinated according to their chronological age from birth (ActHIB 2005). ActHIB vaccine reconstituted with the saline diluent (0.5 mL per dose) should be administered intramuscularly in the outer aspect of the midthigh or deltoid. Do not inject intravenously. It should not be injected into the gluteal area or areas where there may be a nerve trunk. When administering multiple vaccines during a single visit, separate injection sites and syringes should be used. Administer ActHIB vaccine within 24 hours after reconstitution (ActHIB 2005). Two clinical trials supported by the National Institutes of Health (NIH) compared the anti-PRP (polyribosyl-ribitol-phosphate) antibody responses to three Hib conjugate vaccines in racially mixed populations of children. In these trials, ActHIB vaccine consistently produced high rates of seroconversion (83% to 97%) to antibody levels that correlate with long-term protection (>1.0 µg/mL) following the 3-dose primary series (Decker et al., 1992). Consistently high rates of seroconversion (83% to 99%) with an ActHIB vaccine 3-dose primary series were also obtained in 11 non-comparative clinical trials (N=1225) (Fritzell et al., 1992). Additionally, three NIH trials demonstrated that ActHIB vaccine produced consistently high geometric mean anti-PRP antibody titers (Decker et al., 1992). The most common side effects with ActHIB vaccine may include redness, swelling, and tenderness where the injection was given; fever, fussiness, and drowsiness. Other side effects may occur. ActHIB vaccine should not be given to children who have had a serious allergic reaction (anaphylactic reaction) after a previous dose of the vaccine. When administering an intramuscular injection, like ActHIB vaccine, to people with bleeding disorders, caution should be exercised because they may develop a serious bruise or collection of blood at the injection site (ActHIB 2005). COMVAX Haemophilus b Conjugate COMVAX Merck & Co., Inc VO_0000028 Conjugate vaccine Licensed Intramuscular injection (i.m.) USA (License #0002) Immunization of persons 6 weeks to 15 months of age born to hepatitis B surface antigen (HBsAg) negative mothers. aluminum hydroxide adjuvant. Store vaccine at 2-8°C (36-46*F). Storage above or belew the recommended temperature may reduce potency. DO NOT FREEZE since freezing destroys potency.(Comvax) It is grown in complex fermentation media. It is then purified from the culture broth by purification procedures which include ethanol fractionation, enzyme digestion, phenol extraction and diafiltration (FDA: COMVAX). Intramuscular injection (i.m.) No impairment of immune response to individually tested vaccine antigens COMVAX SHOULD NOT BE USED IN INFANTS YOUNGER THAN 6 WEEKS OF AGE.(Comvax) A protective efficacy of 93% was achieved with an anti-PRP level of >l.O mcg/mL in 60% of vaccines and a GMT of 1.43 mcg/mL one to three months after the second dose.(Comvax) Injection site reactions as well as systemic complaints occur. It is a vaccination against invasive disease caused by Haemophilus influenzae type b and against infection caused by all known subtypes of hepatitis B virus in infants 6 weeks to 15 months of age born of HBsAg negative mothers. H. influenzae D15 protein vaccine VO_0011551 Subunit vaccine Research Intramuscular injection (i.m.) Freund's adjuvant Intramuscular injection (i.m.) H. influenzae protective surface antigen D15 Recombinant protein preparation 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). Sprague- Dawley 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). 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). 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). H. influenzae Hap protein vaccine VO_0011499 Subunit vaccine Research Subcutaneous injection Mutant cholera toxin CT-E29H Subcutaneous injection H. influenzae Hap Recombinant protein preparation 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). BALB/c 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). 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). Three weeks after the final immunization, mice were challenged intranasally with approximately ×10^6 CFU of strain TN106.P2 (Liu et al., 2004). H. influenzae Omp26 protein vaccine VO_0011562 Subunit vaccine Research Subserosal injection Incomplete Freund's adjuvant Subserosal injection H. influenzae outer membrane protein 26 Recombinant protein preparation 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). DA 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). 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). 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 × 10^8 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). 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). 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). 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). H. influenzae Type b Capsular Polysaccharide Vaccine VO_0000627 Subunit vaccine H. influenzae type b vaccine, coded as M1 and M2, respectively, was prepared for this trial in Boston and bottled in ten-dose vials (BenVenue Laboratories, Inc., Ohio). The lyophilized vaccine was kept at -20 C; after reconstitution with sterile, pyrogen-free water is was kept at + 4 C and used within eight hours (Peltola et al., 1977). Haemophilus b capsular polysaccharide (polyribosyl-ribitol-phosphate, PRP) The serum antibody response to the H. influenzae type b polysaccharide, measured by radioimmunoassay, was poor in children below 18 months of age and good in those above it. No effect of the vaccine could be seen on the nasopharyngeal carriage of H. influenzae type b, which was approximately 6% in this age group (Peltola et al., 1977). A total of 98,272 Finnish children, or 75.5% of the 130,178 children between the ages of 3 months to 5 years in the area, were vaccinated in three provinces in Finland. The campaign took place within two weeks in November 1974. A booster dose was given three months later to those who had been 3 to 17 months old at the time of the primary vaccination. The participation in the booster vaccination was 73.3%. Every other child (total, 49,295) received the group A meningococcal vaccine, every other (total, 48,977) the H. influenzae type b vaccine, coded as M1 and M2, respectively: All age groups and both sexes were equally represented. The dose of H. influenzae type b was 12.7 ug of polysaccharide in a volume of 0.5 ml except for the smallest infants (ages 3 to 5 months) who received only half of this dose. The subcutaneous injection was given by needle and syringe, usually into the upper part of the right arm. Children with an acute febrile disease, extensive eczema, or symptomatic asthma were not vaccinated (Peltola et al., 1977). The serum antibodies induced by the vaccination proved short-lived (less than 6 months) in the infants younger than 18 months. Elevated serum antibody levels were detectable for 1and half year but less than 3 and half years in the children who were vaccinated when 18 to 35 months old. In the children who were 3 to 5 years old when vaccinated, the elevated anti-H influenzae type b capsular polysaccharide levels persisted for at least 3 and half years (Kayhty et al., 1984). The protection as well as senim antibody response was strongly age-dependent. Among children who had received the H. influenwe type b vaccine when 18 months of age or older, there were no cases of bacteremic disease caused by H. influenzae type b in the first year after vaccination. At the same time 1 1 such cases were seen in the control group of the same age, a highly significant difference. In the second year after vaccination two cases occurred in the H. influenzoe type b-vaccinated group, five in the meningocoecal-group A vaccinated group. No protection Was seen among children who had been younger than 18 months when vaccinatedı even if they received a booster dose of the vaccine. Adverse effects of the vaccine were mild Hiberix Hiberix GlaxoSmithKline VO_0010715 Subunit vaccine Licensed Intramuscular injection (i.m.) Canada Products: Conjugate. Other components: Lactose. Store between 2° and 8°C. Intramuscular injection (i.m.) HIBTITER H. influenzae type b conjugate vaccine (Hb-OC) HIBTITER VO_0000659 conjugate vaccines Linkage of Haemophilus b saccharides to a protein such as CRM197 converts the saccharide (HbO) to a T-dependent (HbOC) antigen, and results in an enhanced antibody response to the saccharide in young infants that primes for an anamnestic response and is predominantly of the IgG class.12 Laboratory evidence indicates that the native state of the CRM197 protein and the use of oligosaccharides in the formulation of HibTITER enhances its immunogenicity (Weinberg et al., 1988). In most cases HibTITER was administered concomitantly with other vaccines including DTP, DTaP, hepatitis B vaccine, IPV, OPV, pneumococcal 7-valent conjugate vaccine, MMR, and/or meningococcal group C conjugate vaccine (not licensed in the US). The oligosaccharides are derived from highly purified capsular polysaccharide, polyribosylribitol phosphate, isolated from Haemophilus b strain Eagan grown in a chemically defined medium (a mixture of mineral salts, amino acids, and cofactors). The oligosaccharides are purified and sized by diafiltrations through a series of ultrafiltration membranes, and coupled by reductive amination directly to highly purified CRM197. CRM197 is a nontoxic variant of diphtheria toxin isolated from cultures of Corynebacterium diphtheriae C7 (β197) grown in a casamino acids and yeast extract-based medium that is ultrafiltered before use. CRM197 is purified through ultrafiltration, ammonium sulfate precipitation, and ion-exchange chromatography to high purity. The conjugate is purified to remove unreacted protein, oligosaccharides, and reagents; sterilized by filtration; and filled into vials (HibTITER 2007). The vaccine is a clear, colorless solution. Each single dose of 0.5 mL is formulated to contain 10 μg of purified Haemophilus b saccharide and approximately 25 μg of CRM197 protein (HibTITER 2007). Haemophilus influenzae type b capsular polyribosyl-ribitol-phosphate(PRP)-Diphtheria CRM197 Protein (HibTITER 2007). Haemophilus b Conjugate Vaccine (Diphtheria CRM197 Protein Conjugate) HibTITER is indicated for the immunization of children 2 months to 71 months of age against invasive diseases caused by H. influenzae type b. HibTITER is for intramuscular use only. For infants 2 to 6 months of age, the immunizing dose is three separate injections of 0.5 mL given at approximately 2-month intervals. Previously unvaccinated infants 12 from 7 through 11 months of age should receive two separate injections approximately 2 months apart. Children from 12 through 14 months of age who have not been vaccinated previously receive one injection. All vaccinated children receive a single booster dose at 15 months of age or older, but not less than 2 months after the previous dose. Previously unvaccinated children 15 to 71 months of age receive a single injection of HibTITER. Preterm infants should be vaccinated with HibTITER according to their chronological age, from birth (AAPC 1991). Data support that HibTITER may be interchanged with other Haemophilus influenzae type b conjugate vaccines for the primary immunization series and booster dose. Each dose of 0.5 mL is formulated to contain 10 μg of purified Haemophilus b saccharide and approximately 25 μg of CRM197 protein. Long-term persistence of the antibody response was observed. More than 80% of 235 infants who received three doses of vaccine had an anti-HbPs antibody level ≥ 1 μg/mL at 2 years of age (Reinholdt et al., 1997). The immunogenicity of HibTITER was evaluated in US infants and children.Infants 1 to 6 months of age at first immunization received three doses at approximately 2-month intervals. Children 7 to 11 and 12 to 14 months of age received 2 doses at the same interval.Children 15 to 23 months of age received a single dose. HibTITER was highly immunogenic in all age groups studied, with 97% to 100% of 1,232 infants attaining titers of ≥ 1 μg/mL and 92% to 100% for bactericidal activity (HibTITER 2007). Postlicensure surveillance of immunogenicity was conducted during the distribution of the first 30 million doses of HibTITER and during the time period over which Haemophilus b disease in children has been decreasing significantly in areas of extensive vaccine usage.After three doses, titers ranged from 2.37 to 8.45 μg/mL with 67% to 94% attaining ≥ 1 μg/mL (HibTITER 2007). A comparative clinical trial was performed in Finland where approximately 53,000 infants received HibTITER at 4 and 6 months of age and a booster dose at 14 months in a trial conducted from January 1988 through December 1990. Only two children developed Haemophilus b disease after receiving the two-dose primary immunization schedule. One child became ill at 15 months of age and the other at 18 months of age; neither child received the scheduled booster at 14 months of age. No vaccine failure has been reported in children who received the two-dose primary series and the booster dose at 14 months of age. Based on more than 32,000 person-years of follow-up time, the estimate of efficacy is about 95% when compared to historical control groups followed between 1985 and 1988.20 Historical controls were used since all infants received one of two Haemophilus b conjugate vaccines during the period of the trial (HibTITER 2007). Side effects associated with a single vaccination of HibTITER include fever, local reactions, rash, diarrhea , vomiting , prolonged crying. Infanrix -IPV/Hib Diphtheria, tetanus, pertussis (acellular component), poliomyelitis (inactivated) and Haemophilus type b conjugate vaccine (adsorbed) Infanrix -IPV/Hib GlaxoSmithKline VO_0010722 Subunit vaccine + Inactivated or "killed" vaccine Licensed Intramuscular injection (i.m.) Canada Products: Proteins + killed virus + conjugate. Other components: Formaldehyde Polysorbate 80, Lactose. Aluminum Hydroxide Store in a refrigerator (2°C – 8°C). Intramuscular injection (i.m.) Infanrix-hexa Combined diphtheria and tetanus toxoids, acellular pertussis, hepatitis B (recombinant), inactivated poliomyelitis and adsorbed conjugated Haemophilus influenzae type b vaccine Infanrix-hexa GlaxoSmithKline VO_0010719 Subunit vaccine + Inactivated or "killed" vaccine Licensed Intramuscular injection (i.m.) Canada Products: Proteins + killed viruses + conjugate. Other components: Yeast protein Formaldehyde, Lactose, Polysorbate 20 and 80. Aluminum phosphate and aluminum hydroxide Should be stored at 2° to 8°C (35° to 46°F). Intramuscular injection (i.m.) Infanrix/Hib Diphtheria-tetanus-acellular pertussis and Haemophilus influenzae type b (Hib) vaccine. Infanrix/Hib GlaxoSmithKline VO_0010720 Subunit vaccine Licensed Intramuscular injection (i.m.) Canada Products: Proteins + conjugate. Other components: Formaldehyde, Lactose. Aluminum hydroxide Store at 2°C – 8°C (in a refrigerator). Intramuscular injection (i.m.) Killed nontypeable H. influenzae whole-cell vaccine VO_0000474 Inactivated or "killed" vaccine Oral immunization Acute exacerbations of bronchitis and chronic obstructive pulmonary disease often occur in people with compromised respiratory function.The bacterial agents responsible for the acute exacerbations are commonly an expansion of the patient's own respiratory tract microbiota often following the arrival of a new strain of normal commensal. Nontypeable Haemophilus influenzae is of particular importance because it is the most commonly isolated bacterium at times of exacerbation (Foxwell et al., 2006). Successful vaccines for infections caused by H. influenzae serotype B (Hib) depend on immunity stimulated against the type-specific polysaccharide capsule of Hib. But this is not effective against infections caused by organisms lacking the polysaccharide, such as nontypeable H. influenzae. The oral whole-cell nontypeable. H. influenzae vaccine is developed to prevent nontypeable H. influenzae infections (Foxwell et al., 2006). no virulence The vaccine was enteric-coated tabletcontaining 10th power numbers of killed H influenzae with sodium tauroglycocholate. The organism was a non-serotypable biotype 2 H influertzae, and was formalin killed. The tablets were quality controlled for acid resistance, release in alkali, and sterility of content (Clancy et al., 1985). Oral immunization whole cell A prospective, double-blind, placebo-controlled trial was performed during the Australian winter of 1983. 50 patients with established COLD were recruited from the chest clinic of the Royal Newcastle Hospital and randomly allocated to three groups. No patient was taking corticosteroids or mmunosuppressive agents, but many took bronchodilator drugs and antibiotics. Three groups were tested: one took enteric-coated glucose tablets; the second took enteric-coated tablets containing 25 mg sodium tauroglycocholate; while the third took enteric-coated tablets each containing 10th power of killed H influenzae with sodium tauroglycocholate. Three courses of tablets were given at 0, 28, and 56 days. Each course consisted of two tablets taken before breakfast each day for 3 consecutive days. Each patient was assessed at 0, 28, 56 and 84 days (Clancy et al., 1985). Over a similar 3-month period through the subsequent winter (1984), during which no tablets were given, 11 of 22 patients available from the combined placebo groups had one or more acute episodes of bronchitis, while 4 of 14 available from the group who had previously taken the H influenzae tablets, had acute episodes. This trend towards continued protection was not significant (Clancy et al., 1985). The vaccine provided a more than 9007o protection rate against clinical episodes of acute bronchitis, with no noticeable effect on the incidence of upper respiratory tract infection. No significant difference in the incidence of acute upper respiratory tract infection was detected between the three groups. There was significant reduction in both the number of subjects with episode(s) of acute bronchitis (p<0 . 005), and the absolute number of episodes of acute bronchitis (p<0 . 002), in the group taking tablets containing H influenzae. If results were analysed according to the relative incidence of infection, there was a tenfold reduction in incidence in those taking the active tablet (p<0 . 001) (Clancy et al., 1985). No significant difference in antibody level to HI/H2 antigen existed between the three groups at zero time or at any point in the trial. Randomised, double-blind, placebo-controlled study of six months duration including winter. 40 patients with chronic bronchitis, 3 withdrawals, 2 from placebo and 1 from active treatment group. 20 given a placebo, 20 vaccinated. Mean age 46.3, sex ratio (m/f) 8/12 for the placebo group and 47.4, sex ratio 11/9 for the vaccine group. Vaccine consisting of 10 to the 11th formalin killed nontypeable Haemophilus infuenzae in 3 courses of enteric coated tablets given at days 0, 28 and 56. Each course consisted of two tablets given over three consecutive days before breakfast. Placebos were enteric coated glucose tablets (Clancy et al., 1990). The vaccine resulted in a marked reduction in the total number of episodes of acute bronchitis and acute wheezy bronchitis concomitant with a reduction in antibiotic use. They also warned that the small group number and a possible favouring of the vaccine group might have occurred. They also demonstrated a prevention of increase of H. inuenzae colonization in the vaccinated group (Clancy et al., 1990). Randomised, double-blind, prospective placebo-controlled study of 12 months duration starting in October. 62 patients with chronic bronchitis or more than 2 episodes of acute bronchitis in 2 years. 32 given a placebo,30 vaccinated. Mean age 53.7, sex ratio (m/f) 15/17 for the placebo group and 52.6, sex ratio 15/17 for the vaccine group: Drop-outs were 11 from vaccine group and 4 from placebo group. Vaccine consisting of 10 to the 11th formalin killed nontypeable Haemophilus influenzae in 3 courses of enteric coated tablets given at days 0, 28 and 56. Each course consisted of two tablets given over three consecutive days before breakfast. Placebos were enteric coated glucose tablets (Lehmann et al., 1991). the vaccine group had significantly lower acute bronchitic episodes post vaccination than the control group in individuals who had less severe but not severe disease. The carriage rate of H. influenzae also declined in the vaccinated group (Lehmann et al., 1991). Randomised, double-blind, placebo-controlled study of 12 months duration starting in March. 77 patients with chronic bronchitis. 10 were withdrawn (7 in placebo group and 3 in vaccine group). 3 from the vaccine group died. 33 given a placebo, 31 vaccinated. Mean age 71.1, sex ratio (m/f) 30/3 for the placebo group and 73.1, sex ratio 22/9 for the vaccine group. Vaccine consisting of 10 to the 11th formalin killed nontypeable Haemophilus influenzae in 3 courses of enteric coated tablets given at days 0, 28 and 56. Each course consisted of two tablets given over three consecutive days before breakfast. Placebos were enteric coated glucose tablets (Tandon et al., 1991). A reduction in acute bronchitic episodes in patients who received the vaccine was observed. They also needed to prescribe fewer antibiotics despite the number of individual's requiring antibiotics being similar. A reduction in H. influenzae colonization also occurred in vaccinated individuals (Tandon et al., 1991). licensed Haemophilus influenzae meningitis human vaccine Generic Unknown VO_0012185 Conjugate vaccine Licensed A generic representation of vaccines used to prevent Haemophilus influenzae type b (Hib) meningitis in humans. These vaccines consist of Hib polysaccharide antigens chemically linked to a carrier protein to enhance immunogenicity, especially in young children. MenHibrix Meningococcal Groups C and Y and Haemophilus b Tetanus Toxoid Conjugate Vaccine MenHibrix GlaxoSmithKline Biologicals VO_0000031 Conjugate vaccine Licensed Intramuscular injection (i.m.) United States Intramuscular injection (i.m.) Neisseria meningitidis serogroup C and Y capsular polysaccharide antigens and Haemophilus b capsular polysaccharide (polyribosyl-ribitol-phosphate [PRP]) (FDA: MenHibrix). Non-typeable H. influenzae dLOS–P6 vaccine VO_0004115 Conjugate vaccine Ribi detoxified 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). NTHi detoxified lipooligosaccharide (dLOS) conjugated with outer membrane protein P6 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). 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). 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). 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). Non-typeable H. influenzae rLP4/rLP6 and Moraxella catarrhalis UspA2 protein vaccine VO_0000473 Subunit vaccine Nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis are two of the leading causes of bacterial otitis media. The immunodominant exposed surface molecules of NTHi, including lipooligosaccharide (LOS) and outer membrane proteins, are extremely variable antigenically and infection with one strain provides immunity only to that strain. Thus, NTHi vaccine efforts have focused on antigenically conserved outer membrane protein such as the P4 and P6 proteins and LOS-conjugates. While the surface of M. catarrhalis appears to be much less antigenically variable, vaccine efforts have also focused on conserved outer membrane proteins and LOS. The ubiquitous cell surface protein A (UspA) of M. catarrhalis is a particularly interesting vaccine candidate (Mason et al., 2004). RC529 (Mason et al., 2004) Recombinant nontypeable H. influenzae LP4 (rLP4) and LP6 (rLP6) were expressed in E. coli strain BLR and purified (Mason et al., 2004). M. catarrhalis UspA was purified from outer membrane vesicles prepared from isolate O35E (Mason et al., 2004). NTHi rLP4/rLP6 and M. catarrhalis UspA2 proteins(Mason et al., 2004) recombinant The P4 protein is an integral outer membrane protein that has a role in acquiring hemin and nucleosides.(Green et al., 1991) recombinant The P6 protein is an integral outer membrane protein and, as a peptidoglycan-associated lipoprotein , is thought to be necessary for the integrity of the bacterium’s outer membrane.(Mason et al., 2004) These proteins combined with RC529-AF administered intranasally to mice elicited (1) significantly increased rLP4/rLP6/UspA2 protein-specific circulating IgG and IgA antibody responses; (2) local rLP4/rLP6/UspA2-specific IgA responses in the respiratory tract. The serum IgG subclass distribution was predominantly IgG2a, representing a Th1 response. At weeks of 0, 1, 3 and 5, groups of 10, 6 to 8-week-old, female BALB/c mice were immunized intranasally with 30 μg purified rLP4/rLP6/UspA2 (10 μg each protein) mixed with or without 10 μg RC529-AF in 15 μl of volume. Control mice received Delbecco's phosphate buffered saline (D-PBS) alone or D-PBS with 10 μg RC529-AF, again in 15 μl volumes. The vaccine was delivered by pipette in a volume of 7.5 μl per nostril. The pipette was positioned so that the tip touched the opening of the nostril and liquid was drawn into the nasopharynx during breathing. Immediately following immunization, mice were placed in a supine position for 3~5 min (Mason et al., 2004). These proteins combined with RC529-AF administered intranasally to mice elicited more than a two log reduction of nasal colonization of NTHi strain SR7332 from the nasal tissues of mice. The antiserum also exhibited bactericidal activities to several strains of M. catarrhalis. Two weeks after the final immunization, animals were challenged intranasally with approximately 1 million colony forming units (CFU) of NTHi strain SR7332.P1. Mice were anesthetized, and 5 μl of bacteria were administered into each nostril. Twenty minutes after the challenge, an aliquot of the bacterial suspension was diluted in D-PBS and plated onto brain-heart infusion XV (BHI-XV) agar to determine the actual inoculum. Three days after challenge, nasal turbinates were harvested, weighed, homogenized, serially diluted and plated on BHI-XV plates containing 100 μg/ml streptomycin. Following incubation of plates overnight, colonies were counted, and CFU per gram of nasal tissue were determined (Mason et al., 2004). Nontypeable H. influenzae cell membrane (CM-Hi) vaccine VO_0000479 Subunit vaccine cholera toxin (CT) 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). Nontypeable Haemophilus influenzae cell membrane 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. Male BALB/c mice 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). Bacterial clearance of a homologous strain of NTHi from the nasal tract was significantly enhanced in the nasal immunization group (Kurono et al., 1999). 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). Nontypeable H. influenzae Hap Protein Vaccine VO_0000432 Subunit vaccine 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). CT-E29H (a mutant cholera toxin) 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). Recombinant proteins corresponding to the C-terminal region of HapS from H. influenzae strains N187, P860295, and TN106. recombinant recombinant BALB/c 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). 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). 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). Nontypeable H. influenzae LOS-TT conjugate vaccine VO_0000480 Conjugate vaccine Nontypeable Haemophilus influenzae (NTHi) accounts for about one-third of purulent otitis media (OM) in children and is a common cause of pulmonary infection in adults with decreased resistance. Lipooligosaccharide(LOS) is both a virulence factor and a potential protective surface antigen. Human antibodies and mouse monoclonal antibodies against LOS are produced and can be bactericidal for NTHi (Gu et al., 2003). Tetanus toxoid (TT) was obtained from Pasteur-Merieux Connaught and purified through an S-300 Sephacryl column. LOS was prepared from NTHi strain 9274. It was used for preparation of dLOS and its derivative with adipic acid dehydrazide (AH–dLOS). The fusion dLOS–TT was further synthesized (Gu et al., 2003). lipooligosaccharide (LOS) All volunteers had pre-existing IgG anti-LOS. The geometric mean (GM) level rose from 14 to 40 at 2 weeks, remained at 35 at 6 weeks (40 or 35 versus 14,P <0.01) and dropped to 27 at 14 weeks after the first injection. There was also a rise 2 weeks after the second injection (27 versus 37,P <0.05 ). A total of 52.5% of subjects showed serum-conversion (greater than four-fold increase) after one and two injections. At 38 weeks, the GM IgG anti-LOS was still higher than before initial injection (20 versus 14,P < 0.05). A similar pattern of reactivity was observed for IgA and IgM anti-LOS (Gu et al., 2003). This is a Phase I clinical trial for this vaccine. Forty healthy adult volunteers of either sex, between 18 and 35 years of age, were recruited and informed consent was obtained. All 40 volunteers received an injection in the deltoid muscle with 0.5 mlof the investigational vaccine (25ug saccharide), and 28 of them also received a second injection in 3–4 months after the first injection. Their injection sites and body temperatures along with other reactions were monitored by a medically credentialled provider or nurse before and 1, 6, 24, and 48 h after each injection. Local and systemic reactions were monitored and sera, taken before and 2, 6, 14, 16, and 38 weeks after injection, were assayed for IgG, IgA, and IgM antibodies to the LOS by ELISA and for bactericidal activity (Gu et al., 2003). Analysis of the frequency and degree of local signs and symptoms at the injection area showed that all reactions were mild or moderate. There were four subjects complained of mild to moderate pain, one subject showed mild to moderate erythema (1–2 cm), two showed mild to moderate induration (1–2 cm). For systemic reactions, two subjects showed temperatures of 37.6 and 37.7 ◦C after the first injection and one subject reached 37.7 ◦C after the second injection. None reported abdominal discomfort or skin rashes. All other complaints were judged to be mild or moderate and medication was rarely required for the symptoms. There was no significant difference in systemic symptoms between two injections except for the incidence of myalgia (P = 0.039) (Gu et al., 2003). Three injections of saline did not elicit a rise of LOS antibodies in controls. In contrast, both conjugates elicited significant levels of anti-LOS IgG and IgM antibodies. The 58 chinchillas animals were randomly assigned to three groups (saline [n = 19], dLOS-TT [n = 20], and dLOS-HMP [n = 19]), and a blood sample was collected from the transverse venous sinus of each chinchilla to assess antibody levels. Three days later, the animals were immunized with three doses of the two conjugates or saline (as a control) at 4-week intervals. Blood samples were also collected from all of the chinchillas 14 days after the first and second immunizations, 10 days after the third immunization, and before sacrifice. The animals were anesthetized with ketamine-HCl (30 mg/kg of body weight given intramuscularly) prior to all operative procedures (Gu et al., 1997). All controls developed OM with culture-positive NTHi effusions up to 21 days postchallenge. In contrast, 60% of chinchillas from both conjugate groups developed OM on day 3, 80% did so on day 7, and 60% did so on day 14. On day 21, no animals in the dLOS-TT group and only 50% of the animals in the dLOS-HMP group showed OM with effusions. The incidence of OM was significantly lower in the dLOS-TT group than in the controls on day 21 and over the whole course. There was no significant difference between the dLOS-TT and dLOS-HMP groups. The animals were challenged by injection of 140 CFU of strain 9274 into the right ME 14 days after the last immunization. Both ears were examined daily by otoscopy for evidence of acute OM for 21 days postchallenge. On days 3, 7, 14, and 21 postchallenge, four or five animals from each group were sacrificed by ketamine injection followed by cervical dislocation and the ME fluids from both ears were cultured for bacterial counting (Gu et al., 1997). Nontypeable H. influenzae NucA Protein vaccine VO_0004038 Subunit vaccine Research Intraperitoneal injection (i.p.) 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. MPL (3-O-deacylated monophosphoryl lipid A. 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). Intraperitoneal injection (i.p.) NucA Protein Recombinant protein preparation Swiss-Webster mice 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). 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. 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). 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). 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). Nontypeable H. influenzae Outer Membrane Protein P1 vaccine VO_0004110 Subunit vaccine OMP P1 (47 kDa) accounts for ~10% of H. influenzae OMP content. Passive immunization with P1 induces protection against bacteremia in the infant rat model. OMP P1 has eight potentially surface-exposed loops, four of which are immunogenic. The availability of several ompP1 sequences was particularly relevant, as it offered the information needed for a broader survey of the sequence conservation of the ompP1 gene across a phylogenically classified collection of >500 typeable H. influenzae and NTHI isolates representing the natural population structure of the species (Bolduc et al., 2000). OmpP1 was cloned from the clinical NTHI strain BCH-3 and expressed P1 in the cytoplasm of E. coli (rP1BCH-3 ). Outer Membrane Protein P1 Recombinant protein preparation rP1BCH-3 was emulsified in RIBI adjuvant R-730 emulsion (RIBI Immunochem Research) to a final rP1BCH-3 concentration of 125 µg/ml. Twenty-eight chinchillas were immunized with two 0.1-ml intramuscular (i.m.) injections in the hindquarters on days 0, 35, 57, 79, 99, 120, and 141. Eight control chinchillas received two 0.1-ml i.m. injections of RIBI adjuvant only on the same days. All chinchillas were bled by cardiac puncture using a 23-gauge 3-ml syringe on days 0 (before receiving the first immunization) and 148 (prior to challenge) (Bolduc et al., 2000). all animals developed antibodies specific for rP1. Immunized animals were protected against disease when challenged with BCH-3, but not with an ompP1 mutant of BCH-3 or a strain (BCH-2) possessing a heterologous P1 (91% identity). Seven days after the final immunization, four nonimmunized chinchillas were added to the experimental group as controls. All animals were examined by otoscopy and tympanometry to document healthy, normal middle ears. Loopfuls of wt strain BCH-3 and the isogenic BCH-3 ompP1 mutant were inoculated separately into 1 ml of supplemented brain heart infusion medium and incubated without agitation for 16 to 18 h at 37°C. Overnight cultures were diluted by 2 × 10-3 in Gey's balanced salt solution (Sigma). The chinchillas were divided into two groups. One group was challenged with wt BCH-3, and the other was challenged with the BCH-3 ompP1 mutant by injecting 0.1 ml of the diluted cultures containing 50 to 60 CFU directly into the right middle-ear cavity through the superior bulla with a tuberculin syringe. Chinchillas were reexamined by otoscopy and tympanometry on days 2, 4, 6, 8, 10, 14, and 18. On these days, the middle-ear cavities were also examined through a small (4 mm in diameter) incision in the superior bulla, leaving the tympanic membrane intact. The right middle-ear cavities were cultured by swabbing the cavity with a calcium alginate swab (Calgiswab type 1; Hardwood Products Company LP) and streaking onto chocolate agar plates (Bolduc et al., 2000). Nontypeable H. influenzae outer membrane recombinant P4 vaccine VO_0004114 Subunit vaccine CT 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). 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). BALB/c mice 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). 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). 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). nontypeable H. influenzae P5 peptide MVF/H3 vaccine VO_0000509 Subunit vaccine incomplete Freund's adjuvant 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) . 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). Wistar rat 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). 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). Nontypeable H. influenzae protein P6 with cholera toxin VO_0000614 Subunit vaccine Cholera toxin 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). outer membrane protein P6 (Sabirov et al., 2004) Intranasal immunization induced P6-specific sinus mucosal and systemic immunological responses, mainly of the IgA and IgG isotype. White Wistar male rats 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). 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). At day 35, 50 μl (10^9 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). Nontypeable H. influenzae rTbpB vaccine VO_0000510 Subunit vaccine 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). incomplete Freund’s adjuvant (IFA) 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). Nontypeable H. influenzae vaccine recombinant transferrin binding protein B Wistar rat 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). 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). 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). Pediacel Diphtheria and Tetanus Toxoids and Acellular Pertussis Vaccine Adsorbed Combined with Inactivated Poliomyelitis Vaccine and Haemophilus b Conjugate Vaccine (Tetanus Protein – Conjugate) Pediacel Sanofi Pasteur Ltd VO_0010730 Subunit vaccine + Inactivated or "killed" vaccine Licensed Intramuscular injection (i.m.) Canada Products: Proteins, killed virus + conjugate. Other components: Bovine serum Formaldehyde, Polysorbate 80. Aluminum phosphate Store at 2° to 8°C (35° to 46°F). Intramuscular injection (i.m.) PedvaxHIB Haemophilus b Conjugate Vaccine (Meningococcal Protein Conjugate) PedvaxHIB Merck & Co, Inc. VO_0000083 Conjugate vaccine Licensed Intramuscular injection (i.m.) USA PedvaxHIB is a highly purified capsular polysaccharide (polyribosylribitol phosphate or PRP) of Haemophilus influenzae type b (Haemophilus b, Ross strain) that is covalently bound to an outer membrane protein complex (OMPC) of the B11 strain of Neisseria meningitidis serogroup B. The covalent bonding of the PRP to the OMPC which is necessary for enhanced immunogenicity of the PRP is confirmed by quantitative analysis of the conjugate’s components following chemical treatment which yields a unique amino acid (PedvasHIB). Nonconjugated PRP vaccines are capable of stimulating B-lymphocytes to produce antibody without the help of T-lymphocytes (T-independent). The responses to many other antigens are augmented by helper T-lymphocytes (T-dependent). PedvaxHIB is a PRP-conjugate vaccine in which the PRP is covalently bound to the OMPC carrier29 producing an antigen which is postulated to convert the T-independent antigen (PRP alone) into a T-dependent antigen resulting in both an enhanced antibody response and immunologic memory (Marburg 1986). Aluminum hydroxide Store vaccine at 2-8°C (36-46°F). No virulence Haemophilus influenzae type b and Neisseria meningitidis serogroup B are grown in complex fermentation media. The PRP is purified from the culture broth by purification procedures which include ethanol ractionation, enzyme digestion, phenol extraction and diafiltration. The OMPC from Neisseria meningitidis is purified by detergent extraction, ultracentrifugation, diafiltration and sterile filtration (PedvasHIB). Intramuscular injection (i.m.) Haemophilus b capsular polyribosyl-ribitol-phosphate( PRP)-Neisseria meningitidis outer membrane protein complex (OMPC) Liquid PedvaxHIB is indicated for routine vaccination against invasive disease caused by Haemophilus influenzae type b in infants and children 2 to 71 months of age. Liquid PedvaxHIB is ready to use and does not require a diluent. Each 0.5 mL dose of Liquid PedvaxHIB is a sterile product formulated to contain: 7.5 mcg of Haemophilus b PRP, 125 mcg of Neisseria meningitidis OMPC and 225 mcg of aluminum as amorphous aluminum hydroxyphosphate sulfate (previously referred to as aluminum hydroxide), in 0.9% sodium chloride, but does not contain lactose or thimerosal. Infants 2 to 14 months of age should receive a 0.5 mL dose of vaccine ideally beginning at 2 months of age followed by a 0.5 mL dose 2 months later (or as soon as possible thereafter). When the primary two-dose regimen is completed before 12 months of age, a booster dose is required . Infants born prematurely, regardless of birth weight, should be vaccinated at the same chronological age and according to the same schedule and precautions as full-term infants and children. Children 15 months of age and older previously unvaccinated against Hib disease should receive a single 0.5 mL dose of vaccine. In infants completing the primary two-dose regimen before 12 months of age, a booster dose (0.5 mL) should be administered at 12 to 15 months of age, but not earlier than 2 months after the second dose (PedvasHIB). A booster dose of PedvaxHIB is required in infants who complete the primary two-dose regimen before 12 months of age. This booster dose will help maintain antibody levels during the first two years of life when children are at highest risk for invasive Hib disease. PedvaxHIB was initially evaluated in 3,486 Native American (Navajo) infants, who completed the primary two-dose regimen in a randomized, double-blind, placebo-controlled study (The Protective Efficacy Study). Each infant in this study received two doses of either placebo or lyophilized PedvaxHIB with the first dose administered at a mean of 8 weeks of age and the second administered approximately two months later. Following the primary two-dose regimen, the protective efficacy of lyophilized PedvaxHIB was calculated to be 93% with a 95% confidence interval of 57%-98% (p=0.001, twotailed).All original participants were then followed two years and nine months from termination of the study. Efficacy for this follow-up period, estimated from persondays at risk, was 96.6% (95 C.I., 72.2-99.9%) in children under 18 months of age and 100% (95 C.I., 23.5-100%) in children over 18 months of age (PedvasHIB). Lyophilized PedvaxHIB induced antibody levels greater than 1.0 mcg/mL in children who were poor responders to nonconjugated PRP vaccines (PedvasHIB). In addition, lyophilized PedvaxHIB has been studied in children at high risk of Hib disease because of genetically-related deficiencies [Blacks who were Km(1) allotype negative and Caucasians who were G2m(23) allotype negative] and are considered hyporesponsive to nonconjugated PRP vaccines on this basis. The hyporesponsive children had anti-PRP responses comparable to those of allotype positive children of similar age range when vaccinated with lyophilized PedvaxHIB. All children achieved anti-PRP levels of >1.0 mcg/mL (PedvasHIB). The most frequently reported (>1%) adverse reactions, in decreasing order of frequency, were irritability, sleepiness, injection site pain/soreness, injection site erythema (≤2.5 cm diameter), injection site swelling/induration, unusual high-pitched crying, prolonged crying (>4 hr), diarrhea, vomiting, crying, pain, otitis media, rash, and upper respiratory infection (PedvasHIB). The use of Haemophilus b Polysaccharide Vaccines and another Haemophilus b Conjugate Vaccine has been associated with the following additional adverse effects: early onset Hib disease and Guillain-Barré syndrome (Meekison et al., 1989). Pentacel Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed, Inactivated Poliovirus and Haemophilus b Conjugate (Tetanus Toxoid Conjugate) Vaccine Pentacel Sanofi Pasteur Ltd VO_0000084 Subunit vaccine + Inactivated or "killed" vaccine Licensed Intramuscular injection (i.m.) USA (License #1726), Canada It consists of a Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed and Inactivated Poliovirus (DTaP-IPV) component and an ActHIB® vaccine component. Pentacel vaccine is indicated for active immunization against diphtheria, tetanus, pertussis, poliomyelitis and invasive disease due to Haemophilus influenzae type b (FDA: Pentacel). Aluminum phosphate Store at 2° to 8°C (35° to 46°F). Do not freeze. Corynebacterium diphtheriae is grown in modified Mueller’s growth medium. After purification by ammonium sulfate fractionation, the diphtheria toxin is detoxified with formaldehyde and diafiltered (FDA: Pentacel). Intramuscular injection (i.m.) ProHIBiT H. influenzae type b conjugate vaccine (PRP-D) ProHIBiT VO_0000660 conjugate vaccines The manufacturing process utilizes a technology of covalent bonding the capsular polysaccharide of Haemophilus influenzae type b to diphtheria toxoid, to produce an antigen which is postulated to convert a T- independent antigen into a T- dependent antigen. The protein carries both its own antigenic determinants and those of the covalently bound polysaccharide. As a result of the conjugation to protein, the polysaccharide is presented as a T- dependent antigen resulting in both an enhanced antibody response and an immunologic memory (Lepow et al., 1987). ProHIBiT was prepared from the purified capsular polysaccharide, a polymer of ribose, ribitol and phosphate (PRP) of the Eagen Haemophilus influenzaetype b strain covalently bound to diphtheria toxoid (D) and dissolved in sodium phosphate buffered isotonic sodium chloride solution. The polysaccharide- protein conjugate molecule is referred to as PRP- D. Thimerosal (mercury derivative) 1:10,000 is added as a preservative. The vaccine is a clear, colorless solution. Each single dose of 0.5 mL is formulated to contain 25 µg of purified capsular polysaccharide and 18 µg of diphtheria toxoid protein (ProHIBiT 2007). Haemophilus b capsular polyribosyl-ribitol-phosphate(PRP)-diphtheria toxoid (D) In studies conducted with ProHIBiT ® in several locations throughout the US, the antibody responses of 18- to 26- month- old children were measured, mean antibody levels induced by ProHIBiT ® in children 18 to 20 months of age are 30- fold higher than those induced by polysaccharide vaccines in the same age group. Following immunization of 16 to 24- month- old children with a single dose of ProHIBiT ® , 89% (109/ 123) had antibody levels ³ 0.15 µg/ mL 12 months post- immunization, compared to 93% one month post- immunization (ProHIBiT 2007). ProHIBiT ® is indicated for immunization against invasive diseases caused by Haemophilus influenzae type b. ProHIBiT ® may be administered as a booster vaccination at 12 to 15 months of age in children who received primary immunization with Haemophilus b Conjugate Vaccine (Meningococcal Protein Conjugate) or Haemophilus b Conjugate Vaccine (Diphtheria CRM 197 Protein Conjugate). This vaccine also may be administered as primary immunization at 15 months of age in children who have not received primary immunization with any licensed Haemophilus b Conjugate Vaccine (AAPC 1991). The immunizing dose is a single injection of 0.5 mL given intramuscularly in the outer aspect area of the vastus lateralis (mid- thigh) or deltoid. Side effects associated with a single vaccination of HibTITER include fever, local reactions, rash, diarrhea , vomiting , prolonged crying. recombinant nontypeable H. influenzae protein P6 with AdDP VO_0000558 Subunit vaccine Adamantylamide Dipeptide 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). outer membrane protein P6 Recombinant protein preparation 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. BALB/c mice 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. Mice vaccinated with rP6 were protected against both pulmonary and middle-ear infections (P < .01). 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). D15 Haemophilus influenzae 86-028NP VO_0011083 3430379 68249503 NTHI1084 CP000057 YP_248615 281310 1030831 1033233 - protective surface antigen D15 8.84 81691.94 800 Similar to: HI0917, D152_HAEIN >NC_007146.2:1030831-1033233 Haemophilus influenzae 86-028NP, complete genome ATTAGAAAGAACCTCCAATACTAAATTGGAACTGTTCGACATCATCATTTTCATATTTTTTAATTGGTTT GGCATAAGAGAATACCAATGGCCCAATAGGAGATTGCCATTGGAATCCGACACCTGTAGAGGCGCGAATA CGGCTTGATTTGCCATAATCGGGTAAGCTTTTTAATACATTGTTATCTAACCCACTCTTATCCGATTTCC ACTTAGTATTCCAAACACTTGCCGCATCAACAAATAGGGAAGTTCGGACCGTATTTTGGCTCTTATCGCT CACAAACGGTGTTGGTACAATAAGTTCTGCACTCGCAGTTGTGATTGCATTACCACCAATCACATCAGAA CTTATCTTATTAAAAGTACCATTACTATTACTATTACTATTACCATTTTGAGCGGCATAAATTGCGTTAG GTCCAATACTACCATAAGCAAAACCACGTAATGAACCGATGCCACCCGCTGTATAAGTTTGATAGAACGG TAAACGCTTGTTTCCAAAACCATTTGCATATCCTGCAGATGCTTTTGCAGATACAACCCAGAGGTGATCT CTGTCTAATGGGTAGAAACCCTGTACATCTGCACTTAGTTTGTAGTATTTGTTATCAGAACCTGGAATAG TAACTCGTCCACCAAGACTTGCTTTAACCCCTTTAGTTGGGAAATAGCCTCTATTAAGGCTGTTATAGTT CCAACCAAAAGAAAAATCAAAGTCATTTGTTTTAATGCCATTACCTTTAAATTTCATTGATTGAATATAT AAATTACGGTTATATTCTAGAGCAAAGTTACTAATTTTATTATAGGTATGGCCTAATCCTACATAATAGG AGTTATTTTCATTTACAGGGAAACCTAAAGTAACATTACTTCCATAAGTCGTACGCTTATAGTTAGAGGA TGTATCACTTTTAGAGTTATCGTAGTTTTCAAAGAAAACATTTCCACCAAGACTTACACCATCTTTAGTA AAATAGGGCTCGGTATAACCCAAATTGACACTCGTACCATAATCATTTTTCGTACCAGCTATACTTACTG CAGCCCCTGTTCCTAAGAAATTATCTTGTTTAACACTTGCTTGATAACTAATACCACTCTCTGTACCGTA ACCAATACCAAAGTTGATACTACCCGTGTTACGTTCTTTGACTTTATATACGACATCCACTTCATCATTG CTACCATTGATAGGATCAATTCGGTTTTCGACTGTTTCGAAGAAACCTGTACGATCTAAGCGAATTTTTC CTAACTCAACTAATTGTGAATTATACCAAGTTCCTTCTTGTTGGCGCATTTCCTGACGTAAAGTGCTATC AGCAGAAACGGTATTTCCTTCAAAGCGAAGTTGGCGAACAGTTAAACGTCGTCCAGCATCAACAACAAGG GTTATCGCTAATGTTTTATTTGCATCATCAAAATCAGGTACTGAATTTACCGTTGCGCTACCGTAACCGC GTTCTCCAAGTTTTGCTTTAATTGCATTTTCTACATCTGCAATATCACTACGGCGGAAAGTATCATTTAA ATGTAATGCTGAAAGTAAAGGTTCAAGCTCGGCAGACATACCTCCCAGATTACCTATAATGCGTGCACTA CGAAGGTCATACTGTAAACCTTCATTTACATCAATGGTTACATTAACTTTTGTTTTTTCATCATTTAGCT GAACATCCGTTTTAGTAATTTGTGCTTTGGCATAGCCATTATTTAAATAATAATCACGAATTGACTGCAA ATCTTTCTCGAATTGCGCACCTTCAAATTTATTTCCCCATAATTTCCACCAAGAATCAGGTTGTAATTCC ATTTGTTCTTGTAATGTACTGCTACTAACAGATTCGTTCCCCTTGAAAGTTAATGATGCCAATTTTGCTT TATCATCTTCATTGATTTGAATTAAAATTTCAGCGCGATTATTTGGTAGCGTATTGACAATAGGTTCAAC GGTTGCGTTATAGCGACCTACACTTGCATAGTGTTCTTTTACACTTTTGGCAAATTCATTTAATTTTTCT CGAATTAAAACATCGCCAACTTTAAACCCGTTAGCATCTAAGTTTTGTTTTAGTGCTTCAGTGGGAATAA TAGAGTTACCTTTGATTTTAACATCTGAAATGATCGATTTAGCCACAACGCTAACAACAAGCACATCGCC TTCTTGATGCGCTTTCACATCATCGAATCGACCACTTACGAATAAAGAGCGGACAATATTAGCCACATCA TTGTCAGTCACACGCTGACCGGCACGAACAGGTAAACTTGCTCGGATTTGTTGTTTTAAGTCACCTTGAA CACCATCCACACGAATATCTTTTGCCACAAAAGGTGCGGCAAACACAGTCGTTGTCGTACCGAATAATAA ACTTGCGATTAGAAGTTTTTTCA >YP_248615.1 protective surface antigen D15 [Haemophilus influenzae 86-028NP] MKKLLIASLLFGTTTTVFAAPFVAKDIRVDGVQGDLKQQIRASLPVRAGQRVTDNDVANIVRSLFVSGRF DDVKAHQEGDVLVVSVVAKSIISDVKIKGNSIIPTEALKQNLDANGFKVGDVLIREKLNEFAKSVKEHYA SVGRYNATVEPIVNTLPNNRAEILIQINEDDKAKLASLTFKGNESVSSSTLQEQMELQPDSWWKLWGNKF EGAQFEKDLQSIRDYYLNNGYAKAQITKTDVQLNDEKTKVNVTIDVNEGLQYDLRSARIIGNLGGMSAEL EPLLSALHLNDTFRRSDIADVENAIKAKLGERGYGSATVNSVPDFDDANKTLAITLVVDAGRRLTVRQLR FEGNTVSADSTLRQEMRQQEGTWYNSQLVELGKIRLDRTGFFETVENRIDPINGSNDEVDVVYKVKERNT GSINFGIGYGTESGISYQASVKQDNFLGTGAAVSIAGTKNDYGTSVNLGYTEPYFTKDGVSLGGNVFFEN YDNSKSDTSSNYKRTTYGSNVTLGFPVNENNSYYVGLGHTYNKISNFALEYNRNLYIQSMKFKGNGIKTN DFDFSFGWNYNSLNRGYFPTKGVKASLGGRVTIPGSDNKYYKLSADVQGFYPLDRDHLWVVSAKASAGYA NGFGNKRLPFYQTYTAGGIGSLRGFAYGSIGPNAIYAAQNGNSNSNSNGTFNKISSDVIGGNAITTASAE LIVPTPFVSDKSQNTVRTSLFVDAASVWNTKWKSDKSGLDNNVLKSLPDYGKSSRIRASTGVGFQWQSPI GPLVFSYAKPIKKYENDDVEQFQFSIGGSF Protective antigen 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 [Ref1096:Loosmore et al., 1997]. hap VO_0010916 AAB03707 CDD:280543 CDD:238653 CDD:214872 727 ? adhesion and penetration protein 9.34 147353.86 1484 Immunoglobulin A1 protease; pfam02395 >AAB03707.1 adhesion and penetration protein [Haemophilus influenzae] MKKTVFRLNFLTACISLGIVSQAWAGHTYFGIDYQYYRDFAENKGKFTVGAQNIKVYNKQGQLVGTSMTK APMIDFSVVSRNGVAALVENQYIVSVAHNVGYTDVDFGAEGNNPDQHRFTYKIVKRNNYKKDNLHPYEDD YHNPRLHKFVTEAAPIDMTSNMNGSTYSDRTKYPERVRIGSGRQFWRNDQDKGDQVAGAYHYLTAGNTHN QRGAGNGYSYLGGDVRKAGEYGPLPIAGSKGDSGSPMFIYDAEKQKWLINGILREGNPFEGKENGFQLVR KSYFDEIFERDLHTSLYTRAGNGVYTISGNDNGQGSITQKSGIPSEIKITLANMSLPLKEKDKVHNPRYD GPNIYSPRLNNGETLYFMDQKQGSLIFASDINQGAGGLYFEGNFTVSPNSNQTWQGAGIHVSENSTVTWK VNGVEHDRLSKIGKGTLHVQAKGENKGSISVGDGKVILEQQADDQGNKQAFSEIGLVSGRGTVQLNDDKQ FDTDKFYFGFRGGRLDLNGHSLTFKRIQNTDEGAMIVNHNTTQAANVTITGNESIVLPNGNNINKLDYRK EIAYNGWFGETDKNKHNGRLNLIYKPTTEDRTLLLSGGTNLKGDITQTKGKLFFSGRPTPHAYNHLNKRW SEMEGIPQGEIVWDHDWINRTFKAENFQIKGGSAVVSRNVSSIEGNWTVSNNANATFGVVPNQQNTICTR SDWTGLTTCQKVDLTDTKVINSIPKTQINGSINLTDNATANVKGLAKLNGNVTLTNHSQFTLSNNATQIG NIRLSDNSTATVDNANLNGNVHLTDSAQFSLKNSHFSHQIQGDKGTTVTLENATWTMPSDTTLQNLTLNN STITLNSAYSASSNNTPRRRSLETETTPTSAEHRFNTLTVNGKLSGQGTFQFTSSLFGYKSDKLKLSNDA EGDYILSVRNTGKEPETLEQLTLVESKDNQPLSDKLKFTLENDHVDAGALRYKLVKNDGEFRLHNPIKEQ ELHNDLVRAEQAERTLEAKQVEPTAKTQTGEPKVRSRRAARAAFPDTLPDQSLLNALEAKQAELTAETQK SKAKTKKVRSKRAVFSDPLLDQSLFALEAALEVIDAPQQSEKDRLAQEEAEKQRKQKDLISRYSNSALSE LSATVNSMLSVQDELDRLFVDQAQSAVWTNIAQDKRRYDSDAFRAYQQKTNLRQIGVQKALANGRIGAVF SHSRSDNTFDEQVKNHATLTMMSGFAQYQWGDLQFGVNVGTGISASKMAEEQSRKIHRKAINYGVNASYQ FRLGQLGIQPYFGVNRYFIERENYQSEEVRVKTPSLAFNRYNAGIRVDYTFTPTDNISVKPYFFVNYVDV SNANVQTTVNLTVLQQPFGRYWQKEVGLKAEILHFQISAFISKSQGSQLGKQQNVGVKLGYRW Protective antigen 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 [Ref345:Liu et al., 2004]. hap VO_0010917 23506944 AF369380 CDD:280543 CDD:238653 CDD:313027 CDD:214872 727 ? adhesin 9.09 146306.58 1466 Immunoglobulin A1 protease; pfam02395 >gi|23506943|gb|AF369380.1| Haemophilus influenzae strain TN106 adhesin (hap) gene, partial cds ATGAAAAAAACTGTATTTCGTCTGAATTTTTTAACCGCTTGCATTTCATTAGGGATAGTATCGCAAGCGT GGGCAGGTCATACTTATTTTGGGATTGACTACCAATATTATCGTGATTTTGCCGAGAATAAAGGGAAGTT TACAGTTGGGGCTCAAGATATTGATATCTACAATAAAAAAGGGGAAATGATAGGTACGATGATGAAAGGT GTGCCTATGCCTGATTTATCTTCCATGGTTCGTGGTGGTTATTCAACATTGATAAGTGAGCAGCATTTAA TTAGCGTCGCACATAATGTAGGGTATGATGTCGTTGATTTTGGTATGGAGGGGGAAAATCCAGACCAACA TCGTTTTAAGTATAAAGTTGTTAAACGATATAATTATAAGAGCGGTGATAGACAATATAATGATTATCAA CATCCAAGATTAGAGAAATTTGTAACGGAAACTGCACCTATTGAAATGGTTTCATATATGGATGGTAATC ATTACAAAAATTTTAATCAATATCCTTTGCGAGTTAGAGTTGGAAGTGGGCATCAATGGTGGAAAGACGA TAATAATAAAACCATTGGAGACTTAGCCTATGGAGGTTCATGGTTAATAGGTGGAAATACCTTTGAAGAT GGACCAGCTGGTAACGGTACATTAGAATTAAATGGGCGAGTACAAAATCCTAATAAATATGGTCCACTAC CTACGGCAGGTTCATTCGGGGATAGTGGTTCTCCAATGTTTATTTATGATAAGGAAGTTAAGAAATGGTT ATTAAATGGCGTGTTACGTGAAGGAAATCCTTATGCTGCAGTAGGAAACAGCTATCAAATTACACGAAAA GATTATTTTCAAGGTATTCTTAATCAAGACATTACAGCTAATTTTTGGGATACTAATGCTGAATATAGAT TTAATATAGGGAGTGACCACAATGGAAGAGTGGCAACAATCAAAAGTACATTACCTAAAAAAGCTATTCA GCCTGAACGAATAGTGGGTCTTTATGATAATAGCCAACTTCATGATGCTAGAGATAAAAATGGCGATGAA TCTCCCTCTTATAAAGGTCCTAATCCATGGTCGCCAGCATTACATCATGGGAAAAGTATTTACTTTGGCG ATCAAGGAACAGGAACTTTAACAATTGAAAATAATATAAATCAAGGTGCAGGTGGATTGTATTTTGAAGG TAATTTTGTTGTAAAAGGCAATCAAAATAATATAACTTGGCAAGGTGCAGGCGTTTCTGTTGGAGAAGAA AGTACTGTTGAATGGCAGGTGCATAATCCAGAAGGCGATCGCTTATCCAAAATTGGGCTGGGAACCTTAC TTGTTAATGGTAAAGGGAAAAACTTAGGAAGCCTGAGTGTCGGTAACGGTTTGGTTGTGTTAGATCAACA AGCAGATGAATCAGGTCAAAAACAAGCCTTTAAAGAAGTTGGCATTGTAAGTGGTAGAGCTACCGTTCAA CTAAATAGTGCAGATCAAGTTGATCCTAACAATATTTATTTCGGCTTTCGTGGTGGTCGCTTAGATCTTA ATGGGCATTCATTAACCTTTGAACGTATCCAAAATACGGATGAAGGCGCGATGATTGTGAACCACAACGC TTCTCAAACCGCAAATATTACGATTACAGGCAACGCAACTATTAATTCAGATAGCAAACAACTTACTAAT AAAAAAGATATTGCATTTAACGGCTGGTTTGGTGAGCAAGATAAAGCTAAAACAAATGGTCGTTTAAATG TGAATTATCAACCAGTTAATGCAGAAAATCATTTGTTGCTTTCTGGGGGGACAAATTTAAACGGCAATAT CACGCAAAATGGTGGTACGTTAGTTTTTAGTGGTCGTCCAACGCCTCATGCTTACAATCATTTAAGAAGA GACTTGTCTAACATGGAAGGTATCCCACAAGGCGAAATTGTGTGGGATCACGATTGGATCAACCGCACAT TTAAAGCTGAAAACTTCCAAATTAAAGGCGGAAGTGCGGTGGTTTCTCGCAATGTTTCTTCAATTGAGGG AAATTGGACAGTCAGCAATAATGCAAATGCCACATTTGGTGTTGTGCCAAATCAGCAAAATACCATTTGC ACGCGTTCAGATTGGACAGGATTAACGACTTGTAAAACAGTTGATTTAACCGATAAAAAAGTTATTAATT CCATACCGACAACACAAATTAATGGTTCTATTAATTTAACTGATAATGCAACAGTGAATATTCATGGTTT AGCAAAACTTAATGGTAATGTCACTTTAATAGATCACAGCCAATTTACATTGAGCAACAATGCCACCCAA ACAGGCAATATCAAACTTTCAAATCACGCAAATGCAACGGTGGACAATGCAAATTTGAACGGTAATGTGA ATTTAATGGATTCTGCTCAATTTTCTTTAAAAAACAGCCATTTTTCGCACCAAATCCAAGGTGGGGAAGA CACAACAGTGATGTTGGAAAATGCGACTTGGACAATGCCTAGCGATACCACATTGCAGAATTTAACGCTA AATAATAGTACTGTTACGTTAAATTCAGCTTATTCAGCTATCTCAAATAATGCGCCACGCCGTCGCCGCC GTTCATTAGAGACGGAAACAACGCCAACATCGGCAGAACATCGTTTCAACACATTGACAGTAAATGGTAA ATTGAGCGGGCAAGGCACATTCCAATTTACTTCATCTTTATTTGGCTATAAAAGCGATAAATTAAAATTA TCCAATGACGCTGAGGGCGATTACACATTATCTGTTCGCAACACAGGCAAAGAACCCGTGACCTTTGGGC AATTAACTTTGGTTGAAAGCAAAGATAATAAACCGTTATCAGACAAACTCACATTCACGTTAGAAAATGA CCACGTTGATGCAGGTGCATTACGTTATAAATTAGTGAAGAATGATGGCGAATTCCGCTTACATAACCCA ATAAAAGAGCAGGAATTGCGCTCTGATTTAGTAAGAGCAGAGCAAGCAGAACGAACATTAGAAGCCAAAC AAGTTGAACAGACTGCTAAAACACAAACAAGTAAGGCAAGAGTGCGGTCAAGAAGAGCGGTGTTTTCTGA TCCCCTGCCTGCTCAAAGCCTGTTAAAAGCATTAGAAGCCAAACAAGCTCTGACTACTGAAACACAAACA AGTAAGGCAAAAAAAGTGCGGTCAAAAAGAGCTGCGAGAGAGTTTTCTGATACCCTGCCTGATCAAATAT TACAAGCCGCACTTGAGGTTATTGATGCCCAACAGCAAGTGAAAAAAGAACCTCAAACTCAAGAGGAAGA AGAGAAAAGACAACGCAAACAAAAAGAATTGATCAGCCGTTACTCAAATAGTGCGTTATCGGAGTTGTCT GCGACAGTAAATAGTATGCTTTCCGTTCAAGATGAATTGGATCGTCTTTTTGTAGATCAAGCACAATCTG CCGTGTGGACAAATATCGCACAGGATAAAAGACGCTATGATTCTGATGCGTTCCGTGCTTATCAGCAGAA AACGAACTTGCGTCAAATTGGGGTGCAAAAAGCCTTAGATAATGGACGAATTGGGGCGGTTTTCTCGCAT AGCCGTTCAGATAATACCTTTGACGAACAGGTTAAAAATCACGCGACATTAGCGATGATGTCGGGTTTTG CCCAATATCAATGGGGCGATTTACAATTTGGTGTAAACGTGGGTGCGGGAATTAGTGCGAGTAAAATGGC TGAAGAACAAAGCCGAAAAATTCATCGAAAAGCGATAAATTATGGTGTGAATGCAAGTTATCAGTTCCGT TTAGGGCAATTGGGTATTCAGCCTTATTTGGGTGTTAATCGATATTTTATTGAACGTGAAAATTATCAAT CTGAAGAAGTGAAAGTGCAAACACCGAGCCTTGTATTTAATCGCTATAATGCTGGCATTCGAGTTGATTA TACATTTACCCCGACAGATAATATCAGCATTAAGCCTTATTTCTTCGTCAATTATGTTGATGTTTCAAAC GCTAACGTACAAACCACTGTAAATCGCACGATGTTGCAACAATCATTTGGGCGTTATTGGCAAAAAGAAG TGGGATTAAAGGCAGAAATTTTACATTTCCAACTTTCCGCTTTTATCTCAAAATCTCAAGGTTCACAACT CGGCAAACAGCAAAATGTGGGCGTGAAATTGGGGTATCGTTGG >AAN37924.1 adhesin, partial [Haemophilus influenzae] MKKTVFRLNFLTACISLGIVSQAWAGHTYFGIDYQYYRDFAENKGKFTVGAQDIDIYNKKGEMIGTMMKG VPMPDLSSMVRGGYSTLISEQHLISVAHNVGYDVVDFGMEGENPDQHRFKYKVVKRYNYKSGDRQYNDYQ HPRLEKFVTETAPIEMVSYMDGNHYKNFNQYPLRVRVGSGHQWWKDDNNKTIGDLAYGGSWLIGGNTFED GPAGNGTLELNGRVQNPNKYGPLPTAGSFGDSGSPMFIYDKEVKKWLLNGVLREGNPYAAVGNSYQITRK DYFQGILNQDITANFWDTNAEYRFNIGSDHNGRVATIKSTLPKKAIQPERIVGLYDNSQLHDARDKNGDE SPSYKGPNPWSPALHHGKSIYFGDQGTGTLTIENNINQGAGGLYFEGNFVVKGNQNNITWQGAGVSVGEE STVEWQVHNPEGDRLSKIGLGTLLVNGKGKNLGSLSVGNGLVVLDQQADESGQKQAFKEVGIVSGRATVQ LNSADQVDPNNIYFGFRGGRLDLNGHSLTFERIQNTDEGAMIVNHNASQTANITITGNATINSDSKQLTN KKDIAFNGWFGEQDKAKTNGRLNVNYQPVNAENHLLLSGGTNLNGNITQNGGTLVFSGRPTPHAYNHLRR DLSNMEGIPQGEIVWDHDWINRTFKAENFQIKGGSAVVSRNVSSIEGNWTVSNNANATFGVVPNQQNTIC TRSDWTGLTTCKTVDLTDKKVINSIPTTQINGSINLTDNATVNIHGLAKLNGNVTLIDHSQFTLSNNATQ TGNIKLSNHANATVDNANLNGNVNLMDSAQFSLKNSHFSHQIQGGEDTTVMLENATWTMPSDTTLQNLTL NNSTVTLNSAYSAISNNAPRRRRRSLETETTPTSAEHRFNTLTVNGKLSGQGTFQFTSSLFGYKSDKLKL SNDAEGDYTLSVRNTGKEPVTFGQLTLVESKDNKPLSDKLTFTLENDHVDAGALRYKLVKNDGEFRLHNP IKEQELRSDLVRAEQAERTLEAKQVEQTAKTQTSKARVRSRRAVFSDPLPAQSLLKALEAKQALTTETQT SKAKKVRSKRAAREFSDTLPDQILQAALEVIDAQQQVKKEPQTQEEEEKRQRKQKELISRYSNSALSELS ATVNSMLSVQDELDRLFVDQAQSAVWTNIAQDKRRYDSDAFRAYQQKTNLRQIGVQKALDNGRIGAVFSH SRSDNTFDEQVKNHATLAMMSGFAQYQWGDLQFGVNVGAGISASKMAEEQSRKIHRKAINYGVNASYQFR LGQLGIQPYLGVNRYFIERENYQSEEVKVQTPSLVFNRYNAGIRVDYTFTPTDNISIKPYFFVNYVDVSN ANVQTTVNRTMLQQSFGRYWQKEVGLKAEILHFQLSAFISKSQGSQLGKQQNVGVKLGYRW Protective antigen IgA Rattus norvegicus 23559227 CDD:209398 CDD:143187 10116 ? immunoglobulin alpha heavy chain 339 Immunoglobulin domain; cl11960 >gi|23559227|emb|CAD52870.1| immunoglobulin alpha heavy chain [Rattus norvegicus] ESAKDPTIYPLRPPPSPSSDPVTIGCLIQNYFPSGTMNVTWGKSGKDISVINFPPAPASGPYTMCSQLTL PAAECPKGTSVKYYVQYNTSPVRELSVECPGPKPSLVCRPRLSLQRPALEDLLLGSEASLTCTLRGLKEP TGAVFTWQPTTGKDAVQKEAVQDSCGCYTVSSVLPGCAERWNNGETFTCTATHPEFETPLTGEIAKVTEN TFPPQVHLLPPPSEELALNELVSLTCLVRGFNPKDVLVRWLQGNEELPSESYLVFEPLREPGEGAITYLV TSVLRVSAETWKQGAQYSCMVGHEALPMSFTQKTIDRLSGKPTNVNVSVIMSEGDGICY Vaximmutor IgG (partial) Rattus norvegicus 204718 CDD:209398 10116 ? immunoglobulin gamma-1 chain 106 Immunoglobulin domain; cl11960 >gi|204718|gb|AAA41373.1| immunoglobulin gamma-1 chain, partial [Rattus norvegicus] RTQVPHVYTMSPTKEEMTQNEVSITCMVKGFYPPDIYVEWQMNGQPQENYKNTPPTMDTDGSYFLYSKLN VKKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK Vaximmutor IgM Rattus norvegicus 299357 M13800 10116 6 134089641 134090090 - immunoglobulin heavy chain 6 Also known as IgM; RGD1359202; MGC125059 >gi|109658137:134089641-134090090 Rattus norvegicus chromosome 6, alternate assembly Rn_Celera, whole genome shotgun sequence GTCAATGTGCTATCCGAGCACAGTAGTATGTGGCAGTATCTGCAGTGTCCACATTGGTGATCTTGAGGAA TGCTTGGTTGTTGGAGGTGTCCTTGGAGATTGTGAGCCGGTTTTTCAGAGATGGATTGTAGTACTTATCA TCATCCCACCAAATGTTTGCCAGCCACTCCAGACCCTTCCCTGAAGGCTGACGAATCCAGCCCACACCCA TACCATAAGTGCTCAGTGAAAACCCAGAGAAAGTGCAAGTCAGACTGAGGGTCTGGGAGGGCTGCAATAT CCCAGGGCCAGACTCTTTCAGAGTAACCTGAGACAGGACATCTGTGGAGAAATGAAAGGGGAGAAGGTCA GGTTTGAACAGGTGAGGATAGAACAGTTCATCACTTAGGAAGCCTTGGTACTCACATGCAGGGACAATCA GCAGTAGGAATGAGGAAGTAAGCCTGTCCA Vaximmutor nucA Haemophilus influenzae strain P860295 VO_0012405 4929317 CDD:211667 727 ? 5'-nucleotidase NucA precursor 7.13 62950.55 681 NAD pyrophosphatase/5'-nucleotidase NadN; TIGR01530 >AAD33949.1 5'-nucleotidase NucA precursor [Haemophilus influenzae] MLLSKKSATFALSVFAMLFTSVALAKEAPQAHKAVELSILHINDHHSYLEPHETRINLNGQQTKVDIGGF SAVNAKLNKLRKKYKNPLVLHAGDAITGTLYFTLFGGSADAAVMNAGNFHYFTLGNHEFDAGNEGLLKLL EPLKIPVLSANVIPDKSSILYNKWKPYDIFTVDGEKIAIIGLDTVNKTVNSSSPGKDVKFYDEIATAQIM ANALKQQGINKIILLSHAGSEKNIEIAQKVNDIDVIVTGDSHYLYGNDELRSLKLPVIYEYPLEFKNPNG EPVFVMEGWAYSAVVGDLGVKFSPEGIASITRKIPHVLMSSHKLQVKNSEGKWAELTGDERKKALDTLKS MKSISLDDHDAKTDKLIAKYKSEKDRLAQEIVGVITGSAMPGGSANRIPNKAGSNPEGSIATRFIAETMY NELKTVDLTIQNAGGVRADILPGNVTFNDAYTFLPFGNTLYTYKMEGSLVKQVLEDAMQFALVDGSTGAF PYGAGIRYEANETPNAEGKRLVSVEVLNKQTQQWEPIDDNKRYLVGTNAYVAGGKDGYKTFGKLFNDPKY EGVDTYLPDAESFIKFMKKHPHFEAYTSSNVKFNASTDALPKK Protective antigen Mouse antiserum directed against the recombinant NucA protein was reactive on Western immunoblots and whole-cell ELISA with all H. influenzae strains tested including Eagan and was bactericidal for two heterologous strains tested. The antiserum also resulted in a log reduction in bacteremia, in an infant-rat protection study with H. influenzae type b as the challenge strain [Ref399:Zagursky et al., 2000]. ompP1 Haemophilus influenzae strain 4-H-1093 VO_0012406 9716645 CDD:224978 727 ? outer membrane protein P1 9.25 46882.94 521 Long-chain fatty acid transport protein [Lipid transport and metabolism]; COG2067 >AAF97590.1 outer membrane protein P1 [Haemophilus influenzae] MKKFNQSLLATAMLLAAGGANAAAFQLAEVSTSGLGRAYAGEAAIADNASVVATNPALMSLFKTAQFSTG GVYIDSRINMSGDVDASIKATAMARTKYGSASQRNVVPGAFVPNLYFVAPVNDKFALGAGMNVNFGLKSE YGDSYDAGIFGGKTDLSAINLNLSGAYRVTEGLSLGLGVNAVYANAQIERNAGIIADSIQDNQIQQALKA VDPQTKIHEYLTSKDKSVVSLQDRAAWGFGWNAGVMYQFNEANRIGLAYHSKVDIDFTDRTATSLGKKDI VAGKTGDLTLTLPDYLELSGFHQLTDKLAVHYSYKYTHWSRLTKLYASYENGEKAFDKELQYSNNSRIAL GASYNLYEKLTLRAGIAYDQAASRHQRSAAIPDTDRTWYSLGATYKFTPNLSVDLGYAYLKGKKVHFKEV KTIGAHIITNANYTSQAHANLYGLNLNYSF Protective antigen When chinchillas were immunized with recombinant P1 (rP1) obtained from one of these isolates (BCH-3), all animals developed antibodies specific for rP1. Immunized animals were protected against disease when challenged with BCH-3, but not with an ompP1 mutant of BCH-3 or a strain (BCH-2) possessing a heterologous P1 (91% identity). We conclude that (i) while P1 induces protection against NTHI-mediated otitis media, development of a polyvalent vaccine reflecting the variability of P1 would be necessary to construct an efficacious vaccine [Ref371:Bolduc et al., 2000]. Pal Haemophilus influenzae 86-028NP VO_0010915 3429820 68248984 NTHI0501 CP000057 YP_248096 281310 470966 471427 - outer membrane protein P6 6.52 14818.12 153 Similar to: HI0381, PAL_HAEIN >NC_007146.2:470966-471427 Haemophilus influenzae 86-028NP, complete genome ATTAGTACGCTAACACTGCACGACGGTTTTTAGAATATGCAGCTTCATCATGACCTAATACTGCAGGTTT TTCTTCACCGTAAGATACTGTGCCTAATTTACCAGCATCAACACCTTTACCAGCTAAATAACCTTTAACT GCATCTGCACGACGTTGGCCTAATGCGATGTTGTATTCTGGTGTACCACGTTCATCAGTGTTACCTTCTA CTAATACTTTAGCAGCTGGCGTTGCATTTAAATATGCAGCGTGCGCATCTAAGATTTGAACGTATTCACC AGTAATGTCATATTTATCAAAACCGAAATAAACGGTATTGTAACGTTGTTGAAGATCAGCAACAGAGTAA CCGCCAAAAGTTTGAGCAGCACCATTGCCTGCAGCATCGTTGTTAGATGAACTACAAGCTGCTAATGCAG CTACAGAACCTGCAACTAATAATGATTTAACAAATTTGTTCA >YP_248096.1 outer membrane protein P6 [Haemophilus influenzae 86-028NP] MNKFVKSLLVAGSVAALAACSSSNNDAAGNGAAQTFGGYSVADLQQRYNTVYFGFDKYDITGEYVQILDA HAAYLNATPAAKVLVEGNTDERGTPEYNIALGQRRADAVKGYLAGKGVDAGKLGTVSYGEEKPAVLGHDE AAYSKNRRAVLAY Protective antigen Recombinant P6 (pal) was capable of eliciting a protective antibody response against live H. influenzae type b challenge in a modified infant rat model of bacteremia [Ref1001:Yang et al., 1997]. These data demonstrate that active immunization with P6 results in the production of NTHi-specific bactericidal antibody in the chinchilla and also affords a reduction in the incidence of NTHi-induced OM [Ref5090:DeMaria et al., 1996]. skp VO_0010918 4574245 4574246 AF109085 AAD23967.1 CDD:225381 CDD:182724 727 ? outer membrane protein 26 7.75 20936.21 264 Periplasmic chaperone for outer membrane proteins, Skp family [Cell wall/membrane/envelope biogenesis, Posttranslational modification, protein turnover, chaperones]; COG2825 >gi|4574245|gb|AF109085.1| Haemophilus influenzae NTHI-289 outer membrane protein 26 (skp) gene, complete cds ATGAAAAACATCGCAAAAGTAACCGCACTTGCTTTAGGTATTGCACTTGCTTCAGGCTATGCTTCCGCTG AAGAAAAAATTGCTTTCATTAATGCAGGTTATATTTTTCAACATCACCCAGATCGCCAAGCGGTAGCAGA TAAACTTGATGCTGAATTTAAACCTGTAGCTGAGAAATTAGCAGCAAGCAAAAAAGAAGTTGATGATAAA ATTGCTGCTGCTCGTAAAAAAGTAGAAGCAAAAGTTGCGGCTTTAGAAAAAGATGCACCTCGCTTACGTC AAGCTGATATTCAAAAACGCCAAGAAGAAATTAATAAATTAGGTGCGGCTGAAGATGCTGAATTACAAAA ATTAATGCAAGAACAAGATAAAAAAGTTCAAGAATTCCAAGCTCAAAATGAAAAACGTCAAGCTGAAGAA CGTGGTAAATTATTAGATAGCATTCAAACTGCGACAAATAATTTAGCAAGAGCAAAAGGTTATACTTATG TGCTTGATGCAAATTCAGTTGTATTTGCGGTAGAGGGTAAAGATATTACTGAAGAAGTATTAAAATCTAT CCCTGCTTCTGAAAAAGCACAAGAGAAAAAATAATAGGTTC >AAD23967.1 outer membrane protein 26 [Haemophilus influenzae] MKNIAKVTALALGIALASGYASAEEKIAFINAGYIFQHHPDRQAVADKLDAEFKPVAEKLAASKKEVDDK IAAARKKVEAKVAALEKDAPRLRQADIQKRQEEINKLGAAEDAELQKLMQEQDKKVQEFQAQNEKRQAEE RGKLLDSIQTATNNLARAKGYTYVLDANSVVFAVEGKDITEEVLKSIPASEKAQEKK Protective antigen 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 [Ref868:Kyd and Cripps, 1998]. yscF Yersinia pestis CO92 VO_0010914 16082743 YPCD1.55 AL117189 NP_395189 3ET4 CDD:131160 214092 41025 41288 + needle complex major subunit 7.35 9570.09 165 type III secretion apparatus needle protein; TIGR02105 >NC_003131.1:41025-41288 Yersinia pestis CO92 plasmid pCD1, complete sequence AATGAGTAACTTCTCTGGATTTACGAAAGGAACCGATATCGCAGACTTAGATGCGGTGGCTCAAACGCTC AAGAAGCCAGCAGACGATGCAAACAAAGCGGTTAATGACTCGATAGCAGCATTGAAAGATAAGCCTGACA ACCCGGCGCTACTTGCTGACTTACAACATTCAATTAATAAATGGTCGGTAATTTACAATATAAACTCAAC CATAGTTCGTAGCATGAAAGACTTAATGCAAGGCATCCTACAGAAGTTCCCATA >NP_395189.1 needle complex major subunit (plasmid) [Yersinia pestis CO92] MSNFSGFTKGTDIADLDAVAQTLKKPADDANKAVNDSIAALKDKPDNPALLADLQHSINKWSVIYNINST IVRSMKDLMQGILQKFP Protective antigen The P4 protein is an integral outer membrane protein that has a role in acquiring hemin and nucleosides [Ref343:Green et al., 1991]. 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