Abstract:
The invention relates to a mutation within the sap operon of an avirulent clone of a nontypeable strain of  Haemophilus influenzae  (NTHi). The invention also relates to the NTHi sap operon genes and the polypeptides encoded by these polynucleotide sequences. The invention also relates to a novel 110 kDa NTHi outer membrane protein and the polynucleotide that encodes this outer membrane protein. Methods of screening for NTHi infection, and treating and preventing NTHi related disorders are also contemplated.

Description:
RELATED APPLICATIONS  
       [0001]    The present application claims priority benefit from U.S. Provisional Application 60/458,234 filed Mar. 27, 2003 which is incorporated herein by reference in its entirety. 
     
    
     
       FIELD OF INVENTION  
         [0002]    The invention relates to a mutation within the sap operon of an avirulent clone of a nontypeable strain of  Haemophilus influenzae  (NTHi). The invention relates to methods of modulating NTHi virulence and NTHi sensitivity to antimicrobial agents. The invention also relates to a novel 110 kDa NTHi outer membrane protein and the polynucleotide that encodes this outer membrane protein. Methods of screening for NTHi infection, and treating and preventing NTHi related disorders are also contemplated.  
         BACKGROUND  
         [0003]    Otitis media (OM) is a highly prevalent pediatric disease worldwide and is the primary cause for emergency room visits by children (Infante-Rivand and Fernandez,  Epidemiol. Rev.,  15: 444-465,1993). Recent statistics indicate that 24.5 million physician office visits were made for OM in 1990, representing a greater than 200% increase over those reported in the 1980&#39;s. While rarely associated with mortality any longer, the morbidity associated with OM is significant. Hearing loss is a common problem associated with this disease, often times affecting a child&#39;s behavior, education and development of language skills (Baldwin,  Am. J. Otol.,  14: 601-604, 1993; Hunter et al.,  Ann. Otol. Rhinol. Laryngol. Suppl.,  163: 59-61, 1994; Teele et al.,  J. Infect. Dis.,  162:685-694, 1990). The socioeconomic impact of OM is also great, with direct and indirect costs of diagnosing and managing OM exceeding $5 billion annually in the U.S. alone (Kaplan et al.,  Pediatr. Infect. Dis. J,  16: S9-11, 1997).  
           [0004]    Whereas antibiotic therapy is common and the surgical placement of tympanostomy tubes has been successful in terms of draining effusions, clearing infection and relieving pain associated with the accumulation of fluids in the middle ear, the emergence of multiple antibiotic-resistant bacteria and the invasive nature associated with tube placement, has illuminated the need for more effective and accepted approaches to the management and preferably, the prevention of OM. Surgical management of chronic OM involves the insertion of tympanostomy tubes through the tympanic membrane while a child is under general anesthesia. While this procedure is commonplace (prevalence rates are ˜13%; Bright et al.,  Am. J. Public Health,  83(7): 1026-8, 1993) and is highly effective in terms of relieving painful symptoms by draining the middle ear of accumulated fluids, it too has met with criticism due to the invasive nature of the procedure and its incumbent risks (Berman et al.,  Pediatrics,  93(3):353-63, 1994; Bright et al., supra.; Cimons,  ASM News,  60: 527-528; Paap,  Ann. Pharmacother.,  30(11): 1291-7, 1996).  
           [0005]    Progress in vaccine development is most advanced for  Streptococcus pneumoniae,  the primary causative agent of acute OM (AOM), as evidenced by the recent approval and release of a seven-valent capsular-conjugate vaccine, PREVNAR® (Eskola and Kilpi,  Pedriatr. Infect. Dis. J.  16: S72-78, 2000). While PREVNAR® has been highly efficacious for invasive pneumococcal disease, coverage for OM has been disappointing (6-8%) with reports of an increased number of OM cases due to serotypes not included in the vaccine (Black et al.,  Pedriatr. Infect. Dis J.,  19: 187-195; Eskola et al.,  Pedriatr. Infect. Dis J.,  19: S72-78, 2000; Eskola et al.,  N. Engl. J. Med.  344: 403-409, 2001; Snow et al.,  Otol. Neurotol.,  23: 1-2, 2002). Less progress has been made for nontypeable  Haemophilus influenzae  (NTHi), the gram-negative pathogen that predominates in chronic OM with effusion (Klein,  Pedriatr. Infect. Dis J.,  16: S5-8, 1997; Spinola et al.,  J. Infect. Dis.,  54: 100-109, 1986). Hampering development of effective vaccines against NTHi, has been the incomplete understanding of the pathogenesis of NTHi-induced middle ear disease. Contributing to this delay was a lack of understanding of the dynamic interplay between microbe-expressed virulence factors and the host&#39;s immune response as the disease progresses from one of host immunogenic tolerance of a benign nasopharyngeal commensal, to that of an active defensive reaction to an opportunistic invader of the normally sterile middle ear space.  
           [0006]    There has been a poor understanding of how NTHi causes OM in children. The identification of putative virulence factors necessary for induction of OM will contribute significantly to the understanding of the host-pathogen interaction and ultimately, the identification of potential vaccine candidates and targets of chemotherapy. There is a tremendous need to develop more effective and accepted approaches to the management and preferably, the prevention of otitis media. Vaccine development is a very promising and cost effective method to accomplish this goal (Giebank,  Pedriair. Infect. Dis J.,  13(11): 1064-8, 1994: Karma et al.,  Int. J. Pedritr. Otorhinolaryngol.,  32(Suppl.): S127-34, 1995).  
         SUMMARY OF INVENTION  
         [0007]    Signature-tagged mutagenesis screening of avirulent NTHi clones using a transbullar chinchilla model of OM identified a mutant that was unable to survive in the environment of the middle ear during OM. This mutant of interest harbored an interruption in the sapF gene within the sap operon. The mutant is denoted herein as sapF::mTn5. This mutant was 3-fold more sensitive to the action of the antimicrobial peptide protamine and displayed a concurrent loss of an approximately 110 kDa outer membrane protein (OMP).  
           [0008]    The sap operon is known to be involved in conferring resistance to the action of antimicrobial peptides. The sap operon was first identified and characterized in  S. typhimurium  where it functions in resistance to the cationic peptide protamine. (Parra-Lopez et al.,  EMBO J.  12: 4053-62, 1993). A search of the available complete and incomplete bacterial genome sequences in NCBI databases revealed sap operons in the genomes of  H. influenzae, Pasteurella multocida, Yersinia pestis, S. typhimurium, S. enterica, E. coli, E. chrysanthemi,  and  V. cholerae.  All of these organisms had the conserved gene order of sapABCDF in the operon. The structure of the gene cluster suggests that all sap genes were co-transcribed as a single polycistronic mRNA. An interesting finding is the presence of sapZ, which encodes a hypothetical transmembrane protein and is unique in  H. influenzae  due to its placement within the sap operon. In other organisms with a comparable sap system, sapZ is not co-transcribed with sapA-F. The sapABCDF gene products are components of an ABC transporter system involved in peptide uptake (Parra-Lopez et al., supra.). The SapA protein is a periplasmic dipeptide binding protein. SapB and SapC are transmembrane proteins embedded in the inner membrane. SapD and SapF are two ATP hydrolyzing proteins localized in cytoplasm presumably associated with SapB and SapC. The sapZ gene product is an as-yet uncharacterized hypothetical protein that is predicted to be a transmembrane protein with gene homologs in sap operon-containing bacteria,  P. multocida, S. typhimurium, S. enterica,  and  E. coli  0157:H7, and in  Neisseria meningitidis  and  Pseudomonas aeriginosa,  which do not contain a sap operon. In bacteria containing the described sap system, however, sapZ is not located near the sap operon in the bacterial genome.  
           [0009]    The present invention provides the sequences of the 6 NTHi sap genes (sapA, sapB, sapC, sapD, sapF and sapZ) set out as SEQ ID NOS: 1-6 respectively. The polypeptide gene products encoded by the 6 NTHi sap genes (SapA, SapB, SapC, SapD, SapF, and SapZ) are set out as SEQ ID NOS: 7-12 respectively. The polynucleotide sequence of the complete NTHi sap operon is set out as SEQ ID NO: 13.  
           [0010]    In vitro phenotypic assays described herein revealed that the sapF mutant was more sensitive to the antimicrobial peptide protamine than the parent strain, in addition to its absence of a 110 kDa OMP. This was the first observation about the NTHi sap gene playing an essential role in survival in the microenvironment of the chinchilla middle ear and in resistance to an antimicrobial peptide. The invention contemplates identifying the relevant host antimicrobial peptides that may be responsible in part for the rapid clearance of the sapF mutant, and determining the identity of the absent OMP, and also the functional linkage between this protein and the SapF protein.  
           [0011]    A non-polar in-frame mutation of the NTHi sap operon, denoted herein as sapA::kan, was more sensitive to chinchilla antimicrobial peptide beta-defensin-1 than the parent strain in vitro. This mutation also attenuated bacterial survival in vivo in the chinchilla middle ear. These studies further demonstrate that the NTHi sap operon is critical to survival in vivo.  
           [0012]    The present invention also provides for the polynucleotide sequences that encodes a portion of the polypeptide sequence of the novel NTHi 110 kDa OMP protein that is set out as SEQ ID NOS: 21-38. Additional sequence analysis identified the full length sequence of the NTHi 110 kDa OMP set out as SEQ ID NO: 41 that is encoded by the nucleic acid set out in SEQ ID NO: 40.  
           [0013]    The present invention also provides for antibodies specific for the NTHi SapA, SapB, SapC, SapD, SapF and SapZ proteins and the NTHi 110 kDa OMP protein of the invention. Methods of detecting NTHi bacteria in a human or in sample, such as serum, sputum, ear fluid, blood, urine, lymphatic fluid and cerebrospinal fluid are contemplated. These methods include detecting a NTHi sap polynucleotides or the NTHi 110 kDa OMP polynucleotide with specific polynucleotide probes or detecting an NTHi Sap protein or the NTHi 110 kDa OMP protein with specific antibodies. The invention also contemplates diagnostic kits which utilize these methods of detecting NTHi bacteria.  
           [0014]    According to the present invention, the presence of the functional NTHi Sap proteins and/or the NTHi 110 kDa OMP protein is associated with survivability of the NTHi bacterium within the middle ear. The sapA gene has been shown to be upregulated during OM infection of the middle ear in the chinchilla. Expression of SapZ protein as part of the sap operon is unique to NTHi and therefore is contemplated to be a target for therapies to infections caused by NTHI. Therefore, the NTHi SapA, SapB, SapC, SapD, SapF, SapZ and 110 kDa OMP protein are contemplated as vaccine candidates and/or targets of chemotherapy. The present invention also contemplates methods of eliciting an immune response to one or more of the NTHi SapA, SapB, SapC, SapD, SapF, SapZ and 110 kDa OMP protein of the invention by administering one or more of those proteins or peptides thereof. In one aspect, these methods involve administering one or more of the NTHi SapA, SapB, SapC, SapD, SapF, SapZ and 110 kDa OMP protein or a peptide thereof as a vaccine for treatment and/or prevention of diseases caused by NTHi infection, such as OM.  
           [0015]    As a method of treating or preventing NTHi infection, the present invention contemplates administering a molecule that inhibits expression or the activity of one or more of the NTHi SapA, SapB, SapC, SapD, SapF, SapZ and/or 110 kDa OMP proteins. In particular, the invention contemplates methods of treating or preventing NTHi infection comprising modulating expression of one or more of the NTHi SapA, SapB, SapC, SapD, SapF, SapZ and/or 110 kDa OMP protein by administering an antisense oligonucleotide that specifically binds to prevent expression of the appropriate NTHi genes. The invention also contemplates methods of treating or preventing NTHi infection comprising administering antibodies or small molecules that modulate the activity of one or more of the NTHi SapA, SapB, SapC, SapD, SapF, SapZ and 110 kDa OMP protein.  
           [0016]    The invention also provides for methods of modulating the virulence of the NTHi bacterium or increasing NTHi sensitivity to antimicrobial agents. These methods include mutating the NTHi genes within the sap operon. The sap operon is known to be associated with resistance to antimicrobial agents, and a disruption or mutation within this operon is contemplated to decrease virulence. These method include utilizing methods of intercalating or disrupting the DNA within the sap operon.  
           [0017]    Polynucleotides and Polypeptides of the Invention  
           [0018]    The present invention provides polynucleotide sequences of the NTHi sap operon genes (sapA, sapB, sapC, sapD, sapF and sapZ) set out as SEQ ID NOS: 1-6, respectively. The present invention also provides for the polypeptides encoded by the sap operon polynucleotides of the present invention. In addition, the invention provides for the polynucleotide sequence encoding the NTHi 110 kDa OMP set out in SEQ ID NO: 40. The invention provides for polynucleotides that hybridize under stringent conditions to (a) the complement of the nucleotide sequence of SEQ ID NOS: 1-6, (b) the complement of the nucleotide sequence encoding the SEQ ID NO: 40, (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the NTHi polypeptides of the present invention.  
           [0019]    The NTHi polynucleotides of the invention also include nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94% and even more typically at least 95%, 96%, 97%, 98% or 99% sequence identity to one of the NTHi sap operon polynucleotides or the polynucleotide encoding the NTHi 110 kDa OMP recited above.  
           [0020]    Included within the scope of the nucleic acids of the invention are nucleic acid fragments that hybridize under stringent conditions to one of the NTHi sap operon polynucleotides of SEQ ID NOS: 1-6 or polynucleotides encoding the NTHi 110 kDa OMP (SEQ ID NO: 40), or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides in length. Fragments of, e.g., 15, 17, or 20 nucleotides or more that are selective for (i.e., specifically hybridize to any one of the polynucleotides of the invention) are contemplated. Probes capable of specifically hybridizing to a polynucleotide can differentiate NTHi polynucleotides of the invention from other polynucleotides in the same family of genes or can differentiate NTHi genes from other bacterial genes, and are preferably based on unique nucleotide sequences.  
           [0021]    The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42° C. See Sambrook et al.,  Molecular Cloning: A Laboratory Manual,  2 nd  Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used, however, the rate of hybridization will be affected. In instances wherein hybridization of deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).  
           [0022]    Other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO 4 , (SDS), ficoll, Denhardt&#39;s solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4, however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al.,  Nucleic Acid Hybridisation: A Practical Approach,  Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art in order to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids.  
           [0023]    The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NOS: 1-6, the nucleic acid sequence encoding the NTHi 110 kDa OMP polypeptide (SEQ ID NO: 40), a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NOS: 1-6 or SEQ ID NO: 40, with a sequence from another isolate of the same species. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al.,  Nucl. Acid. Res.,  12: 387, 1984; Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al.,  J. Mol. Biol.,  215: 403-410, 1990). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources ( BLAST Manual,  Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity.  
           [0024]    Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific open reading frames (ORF) disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated. The present invention further provides isolated NTHi polypeptides encoded by the NTHi nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. The term “degenerate variant” refers to nucleotide fragments that differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical NTHi polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.  
           [0025]    The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acids encoded by the nucleotide sequences SEQ ID NOS: 7-12, the nucleotide sequence encoding NTHi 110 kDa OMP (SEQ ID NO: 41), or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides preferably with biological or immunogenic activity that are encoded by: (a) a polynucleotide having the nucleotide sequences set forth in SEQ ID NOS: 1-6 or SEQ ID NO: 40 or (b) polynucleotides encoding the amino acid sequence set forth as SEQ ID NOS: 7-12 or (c) a polynucleotide having the nucleotide sequence encoding the amino acid sequences set forth as SEQ ID NO: 41, (d) polynucleotides that hybridize to the complement of the polynucleotides of either (a), (b) or (c) under stringent hybridization conditions.  
           [0026]    The invention also provides biologically active or immunogenically active variants of the polypeptides of the present invention; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological and/or immunogenic activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to one of the polypeptides encoded by the polynucleotides comprising SEQ ID NOS: 1-6 or the (NTHi 110 kDa OMP polypeptide (SEQ IS NO: 41).  
           [0027]    The invention also provides for NTHi polypeptides with one or more conservative amino acid substitutions that do not affect the biological and/or immunogenic activity of the polypeptide. Alternatively, the NTHi polypeptides of the invention are contemplated to have conservative amino acids substitutions that may or may not alter biological activity. The term “conservative amino acid substitution” refers to a substitution of a native amino acid residue with a nonnative residue, including naturally occurring and nonnaturally occurring amino acids, such that there is little or no effect on the polarity or charge of the amino acid residue at that position. For example, a conservative substitution results from the replacement of a non-polar residue in a polypeptide with any other non-polar residue. Further, any native residue in the polypeptide may also be substituted with alanine, according to the methods of “alanine scanning mutagenesis”. Naturally occurring amino acids are characterized based on their side chains as follows: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; uncharged polar: glutamine, asparagine, serine, threonine, tyrosine; and non-polar: phenylalanine, tryptophan, cysteine, glycine, alanine, valine, proline, methionine, leucine, norleucine, isoleucine General rules for amino acid substitutions are set forth in Table 1 below.  
                             TABLE 1                           Amino Acid Substitutions            Original Residues   Exemplary Substitutions   Preferred Substitutions               Ala   Val, Leu, Ile   Val       Arg   Lys, Gln, Asn   Lys       Asn   Gln   Gln       Asp   Glu   Glu       Cys   Ser, Ala   Ser       Gln   Asn   Asn       Glu   Asp   Asn       Gly   Pro, Ala   Ala       His   Asn, Gln, Lys, Arg   Arg       Ile   Leu, Val, Met, Ala, Phe,   Leu       Leu   Norleucine, Ile, Val, Met,   Leu       Lys   Arg, 1,4 Diaminobutyric   Arg       Met   Leu, Phe, Ile   Leu       Phe   Leu, Val, Ile, Ala, Tyr   Arg       Pro   Ala   Gly       Ser   Thr, Ala, Cys   Thr       Thr   Ser   Ser       Trp   Tyr, Phe   Tyr       Tyr   Trp, Phe, Thr, Ser   Phe       Val   Ile, Met, Leu, Phe, Ala,   Leu                  
 
           [0028]    Antisense polynucleotides complementary to the polynucleotides encoding one of the NTHi sap operon proteins and NTHi 110 kDa OMP protein are also provided.  
           [0029]    Antisense technology may be employed to inhibit the activity of NTHi SapA, SapB, SapC, SapD, SapF, SapZ or NTHi 110 kDa OMP protein. Such inhibition may be effected by nucleic acid molecules which are complementary to and hybridize to expression control sequences (triple helix formation) or to sap operon mRNA or the 110 kDa OMP mRNA. For example, antisense DNA, RNA or RNAi molecules, which have a sequence that is complementary to at least a portion of the selected gene(s) can be introduced into the cell. Antisense probes may be designed by available techniques using the nucleotide sequence of NTHi sap operon or the gene that encodes the NTHi 110 kDa OMP protein disclosed herein. Typically, each such antisense molecule will be complementary to the start site (5′ end) of each selected sap operon gene or the gene encoding the 110 kDa OMP protein. When the antisense molecule then hybridizes to the corresponding mRNA, translation of this mRNA is prevented or reduced.  
           [0030]    Alternatively, gene therapy may be employed to create a dominant-negative inhibitor of one of the NTHi sap operon gene products or the NTHi 110 kDa OMP protein. The DNA encoding a mutant polypeptide of these polypeptides can be prepared and introduced into the cells of a patient using either viral or non-viral methods. Each such mutant is typically designed to compete with endogenous polypeptide in its biological role.  
           [0031]    In some cases, it may be desirable to prepare nucleic acid molecules encoding variants of the sap operon gene product or the NTHi 110 kDa OMP protein. Nucleic acid molecules encoding variants may be produced using site directed mutagenesis, PCR amplification, or other appropriate methods, where the primer(s) have the desired point mutations (see Sambrook et al., supra, and Ausubel et al., supra, for descriptions of mutagenesis techniques). Chemical synthesis using methods described by Engels et al., supra, may also be used to prepare such variants. Other methods known to the skilled artisan may be used as well.  
           [0032]    Homologous recombination may also be used to introduce mutations in genes of interest. The basic technique was developed as a method for introducing specific mutations into specific regions of the mammalian genome (Thomas et al.,  Cell,  44:419-428, 1986; Thomas and Capecchi,  Cell,  51:503-512, 1987; Doetschman et al.,  Proc. Natl. Acad. Sci.,  85:8583-8587, 1988) or to correct specific mutations within defective genes (Doetschman et al.,  Nature,  330:576-578, 1987). Exemplary homologous recombination techniques are described in U.S. Pat. No. 5,272,071.  
           [0033]    Through homologous recombination, the DNA sequence to be inserted into the genome can be directed to a specific region of the gene of interest by attaching it to targeting DNA. The targeting DNA is a nucleotide sequence that is complementary (homologous) to a region of the genomic DNA. Small pieces of targeting DNA that are complementary to a specific region of the genome are put in contact with the parental strand during the DNA replication process. It is a general property of DNA that has been inserted into a cell to hybridize, and therefore, recombine with other pieces of endogenous DNA through shared homologous regions. If this complementary strand is attached to an oligonucleotide that contains a mutation or a different sequence or an additional nucleotide, it too is incorporated into the newly synthesized strand as a result of the recombination. As a result of the proofreading function, it is possible for the new sequence of DNA to serve as the template. Thus, the transferred DNA is incorporated into the genome.  
           [0034]    Attached to these pieces of targeting DNA are regions of DNA which may interact with or control the expression of a sap operon gene product of the NTHi 110 kDa OMP, e.g., flanking sequences. For example, a promoter/enhancer element, a suppresser, or an exogenous transcription modulatory element is inserted in the genome of the intended host cell in proximity and orientation sufficient to influence the transcription of DNA encoding the desired NTHi polypeptide. The control element controls a portion of the DNA present in the host cell genome. Thus, the expression of the desired NTHi polypeptide may be achieved not by transfection of DNA that encodes NTHi polypeptide itself, but rather by the use of targeting DNA (containing regions of homology with the endogenous gene of interest) coupled with DNA regulatory segments that provide the endogenous gene sequence with recognizable signals for transcription of an NTHi polypeptide.  
           [0035]    The invention contemplates that polynucleotides of the invention may be inserted in a vector for amplification or expression. For expression, the polynucleotides are operatively linked to appropriate expression control sequence such as a promoter and polyadenylation signal sequences. Further provided are cells containing polynucleotides of the invention. Exemplary prokaryotic hosts include bacteria such as  E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella  and  Serratia.    
           [0036]    The term “isolated” refers to a substance removed from, and essentially free of, the other components of the environment in which it naturally exists. For example, a polypeptide is separated from other cellular proteins or a DNA is separated from other DNA flanking it in a genome in which it naturally occurs.  
           [0037]    Antibodies and Methods for Eliciting an Immune Response  
           [0038]    The invention provides antibodies which bind to antigenic epitopes unique to one of the NTHi SapA, SapB, SapC; SapD, SapF, SapZ and NTHi 110 kDa OMP polypeptides. Also provided are antibodies that bind to antigenic epitopes common among multiple  H. influenzae  subtypes but unique with respect to any other antigenic epitopes. The antibodies may be polyclonal antibodies, monoclonal antibodies, antibody fragments which retain their ability to bind their unique epitope (e.g., Fv, Fab and F(ab)2 fragments), single chain antibodies and human or humanized antibodies. Antibodies may be generated by techniques standard in the art.  
           [0039]    In vitro complement mediated bactericidal assay systems (Musher et al.,  Infect. Immun.  39: 297-304, 1983; Anderson et al.,  J. Clin. Invest.  51: 31-38, 1972) may be used to measure the bactericidal activity of antibodies that specifically bind to NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 kDa OMP polypeptides. Further data on the ability of NTHi 110 kDa OMP protein and peptides thereof elicit a protective antibody response may be generated by using animal models of infection such as the chinchilla model system described herein.  
           [0040]    The present invention provides for antibodies specific for the NTHi polypeptides of the present invention and fragments thereof, which exhibit the ability to kill both  H. influenzae  bacteria and to protect humans from NTHi infection. The present invention also provides for antibodies specific for the NTHi polypeptides of the invention that reduce the virulence, inhibit adherence, inhibit cell division, and/or inhibit penetration of  H. influenzae  bacteria into the epithelium or enhance phagocytosis of the  H. influenzae  bacteria.  
           [0041]    It is also possible to confer short-term protection to a host by passive immunotherapy by the administration of pre-formed antibody against an epitope or epitopes of the NTHi SapA, SapB, SapC, SapD, SapF, SapZ proteins and NTHi 110 kDa OMP protein. Thus, the contemplated vaccine formulations can be used to produce antibodies for use in passive immunotherapy. Human immunoglobulin is preferred in human medicine because a heterologous immunoglobulin may provoke an immune response to its foreign immunogenic components. Such passive immunization could be used on an emergency basis for immediate protection of unimmunized individuals exposed to special risks. Alternatively, these antibodies can be used in the production of anti-idiotypic antibody, which in turn can be used as an antigen to stimulate an immune response against one or more of the NTHi SapA, SapB, SapC, SapD, SapF, SapZ proteins and NTHi 110 kDa OMP protein.  
           [0042]    The invention contemplates methods of eliciting an immune response to NTHi in an individual. These methods include immune responses that kill the NTHi bacteria and immune responses which block  H. influenzae  attachment to cells or  H. influenzae  proliferation. In one embodiment, the methods comprise a step of administering an immunogenic dose of a composition comprising one or more of the NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 kDa OMP proteins or peptides thereof. In another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a cell expressing one or more of the NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 kDa OMP proteins or peptides thereof. In yet another embodiment, the methods comprise administering an immunogenic dose of a composition comprising a polynucleotide encoding one or more of the NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 kDa OMP proteins or peptides thereof. The polynucleotide may be a naked polynucleotide not associated with any other nucleic acid or may be in a vector such as a plasmid or viral vector (e.g., adeno-associated virus vector or adenovirus vector). Administration of the compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal. The methods may be used in combination in a single individual. The methods may be used prior or subsequent to NTHi infection of an individual.  
           [0043]    An “immunogenic dose” of a composition of the invention is one that generates, after administration, a detectable humoral and/or cellular immune response in comparison to the immune response detectable before administration or in comparison to a standard immune response before administration. The invention contemplates that the immune response resulting from the methods may be protective and/or therapeutic. For example, an “immunogenic dose” is a dose that is adequate to produce antibody and/or T cell immune responses to NTHi. In some embodiments the immune response protects said individual from NTHi infection, particularly NTHi infection of the middle ear, nasopharynx and/or lower airway. Also provided are methods whereby such immune response slows bacterial replication. The immune response may be induced therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells. The NTHi protein or an antigenic peptide thereof may be fused with co-protein which may not by itself induce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, may further comprise an antigenic co-protein, such as glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins that solubilize the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein.  
           [0044]    The invention correspondingly provides compositions suitable for eliciting an immune response to NTHi infection, wherein antibodies elicited block binding of NTHi bacterium to the host&#39;s cells, reduce the virulence, inhibit adherence, inhibit cell division, and/or inhibit penetration of  H. influenzae  bacteria into the epithelium or enhance phagocytosis of the  H. influenzae  bacteria. The compositions comprise one or more NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 kDa OMP proteins or peptides thereof, cells expressing one or more NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 kDa OMP proteins, or polynucleotides encoding one or more NTHi SapA, SapB, SapC, SapD, SapF, SapZ proteins and NTHi 110 kDa OMP protein. The compositions may also comprise other ingredients such as carriers and adjuvants.  
           [0045]    The invention includes methods of blocking binding of NTHi bacteria to host cells in an individual. The methods comprise inducing and/or administering antibodies of the invention that block binding of NTHi cellular attachment, reduce the virulence, inhibit adherence, inhibit cell division, and/or inhibit penetration of  H. influenzae  bacteria into the epithelium or enhance phagocytosis of the  H. influenzae  bacteria. Alternatively, administration of one or more small molecules that block binding of NTHi cell attachment is contemplated. In vitro assays may be used to demonstrate the ability of an antibody, polypeptide or small molecule of the invention to block NTHi cell attachment.  
           [0046]    Pharmaceutical compositions comprising antibodies of the invention, or small molecules of the invention that block NTHi cellular attachment, reduce the virulence, inhibit adherence, inhibit cell division, and/or inhibit penetration of  H. influenzae  bacteria into the epithelium or enhance phagocytosis of the  H. influenzae  bacteria are provided. The pharmaceutical compositions may consist of one of the foregoing active ingredients alone, may comprise combinations of the foregoing active ingredients or may comprise additional active ingredients used to treat bacterial infections. The pharmaceutical compositions may comprise one or more additional ingredients such as pharmaceutically effective carriers. Dosage and frequency of the administration of the pharmaceutical compositions are determined by standard techniques and depend, for example, on the weight and age of the individual, the route of administration, and the severity of symptoms. Administration of the pharmaceutical compositions may be by routes standard in the art, for example, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, or vaginal.  
           [0047]    Also provided by the invention are methods for detecting NTHi infection in an individual. In one embodiment, the methods comprise detecting one or more NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 kDa OMP proteins in a sample using primers or probes that specifically bind to the polynucleotides. Detection of the polynucleotides may be accomplished by numerous techniques routine in the art involving, for example, hybridization and PCR.  
           [0048]    The antibodies of the present invention may also be used to provide reagents for use in diagnostic assays for the detection of one or more NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 kDa OMP proteins or peptides thereof in various body fluids of individuals suspected of  H. influenzae  infection. In another embodiment, the NTHi SapA, SapB, SapC, SapD, SapF, SapZ or NTHi 110 kDa OMP protein or peptides thereof of the present invention may be used as antigens in immunoassays for the detection of NTHi in various patient tissues and body fluids including, but not limited to: blood, serum, ear fluid, spinal fluid, sputum, urine, lymphatic fluid and cerebrospinal fluid. The antigens of the present invention may be used in any immunoassay system known in the art including, but not limited to: radioimmunoassays, ELISA assays, sandwich assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays.  
           [0049]    Vaccines and Chemotherapeutic Targets  
           [0050]    As noted above, an aspect of the invention relates to a method for inducing an immune response in an individual, particularly a mammal, that comprises inoculating the individual with one or more NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 IcDa OMP proteins or an antigenic peptides thereof. The present invention also provides for vaccine formulations that comprise one or more immunogenic recombinant NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 kDa OMP proteins or peptides thereof together with a suitable carrier. The NTHi SapA, SapB, SapC, SapD, SapF, SapZ or NTHi 110 kDa OMP protein or peptides thereof are contemplated as vaccine candidates and/or targets of chemotherapy.  
           [0051]    Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.  
           [0052]    A. Peptide Vaccines  
           [0053]    Peptide therapeutic agents, such as peptide vaccines, are well known in the art and are of increasing use in the pharmaceutical arts. Consistent drawbacks to the parenteral administration of such peptide compounds have been the rapidity of breakdown or denaturation. Infusion pumps, as well as wax or oil implants, have been employed for chronic administration of therapeutic agents in an effort to both prolong the presence of peptide-like therapeutic agents and preserve the integrity of such agents. Furthermore, the peptide-like agent should (with particular reference to each epitope of the peptide-like agent) ideally maintain native state configuration for an extended period of time and additionally be presented in a fashion suitable for triggering an immunogenic response in the challenged animal.  
           [0054]    The NTHi polypeptides or peptides thereof of the invention can be prepared in a number of conventional ways. The short peptides sequences can be prepared by chemical synthesis using standard means. Particularly convenient are solid phase techniques (see, e.g., Erikson et al.,  The Proteins  (1976) v. 2, Academic Press, New York, p. 255). Automated solid phase synthesizers are commerically available. In addition, modifications in the sequence are easily made by substitution, addition or omission of appropriate residues. For example, a cysteine residue may be added at the carboxy terminus to provide a sulfhydryl group for convenient linkage to a carrier protein, or spacer elements, such as an additional glycine residue, may be incorporated into the sequence between the linking amino acid at the C-terminus and the remainder of the peptide. The short NTHi peptides can also be produced by recombinant techniques. The coding sequence for peptides of this length can easily be synthesized by chemical techniques, e.g., the phosphotriester method described in Matteucci et al.,  J. Am Chem Soc.,  103: 3185 (1981).  
           [0055]    Where some of the NTHi peptide sequences contemplated herein may be considered too small to be immunogenic, they may be linked to carrier substances in order to confer this property upon them. Any method of creating such linkages known in the art may be used. Linkages can be formed with heterobifunctional agents that generate a disulfide link at one functional group end and a peptide link at the other, such as a disulfide amide forming agent, e.g., N-succidimidyl-3-(2-pyridyldithio)proprionate (SPDP) (See, e.g., Jansen et al.,  Immun. Rev.  62:185, 1982) and bifunctional coupling agents that form a thioether rather than a disulfide linkage such as reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid and the like, and coupling agents which activate carboxyl groups by combining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt such as succinimmidyl 4-(N-maleimido-methyl)cyclohexane-1-carobxylate (SMCC).  
           [0056]    B. Vaccine Compositions and Administration  
           [0057]    A priming dose of an immunogenic composition of the invention may be followed by one or more booster exposures to the immunogen. (Kramp et al.,  Infect. Immun.,  25: 771-773, 1979; Davis et al.,  Immunology Letters,  14: 341-8 1986 1987). moreover, examples of proteins or polypeptides that could beneficially enhance the immune response if co-administered include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g. Leaf) or costimulatory molecules. Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment different than the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.  
           [0058]    Ideally, an immunogen will exhibit two properties; the capacity to stimulate the formation of the corresponding antibodies and the propensity to react specifically with these antibodies. Immunogens bear one or more epitopes that are the smallest part of an immunogen recognizable by the combing site of an antibody. In particular instances, immunogen, fractions of immunogens or conditions under which the immunogen is presented are inadequate to precipitate the desired immune response resulting in insufficient immunity. This is often the case with peptides or other small molecules used as immunogens. Other substances such as immunomodulators (e.g., cytokines such as the interleukins) may be combined in vaccines as well.  
           [0059]    The vaccine art recognizes the use of certain substances called adjuvants to potentate an immune response when used in conjunction with an immunogen. Adjuvants are further used to elicit an immune response that is faster or greater than would be elicited without the use of the adjuvant. In addition, adjuvants may be used to create an immune response using less immunogen than would be needed without the inclusion of adjuvant, to increase production of certain antibody subclasses that afford immunogenic protection or to enhance components of the immune response (e.g., humoral, cellular). Known adjuvants include emulsions such as Freund&#39;s Adjuvants and other oil emulsions,  Bordetella pertussis,  MF59, purified saponin from  Quillaja saponin  (QS2 1), aluminum salts such as hydroxide, phosphate and alum, calcium phosphate, (and other metal salts), gels such as aluminum hydroxide salts, mycobacterial products including muramyl dipeptides, solid materials, particles such as liposomes and virosomes. Examples of natural and bacterial products known to be used as adjuvants include monophosphoryl lipid A (MPL), RC-529 (synthetic MPL-like acylated monosaccharide), OM-174 which is a lipid A derivative from  E. coli.,  holotoxins such as cholera toxin (CT) or one of its derivatives, pertussis toxin (PT) and heat-labile toxin (LT) of  E. coli  or one of its derivatives, and CpG oligonucleotides. Adjuvant activity can be affected by a number of factors, such as carrier effect, depot formation, altered lymphocyte recirculation, stimulation of T-lymphocytes, direct stimulation of B-lymphocytes and stimulation of macrophages.  
           [0060]    Vaccines are typically prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, e.g., water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccine. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.  
           [0061]    Vaccines may also be administered through transdermal routes utilizing jet injectors, microneedles, electroporation, sonoporation, microencapsulation, polymers or liposomes, transmucosal routes and intranasal routes using nebulizers, aerosols and nasal sprays. Microencapsulation using natural or synthetic polymers such as starch, alginate and chitosan, D-poly L-lactate (PLA), D-poly DL-lactic-coglycolic microspheres, polycaprolactones, polyorthoesters, polyanhydrides and polyphosphazanes are useful for both transdermal and transmucosal administration. Polymeric complexes comprising synthetic poly-ornithate, poly-lysine and poly-arginine or amiphipathic peptides are useful for transdermal delivery systems. In addition, due to their amphipathic nature, liposomes are contemplated for transdermal, transmucosal and intranasal vaccine delivery systems. Common lipids used for vaccine delivery include N-(1)2,3-dioleyl-dihydroxypropyl)-N,N,N,-trimethylammonium-methyl sulfate (DOTAP), dioleyloxy-propyl-trimethylammonium chloride (DOTMA), dimystyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), dimethyldioctadecyl ammonium bromide (DDAB) and 9N(N′,N-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol). The combination of helper lipids and liposomes will enhance up-take of the liposomes through the skin. These helper lipids include, dioeolphosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine (DLPE), dimystristoylphosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE). In addition, triterpenoid glycosides or saponins derived from the Chilean soap tree bark ( Quillaja saponaria ) and chitosan (deacetylated chitan) have been contemplated as useful adjuvants for intranasal and transmucosal vaccine delivery.  
           [0062]    The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, e.g., hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, and procaine.  
           [0063]    The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, capacity of the subject&#39;s immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or three month intervals by a subsequent injection or other administration.  
           [0064]    Upon immunization with a vaccine composition as described herein, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection. Vaccine compositions containing one or more NTHi SapA, SapB, SapC, SapD, SapF, SapZ and NTHi 110 kDa OMP proteins or peptides thereof are administered to a patient susceptible to or otherwise at risk of bacterial infection or cancer to elicit an immune response against the antigen and thus enhance the patient&#39;s own immune response capabilities. Such an amount is defined to be an “immunogenically effective dose.” In this use, the precise amounts again depend on the patient&#39;s state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 μg to about 5000 per 70-kilogram patient, more commonly from about 10 to about 500 mg per 70 kg of body weight. For therapeutic or immunization purposes, the NTHi SapA, SapB, SapC, SapD, SapF, SapZ or NTHi 110 kDa OMP protein or peptides thereof may also be expressed by attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response.  
           [0065]    Humoral immune response may be measured by many well-known methods, such as Single Radial Immunodiffussion Assay (SRID), Enzyme Immunoassay (EIA) and Hemagglutination Inhibition Assay (HAT). In particular, SRID utilizes a layer of a gel, such as agarose, containing the immunogen being tested. A well is cut in the gel and the serum being tested is placed in the well. Diffusion of the antibody out into the gel leads to the formation of a precipitation ring whose area is proportional to the concentration of the antibody in the serum being tested. EIA, also known as ELISA (Enzyme Linked Immunoassay), is used to determine total antibodies in the sample. The immunogen is adsorbed to the surface of a microtiter plate. The test serum is exposed to the plate followed by an enzyme linked immunoglobulin, such as IgG. The enzyme activity adherent to the plate is quantified by any convenient means such as spectrophotometry and is proportional to the concentration of antibody directed against the immunogen present in the test sample. HAI utilizes the capability of an immunogen such as viral proteins to agglutinate chicken red blood cells (or the like). The assay detects neutralizing antibodies, i.e., those antibodies able to inhibit hemagglutination. Dilutions of the test sera are incubated with a standard concentration of immunogen, followed by the addition of the red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of the red blood cells by the immunogen. Tests to measure cellular immune response include determination of delayed-type hypersensitivity or measuring the proliferative response of lymphocytes to target immunogen.  
           [0066]    Assays for measuring T-cell response are well known in the art. For example, T-cell response can be measured using delayed-type hypersensitivity testing, flow cytometry using peptide major histocompatibility complex tetramers, lymphoproliferation assay, enzyme-linked immunosorbant assay, enzyme-linked immunospot assay, cytokine flow cytometry, direct cytotoxicity assay, measurement of cytokine mRNA by quantitative reverse transcriptase polymerase chain reaction, and limiting dilution analysis. (See Lyerly,  Semin Oncol.,  30(3 Suppl 8):9-16, 2003).  
           [0067]    Nontypeable  Haemophilus influenzae  (NTHi)  
           [0068]    [0068] H. influenzae  is a small, nonmotile gram negative bacterium. Unlike other  H. influenzae  strains, the nontypable  H. influenzae  (NTHi) strains lack a polysaccharide capsule and are sometimes denoted as “nonencapsulated.” NTHi strains are genetically distinct from encapsulated strains and are more heterogenous than the type b  H. influenzae  isolates. NTHi presents a complex array of antigens to the human host. Possible antigens that may elicit protection include OMPs, liposaccharises, lipoproteins, adhesion proteins and noncapsular proteins.  
           [0069]    Humans are the only host for  H. influenzae.  NTHi strains commonly reside in the middle ear, upper respiratory tract including the nasopharynx and the posterior oropharynx, the lower respiratory tract and the female genital tract. NTHi causes a broad spectrum of diseases in humans, including but not limited to, otitis media, pneumonia, sinusitis, septicemia, endocarditis, epiglottitis, septic arthritis, meningitis, postpartum and neonatal infections, postpartum and neonatal sepsis, acute and chromic salpingitis, epiglottis, pericardis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intraabdominal infections, urinary tract infection, mastoiditis, aortic graft infection, conjunctitivitis, Brazilian purpuric fever, occult bacteremia and exacerbation of underlying lung diseases such as chronic bronchitis, bronchietasis and cystic fibrosis.  
           [0070]    Epidemiologic studies of NTHi have indicated that the strains are heterogeneous with respect to outer membrane protein profiles (Barenkamp et al.,  Infect. Immun.,  36: 535-40, 1982), enzyme allotypes (Musser et al.,  Infect. Immun.,  52: 183-191, 1986), and other commonly used epidemiologic tools. There have been several attempts to subtype NTHi, but none of the methodologies have been totally satisfactory. The outer-membrane protein composition of NTHi consists of approximately 20 proteins. All NTHi strains contain two common OMP&#39;s with molecular weights of 30,000 and 16,600 daltons. NTHi strains may be subtyped based on two OMP&#39;s within the 32,000-42,000 dalton range. The NTHi lipopolysaccharide profile is fundamentally different than the enteric Gram-negative bacteria and separates into several distinct bands less than 20,000 daltons in size.  
           [0071]    A prototype NTHi isolate is the low passage isolate 86-028NP which was recovered from a child with chronic otitis media. This strain has been well characterized in vitro (Bakaletz et al.,  Infect. Immun.,  53: 331-5, 1988; Holmes et al.,  Microb. Pathog.,  23: 157-66, 1997) as well as in the chinchilla OM model (described herein) (Bakaletz et al.,  Vaccine,  15: 955-61, 1997; Suzuki et al.,  Infect. Immun.,  62: 1710-8, 1994; DeMaria et al.,  Infect. Immun.,  64: 5187-92, 1996). The 86-028NP strain was used, as described herein, to identify genes that are up-regulated in expression in the chinchilla model of otitis media and genes that are necessary for NTHi survival in the chinchilla middle ear.  
           [0072]    The NTHi strain 86-026NP was deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110, on Oct. 16, 2001 and assigned accession no. PTA-4764.  
           [0073]    Signature-Tag Mutagenesis Strategy  
           [0074]    The signature tag mutagenesis strategy (STM) has been employed to identify genes that are required for bacterial survival during infection in a number of systems. In this strategy, a series of mutants are constructed by random transposon mutagenesis. Each mutant was uniquely tagged with an oligonucleotide sequence that enables tag-specific identification of genes that alter virulence properties of a microorganism when mutated. The pool of mutants is then used to infect the experimental animal (‘input pool’). After an appropriate period of time has elapsed, the surviving infecting organisms are recovered (‘recovery pool’).  
           [0075]    Herein, the Tn903 kanamycin resistance gene was cloned into the EcoRI site of the pUC-based mini-Tn5 construction vector EZ::TN pMOD-2 (Epicentre). Oligonucleotide tags were prepared using the strategy of Nelson et al. ( Genetics,  157: 935-47, 2001) and cloned into the KpnI site of the modified EZ::TN pMOD-2 vector. Individual tags were characterized to confirm that they hybridized uniquely. Seventy-eight unique tags were saved. Chromosomal DNA from strain 86-028NP was mutagenized with 38 individual tag-containing mini-Tn5 elements in vitro, gaps repaired with T4 polymerase and ligase, then mutagenized DNA was transformed back into strain 86-028NP using the M-IV method. Mutants were selected for growth on kanamycin-containing media. A signature tagged library containing 2500 clones was screened for mutants defective in their ability to survive in the chinchilla middle ear. The genes disrupted by the mini-Tn5 elements in avirulent mutants were identified by sequencing DNA flanking the mini-Tn5 elements. Template was prepared using single primer PCR strategy.  
           [0076]    This analysis identified an avirulent clone containing a mutation in sapF. The sap operon has been shown in other systems to confer resistance to cationic antimicrobial peptides (Lopez-Solanilla et al.,  Plant Cell,  10(6): 917-24, 1998; McCoy et al.,  Antimicrobiol. Agent Chemother.,  45(7): 2030-7, 2001: Parra-Lopez et al.,  EMBO J.,  12(11): 4053-62, 1993). In vitro, the  H. influenzae  sapF mutant is more sensitive to cationic peptides suggesting that resistance to cationic peptides involved in innate immunity may be an important virulence determinant for  H. influenzae  in otitis media.  
           [0077]    DFI Strategy  
           [0078]    A differential fluorescence induction (DFI) strategy may be used to identify NTHi genes induced during OM in a chinchilla animal model. Several methods have been developed to identify bacterial genes that contribute to the virulence of an organism during infection. Such methods include in vivo expression technology (IVET) in which bacterial promoters regulate the expression of gene(s) required for synthesis of essential nutrients required for survival in the host; DNA microarray technology to globally screen for transcriptionally active genes, and DFI which uses FACS analysis to select for transcriptionally active promoters (Chiang et al.,  Annu. Rev. Microbiol.,  53: 129-154, 1999). DFI is a high-throughput method that allows for the identification of differentially regulated genes regardless of the basal level of expression and does not exclude those that are essential for growth in vitro.  
           [0079]    DFI has been successfully utilized in many microorganisms. For example, a GFP reporter system and flow cytometry was used to study mycobacterial gene expression upon interaction with macrophages (Dhandayuthapani et al.,  Mol. Microbiol.,  17: 901-912, 1995). A promoter trap system was used to identify genes whose transcription was increased when  Salmonellae  were subjected to environments simulating in vivo growth and when internalized by cultured macrophage-like cells (Valdivia and Falkow,  Mol. Microbiol.,  22: 367-378, 1996; Valdivia and Falkow,  Science,  277: 2007-2011, 1997; Valdivia and Falkow,  Curr. Opin. Microbiol.,  1: 359-363, 1998). In addition, DFI has been used to identify promoters expressed in  S. pneumoniae  and  S. aureus  when grown under varied in vitro conditions simulating infection (Marra et al.,  Infect. Immzun.,  148: 1483-1491, 2002; Schneider et al.,  Proc. Natl. Acad. Sci. U.S.A.,  97: 1671-1676, 2000). In addition, DFI has been utilized to study gene regulation in  Bacillus cereus  in response to environmental stimuli (Dunn and Handelsman,  Gene,  226: 297-305, 1999), in  S. pneumoniae  in response to a competence stimulatory peptide (Bartilson et al.,  Mol. Microbiol.,  39: 126-135, 2001), and upon interaction with and invasion of host cells in  Bartonella henselae  (Lee and Falkow,  Infect. Immun.,  66: 3964-3967, 1998),  Listeria monocytogenes  (Wilson et al.,  Infect. Immun.,  69: 5016-5024, 2001),  Brucella abortus  (Eskra et al.,  Infect. Immun.,  69: 7736-7742, 2001), and  Escherichia coli  (Badger et al.,  Mol. Microbiol.,  36: 174-182, 2000).  
           [0080]    Animal Model  
           [0081]    The chinchilla model is a widely accepted experimental model for OM. In particular, a chinchilla model of NTHi-induced OM has been well characterized (Bakaletz et al.,  J. Infect. Dis.,  168: 865-872, 1993; Bakaletz and Holmes,  Clin. Diagn. Lab. Immunol., 4: 223-225, 1997; Suzuki and Bakaletz,  Infect. Immun.,  62: 1710-1718, 1994), and has been used to determine the protective efficacy of several NTHi outer membrane proteins, combinations of outer membrane proteins, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against OM (Bakaletz et al.,  Vaccine,  15: 955-961, 1997; Bakaletz et al.,  Infect. Immun.,  67: 2746-2762, 1999; Kennedy et al.,  Infect. Immun.,  68: 2756-2765, 2000).  
           [0082]    In particular, there is a unique in vivo model wherein adenovirus predisposes chinchillas to  H. influenzae -induced otitis media, which allowed for the establishment of relevant cell, tissue and organ culture systems for the biological assessment of NTHi (Bakaletz et al.,  J. Infect. Dis.,  168: 865-72, 1993; Suzuki et al.,  Infect. Immunity  62: 1710-8, 1994). Adenovirus infection alone has been used to assess for the transudation of induced serum antibodies into the tympanum (Bakaletz et al.,  Clin. Diagnostic Lab Immunol.,  4(2): 223-5, 1997) and has been used as a co-pathogen with NTHi, to determine the protective efficacy of several active and passive immunization regimens targeting various NTHi outer membrane proteins, combinations of OMPs, chimeric synthetic peptide vaccine components, and adjuvant formulations as vaccinogens against otitis media (Bakaletz et al.,  Infect Immunity,  67(6): 2746-62, 1999; Kennedy et al.,  Infect Immun.,  68(5): 2756-65, 2000; Novotny et al.,  Infect Immunity  68(4): 2119-28, 2000; Poolman et al,;  Vaccine  19 (Suppl. 1): S108-15, 2000). 
       
    
    
     BRIEF DESCRIPTION OF FIGURES  
       [0083]    [0083]FIG. 1 depicts identification of attenuated A1 clone (circled) by comparative hybridization of signature tags present in the input pool (A) and the recovery pool (B).  
         [0084]    [0084]FIG. 2 depicts that interruption of the sapF gene by the miniTn5 transposon had no polar effect on the downstream sapZ gene in the sapF::mTn5 mutant. RT-PCR analysis showing transcription of the sapZ gene (A). Insertion of miniTn5 in the sapF gene near the 3′ end (B). Short arrows are RT-PCR primers. Lines represent RT-PCR products.  
         [0085]    [0085]FIG. 3 depicts RT-PCR analysis showing cotranscription of the sapABCDFZ genes as a single polycistronic mRNA. Transcriptional profile of the NTHi sap genes when grown in the sBHI media (A), and the computer predicted NTHi sap operon (B). Short arrows are RT-PCR primers. Lines represent RT-PCR products.  
         [0086]    [0086]FIG. 4 depicts the gene order of the NTHi sap gene cluster. RT-PCR analysis demonstrates these genes are transcribed as an operon.  
         [0087]    [0087]FIG. 5 depicts the sensitivity of NTHI bacterium with the sapA::kan mutation or the parental NTHi strain to killing induced by recombinant chinchilla beta-defensin-1 (cBD-1).  
         [0088]    [0088]FIG. 6 depicts the relative bacterial counts in the chinchilla middle ear after inoculation of equal parts sapA::kkan mutant NTHi and the parental NTHi strain. This plot depicts the inability of the sapA::kan mutant to survive in the middle ear while the parental strain maintained high bacterial counts.  
         [0089]    [0089]FIG. 7 depicts the ability of the sapA::kan mutant to survive when inoculated alone in the chinchilla middle ear (top panel) or in the chinchilla nasopharynx (bottom panel). These plots depict the inability of the sapA::kan mutant to survive in vivo while the parental strain maintained high bacterial counts. 
     
    
     DETAILED DESCRIPTION  
       [0090]    The following examples illustrate the invention wherein Example 1 describes construction of a signature-tagged mutagenesis library and identification of avirulent NTHi clones, Example 2 describes the characterization of the avirulent NTHi clone A1, Example 3 describes the in vitro phenotypic characterization of the NTHi sapF::mTn5 mutant, and Example 4 describes the OMP profile of the NTHi sapF::mTn5 mutant.  
       EXAMPLE 1  
     Construction of the STM Library  
       [0091]    An attenuated NTHi mutant was identified by signature-tagged mutagenesis (STM) using the transbullar chinchilla model of OM. The NTHi, strain 86-028NP, was mutagenized by miniTn5 transposons marked with unique signature tags to construct an STM library. A panel of signature-tagged miniTn5 transposons was constructed by cloning an EcoRI cassette containing the Tn903 kanamycin resistance gene into the EcoRI site and a signature tag sequence into the KpnI site within the transposon of the Epicentre EZ::TN pMOD&lt;MCS&gt; Transposon Construction Vector. To ensure that the signature tag sequences give a strong hybridization signal and do not cross hybridize to other tags, the signature tag sequences were screened by dot blot hybridization. To adapt the Epicentre miniTn5 in vitro transposition mutagenesis system to strain 86-028NP, single stranded gaps generated by the transposase in the chromosomal DNA were repaired using DNA polymerase and ligase. The transposon inserted DNA was transformed back into the parent strain using M-IV transformation method described in Herriott et al. ( J. Bacterial.  101: 513-6, 1970). The individual kanamycin resistant clones with unique tags were assembled into 96 well plates for animal screening. Southern blot analysis was performed to confirm random and single insertion of the transposon in the STM mutants.  
         [0092]    A pool of 38 STM mutants containing unique signature tags were directly inoculated into the middle ear cavity of a chinchilla at a concentration of 1.0×10 6  cfu/ear. The chinchilla was monitored for OM development and formation of effusions in the middle ear over a period of 48 hours by otoscopy and tympanometry. Effusions were removed by epitympanic taps and plated on chocolate agar plates supplanted with kanamycin to recover the NTHi mutants that survived in the middle ear. Bacteria recovered after two days of inoculation were selected as the recovery pool, at which time point the proliferation of NTHi cells in the middle ear reached a peak level during the course of OM development.  
         [0093]    Bacterial genomic DNA isolated from the input and recovery pool was used as template for PCR amplification of signature tags. The input and recovery probes were hybridized to membranes spotted with each signature tag PCR product or oligonucleotide in quadruplicate. By comparing the input and recovery hybridization patterns as depicted in FIG. 1, attenuated mutants containing signature tags were identified within the input pool but not in the recovery pool. The mutant carrying the A1 tag (circled in FIG. 1) was cleared from the middle ear in two other independent STM animal experiments confirming that this mutant was attenuated in vivo. This mutant was subjected for further characterization as described below.  
       EXAMPLE 2  
     Characterization of the Attenuated A1 Clone  
       [0094]    Sequence analysis was carried out on the transposon interrupted DNA locus in the attenuated strain using standard methods in the art. Southern blot analysis showed that a 6 kb EcoRV restricted genomic DNA fragment of the mutant of interest contained the transposon interrupted gene. The EcoRV restricted genomic DNA fragments were cloned into the pBluescript plasmid, and the transposon containing clone, designated pBlueA1, was isolated using marker rescue from LB agar plates supplemented with kanamycin. The 6 kb insert of the pBlueA1plasmid was sequenced and the resulting DNA sequence data were searched against NCBI databases using the BLASTX and BLASTN algorithms. Contigs were assembled using SeqmanII software (DNASTAR Inc.). As shown in FIG. 2, sequence analysis indicated that the transposon was inserted 165 bp from the 3′-end of the sapF gene, thus this attenuated mutant was designated as “sapF::mTn5.” The coding sequence of the kanamycin resistance gene is in the same orientation as the sapF gene.  
         [0095]    A search of the  H. influenzae  Rd genome using the sapF DNA, identified the  Haemophilus  sap gene cluster containing 6 open reading frames (ORFs) in the order of sapABCDFZ, where the sapF was the fifth gene of the cluster followed by a hypothetical protein H11643 which we designated “sapZ” in this study. This study, utilized the genomic sequencing NTHi strain 86-028NP and a three-fold coverage contig assembly. Part of the sap operon was present in the contigs (Contigs 512 and 324; SEQ ID NO: 16-17). A pair of primers were designed according to the contig sequences to PCR amplify the whole sap operon from strain 86-028NP. Sequence comparison analysis showed that the sap operon of strain 86-028NP had 98% identity as that of strain Rd, and the sap genes were organized in the same way. The polynucleotide sequence of the sap operon genes (sapA, sapB, sap C, sapD, sapF, and sapZ) are set out as SEQ ID NOS: 1-6, respectively. The amino acid sequences of the sap operon gene products, SapA, SapB, SapC, SapD, SapF and SapZ, are set out as SEQ ID NOS: 7-12 respectively.  
         [0096]    The sapF gene contains an ATP-binding domain and may share translocation ATPase activity with the sapD gene, shown to be up-regulated in response to iron and may play a role in potassium uptake via the TRK system (Harms et al.,  Microbiology  147: 2991-3003, 2001; Paustian et al.  J. Bacteriol,  184:6714-20, 2002) The sapZ gene is unique to  Haemophilus.  SapZ is predicted to be a transmembrane protein with gene homologs in sap operon-containing bacteria,  P. multocida, S. typhimurium, S. enterica,  and  E. coli  0157:H7, and in  Neisseria meningitidis  and  Pseudomonas aeruginosa,  which do not contain a sap operon. In bacteria containing the described sap system, however, sapZ is not located near the sap operon in the bacterial genome. The NTHi sap operon locus is organized as a single operon containing 6 genes as displayed in FIG. 4 and this gene locus was upregulated in vivo as determined by quantitative RT-PCR.  
         [0097]    DNA sequence analysis indicated that the coding sequences of the 86-028NP 6 sap genes were located on the same DNA strand with very few non-coding bases between the ORFs (FIG. 3). When the sap gene cluster was scanned for transcriptional terminators (GCG Wisconsin package v. 10), one typical rho-independent terminator as a stem-loop structure followed by polyU sequence was found downstream of the sapZ gene. Therefore, the 6 NTHi sap genes were predicted to be organized in an operon structure and presumed to be co-transcribed as one polycistronic mRNA. The sapZ gene begins 11 nucleotides downstream of the end of the sapF gene and therefore it is highly likely that is co-transcribed with the sap gene cluster. To confirm this organization, RT-PCR was used to determine whether the region between the sap genes was transcribed. Each RT-PCR reaction utilized a primer from the 3′ end of one gene and a primer from the 5′ end of the following gene. If there was a PCR product, the two adjacent genes were cotranscribed. As amplicons were obtained from each junction region, all 6 sap genes were co-transcribed as one polycistronic mRNA (FIG. 3, upper panel), which was in agreement with the transcriptional property of the sap operon in  S. typhimurium  (Parra-Lopez et al, supra).  
         [0098]    In order to determine whether insertion of the transposon prevented transcription of the downstream sapZ gene in the sapF::mTn5 mutant, a similar RT-PCR strategy using primers which annealed to the 3′-end of the sapF gene or the miniTn5 transposon and a primer which annealed to the 5′-end of the sapZ gene was employed. As depicted in FIG. 2, both primer sets gave positive results using sapF::mTn5 RNA as template demonstrating that there was detectable sapZ mRNA produced in the sapF::mTn5 mutant. The sapZ transcript in the mutant is presumably due to the absence of a transcriptional terminator downstream of the kanamycin resistance gene in the miniTn5 transposon. Thus, the attenuated phenotype of strain sapF::mTn5 was likely due to the sapF mutation but not the result of polar effect on the downstream sapZ gene.  
       EXAMPLE 3  
     In vitro Phenotypic Characterization of the sapF::mTn5 Mutant  
       [0099]    To ensure no secondary mutation in the original sapF::mTn5 mutant contributed to the various phenotypes of this mutant, the parent strain 86-028NP was transformed with the 6 kb EcoRV fragment containing the sapF::mTn5 allele from the pBlueA1 plasmid. The wild type sapF gene was replaced in this strain by homologous recombination with the sapF::mTn5 allele. One Km resistant clone was confirmed to harbor a miniTn5 interrupted sapF gene by PCR and Southern blot analysis. This clone was further characterized together with the sapF::mTn5 strain and designated RcsapF::mTn5.  
         [0100]    Since the sap mutants of  S. typhimurium  and  E. chrysanthemi  were reported to be hypersensitive to certain antimicrobial peptides, sensitivity to several commercial available cationic peptides against the NTHi parent and the sapF mutant strains was analyzed. Protamine displayed differential killing effect on the sapF mutants comparing to the parent strain. Broth minimal inhibitory concentration (MIC) analyses for protamine determined that the MIC of protamine for the sapF::mTn5 mutants was lower than that for the parent strain (0.2 mg/ml versus 0.4 mg/ml). Growth curve measurement under the same growth condition (aerobic growth in sBHI broth) demonstrated that the growth curves of the two mutant strains and the parent strain were identical. This analysis suggests that the two mutant strains do not possess a growth defect. Thus, the sapF gene product is not required for growth in enriched media, and the lack of growth of the sapF mutants at the lower protamine concentrations in sBHI broth was not due to a growth defect. Therefore, the sapF mutation may be responsible for the phenotype of increased sensitivity to protamine, and the in vivo attenuation property of the sapF mutant.  
       EXAMPLE 4  
     OMP Profile for the sapF::mTn5 Mutant NTHI Strain  
       [0101]    The sapF mutant displayed a minor variation of OMP profile in comparison with the parent strain. Sarkosyl insoluble OMPs of the three strains were prepared using differential detergent extraction as described in Filip et al., ( J. Bacteriol.  115: 717-722, 1973), and separated in a 10% SDS-PAGE. Absence of a 110 kDa OMP band was consistently observed from several OMP preparations in both mutant strains compared to the parent strain. Both the original and reconstructed mutant exhibited this minor change of the OMP profile, suggesting that the loss of the high molecular protein in the outer membrane was not due to a secondary mutation in the original sapF::mTn5 mutant.  
         [0102]    To determine the amino acid sequence of the 110 kDa OMP protein, a tryptic digest was performed. The 110 kDa protein was digested overnight at 37° C. Subsequently the peptides (SEQ ID NOS: 22-39) were extracted, desalted (10%) using C18ziptip (Millipore), and analyzed by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The peptide information is set out below in Table 1. The MALDI monoisotopic peaks were then searched in the NCBInr database using the Profound computer program.  
                                                             TABLE 1                           SEQ                           ID       Computed   Meaured       Peptide sequence   NO:   Residues   mass   mass                                FYAPGR   22    998-1003   709.354   709.342                   LWQER   23   530-534   730.376   730.372               FGQSGFAIR   24   122-130   981.503   981.492               AGVYNLTNR   25   959-967   1006.519   1006.512               YITWDSAR   26   969-976   1010.482   1010.482               KYITWDSAR   27   968-976   1138.577   1138.582               EFARINNGTR   28   504-513   1176.599   1176.552               YDNIHYQPK   29   659-667   1176.556   1176.552               LSFNPTENHR   30   292-301   1213.583   1213.602               SRGQDLSYTLK   31   313-323   1266.656   1266.692               YETGVTVVEAGR   32   110-121   1279.640   1279.682               NPEDTYDIYAK   33   914-924   1327.593   1327.632               FTLAADLYEHR   34   302-312   1334.661   1334.722               ELFEGYGNFNNTR   35   157-169   1559.700   1559.802               TMVYGLGYDHPSQK   36   887-900   1594.744   1594.892               VEHNLQYGSSYNTTMK   37   556-571   1870.851   1870.972               GYATENNQSFNTLTLAGR   38   223-240   1955.933   1956.082               KGYATENNQSFNTLTLAGR   39   222-240   2084.028   2084.172                  
 
         [0103]    This analysis identified the 110 kDa OMP protein as  H. influenzae  hemoglobin binding protein (HGBA_HAEIN; Genebank Accession No. Q9KIV2 or closely related homologue) by the Emory Microchemical Facility. The amino acid sequence from HGBA_HAEIN (Q9KIV2) (SEQ ID NO: 15) was employed to query the 86-028NP genomic contig set using the TBLASTN algorithm. The translation of the compliment of nucleotides 2623 to 5358 of contig 516 (SEQ ID NO: 18) was a translated sequence that is closely related to amino acids 94 to 1013 of HGBA_HAEIN (SEQ ID NO: 15). Similarly, contig 411 (SEQ ID NO: 19) contains nucleic acid sequences whose translation is highly related to amino acids 59 to 148 of HGBA_HAEIN and less closely related to amino acids 147-969 of HGBA_HAEIN. Contig 2 (SEQ ID NO: 39) contains nucleic acid sequences whose translation is highly related to amino acids 1 to 122 of HGBA_HAEIN (SEQ ID NO: 15). Contigs 469 and 497 (SEQ ID NOS: 20 and 21) also contain sequences with homology to HGBA_HAEIN. The sequence similarity is summarized in Table 2 below. Additional sequence analysis identified the full length sequence of the NTHi 110 kDa OMP set out as SEQ ID NO: 41 that is encoded by the nucleic acid set out in SEQ ID NO: 40.  
                               TABLE 2                           Translation of                   NTHi   Nucleotides of   Identity to   Total of number       Contig   Contig with   Amino acids of   of amino acids   Percent       #   identity   SEQ ID NO: 15   with identity   Identity                   516   complement of    94-1013   752/928   81%           2623-5358       469   complement of    59-1013    464/1043   44%            427-3462       411    651-3263   147-969   358/900   39%       411   388-657    59-148   82/90   91%       497   3377-4069    60-286    71/235   30%        2    79-396    1-122    61/122   50%                  
 
         [0104]    The sapF gene is 810 base pairs in length (SEQ ID NO: 5) and encodes a 269 amino acid protein (SEQ ID NO: 11) with a predicted mass of a 30 kDa soluble cytoplasmic protein with a an isoelectric point of 6.5. Therefore it is unlikely that the biosynthesis or secretion of this 110 kDa high molecular mass OMP is associated with the sapF gene product. Many OMPs of gram negative pathogens are important virulent factors playing roles in different pathogenesis aspects, such as host cells interaction, adhesion, iron acquisition, antigenic drift. The absence of the 110 kDa OMP may also contribute to the lost virulence of the sapF: :Tn5 mutant.  
       EXAMPLE 5  
     Generation of a Non-Polar, In-Frame Mutant of NTHI sap operon  
       [0105]    A set of clones with putative promoter activity in vivo were identified by differential fluorescence induction, and upregulated in vivo expression was confirmed by quantitative RT-PCR analysis as described in Mason et al. ( Infection and Immunity  71: 3454-3462, 2003). A clone that contained sequence upstream of the sapA gene was isolated. This clone demonstrated up-regulated GFP fluorescence in vivo indicating increased transcription of the sap operon. SapA was predicted to localize to the periplasm due to its signal sequence and its sequence identity to periplasmic solute binding proteins involved in peptide transport. (Parra-Lopez et al.,  EMBO J.  12: 4053-62, 1993) It was predicted that a mutation in the sapa gene would disrupt the function of the sap operon, thereby demonstrating the involvement of SapA in survival in a chinchilla model of otitis media.  
         [0106]    A non-polar mutation in the sapA gene was generated by insertion of a promoterless kanamycin resistance cassette as described in Menard et al. ( J. Bacterial.,  175: 5899-906, 1993). The mutant construction was verified by Southern blot analysis and the resulting mutant is denoted herein as “sapA::kan mutant”.  
       EXAMPLE 6  
     Properties of the sapA::kan Mutant  
       [0107]    Defensins are known as important elements of innate immunity against microbial infections. In particular, beta-defensins function to protect the host against microbial infections such as Gram-negative bacteria infections. Recombinant chinchilla beta-defensin-1 (cBD-1), an antimicrobial peptide with homology to human beta-defensin-3, was used to assess the sensitivity of the sapA::kan mutant to antimicrobial protection.  
         [0108]    For microbicidal assays, NTHI strain 86-028NP or its isogenic sapA::kan mutant were cultured to mid-log phase in brain heart infusion (BHI) broth supplemented with 2 μg NAD/ml and 2 μg hemin/ml (sBHI) or on chocolate agar. Static cultures of NTHI,  S. pneumoniae  and  E. coli  were incubated in 5% CO 2  at 37° C. Various concentrations of recombinant cBD-1 (2.5, 5.0, 10.0 and 20 μg/ml) were incubated for 1 hour at 37° C. in 5% CO 2  with 1×10 4  microorganisms in 100 μl of 10 mM sodium phosphate buffer containing either 1% sBHI. Bacteria were serially diluted and plated onto chocolate agar and the CFU of surviving microorganisms per ml was determined following overnight incubation at 37° C. in 5% CO 2 . Percent killing of the bacteria from a minimum of three replicate assays per strain are presented as mean percent survival (±SD) relative to concentration of (r)cBD-1 in FIG. 5. As shown in FIG. 5, the sapA::kan mutant strain had enhanced sensitivity to killing induced by recombinant chinchilla beta-defensin-1 as compared to the parental NTHi strain.  
         [0109]    Survival of the sapA::kan mutant was also assessed in vivo. To conduct these studies, a small inoculum of either the parental NTHI strain alone, the sapA::kan mutant alone or a mixture of these two was inoculated into either the nasopharynx or the middle ears of a chinchilla ( Chinchilla lanigera ). At periodic time points following inoculation, a nasal lavage or middle ear tapping procedure is done in order to determine the number of bacteria (in colony forming units per milliliter fluid) present in each of these anatomic sites within the uppermost airway that are extremely relevant to the disease course of otitis media.  
         [0110]    In the competitive study wherein the parental NTHi strain and the sapA::kan mutant were mixed in equal parts and inoculated into chinchilla middle ears, as shown in FIG. 6, the ability of the sapA::kan mutant to survive in the middle ear was dramatically attenuated as compared to the parental strain. The parental strain behaved typically and was present at a very high bacterial load in the middle ears out to eight days after the challenge.  
         [0111]    In addition, the sapA:.kan mutant was unable to survive when inoculated in the chinchilla middle ear alone as compared to the parental strain inoculated alone. As demonstrated in FIG. 7, in both animals challenged with the sapA::kan mutant, the bacteria were cleared from both ears of both animals by day 19 or 27 respectively. The parental isolate continued to be culturable at high numbers from the middle ear at these time points (FIG. 7; top panel). Similarly, the sapA::kan mutant was unable to survive when inoculated alone into the nasopharynx of a chinchilla (FIG. 7; bottom panel). Whereas the parental isolate maintained stable colonization of the nasopharynx, the sapA::kan mutant was cleared 12 days after challenge.  
         [0112]    While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on the invention.  
     
       
       
         1 
         
           
             41  
           
           
             1  
             1695  
             DNA  
             H. influenzae  
           
            1 

atgttacgtc taaatctgag atttttatct tttctgctct gtataagcca aagtgtagaa     60 

ttacaggctg cgccaagtgt tccaacattt ttaactgaaa atggcttaac ttattgcacc    120 

cacgcttcag gtttttcatt taatccgcaa acagcagatg caggaaccag tatgaatgtg    180 

gtcacggaac aaatttataa caaattattt gatataaaaa atcacagtgc aacattaaca    240 

ccaatgctgg cacaatctta ttccatttca gctgatggta aagaaatttt attaaattta    300 

cgtcacggcg taaaatttca ccaaacccct tggtttaccc caacacgtga ttttaacgct    360 

gaagacgtag tattttcgat taatcgtgta ttagggcata atacttattt accaacctta    420 

gcagaggcga atgttaccta tagtaatcca caatatagag tgtttcacga acaagcaaga    480 

aaagtgcgtt ttccttattt tgatagcatt aaacttaacg aaaaaatcaa atctgtgacc    540 

gcactttcgc cttatcaagt aaaaattgaa ttatttgcac cagattcctc cattttgtcg    600 

catcttgcca gccagtatgc cattattttt tcacaagaat atgcctatca attaagcgca    660 

gatgacaacc ttgctcaatt agatacccac ccagtaggca cagggcctta tcaagtaaaa    720 

gattatgtat ataaccaata tgttcgctta gtgcgtaacg aaaactattg gaaaaaagaa    780 

gccaagatag aacatattat tgtggatctt tctactgatc gcagcggacg tttagtcaaa    840 

tttttcaata atgaatgtca aatcgcctct tatcctgaag taagccaaat tggcttatta    900 

aaaaatgatg acaaacatta ttatatgcaa tctactgatg gtatgaattt agcctattta    960 

gcgtttaatt ttgataagcc attaatgcga gatcacgaaa tccgtgctgc tatttcacaa   1020 

agtttaaacc gagctcgaat cattcatagc atttaccata acacagcaac tgttgctaat   1080 

aacattattc ctgaagtgtc ttgggcttca actgtcaata cgccagaatt tgagtttgat   1140 

taccatccca aaatcgccaa aaataaatta gcagataaaa accttttgtt aaatttatgg   1200 

gtaattaatg aagaacaagt ctataatcca gcacctttta aaatggctga aatgatcaaa   1260 

tgggatttag ctcaagcggg tgtgaaagtt aaagtgcgtg ccgtaactcg tccattttta   1320 

actgcacaat tacgcaatca atcggaaaat tatgatttga ttctatctgg ttggttagct   1380 

ggtaatcttg atcctgatgg ttttatgcgt ccaattttaa gctgtggaac aaaaaatgaa   1440 

ctcactaatt tatctaattg gtgtaatgaa gaatttgatc aatttatgga tcgtgccatt   1500 

accacctcac atttaagttc acgcgcaaaa gcctataatg aagcccaaga actcgtttta   1560 

cgtgaattac ccattattcc tattgccaat gtaaaacgaa ttttagtcgc aaatagtcgt   1620 

gtgaaaggag taaaaatgac gccttttggt agcttagatt tttccacctt atattttatt   1680 

caggagaaac actaa                                                    1695 

 
           
             2  
             966  
             DNA  
             H. influenzae  
           
            2 

atgttctggt cggttcttcg ccatattctg tgggtggcat tattattact cgtattatcg     60 

ctattaggct ttgttatttt attgcgcgat cctcttaatg cgaatcttgt tacacaaaac    120 

atttatatcg gctatttcca ttatttaggc accttgttac aaggtgattt tggcattacc    180 

tataacggtg gaaaatcatt aatgaacctt attcttacgg ttcttcctcc cacattggaa    240 

ctttgtttca ttacattgtt tttggcattt atttttggtt tgccacttgg cattataagt    300 

gcggtcaatt ctgaacaagt ttttgcaaaa agtttacaaa tcctatctta tgtagggcta    360 

tctattccaa tattttggtt agcccccatt ttactgtatg ttgccgcgct ctcacattgg    420 

gaaattgccg ctattggaca atataatttg ctttacgaaa ttaaacccat tacgggattt    480 

cctgttattg atatgtggtt tatggaagta ccttatcgta caaaaatcgt acaaaacata    540 

ttgcaacatt tagccttacc aacattggta ttgtgtattt tgccaacaat ggaaattatc    600 

cgtattattc atcaacgagc agaatatatt ttgaatcaaa atttttctaa agtagcgaca    660 

acacggggtt ggtcaaaatg gaaaattctc catcaatatg tattccgtaa tacttttccc    720 

ctgcttgttc cacaagtacc acgtgtattc acattagtat taacgcaatg tatgttggta    780 

gaaacggctt taggttggcc tggcattggt cgttggttaa ttaatgccgt aaatgaacaa    840 

gattacaaca gcattgccgc aggtgtaatt gttattggtg tatgtattat tttgattgat    900 

acattcacta aaatattcac ttttatactc gatccattta aaaagaaagg ttggtatgca    960 

agataa                                                               966 

 
           
             3  
             888  
             DNA  
             H. influenzae  
           
            3 

atgcaagata aagaacctga tgaattccgc gaaagcacct caatctttca aatttggtta     60 

cgctttcgtc aaaataccat cgcacttttt agcttttatt tattaatcgc attaattttt    120 

accgcacttt ttgctagtta tcttgcacct tatgctgata atcgacaatt tattgggcaa    180 

gaattaatgc ctccttcttg ggtagataga ggaaaaattg cttttttctt tggtactgat    240 

gatttaggtc gcgacatatt aagtcgttta attatgggta ctcgttatac cttaggttct    300 

gctttactgg ttgtcttttc agtggcaata ataggcggcg cactaggaat tattgcagga    360 

ctactgaaag gtattaaagc tcgttttgtc gggcatattt ttgatgcttt tttatcgtta    420 

cctattctat taattgccgt tgttatttca acattaatgg aaccaagttt atggaatgca    480 

atgtttgcta cgctattagc aattttgcct tatttcattc acactatcta tcgcgctatt    540 

caaaaagaat tagaaaagga ttatgttgta atgctaaaac ttgaaggcat ttccaatcaa    600 

accttattaa aaagcactat tttaccgaat attactgtta tttatattca agaagtggct    660 

catgcttttg ttatagccgt gttggatatt agcgcattaa gttttatttc tcttggtgca    720 

caacgaccta caccagaatg gggggcaatg ataaaagact ctttggaact actttatctt    780 

gcaccttgga cagtactttt acccggtttc gctattattt ttactatttt attaagtatt    840 

attttcagta atggcttaac taaagccatc aatcaacatc aagaatag                 888 

 
           
             4  
             1050  
             DNA  
             H. influenzae  
           
            4 

atggcacttt tagacatttg taacctcaat attgaaattc aaacctccaa tggacgtata     60 

aaaattgtag atggcgtcaa tctttccctt aacgaagggg aaatcagtgg attagttggc    120 

gaatcaggct caggaaaaag cttaatcgct aaagtcattt gtaatgcaat caaagaaaat    180 

tggattatta ctgccgatcg ctttcgtttt cacgatatcg aattactaaa actcagtcct    240 

aataaacgac gtaagattgt cggcaaagaa atatccatga ttttccaaaa tcccttatct    300 

tgccttgatc caagtcgaaa aatagggaaa caactcatcc aaaatattcc taattggaca    360 

tttaaaaata aatggtggaa atggtttggg tggaaaaaaa gacgtgctat tgaattgtta    420 

catcgcgtag gaattaaaga tcatcgtgat attatggcaa gctatcctaa cgaactgaca    480 

gaaggcgaag gacaaaaagt tatgatcgca atggctgtcg ctaatcagcc acgtttatta    540 

atcgcagatg aaccaacaaa tacattagaa tcaaccactg ccctacaagt ttttcgttta    600 

ctttccagta tgaaccaaaa tcagggaaca acaattttac ttacgagtaa cgatattaaa    660 

agtattagtg aatggtgcga tcaaatttca gtgctttatt gtgggcaaaa taccgaatct    720 

gccccgactg aaatattaat cgaaagtccc catcatcctt atacccaagc cttaattaat    780 

gcagtacccg attttactca acctttgggg tttaaaacta aattgggtac gttagaaggc    840 

accgcgccta ttttagagca aatgccaatt ggctgtcgtc ttggcccaag atgccctttt    900 

gcacaaaaaa aatgtatgga aaaaccaaga cgattgaaaa taaaacaaca cgaattttct    960 

tgtcattatc ctattaattt acgagaaaaa aatttcaaag aaaaaacaac cgccacccct   1020 

tttatactta attgcaaagg aaatgaataa                                    1050 

 
           
             5  
             810  
             DNA  
             H. influenzae  
           
            5 

atgcccttat tacaagtgga agatttaact aaaactttta aaggtcacgc cagtttattt     60 

ggtcgaaatc aattcaatgc agtggataaa gtgagtttta cccttgaacg taaacaaaca    120 

cttgcaatca ttggcaataa tggctctggt aaatcaactc tagtgaaaat gatagcgggc    180 

attattccgc caacttctgg tcgaatttta tttaatgatc gagaattaca atatcaggat    240 

gcccaatcta gagctaaaca tattcgtatg gttttccaag atgccaactc tgcatttaat    300 

ccacgtttaa atattggaca aatattagac gaaccattaa gcctagcgac agattggaca    360 

gaaacacaac gtaatgaaaa aatctttgag accctctctc ttgttggact ttatcctgat    420 

tacacaaatc tcaatattaa gcatctctct atcagccaaa agcagcgggt tgccctagca    480 

cgcgcattaa ttttagcacc agaaattatt ataatagatg atgcaattgg caatttagat    540 

gcttctgtac gtattcaatt gcttaattta acccttgatt tacaacaacg tttaggtata    600 

tcttatattt atgtgggaca ggatctcggt gtaattaaac atattgcaga tacgattatc    660 

gtaatggatg acggaaaaat gattgaatat ggcagccctc aaaatctttt tactgatcca    720 

caaactgatg ttactcgtcg cttagtcgaa agctattttg gcaaaatttt agatgaaacc    780 

gcttgggtaa aagacaaaaa cactcactaa                                     810 

 
           
             6  
             1017  
             DNA  
             H. influenzae  
           
            6 

atgaacactc gtccctttta tttcggactt atatttattg cgattatcgc tatacttgct     60 

cactatttag gaaacactga tttttcccat cattatcata tcagtgctct aattattgcc    120 

atcttgctgg gaatggcaat cggcaatacc atttatccgc aattttcaac acaagtggaa    180 

aaaggcgtgt tatttgcgaa aggcacgctt cttcgcactg gcattgtgct gtatggtttt    240 

cgccttactt ttggcgatat tgccgatgtt ggcttaaatg ctgttgtcac tgatgcgatt    300 

atgctaattt caaccttttt tcttaccgca cttttgggca ttcgttatct aaaaatggat    360 

aaacaattgg tttatctcac tggggctgga tgtagtattt gtggtgcggc agcggttatg    420 

gcggcagagc ctgttaccaa agcagaatct cataaagttt cagtagcgat tgccgtagtg    480 

gtcattttcg ggacgcttgc tatttttact taccccttgt tctacacgtg gtcacaagat    540 

ttaattaacg cccatcaatt cggtatttat gttggttcta gtgtacacga agtggctcaa    600 

gtgtatgcga ttggggaaaa tattgatcct atcgtggcga atactgccgt catttccaaa    660 

atgatccgag tgatgatgct cgcaccattt ttattaatgc tttcttggtt attaacacgt    720 

agtaatggag tatcagaaaa tacatcacac aaaattacaa ttccttggtt tgctgtactt    780 

tttattggcg ttgcgatttt taattctttt gatttattac caaaagaact cgtgaaatta    840 

ttagttgaaa tcgattcttt cttattaatt tcagcgatgg ctgcccttgg cttaacgaca    900 

caagcaagcg caatcaaaaa ggcaggatta aaaccacttg ttttaggaac actaatttat    960 

ttatggctaa tggttggtgg atttttagtg aattatggaa tatcaaaatt aatataa      1017 

 
           
             7  
             564  
             PRT  
             Homo sapiens  
           
            7 

Met Leu Arg Leu Asn Leu Arg Phe Leu Ser Phe Leu Leu Cys Ile Ser 
1               5                   10                  15 

Gln Ser Val Glu Leu Gln Ala Ala Pro Ser Val Pro Thr Phe Leu Thr 
            20                  25                  30 

Glu Asn Gly Leu Thr Tyr Cys Thr His Ala Ser Gly Phe Ser Phe Asn 
        35                  40                  45 

Pro Gln Thr Ala Asp Ala Gly Thr Ser Met Asn Val Val Thr Glu Gln 
    50                  55                  60 

Ile Tyr Asn Lys Leu Phe Asp Ile Lys Asn His Ser Ala Thr Leu Thr 
65                  70                  75                  80 

Pro Met Leu Ala Gln Ser Tyr Ser Ile Ser Ala Asp Gly Lys Glu Ile 
                85                  90                  95 

Leu Leu Asn Leu Arg His Gly Val Lys Phe His Gln Thr Pro Trp Phe 
            100                 105                 110 

Thr Pro Thr Arg Asp Phe Asn Ala Glu Asp Val Val Phe Ser Ile Asn 
        115                 120                 125 

Arg Val Leu Gly His Asn Thr Tyr Leu Pro Thr Leu Ala Glu Ala Asn 
    130                 135                 140 

Val Thr Tyr Ser Asn Pro Gln Tyr Arg Val Phe His Glu Gln Ala Arg 
145                 150                 155                 160 

Lys Val Arg Phe Pro Tyr Phe Asp Ser Ile Lys Leu Asn Glu Lys Ile 
                165                 170                 175 

Lys Ser Val Thr Ala Leu Ser Pro Tyr Gln Val Lys Ile Glu Leu Phe 
            180                 185                 190 

Ala Pro Asp Ser Ser Ile Leu Ser His Leu Ala Ser Gln Tyr Ala Ile 
        195                 200                 205 

Ile Phe Ser Gln Glu Tyr Ala Tyr Gln Leu Ser Ala Asp Asp Asn Leu 
    210                 215                 220 

Ala Gln Leu Asp Thr His Pro Val Gly Thr Gly Pro Tyr Gln Val Lys 
225                 230                 235                 240 

Asp Tyr Val Tyr Asn Gln Tyr Val Arg Leu Val Arg Asn Glu Asn Tyr 
                245                 250                 255 

Trp Lys Lys Glu Ala Lys Ile Glu His Ile Ile Val Asp Leu Ser Thr 
            260                 265                 270 

Asp Arg Ser Gly Arg Leu Val Lys Phe Phe Asn Asn Glu Cys Gln Ile 
        275                 280                 285 

Ala Ser Tyr Pro Glu Val Ser Gln Ile Gly Leu Leu Lys Asn Asp Asp 
    290                 295                 300 

Lys His Tyr Tyr Met Gln Ser Thr Asp Gly Met Asn Leu Ala Tyr Leu 
305                 310                 315                 320 

Ala Phe Asn Phe Asp Lys Pro Leu Met Arg Asp His Glu Ile Arg Ala 
                325                 330                 335 

Ala Ile Ser Gln Ser Leu Asn Arg Ala Arg Ile Ile His Ser Ile Tyr 
            340                 345                 350 

His Asn Thr Ala Thr Val Ala Asn Asn Ile Ile Pro Glu Val Ser Trp 
        355                 360                 365 

Ala Ser Thr Val Asn Thr Pro Glu Phe Glu Phe Asp Tyr His Pro Lys 
    370                 375                 380 

Ile Ala Lys Asn Lys Leu Ala Asp Lys Asn Leu Leu Leu Asn Leu Trp 
385                 390                 395                 400 

Val Ile Asn Glu Glu Gln Val Tyr Asn Pro Ala Pro Phe Lys Met Ala 
                405                 410                 415 

Glu Met Ile Lys Trp Asp Leu Ala Gln Ala Gly Val Lys Val Lys Val 
            420                 425                 430 

Arg Ala Val Thr Arg Pro Phe Leu Thr Ala Gln Leu Arg Asn Gln Ser 
        435                 440                 445 

Glu Asn Tyr Asp Leu Ile Leu Ser Gly Trp Leu Ala Gly Asn Leu Asp 
    450                 455                 460 

Pro Asp Gly Phe Met Arg Pro Ile Leu Ser Cys Gly Thr Lys Asn Glu 
465                 470                 475                 480 

Leu Thr Asn Leu Ser Asn Trp Cys Asn Glu Glu Phe Asp Gln Phe Met 
                485                 490                 495 

Asp Arg Ala Ile Thr Thr Ser His Leu Ser Ser Arg Ala Lys Ala Tyr 
            500                 505                 510 

Asn Glu Ala Gln Glu Leu Val Leu Arg Glu Leu Pro Ile Ile Pro Ile 
        515                 520                 525 

Ala Asn Val Lys Arg Ile Leu Val Ala Asn Ser Arg Val Lys Gly Val 
    530                 535                 540 

Lys Met Thr Pro Phe Gly Ser Leu Asp Phe Ser Thr Leu Tyr Phe Ile 
545                 550                 555                 560 

Gln Glu Lys His 

 
           
             8  
             320  
             PRT  
             H. influenzae  
           
            8 

Met Phe Trp Ser Val Leu Arg His Ile Leu Trp Val Ala Leu Leu Leu 
1               5                   10                  15 

Leu Val Leu Ser Leu Leu Gly Phe Val Ile Leu Leu Arg Asp Pro Leu 
            20                  25                  30 

Asn Ala Asn Leu Val Thr Gln Asn Ile Tyr Ile Gly Tyr Phe His Tyr 
        35                  40                  45 

Leu Gly Thr Leu Leu Gln Gly Asp Phe Gly Ile Thr Tyr Asn Gly Gly 
    50                  55                  60 

Lys Ser Leu Met Asn Leu Ile Leu Thr Val Leu Pro Pro Thr Leu Glu 
65                  70                  75                  80 

Leu Cys Phe Ile Thr Leu Phe Leu Ala Phe Ile Phe Gly Leu Pro Leu 
                85                  90                  95 

Gly Ile Ile Ser Ala Val Asn Ser Glu Gln Val Phe Ala Lys Ser Leu 
            100                 105                 110 

Gln Ile Leu Ser Tyr Val Gly Leu Ser Ile Pro Ile Phe Trp Leu Ala 
        115                 120                 125 

Pro Ile Leu Leu Tyr Val Ala Ala Leu Ser His Trp Glu Ile Ala Ala 
    130                 135                 140 

Ile Gly Gln Tyr Asn Leu Leu Tyr Glu Ile Lys Pro Ile Thr Gly Phe 
145                 150                 155                 160 

Pro Val Ile Asp Met Trp Phe Met Glu Val Pro Tyr Arg Thr Lys Ile 
                165                 170                 175 

Val Gln Asn Ile Leu Gln His Leu Ala Leu Pro Thr Leu Val Leu Cys 
            180                 185                 190 

Ile Leu Pro Thr Met Glu Ile Ile Arg Ile Ile His Gln Arg Ala Glu 
        195                 200                 205 

Tyr Ile Leu Asn Gln Asn Phe Ser Lys Val Ala Thr Thr Arg Gly Trp 
    210                 215                 220 

Ser Lys Trp Lys Ile Leu His Gln Tyr Val Phe Arg Asn Thr Phe Pro 
225                 230                 235                 240 

Leu Leu Val Pro Gln Val Pro Arg Val Phe Thr Leu Val Leu Thr Gln 
                245                 250                 255 

Cys Met Leu Val Glu Thr Ala Leu Gly Trp Pro Gly Ile Gly Arg Trp 
            260                 265                 270 

Leu Ile Asn Ala Val Asn Glu Gln Asp Tyr Asn Ser Ile Ala Ala Gly 
        275                 280                 285 

Val Ile Val Ile Gly Val Cys Ile Ile Leu Ile Asp Thr Phe Thr Lys 
    290                 295                 300 

Ile Phe Thr Phe Ile Leu Asp Pro Phe Lys Lys Lys Gly Trp Tyr Ala 
305                 310                 315                 320 

 
           
             9  
             295  
             PRT  
             H. influenzae  
           
            9 

Met Gln Asp Lys Glu Pro Asp Glu Phe Arg Glu Ser Thr Ser Ile Phe 
1               5                   10                  15 

Gln Ile Trp Leu Arg Phe Arg Gln Asn Thr Ile Ala Leu Phe Ser Phe 
            20                  25                  30 

Tyr Leu Leu Ile Ala Leu Ile Phe Thr Ala Leu Phe Ala Ser Tyr Leu 
        35                  40                  45 

Ala Pro Tyr Ala Asp Asn Arg Gln Phe Ile Gly Gln Glu Leu Met Pro 
    50                  55                  60 

Pro Ser Trp Val Asp Arg Gly Lys Ile Ala Phe Phe Phe Gly Thr Asp 
65                  70                  75                  80 

Asp Leu Gly Arg Asp Ile Leu Ser Arg Leu Ile Met Gly Thr Arg Tyr 
                85                  90                  95 

Thr Leu Gly Ser Ala Leu Leu Val Val Phe Ser Val Ala Ile Ile Gly 
            100                 105                 110 

Gly Ala Leu Gly Ile Ile Ala Gly Leu Leu Lys Gly Ile Lys Ala Arg 
        115                 120                 125 

Phe Val Gly His Ile Phe Asp Ala Phe Leu Ser Leu Pro Ile Leu Leu 
    130                 135                 140 

Ile Ala Val Val Ile Ser Thr Leu Met Glu Pro Ser Leu Trp Asn Ala 
145                 150                 155                 160 

Met Phe Ala Thr Leu Leu Ala Ile Leu Pro Tyr Phe Ile His Thr Ile 
                165                 170                 175 

Tyr Arg Ala Ile Gln Lys Glu Leu Glu Lys Asp Tyr Val Val Met Leu 
            180                 185                 190 

Lys Leu Glu Gly Ile Ser Asn Gln Thr Leu Leu Lys Ser Thr Ile Leu 
        195                 200                 205 

Pro Asn Ile Thr Val Ile Tyr Ile Gln Glu Val Ala His Ala Phe Val 
    210                 215                 220 

Ile Ala Val Leu Asp Ile Ser Ala Leu Ser Phe Ile Ser Leu Gly Ala 
225                 230                 235                 240 

Gln Arg Pro Thr Pro Glu Trp Gly Ala Met Ile Lys Asp Ser Leu Glu 
                245                 250                 255 

Leu Leu Tyr Leu Ala Pro Trp Thr Val Leu Leu Pro Gly Phe Ala Ile 
            260                 265                 270 

Ile Phe Thr Ile Leu Leu Ser Ile Ile Phe Ser Asn Gly Leu Thr Lys 
        275                 280                 285 

Ala Ile Asn Gln His Gln Glu 
    290                 295 

 
           
             10  
             349  
             PRT  
             H. influenzae  
           
            10 

Met Ala Leu Leu Asp Ile Cys Asn Leu Asn Ile Glu Ile Gln Thr Ser 
1               5                   10                  15 

Asn Gly Arg Ile Lys Ile Val Asp Gly Val Asn Leu Ser Leu Asn Glu 
            20                  25                  30 

Gly Glu Ile Ser Gly Leu Val Gly Glu Ser Gly Ser Gly Lys Ser Leu 
        35                  40                  45 

Ile Ala Lys Val Ile Cys Asn Ala Ile Lys Glu Asn Trp Ile Ile Thr 
    50                  55                  60 

Ala Asp Arg Phe Arg Phe His Asp Ile Glu Leu Leu Lys Leu Ser Pro 
65                  70                  75                  80 

Asn Lys Arg Arg Lys Ile Val Gly Lys Glu Ile Ser Met Ile Phe Gln 
                85                  90                  95 

Asn Pro Leu Ser Cys Leu Asp Pro Ser Arg Lys Ile Gly Lys Gln Leu 
            100                 105                 110 

Ile Gln Asn Ile Pro Asn Trp Thr Phe Lys Asn Lys Trp Trp Lys Trp 
        115                 120                 125 

Phe Gly Trp Lys Lys Arg Arg Ala Ile Glu Leu Leu His Arg Val Gly 
    130                 135                 140 

Ile Lys Asp His Arg Asp Ile Met Ala Ser Tyr Pro Asn Glu Leu Thr 
145                 150                 155                 160 

Glu Gly Glu Gly Gln Lys Val Met Ile Ala Met Ala Val Ala Asn Gln 
                165                 170                 175 

Pro Arg Leu Leu Ile Ala Asp Glu Pro Thr Asn Thr Leu Glu Ser Thr 
            180                 185                 190 

Thr Ala Leu Gln Val Phe Arg Leu Leu Ser Ser Met Asn Gln Asn Gln 
        195                 200                 205 

Gly Thr Thr Ile Leu Leu Thr Ser Asn Asp Ile Lys Ser Ile Ser Glu 
    210                 215                 220 

Trp Cys Asp Gln Ile Ser Val Leu Tyr Cys Gly Gln Asn Thr Glu Ser 
225                 230                 235                 240 

Ala Pro Thr Glu Ile Leu Ile Glu Ser Pro His His Pro Tyr Thr Gln 
                245                 250                 255 

Ala Leu Ile Asn Ala Val Pro Asp Phe Thr Gln Pro Leu Gly Phe Lys 
            260                 265                 270 

Thr Lys Leu Gly Thr Leu Glu Gly Thr Ala Pro Ile Leu Glu Gln Met 
        275                 280                 285 

Pro Ile Gly Cys Arg Leu Gly Pro Arg Cys Pro Phe Ala Gln Lys Lys 
    290                 295                 300 

Cys Met Glu Lys Pro Arg Arg Leu Lys Ile Lys Gln His Glu Phe Ser 
305                 310                 315                 320 

Cys His Tyr Pro Ile Asn Leu Arg Glu Lys Asn Phe Lys Glu Lys Thr 
                325                 330                 335 

Thr Ala Thr Pro Phe Ile Leu Asn Cys Lys Gly Asn Glu 
            340                 345 

 
           
             11  
             269  
             PRT  
             Homo sapiens  
           
            11 

Met Pro Leu Leu Gln Val Glu Asp Leu Thr Lys Thr Phe Lys Gly His 
1               5                   10                  15 

Ala Ser Leu Phe Gly Arg Asn Gln Phe Asn Ala Val Asp Lys Val Ser 
            20                  25                  30 

Phe Thr Leu Glu Arg Lys Gln Thr Leu Ala Ile Ile Gly Asn Asn Gly 
        35                  40                  45 

Ser Gly Lys Ser Thr Leu Val Lys Met Ile Ala Gly Ile Ile Pro Pro 
    50                  55                  60 

Thr Ser Gly Arg Ile Leu Phe Asn Asp Arg Glu Leu Gln Tyr Gln Asp 
65                  70                  75                  80 

Ala Gln Ser Arg Ala Lys His Ile Arg Met Val Phe Gln Asp Ala Asn 
                85                  90                  95 

Ser Ala Phe Asn Pro Arg Leu Asn Ile Gly Gln Ile Leu Asp Glu Pro 
            100                 105                 110 

Leu Ser Leu Ala Thr Asp Trp Thr Glu Thr Gln Arg Asn Glu Lys Ile 
        115                 120                 125 

Phe Glu Thr Leu Ser Leu Val Gly Leu Tyr Pro Asp Tyr Thr Asn Leu 
    130                 135                 140 

Asn Ile Lys His Leu Ser Ile Ser Gln Lys Gln Arg Val Ala Leu Ala 
145                 150                 155                 160 

Arg Ala Leu Ile Leu Ala Pro Glu Ile Ile Ile Ile Asp Asp Ala Ile 
                165                 170                 175 

Gly Asn Leu Asp Ala Ser Val Arg Ile Gln Leu Leu Asn Leu Thr Leu 
            180                 185                 190 

Asp Leu Gln Gln Arg Leu Gly Ile Ser Tyr Ile Tyr Val Gly Gln Asp 
        195                 200                 205 

Leu Gly Val Ile Lys His Ile Ala Asp Thr Ile Ile Val Met Asp Asp 
    210                 215                 220 

Gly Lys Met Ile Glu Tyr Gly Ser Pro Gln Asn Leu Phe Thr Asp Pro 
225                 230                 235                 240 

Gln Thr Asp Val Thr Arg Arg Leu Val Glu Ser Tyr Phe Gly Lys Ile 
                245                 250                 255 

Leu Asp Glu Thr Ala Trp Val Lys Asp Lys Asn Thr His 
            260                 265 

 
           
             12  
             338  
             PRT  
             H. influenzae  
           
            12 

Met Asn Thr Arg Pro Phe Tyr Phe Gly Leu Ile Phe Ile Ala Ile Ile 
1               5                   10                  15 

Ala Ile Leu Ala His Tyr Leu Gly Asn Thr Asp Phe Ser His His Tyr 
            20                  25                  30 

His Ile Ser Ala Leu Ile Ile Ala Ile Leu Leu Gly Met Ala Ile Gly 
        35                  40                  45 

Asn Thr Ile Tyr Pro Gln Phe Ser Thr Gln Val Glu Lys Gly Val Leu 
    50                  55                  60 

Phe Ala Lys Gly Thr Leu Leu Arg Thr Gly Ile Val Leu Tyr Gly Phe 
65                  70                  75                  80 

Arg Leu Thr Phe Gly Asp Ile Ala Asp Val Gly Leu Asn Ala Val Val 
                85                  90                  95 

Thr Asp Ala Ile Met Leu Ile Ser Thr Phe Phe Leu Thr Ala Leu Leu 
            100                 105                 110 

Gly Ile Arg Tyr Leu Lys Met Asp Lys Gln Leu Val Tyr Leu Thr Gly 
        115                 120                 125 

Ala Gly Cys Ser Ile Cys Gly Ala Ala Ala Val Met Ala Ala Glu Pro 
    130                 135                 140 

Val Thr Lys Ala Glu Ser His Lys Val Ser Val Ala Ile Ala Val Val 
145                 150                 155                 160 

Val Ile Phe Gly Thr Leu Ala Ile Phe Thr Tyr Pro Leu Phe Tyr Thr 
                165                 170                 175 

Trp Ser Gln Asp Leu Ile Asn Ala His Gln Phe Gly Ile Tyr Val Gly 
            180                 185                 190 

Ser Ser Val His Glu Val Ala Gln Val Tyr Ala Ile Gly Glu Asn Ile 
        195                 200                 205 

Asp Pro Ile Val Ala Asn Thr Ala Val Ile Ser Lys Met Ile Arg Val 
    210                 215                 220 

Met Met Leu Ala Pro Phe Leu Leu Met Leu Ser Trp Leu Leu Thr Arg 
225                 230                 235                 240 

Ser Asn Gly Val Ser Glu Asn Thr Ser His Lys Ile Thr Ile Pro Trp 
                245                 250                 255 

Phe Ala Val Leu Phe Ile Gly Val Ala Ile Phe Asn Ser Phe Asp Leu 
            260                 265                 270 

Leu Pro Lys Glu Leu Val Lys Leu Leu Val Glu Ile Asp Ser Phe Leu 
        275                 280                 285 

Leu Ile Ser Ala Met Ala Ala Leu Gly Leu Thr Thr Gln Ala Ser Ala 
    290                 295                 300 

Ile Lys Lys Ala Gly Leu Lys Pro Leu Val Leu Gly Thr Leu Ile Tyr 
305                 310                 315                 320 

Leu Trp Leu Met Val Gly Gly Phe Leu Val Asn Tyr Gly Ile Ser Lys 
                325                 330                 335 

Leu Ile 

 
           
             13  
             6427  
             DNA  
             H. influenzae  
           
            13 

atgttacgtc taaatctgag atttttatct tttctgctct gtataagcca aagtgtagaa     60 

ttacaggctg cgccaagtgt tccaacattt ttaactgaaa atggcttaac ttattgcacc    120 

cacgcttcag gtttttcatt taatccgcaa acagcagatg caggaaccag tatgaatgtg    180 

gtcacggaac aaatttataa caaattattt gatataaaaa atcacagtgc aacattaaca    240 

ccaatgctgg cacaatctta ttccatttca gctgatggta aagaaatttt attaaattta    300 

cgtcacggcg taaaatttca ccaaacccct tggtttaccc caacacgtga ttttaacgct    360 

gaagacgtag tattttcgat taatcgtgta ttagggcata atacttattt accaacctta    420 

gcagaggcga atgttaccta tagtaatcca caatatagag tgtttcacga acaagcaaga    480 

aaagtgcgtt ttccttattt tgatagcatt aaacttaacg aaaaaatcaa atctgtgacc    540 

gcactttcgc cttatcaagt aaaaattgaa ttatttgcac cagattcctc cattttgtcg    600 

catcttgcca gccagtatgc cattattttt tcacaagaat atgcctatca attaagcgca    660 

gatgacaacc ttgctcaatt agatacccac ccagtaggca cagggcctta tcaagtaaaa    720 

gattatgtat ataaccaata tgttcgctta gtgcgtaacg aaaactattg gaaaaaagaa    780 

gccaagatag aacatattat tgtggatctt tctactgatc gcagcggacg tttagtcaaa    840 

tttttcaata atgaatgtca aatcgcctct tatcctgaag taagccaaat tggcttatta    900 

aaaaatgatg acaaacatta ttatatgcaa tctactgatg gtatgaattt agcctattta    960 

gcgtttaatt ttgataagcc attaatgcga gatcacgaaa tccgtgctgc tatttcacaa   1020 

agtttaaacc gagctcgaat cattcatagc atttaccata acacagcaac tgttgctaat   1080 

aacattattc ctgaagtgtc ttgggcttca actgtcaata cgccagaatt tgagtttgat   1140 

taccatccca aaatcgccaa aaataaatta gcagataaaa accttttgtt aaatttatgg   1200 

gtaattaatg aagaacaagt ctataatcca gcacctttta aaatggctga aatgatcaaa   1260 

tgggatttag ctcaagcggg tgtgaaagtt aaagtgcgtg ccgtaactcg tccattttta   1320 

actgcacaat tacgcaatca atcggaaaat tatgatttga ttctatctgg ttggttagct   1380 

ggtaatcttg atcctgatgg ttttatgcgt ccaattttaa gctgtggaac aaaaaatgaa   1440 

ctcactaatt tatctaattg gtgtaatgaa gaatttgatc aatttatgga tcgtgccatt   1500 

accacctcac atttaagttc acgcgcaaaa gcctataatg aagcccaaga actcgtttta   1560 

cgtgaattac ccattattcc tattgccaat gtaaaacgaa ttttagtcgc aaatagtcgt   1620 

gtgaaaggag taaaaatgac gccttttggt agcttagatt tttccacctt atattttatt   1680 

caggagaaac actaatgttc tggtcggttc ttcgccatat tctgtgggtg gcattattat   1740 

tactcgtatt atcgctatta ggctttgtta ttttattgcg cgatcctctt aatgcgaatc   1800 

ttgttacaca aaacatttat atcggctatt tccattattt aggcaccttg ttacaaggtg   1860 

attttggcat tacctataac ggtggaaaat cattaatgaa ccttattctt acggttcttc   1920 

ctcccacatt ggaactttgt ttcattacat tgtttttggc atttattttt ggtttgccac   1980 

ttggcattat aagtgcggtc aattctgaac aagtttttgc aaaaagttta caaatcctat   2040 

cttatgtagg gctatctatt ccaatatttt ggttagcccc cattttactg tatgttgccg   2100 

cgctctcaca ttgggaaatt gccgctattg gacaatataa tttgctttac gaaattaaac   2160 

ccattacggg atttcctgtt attgatatgt ggtttatgga agtaccttat cgtacaaaaa   2220 

tcgtacaaaa catattgcaa catttagcct taccaacatt ggtattgtgt atthtgccaa   2280 

caatggaaat tatccgtatt attcatcaac gagcagaata tattttgaat caaaattttt   2340 

ctaaagtagc gacaacacgg ggttggtcaa aatggaaaat tctccatcaa tatgtattcc   2400 

gtaatacttt tcccctgctt gttccacaag taccacgtgt attcacatta gtattaacgc   2460 

aatgtatgtt ggtagaaacg gctttaggtt ggcctggcat tggtcgttgg ttaattaatg   2520 

ccgtaaatga acaagattac aacagcattg ccgcaggtgt aattgttatt ggtgtatgta   2580 

ttattttgat tgatacattc actaaaatat tcacttttat actcgatcca tttaaaaaga   2640 

aaggttggta tgcaagataa agaacctgat gaattccgcg aaagcacctc aatctttcaa   2700 

atttggttac gctttcgtca aaataccatc gcacttttta gcttttattt attaatcgca   2760 

ttaattttta ccgcactttt tgctagttat cttgcacctt atgctgataa tcgacaattt   2820 

attgggcaag aattaatgcc tccttcttgg gtagatagag gaaaaattgc ttttttcttt   2880 

ggtactgatg atttaggtcg cgacatatta agtcgtttaa ttatgggtac tcgttatacc   2940 

ttaggttctg ctttactggt tgtcttttca gtggcaataa taggcggcgc actaggaatt   3000 

attgcaggac tactgaaagg tattaaagct cgttttgtcg ggcatatttt tgatgctttt   3060 

ttatcgttac ctattctatt aattgccgtt gttatttcaa cattaatgga accaagttta   3120 

tggaatgcaa tgtttgctac gctattagca attttgcctt atttcattca cactatctat   3180 

cgcgctattc aaaaagaatt agaaaaggat tatgttgtaa tgctaaaact tgaaggcatt   3240 

tccaatcaaa ccttattaaa aagcactatt ttaccgaata ttactgttat ttatattcaa   3300 

gaagtggctc atgcttttgt tatagccgtg ttggatatta gcgcattaag ttttatttct   3360 

cttggtgcac aacgacctac accagaatgg ggggcaatga taaaagactc tttggaacta   3420 

ctttatcttg caccttggac agtactttta cccggtttcg ctattatttt tactatttta   3480 

ttaagtatta ttttcagtaa tggcttaact aaagccatca atcaacatca agaatagcct   3540 

atggcacttt tagacatttg taacctcaat attgaaattc aaacctccaa tggacgtata   3600 

aaaattgtag atggcgtcaa tctttccctt aacgaagggg aaatcagtgg attagttggc   3660 

gaatcaggct caggaaaaag cttaatcgct aaagtcattt gtaatgcaat caaagaaaat   3720 

tggattatta ctgccgatcg ctttcgtttt cacgatatcg aattactaaa actcagtcct   3780 

aataaacgac gtaagattgt cggcaaagaa atatccatga ttttccaaaa tcccttatct   3840 

tgccttgatc caagtcgaaa aatagggaaa caactcatcc aaaatattcc taattggaca   3900 

tttaaaaata aatggtggaa atggtttggg tggaaaaaaa gacgtgctat tgaattgtta   3960 

catcgcgtag gaattaaaga tcatcgtgat attatggcaa gctatcctaa cgaactgaca   4020 

gaaggcgaag gacaaaaagt tatgatcgca atggctgtcg ctaatcagcc acgtttatta   4080 

atcgcagatg aaccaacaaa tacattagaa tcaaccactg ccctacaagt ttttcgttta   4140 

ctttccagta tgaaccaaaa tcagggaaca acaattttac ttacgagtaa cgatattaaa   4200 

agtattagtg aatggtgcga tcaaatttca gtgctttatt gtgggcaaaa taccgaatct   4260 

gccccgactg aaatattaat cgaaagtccc catcatcctt atacccaagc cttaattaat   4320 

gcagtacccg attttactca acctttgggg tttaaaacta aattgggtac gttagaaggc   4380 

accgcgccta ttttagagca aatgccaatt ggctgtcgtc ttggcccaag atgccctttt   4440 

gcacaaaaaa aatgtatgga aaaaccaaga cgattgaaaa taaaacaaca cgaattttct   4500 

tgtcattatc ctattaattt acgagaaaaa aatttcaaag aaaaaacaac cgccacccct   4560 

tttatactta attgcaaagg aaatgaataa tgcccttatt acaagtggaa gatttaacta   4620 

aaacttttaa aggtcacgcc agtttatttg gtcgaaatca attcaatgca gtggataaag   4680 

tgagttttac ccttgaacgt aaacaaacac ttgcaatcat tggcaataat ggctctggta   4740 

aatcaactct agtgaaaatg atagcgggca ttattccgcc aacttctggt cgaattttat   4800 

ttaatgatcg agaattacaa tatcaggatg cccaatctag agctaaacat attcgtatgg   4860 

ttttccaaga tgccaactct gcatttaatc cacgtttaaa tattggacaa atattagacg   4920 

aaccattaag cctagcgaca gattggacag aaacacaacg taatgaaaaa atctttgaga   4980 

ccctctctct tgttggactt tatcctgatt acacaaatct caatattaag catctctcta   5040 

tcagccaaaa gcagcgggtt gccctagcac gcgcattaat tttagcacca gaaattatta   5100 

taatagatga tgcaattggc aatttagatg cttctgtacg tattcaattg cttaatttaa   5160 

cccttgattt acaacaacgt ttaggtatat cttatattta tgtgggacag gatctcggtg   5220 

taattaaaca tattgcagat acgattatcg taatggatga cggaaaaatg attgaatatg   5280 

gcagccctca aaatcttttt actgatccac aaactgatgt tactcgtcgc ttagtcgaaa   5340 

gctattttgg caaaatttta gatgaaaccg cttgggtaaa agacaaaaac actcactaag   5400 

gaaaggaaaa atgaacactc gtccctttta tttcggactt atatttattg cgattatcgc   5460 

tatacttgct cactatttag gaaacactga tttttcccat cattatcata tcagtgctct   5520 

aattattgcc atcttgctgg gaatggcaat cggcaatacc atttatccgc aattttcaac   5580 

acaagtggaa aaaggcgtgt tatttgcgaa aggcacgctt cttcgcactg gcattgtgct   5640 

gtatggtttt cgccttactt ttggcgatat tgccgatgtt ggcttaaatg ctgttgtcac   5700 

tgatgcgatt atgctaattt caaccttttt tcttaccgca cttttgggca ttcgttatct   5760 

aaaaatggat aaacaattgg tttatctcac tggggctgga tgtagtattt gtggtgcggc   5820 

agcggttatg gcggcagagc ctgttaccaa agcagaatct cataaagttt cagtagcgat   5880 

tgccgtagtg gtcattttcg ggacgcttgc tatttttact taccccttgt tctacacgtg   5940 

gtcacaagat ttaattaacg cccatcaatt cggtatttat gttggttcta gtgtacacga   6000 

agtggctcaa gtgtatgcga ttggggaaaa tattgatcct atcgtggcga atactgccgt   6060 

catttccaaa atgatccgag tgatgatgct cgcaccattt ttattaatgc tttcttggtt   6120 

attaacacgt agtaatggag tatcagaaaa tacatcacac aaaattacaa ttccttggtt   6180 

tgctgtactt tttattggcg ttgcgatttt taattctttt gatttattac caaaagaact   6240 

cgtgaaatta ttagttgaaa tcgattcttt cttattaatt tcagcgatgg ctgcccttgg   6300 

cttaacgaca caagcaagcg caatcaaaaa ggcaggatta aaaccacttg ttttaggaac   6360 

actaatttat ttatggctaa tggttggtgg atttttagtg aattatggaa tatcaaaatt   6420 

aatataa                                                             6427 

 
           
             14  
             1013  
             PRT  
             H. influenzae  
           
            14 

Met Thr Asn Phe Lys Phe Ser Leu Leu Ala Cys Ser Ile Ala Phe Ala 
1               5                   10                  15 

Leu Asn Ala Ser Thr Ala Tyr Ala Ala Gln Pro Thr Asn Gln Pro Thr 
            20                  25                  30 

Asn Gln Pro Thr Asn Gln Pro Thr Asn Gln Pro Thr Asn Gln Pro Thr 
        35                  40                  45 

Asn Gln Pro Thr Asn Gln Pro Thr Asn Gln Asp Ser Asn Leu Ser Glu 
    50                  55                  60 

Gln Leu Glu Gln Ile Asn Val Ser Gly Ser Thr Glu Asn Ser Asp Ser 
65                  70                  75                  80 

Lys Thr Pro Pro Lys Ile Ala Glu Thr Val Lys Thr Ala Lys Thr Leu 
                85                  90                  95 

Glu Arg Glu Gln Ala Asn Asn Ile Lys Asp Ile Val Lys Tyr Glu Thr 
            100                 105                 110 

Gly Val Thr Val Val Glu Ala Gly Arg Phe Gly Gln Ser Gly Phe Ala 
        115                 120                 125 

Ile Arg Gly Val Asp Glu Asn Arg Val Ala Ile Asn Ile Asp Gly Leu 
    130                 135                 140 

Arg Gln Ala Glu Thr Leu Ser Ser Gln Gly Phe Lys Glu Leu Phe Glu 
145                 150                 155                 160 

Gly Tyr Gly Asn Phe Asn Asn Thr Arg Asn Gly Ala Glu Ile Glu Thr 
                165                 170                 175 

Leu Lys Glu Val Asn Ile Thr Lys Gly Ala Asn Ser Ile Lys Ser Gly 
            180                 185                 190 

Ser Gly Ser Leu Gly Gly Ser Val Ile Tyr Lys Thr Lys Asp Ala Arg 
        195                 200                 205 

Asp Tyr Leu Leu Asn Lys Asp Tyr Tyr Val Ser Tyr Lys Lys Gly Tyr 
    210                 215                 220 

Ala Thr Glu Asn Asn Gln Ser Phe Asn Thr Leu Thr Leu Ala Gly Arg 
225                 230                 235                 240 

Tyr Lys Lys Phe Asp Val Leu Val Val Thr Thr Ser Arg Asn Gly His 
                245                 250                 255 

Glu Leu Glu Asn Tyr Gly Tyr Lys Asn Tyr Asn Asp Lys Ile Gln Gly 
            260                 265                 270 

Lys Arg Arg Glu Lys Ala Asp Pro Tyr Lys Ile Glu Gln Asp Ser Thr 
        275                 280                 285 

Leu Leu Lys Leu Ser Phe Asn Pro Thr Glu Asn His Arg Phe Thr Leu 
    290                 295                 300 

Ala Ala Asp Leu Tyr Glu His Arg Ser Arg Gly Gln Asp Leu Ser Tyr 
305                 310                 315                 320 

Thr Leu Lys Tyr Leu Lys Thr Leu Pro Asp Leu Pro Glu Val Asp Ser 
                325                 330                 335 

Arg His Thr Asn Asp Lys Thr Lys Arg His Asn Ile Ser Phe Ser Tyr 
            340                 345                 350 

Glu Asn Phe Ser Gln Thr Pro Phe Trp Asp Thr Leu Lys Ile Thr Phe 
        355                 360                 365 

Ser Lys Gln Lys Ile Lys Thr Arg Ala Arg Thr Asp Glu Tyr Cys Asp 
    370                 375                 380 

Ala Gly Val Arg Tyr Cys Glu Gly Thr Ala Asn Pro Ala Gly Leu Lys 
385                 390                 395                 400 

Leu Lys Asn Gly Glu Ile Thr Arg Arg Asp Gly Thr Pro Leu Gln Phe 
                405                 410                 415 

Lys Glu Ile Asn Asn Thr Thr Thr Pro Asn Ser Asn Ser Asn Lys Asp 
            420                 425                 430 

Lys Thr Tyr Asp Phe Ser Lys Leu Ile Asp Thr Asn Gly Lys Glu Ile 
        435                 440                 445 

Glu Ser Gly Ile Thr Arg Ser Asn Asp Thr Phe Trp Tyr Asp Cys Ser 
    450                 455                 460 

Ile Phe Asp Cys Glu Asn Pro Gly Lys Met Lys Val Ala Glu Gly Lys 
465                 470                 475                 480 

Thr Tyr Tyr Arg Tyr Asp Gly Thr Trp Lys Asn Asn Val Gln Leu Glu 
                485                 490                 495 

Lys Lys Val Leu Asn Gly Lys Glu Phe Ala Arg Ile Asn Asn Gly Thr 
            500                 505                 510 

Arg Gly Lys Thr Phe Pro Ile Leu Pro Ser Ser Leu Gly Tyr Leu Glu 
        515                 520                 525 

Arg Leu Trp Gln Glu Arg Asp Leu Asp Thr Asn Thr Gln Gln Leu Asn 
    530                 535                 540 

Leu Asp Leu Thr Lys Asp Phe Lys Thr Trp Arg Val Glu His Asn Leu 
545                 550                 555                 560 

Gln Tyr Gly Ser Ser Tyr Asn Thr Thr Met Lys Arg Met Val Asn Arg 
                565                 570                 575 

Ala Gly Tyr Asp Ala Thr Asp Val Gln Trp Trp Ala Lys Arg Thr Leu 
            580                 585                 590 

Gly Thr Arg Phe Asp Phe Leu Lys Asn Glu Glu Ile Val Glu Thr Cys 
        595                 600                 605 

Ala Thr Thr Phe Gly Trp Asn Ala Phe Leu Cys Pro Arg Val Asp Pro 
    610                 615                 620 

Glu Phe Ser Tyr Leu Leu Pro Ile Lys Thr Lys Glu Lys Ser Val Tyr 
625                 630                 635                 640 

Leu Phe Asp Asn Val Val Ile Thr Asp Tyr Leu Ser Phe Asp Leu Gly 
                645                 650                 655 

Tyr Arg Tyr Asp Asn Ile His Tyr Gln Pro Lys Tyr Lys His Gly Val 
            660                 665                 670 

Thr Pro Lys Leu Pro Asp Asp Ile Val Lys Glu Leu Phe Ile Pro Leu 
        675                 680                 685 

Lys Ser Gly Gln Asn Asn Asn Asp Ala Glu Val Lys Lys Asn Val Gln 
    690                 695                 700 

Glu Asn Ile Asp Tyr Ile Ala Lys Gln Asn Lys Lys Tyr Lys Ala His 
705                 710                 715                 720 

Ser Tyr Ser Phe Val Ser Thr Ile Asp Pro Thr Ser Phe Leu Arg Leu 
                725                 730                 735 

Gln Leu Lys Tyr Ser Lys Gly Phe Arg Ala Pro Thr Ser Asp Glu Met 
            740                 745                 750 

Tyr Phe Thr Phe Lys His Pro Asp Phe Thr Ile Leu Pro Asn Thr His 
        755                 760                 765 

Leu Lys Pro Glu Ile Ala Lys Thr Lys Glu Ile Ala Phe Thr Leu His 
    770                 775                 780 

His Asp Asp Trp Gly Phe Ile Ser Thr Ser Leu Phe Lys Thr Asn Tyr 
785                 790                 795                 800 

Arg Asp Phe Ile Asp Leu Val Tyr Lys Gly Glu Arg Glu Phe Glu Val 
                805                 810                 815 

Gly Asn Pro Asn Asn Arg Gly Lys Ile Ser Phe Asp Thr Phe Gln Asn 
            820                 825                 830 

Ile Asn Arg Asp Ser Ala Val Val Lys Gly Ile Glu Ile Asn Ser Lys 
        835                 840                 845 

Val Phe Leu Gly Lys Met Ala Lys Phe Met Asp Gly Phe Asn Leu Ser 
    850                 855                 860 

Tyr Lys Tyr Thr Tyr Gln Lys Gly Arg Met Asp Gly Asn Ile Pro Met 
865                 870                 875                 880 

Asn Ala Ile Gln Pro Lys Thr Met Val Tyr Gly Leu Gly Tyr Asp His 
                885                 890                 895 

Pro Ser Gln Lys Phe Gly Phe Asn Phe Tyr Thr Thr His Val Ala Ser 
            900                 905                 910 

Lys Asn Pro Glu Asp Thr Tyr Asp Ile Tyr Ala Lys Asp Lys Asn Gln 
        915                 920                 925 

Thr Asn Thr Ser Ile Lys Trp Arg Ser Lys Ser Tyr Thr Ile Leu Asp 
    930                 935                 940 

Leu Ile Gly Tyr Val Gln Pro Ile Lys Asn Leu Thr Ile Arg Ala Gly 
945                 950                 955                 960 

Val Tyr Asn Leu Thr Asn Arg Lys Tyr Ile Thr Trp Asp Ser Ala Arg 
                965                 970                 975 

Ser Ile Arg Ser Phe Gly Thr Ser Asn Val Ile Asp Gln Lys Thr Gly 
            980                 985                 990 

Gln Gly Ile Asn Arg Phe Tyr Ala  Pro Gly Arg Asn Tyr  Lys Met Ser 
        995                 1000                 1005 

Val Gln  Phe Glu Phe 
    1010 

 
           
             15  
             6125  
             DNA  
             H. influenzae  
             
               misc_feature  
               (6098)..(6098)  
               n = a, c, g, or t  
             
           
            15 

tgcagattcc ggtatttgcc ccccaataaa ggcactgaaa ttttgatcgc cccattcact     60 

aatactttta atatcgttac tcgtaagtaa aattgttgtt ccctgatttg gttcatactg    120 

gaaagtaacc aaaacctgtg acggcagggg tgattccaaa ggtattgttg ggtcatccgc    180 

gaatataaac gtgggcggat tagcgacagc cattgcgatc aaaccttttt gtccttcgcc    240 

ttctgtcagt tcgttaggat agcttgccat aatatcacga tgatctttaa ttcctacgcg    300 

atgtaacaat tcaatagcac gtcttttttt ccacccaaac catttccacc atttattttt    360 

aaatgtccaa ttaggaatat tttggatgag ttgtttccct atttttcgac ttggatcaag    420 

gcaagataag ggattttgga aaatcatgga tatttctttg ccgacaatct tacgtcgttt    480 

attaggactg agttttagta attcgatatc gtgaaaacga aagcgatcgg cagtaataat    540 

ccaattttct ttgattgcat tacaaatgac tttagcgatt aagctttttc ctgagcctga    600 

ttcgccaact aatccactga tttccccttc gttaagggaa agattgacgc catctacaat    660 

ttttatacgt ccattggagg tttgaatttc aatattgagg ttacaaatgt ctaaaagtgc    720 

cataggctat tcttgatgtt gattgatggc tttagttaag ccattactga aaataatact    780 

taataaaata gtaaaaataa tagcgaaacc gggtaaaagt actgtccaag gtgcaagata    840 

aagtagttcc aaagagtctt ttatcattgc cccccattct ggtgtaggtc gttgtgcacc    900 

aagagaaata aaacttaatg cgctaatatc caacacggct ataacaaaag catgagccac    960 

ttcttgaata taaataacag taatattcgg taaaatagtg ctttttaata aggtttgatt   1020 

ggaaatgcct tcaagtttta gcattacaac ataatccttt tctaattctt tttgaatagc   1080 

gcgatagata gtgtgaaatg aaataaggca aaattgctaa tagcgtagca aacattgcat   1140 

tccataaact tggttccatt aatggttgaa ataacaacgg caattaatag aataggtaac   1200 

gataaaaaag catcaaaaat atgcccgaca aaacgagctt taataccttt cagtagtcct   1260 

gcaataattc ctagtgcgcc gcctattatt gccactgaaa agacaaccag taaagcagaa   1320 

cctaaggtat aacgagtacc cataattaaa cgacttaata tgtcgcgacc tatatcatca   1380 

gtaccaaaga aaaaagcaat ttttcctcta tctacccaag aaggaggcat taattcttgc   1440 

ccaataaatt gtcgattatc agcataaggt gcaagataac tagcaaaaag tgcggtaaaa   1500 

attaatgcga ttaataaata aaagctaaaa agtgcgatgg tattttgacg aaagcgtaac   1560 

caaatttgaa agattgaggt gctttcgcgg aattcatcag gttctttatc ttgcatacca   1620 

acctttcttt ttaaatggat cgagtataaa agtgaatatt ttagtgaatg tatcaatcaa   1680 

aataatacat acaccaataa caattacacc tgcggcaatg ctgttgtaat cttgttcatt   1740 

tacggcatta attaaccaac gaccaatgcc aggccaacct aaagccgttt ctaccaacat   1800 

acattgcgtt aatactaatg tgaatacacg tggtacttgt ggaacaagca ggggaaaagt   1860 

attacggaat acatattgat ggagaatttt ccattttgac caaccccgtg ttgtcgctac   1920 

tttagaaaaa ttttgattca aaatatattc tgctcgttga tgaataatac ggataatttc   1980 

cattgttggc aaaatacaca ataccaatgt tggtaaggct aaatgttgca atatgttttg   2040 

tacgattttt gtacgataag gtacttccat aaaccacata tcaataacag gaaatcccgt   2100 

aatgggttta atttcgtaaa gcaaattata ttgtccaata gcggcaattt cccaatgtga   2160 

gagcgcggca acatacagta aaatgggggc taaccaaaat attggaatag atagccctac   2220 

ataagatagg atttgtaaac tttttgcaaa aacttgttca gaattgaccg cacttataat   2280 

gccaagtggc aaaccaaaaa taaatgccaa aaacaatgta atgaaacaaa gttccaatgt   2340 

gggaggaaga accgtaagaa taaggttcat taatgatttt ccaccgttat aggtaatgcc   2400 

aaaatcacct tgtaacaagg tgcctaaata atggaaatag ccgatataaa tgttttgtgt   2460 

aacaagattc gcattaagag gatcgcgcaa taaaataaca aagcctaata gcgataatac   2520 

gagtaataat aatgccaccc acagaatatg gcgaagaacc gaccagaaca ttagtgtttc   2580 

tcctgaataa aataaaaggt ggaaaaatct aagctaccaa aaggcgtcat ttttactcct   2640 

ttcacacgac tatttgcgac taaaattcgt tttacattgg caataggaat aatgggtaat   2700 

tcacgtagaa cgagttcttg ggcttcatta taggcttttg cgcgtgaact taaatgtgag   2760 

gtggtaatgg cacgatccat aaattgatca aattcttcat tacaccaatt agataaatta   2820 

gtgagttcat tttttgttcc acagcttaaa attggacgca taaaaccatc aggatcaaga   2880 

ttaccagcta accaaccaga tagaatcaaa tcataatttt ccgattgatt gcgtaattgt   2940 

gcagttaaaa atggacgagt tacggcacgc actttaactt tcacacccgc ttgagctaaa   3000 

tcccatttga tcatttcagc cattttaaaa ggtgctggat tatagacttg ttcttcatta   3060 

attacccata aatttaacaa aaggttttta tctgctaatt tatttttggc gattttggga   3120 

tggtaatcaa actcaaattc tggcgtattg acagttgaag cccaagacac ttcaggaata   3180 

atgttattag caacagttgc tgtgttatgg taaatgctat gaatgattcg agctcggttt   3240 

aaactttgtg aaatagcagc acggatttcg tgatctcgca ttaatggctt atcaaaatta   3300 

aacgctaaat aggctaaatt cataccatca gtagattgca tataataatg tttgtcatca   3360 

ttttttaata agccaatttg gcttacttca ggataagagg cgatttgaca ttcattattg   3420 

aaaaatttga ctaaacgtcc gctgcgatca gtagaaagat ccacaataat atgttctatc   3480 

ttggcttctt ttttccaata gttttcgtta cgcactaagc gaacatattg gttatataca   3540 

taatctttta cttgataagg ccctgtgcct actgggtggg tatctaattg agcaaggttg   3600 

tcatctgcgc ttaattgata ggcatattct tgtgaaaaaa taatggcata ctggctggca   3660 

agatgcgaca aaatggagga atctggtgca aataattcaa tttttacttg ataaggcgaa   3720 

agtgcggtca cagatttgat tttttcgtta agtttaatgc tatcaaaata aggaaaacgc   3780 

acttttcttg cttgttcgtg aaacactcta tattgtggat tactataggt aacattcgcc   3840 

tctgctaagg ttggtaaata agtattatgc cctaatacac gattaatcga aaatactacg   3900 

tcttcagcgt taaaatcacg tgttggggta aaccaagggg tttggtgaaa ttttacgccg   3960 

tgacgtaaat ttaataaaat ttctttacca tcagctgaaa tggaataaga ttgtgccagc   4020 

attggtgtta atgttgcact gtgatttttt atatcaaata atttgttata aatttgttcc   4080 

gtgaccacat tcatactggt tcctgcatct gctgtttgcg gattaaatga aaaacctgaa   4140 

gcgtgggtgc aataagttaa gccattttca gttaaaaatg ttggaacact tggcgcagcc   4200 

tgtaattcta cactttggct tatacagagc agaaaagata aaaatctcag atttagacgt   4260 

aacataacaa atgcattgtg ataaattatg tgtcaaattg taaggcatat tagtaaaaat   4320 

ggctaggata ttgaatgttt aatcgggttc aaaaggaaat caatcaaatt attaatcgtg   4380 

gttttgatcg cactttgcgt ttagcggtaa cagggttaag tcggagtgga aaaacggcgt   4440 

ttattacaag tttaatcaat caacttctct ccattaatca acattcatca cagaatttgc   4500 

ccttgtttga agcagcgaga aatggtgcga tcttggcagt caaacgagta tcccaacaag   4560 

atctcagcgt gccacgtttt gattatgaaa gtaatttaaa tgatttgtca caaaatccgc   4620 

ctcaatggat tcaatctact cgtggcgtga gtgaaacgcg tttagccatt cgttttcaac   4680 

gccaatctgg cttgctacgc catttgaaag aacgaggcac gctttatcta gatatttttg   4740 

attatccagg ggaatggctg atcgatttgc cgttattaaa tctagatttt caacaatggt   4800 

cacaagagca aattaaggta acaacaggca ttcgtgaaga attggcggag aattggctcg   4860 

ctatgttgca ggatttggat ttaagtgcgg tcgcaaatga agatgtttta gccaagatag   4920 

cgaaaagtta tacggattat ttacatcaat gcaaagtgca aggcatgcaa tttattcagc   4980 

ctgggcgatt tgtattgccg agtgatttag agggcacgcc cgcattacaa tttttcccat   5040 

taattcatct ttcagaagaa cagtggcgaa ccttgaaaaa aacagcaaaa tcaaatagct   5100 

attttgctgt gctgacaaaa cgttatgatt attatcgcaa taaaattgtg aaaggttttt   5160 

acgaaaatta tttttctacc tttgatcgtc aagttatttt ggcggattgt ttaacgcctt   5220 

taaatcacag tcagcaagcc tttttagata tgcaaatggg cttaaatcag ttatttaata   5280 

atttccatta tggcagcaga aattttcttc atcgtttgtt ttctccgcga attgatcgat   5340 

taatgtttgt tgcgacaaag gcggatcata ttactcgtga tcaaattcct aatttagtaa   5400 

gtttaatgcg ccaaattgtg caagagggtg gtcgccatgt ggaatttgaa ggaatcgata   5460 

cggaatatac cgccattgcg gctgttcgta ccacaaagca agtgattgtg aatcagcaag   5520 

gaaaagaaat taaagcaatt caaggggttc gttctattga taaacagctg attacacttt   5580 

atccgggaac ggtgccgagc aaattaccaa gagcagaatt ttggcaaaaa caaccgcact   5640 

ttgattttga tagttttgaa cctcagcctt tagaacaagg ggagagcatt cctcatttga   5700 

gaatggatgc ggttttacaa tttttattaa gtgatcgatt tgaataaaaa gtgcggaaaa   5760 

ttttccgcac ttttttcatc tttctagcct gtattgcgca tcccagccgc gatacctgtg   5820 

atggtaatca tcaatgcttg ttccacatcg ggattgactt gctctggatt ttcacggaaa   5880 

cggtgaagca attccacttg cagtagattg agtggatccg tgtagatatt acgtaatgca   5940 

attgaatctg caatccaagg taaatcagac atcaattcac tttggtgaga aagtgaaagc   6000 

acagtttgaa tatcatcttc aagttgctta cgtaaatttt cacctaaata ccaaagctct   6060 

ttttcactaa tcgttgatca tattgtggga aaagggtnac cgaggcccgn aattncggat   6120 

accat                                                               6125 

 
           
             16  
             3520  
             DNA  
             H. influenzae  
             
               misc_feature  
               (2522)..(2522)  
               n = a, c, g, or t  
             
           
            16 

ctttattaag ccaaaaggta aaaaatgaaa attgcattag gcattgagta taacgggcaa     60 

aattattatg gttggcagag acaagaaaaa gtccgtagtg tacaagaaga attagaaaag    120 

gcactttctc acattgcaaa tgaaaaaatt gagatatttt gtgcaggcag aacggattct    180 

ggcgtaagtg gaacgggtca ggttgttcat tttgaaacca atgcggttcg tccagagaag    240 

gcttgggctt ttggtacgaa tgctcattta cctgatgaca ttgcggtggc ttgggcaaaa    300 

caagtcgatg atgaatttca cgccagattt tccgcaacag cacgccgtta ccgctatatt    360 

ctttattgta ataaattacg ctctgcgatt ttagcgggag gaataaccca ttgccattta    420 

gatttagatg cggaaaaaat gcatcaggca gggcaatgtt tacttggcga acaggatttt    480 

tcctctttcc gtgcggcaca atgtcagtct catacgcctt ggcgtaatgt gcatcatttg    540 

aatgtgtctc gtatcggaaa atatattatt gttgatattc aggcgaacgc ttttgtgcat    600 

catatggtgc gcaatattgt gggaagtttg attgaggtcg gtgctggaaa tcagccgatt    660 

gaatggatgc aatggctact tgagcagaaa aatcgtcagc ttgctgcacc aacagcaaaa    720 

ccagatggat tgtatttggt tgatgtgatt tatccacaaa agtttgatat tcctaaacgc    780 

ccgattggtc ctttattttt agaggatggt ttattaaatc gtactttgaa gtaaagcgta    840 

atttcatgtt ttaaataacc atatctgaaa atattcttca taaaaaaaga ccgcacttta    900 

aaagtgcggt caatcttaag tgagttttat attaattttg atattccata attcactaaa    960 

aatccaccaa ccattagcca taaataaatt agtgttccta aaacaagtgg ttttaatcct   1020 

gcctttttga ttgcgcttgc ttgtgtcgtt aagccaaggg cagccatcgc tgaaattaat   1080 

aagaaagaat cgatttcaac taataatttc acgagttctt ttggtaataa atcaaaagaa   1140 

ttaaaaatcg caacgccaat aaaaagtaca gcaaaccaag gaattgtaat tttgtgtgat   1200 

gtattttctg atactccatt actacgtgtt aataaccaag aaagcattaa taaaaatggt   1260 

gcgagcatca tcactcggat cattttggaa atgacggcag tattcgccac gataggatca   1320 

atattttccc caatcgcata cacttgagcc acttcgtgta cactagaacc aacataaata   1380 

ccgaattgat gggcgttaat taaatcttgt gaccacgtgt agaacaaggg gtaagtaaaa   1440 

atagcaagcg tcccgaaaat gaccactacg gcaatcgcta ctgaaacttt atgagattct   1500 

gctttggtaa caggctctgc cgccataacc gctgccgcac cacaaatact acatccagcc   1560 

ccagtgagat aaaccaattg tttatccatt tttagataac gaatgcccaa aagtgcggta   1620 

agaaaaaagg ttgaaattag cataatcgca tcagtgacaa cagcatttaa gccaacatcg   1680 

gcaatatcgc caaaagtaag gcgaaaacca tacagcacaa tgccagtgcg aagaagcgtg   1740 

cctttcgcaa ataacacgcc tttttccact tgtgttgaaa attgcggata aatggtattg   1800 

ccgattgcca ttcccagcaa gatggcaata attagagcac tgatatgata atgatgggaa   1860 

aaatcagtgt ttcctaaata gtgagcaagt atagcgataa tcgcaataaa tataagtccg   1920 

aaataaaagg gacgagtgtt catttttcct ttccttagtg agtgtttttg tcttttaccc   1980 

aagcggtttc atctaaaatt ttgccaaaat agctttcgac taagcgacga gtaacatcag   2040 

tttgtggatc agtaaaaaga ttttgagggc tgccatattc aatcattttt ccgtcatcca   2100 

ttacgataat cgtatctgca atatgtttaa ttacaccgag atcctgtccc acataaatat   2160 

aagatatacc taaacgttgt tgtaaatcaa gggttaaatt aagcaattga atacgtacag   2220 

aagcatctaa attgccaatt gcatcatcta ttataataat ttctggtgct aaaattaatg   2280 

cgcgtgctag ggcaacccgc tgcttttggc tgatagagag atgcttaata ttgagatttg   2340 

tgtaatcagg ataaagtcca acaagagaga gggtctcaaa gattttttca ttacgttgtg   2400 

tttctgtcca atctgtcgct aggcttaatg gttcgtctaa tatttgtcca atatttaaac   2460 

gtggattaaa tgcagagttg gcatcttgga aaaccatacg aatatgttta gctctagatt   2520 

gngcatcctg atattgtaat tctcgatcat taaataaaat tcgacccaga gttggcggaa   2580 

taatgcccgc tatcattntc actagaggtg atttaccaga gccattattg ccaatgattg   2640 

caagtgtttg tttactgttc aagggtaaaa ctcactttat ccactgcatt gaattgattt   2700 

cgaccaaata aactggcgtg acctttaaaa gttttagtta aatcttccac ttgtaataag   2760 

ggcattattc atttcctttg caattaagta taaaaggggt ggcggttgtt ttttctttga   2820 

aatttttttc tcggagatta ataggataat gacaagaaaa ttcgtgttgt tttattttca   2880 

atcgtcttgg tttttccata catttttttt gcgcaaaagg gcatcttgga ccaagactat   2940 

ctgccttcgg tatttgctct ataacattcc ctgtaccttc ctccgttccc tttttatctt   3000 

aaatttcata tctttttttc ttttctattt ctcttttttt ttattttttt acagcgttcc   3060 

ctcttgattc accgcccctt atccttccag catgcccggc ttgtattttc atttgcccct   3120 

tatcccggtc ttttctgcct gttttttcct tctgtttttt cttccccctc ttttctcccc   3180 

tctctccggc ctcctcgttc ttttcgcttt tttcccttgc ccatcttttt tttcttatct   3240 

ttccacatcc ctttgtatat tgttatcttc tctctattct ttcccccgtt attctcccgt   3300 

tttctttcct cccctccctc cctttcttat tgtttttttt cttttttgtc attttctttt   3360 

atttctctct tttcactccg ttatcttttt attttttata tttctctttt ttttttttga   3420 

tttcttcttc ttttttgtgt ctattcttat ttttcttttt attctttctt ctatccttgg   3480 

agtgtttctt attgttacat tttttgtttc ttcctctttt                         3520 

 
           
             17  
             5562  
             DNA  
             H. influenzae  
           
            17 

ccccgctgca gtttgttggc gtaactctgc ttccggcgtt tttgttcctt acgattttga     60 

accgaatttt cattatcgcc cactttttcc gaaactttat tttcagatgt gctattttgt    120 

tcattcagcc atttttgata gtcttctaaa tcgcctttaa attcttccac ttttttatcg    180 

tgaactaaat aaaattcttc cacggtattg cgtaataaat gacgatcgtg cgacaccacc    240 

accaaagaac cttcgtaatc taccaatgct tccgttaatg cttgacgcat atccaaatcc    300 

aaatggttag tcggttcatc aagtagtaat aaattcgggc gttgccaaac aatcaaagcc    360 

aacaccaaac gagctttttc tcctccagaa aaagatttca ctgcttgatt tactttatcg    420 

ccgtgaaacg caaaactgcc taaataatct cgaacttgtt gctccgtttg ttctggtgcg    480 

agtttttgca tatgccacag agcagattcg tctgcgcgta aagtatctaa ttgatgctga    540 

gcaaaatagc caagctgcac gccttttgcc aactgcactg tgcctgaaag tgcggtcagt    600 

tctcccgcta aaagtttaat caaggttgat tttcctgcac catttttccc gagtaaacca    660 

atgcgcgaac ctggcactaa attcagttta attttactta aaatttctac cgcactttct    720 

ccgctgccat aacctgcact tgcctgttca atcatcacta agggattcgg caaggattgc    780 

ggtggacgaa atttaaaagt aaaaggatta tccacataag ctggtgcaat cagctccatt    840 

ctttctagtg ctttcatacg gctttgtgcc tgtttggctt tagtggcttt ggctttaaag    900 

cgatcaatat atttttgtaa atgggaaatc ttttgttgtt gctgacgata catcgctgtt    960 

tgttgtgcca atttagtggc tcgttgcact tcaaaggaag aataatcgcc cgtgtattcg   1020 

ttgagcttct gattttcgat atggaggatt tttgtcacaa tcggatcgag aaaatcacga   1080 

tcgtgagaaa ttaataccaa ggtgccttga tattgtacta gccaacgctc taaccaaata   1140 

accgcatcca aatccaaatg gttggttggc tcatccagta ataataaatc tgatggacaa   1200 

agcagagctt gtgccaaatt caaacggatc cgccaaccgc ccgaaaaggc tttcactggc   1260 

tgggttgttt cttcttgact aaatcctaaa ccattcaata acgaagcggc acgagattga   1320 

attgtccacg catccaaggt ttctaattgc ccgtgaatac gtgcaatggc gttaccgtca   1380 

ttgcattcat ttgcttgttc aagctcttgt tgcaaacggc aatattcacg atccccttga   1440 

attacataat caattgcaga aatatccaat gcaggcgttt cttgattcac ccaagatacc   1500 

cgccaatttg ctggataatt tacctcgccg ccctctggcg ttaattcttt ttttaataag   1560 

gcaaaaagcg aagattttcc acaaccattt ttccccacca agccgacttt ttgcttagga   1620 

ttaatggtag cagaagcatt ttcgagaagc tccgtttgcc ctcgttttaa ggacagatta   1680 

ctaaatacaa tcatttttct acaataggtc ttaatttgag gctattttgc aatatttttt   1740 

cttttctcgg aacagtctat tccgatttta tcgattttct agtcaaaaaa gccaggtata   1800 

ataaggtgca ataaaaacta tttattgaga aaacttaatg aaaatataac aagtcaatat   1860 

gaaaataatg ggtatatctt aggcattttt tacccttaga ggcacaatcg acagggtttt   1920 

ccctaaagga gcgagcaatt ttaacaacgt ttcaagttga ggattcgttt gtcctttttc   1980 

aatgcgtgca atcataggtt gcttcacacc gcttaaggtt tcaagttgtt tttgggaaat   2040 

cccaagttgc tggcgagaag taatcaattc tttaattaag gctacacgta aattactttc   2100 

gcggatttct tcttcattga aaatttgctg ttcaaactca ttccaatttg aacctaatgg   2160 

gctgatttta ttcatttttc aatctctctt ttaattcact taagcgttgt tgggttgttt   2220 

ggattacata atctcgaatt ttattcagtt taattcgact atctttgctt tcattttgag   2280 

ccagcgatag cagatattct ttcactggct caatgtcatt ttggtctctg taaaagagaa   2340 

tctcgtacat aatttatcct atttacttat ttatttcaat aactaataag ttattgattt   2400 

ttgatgtgta agcataaata gaaactaaaa atttggcgag atagtcattt caatctttcg   2460 

atcgagatcg caaaaatagc aaaaagaggt tgattttcaa ggagattttg cacaaatgcc   2520 

gatgtttgaa cattagcttt gaaagagaga gtaaaaggta gatcctttct taagaaaaag   2580 

tgcggtaaaa atttaccgca cttttgattt tagacaggtt taaaactcaa attgaactga   2640 

catcttataa tttctacctg gtgcgtagaa gcggttaatg ccttgacctg tcttttgatc   2700 

tataacatta cttgtaccaa atgaacgaat tgaacgcgca gaatcccaag tgatgtattt   2760 

acggtttgta agattatata cgccggctct tatggttaaa tttttaattg gttgcacata   2820 

tccaattaaa tctagaatag tataagattt actgcgccat tttatgctag tgttggtttg   2880 

gtttttatct ttcgcataaa tatcataagt atcttctgga tttttacttg ctacgtgggt   2940 

agtgtagaaa ttaaatccaa atttttggct tgggtggtca tagcctaagc catacaccat   3000 

cgttttaggc tgaattgcat tcataggaat attgccatcc attcttcctt tttgataggt   3060 

atatttatag cttaggttaa atccatccat aaattttgcc attttaccaa ggaatacttt   3120 

tgaattaatt tctattcctt ttactaccgc actatctcta ttaatatttt gataaagaga   3180 

aaatggtaat gtgctacctc cgctaactaa tttaaaatct ttttctcctt taaatattag   3240 

gtcgataaag tttttatagt tggttttaaa tagacttgtg gagataaaac cccaatcatc   3300 

attatgtaat gtaaaagcaa tttcttttgt ttttgctatc tctggtttta gattagtatt   3360 

tggcaaaata gtgaaatcag ggtgtttaaa ggtgaaatac atttcatctg aagttggtgc   3420 

tctaaaacct tttgaatatt ttagttgtaa acgaagaaaa ctcgttggat caatcgttga   3480 

aacaaaactg taagaatgtg ctttatattt tttgttttgt ttagcgatat agtcaatatt   3540 

ttgttgtacg tttttcttaa cttcaggatc atcattatta tttttaccac ttggtaatgg   3600 

aataaacaat cctttcacaa tatcatcagg taatttcggt gtaacgccgt gtttatattt   3660 

tggttgataa tggatattgt cataacgata acccaaatca aaagataaat aatcagttat   3720 

aacaacatta tcaaagagat agactgattt ttcttttgtt ttaatgggta ataagaatga   3780 

aaatttagga tcaactcgag ggcaaaggtt agctttccac cccccataag cggttttaca   3840 

agtgtgaggt ttatcataca acaaactgta accaagtgta ggctctgccc accattgcac   3900 

atcagaagca tcattaccag cacgattaac cattcgcttc atcgttgtat tatatgagct   3960 

accatattgt agattatgtt caacacgcca agttttgaag tctttggtta aatctaaatt   4020 

taattgttgg gtgttggtgt ctaaatctcg ctcttgccag aggcgttcta aataacctgg   4080 

agatgaggga agaatagagt atgttttatt attcgcatcc tttactctag cgaaattttt   4140 

gtcatttaat tcttttattt caagttcaaa gtcttttttc cacgttccag tagtgccata   4200 

gccataaaga tgttcagttt caaaaaattt tcattttttg gttttatctt tacaatcaaa   4260 

atattgaaac aatcatacca agttccagag gatctccttt aggcctagtt taacctctat   4320 

tactcgatca tcagtatcaa taaatttatc gaagtcctag atgctggtat caatatttta   4380 

actttttttt caaatgaagt tctgaacatc tccgcggggt tatttcccat ctgttatttt   4440 

tagtcccgca ggatttgcag ttccctcaca atatcttaca cctgcatcac aatagtcatc   4500 

tgtacgtgcg cgagttttaa tacgttgatc tgaataagtg atttttaatg tatcccaaaa   4560 

tggcgtttga gagaaatttt catagctaaa ggaaatatta cgtctctttg ttttatcatt   4620 

ggtgtgtcta gaatcaacct cgagtaaatt aggatctgtt ttttgatatt ttagtgtata   4680 

ggataaatct tgcccacgag aacgatgttc atataaatct gctgcaaggg taaaacgatg   4740 

attttctgta gggttaaaag ataattttaa taatgtacta tcttgttcaa ttttgtatgg   4800 

gtctgctttt tctctttttt taccttgagt aaggctattt gcatttttgt aatcatagtt   4860 

ttcaagttcg tgtccatttc tgcttgttgt aaccactaag gcatcaaact ttttataacg   4920 

tcctgcaaga gtaagggtat tgaatgattg attattttct gtagcgtatc cctttttgta   4980 

gcttacatag taatccttgt taaggagata atctctcgca tcttttgttt tataaattac   5040 

agatccacct aaggaaccac taccactttt gattgaattt gccccttttg taatatttac   5100 

ttcttttaaa gtttcaattt ctgcaccatt acgcgtatta ttgaagttac cataaccctc   5160 

aaaaagctct ttaaagcctt gagaagatag ggtttcagct tgacgtaatc catcaatatt   5220 

aatcgctaca cggttttcat ctacaccacg aatggcaaaa ccgctttgcc caaaacgccc   5280 

agcttcaaca acagtaacgc ccgtctcgta tttaacgatg cctttaatat tggtttgttt   5340 

gttccttttc catcgtttta acctcgtttt accgtttctt tatattctcc gtcggctctt   5400 

ttctattctc gtcttcttgc cgcaccgcta cgtcgatttc ctcttatctt tctataattc   5460 

ttatttctct tgtcttttct tgtctctcat ctatactact tctcatactc tttttgcttt   5520 

cttcacctcc tctttccacc ctttgcctta aagcaccctt ta                      5562 

 
           
             18  
             3318  
             DNA  
             H. influenzae  
             
               misc_feature  
               (3281)..(3281)  
               n = a, c, g, or t  
             
           
            18 

gagaccccgg gctaagcccc caaaatatca cgattgtttg ttggtgggct aaagcccact     60 

ctacaactac taatcaacaa tcaacggctc gcattatagg aaacaagcag gtttaaatac    120 

agaaaataat aagaatagtt tttatttctt gatttttatc aaaaatcaat ctttattgtt    180 

gtgagaaaat gcttcctcgt ataatatatt tgagaattat tattattttt ttataggatt    240 

aaatatgacc aattttagat taaacgtgct tgcctattcc gttatgcttg ggctaacggc    300 

aagtgttgct tatgcagagc caaccaacca accaaccaac caaccaacca accaaccaac    360 

caaccaacca accaaccaac caaccaaaat agtaatgctt ctgaacaact agaacaaata    420 

aatgtatctg gctctaccga aaatactgat acgaaaactc cgccaaaaat tgccgaaact    480 

gtaaaaacgg ctaaaacgct agaaagagaa caagcaaaca acattaaaga catcgttaaa    540 

tacgagactg gcgttactgt tgttgaagct gggcgttttg gtcaaagcgg ttttgcgatt    600 

agaggggtag atgaaaatcg tgttgcaatt acggttgatg gcgttgctca agcagaacat    660 

tatcttccca aggttttaaa gacctgtttg aaggatatgg taattttaat aacacgcgta    720 

atggaattga aattgaaact ttatctgatg ccaaaattac caaaggtgca gattctctca    780 

tgtctggtag tggtgcattg ggtggctccg tcatctataa aactaaagat gcaagagatc    840 

ttctgcttaa caaaaactac gcgtttaaat ataaaactgg ttttaccagc gagaatgatg    900 

aaagattaaa ttctattact tttgcaggaa aagcaagtat attcgatgta cttgctgtcg    960 

gcacttggcg taatggtcat gaaatcaaaa attatgatta caaatctgca gacgacattc   1020 

taggaaaact cagagaaaag accgatcctt ataataaaaa agaccgcagt cttttattga   1080 

aaattggtac aaatcttggt gaaaataatc gcattgccgt agcctatgat agaagacggg   1140 

ttgaaaataa aggtctagac aaatcttact cattacatgg atgcacgaaa tatgtttgtg   1200 

atgataatga aatagatact cgccatactc atgatgaaag cattagaacc agtaaatcta   1260 

tagcatttga aaatacaaat ataaacccac tttgggatac cctaaaactc tcttatacag   1320 

atcaaagtat tactcaacga gcaagaagtg acgaacattg tgatggtgaa cggtgtcctg   1380 

gggtacaaaa ccccatagga ctacattata acaacgataa taaacttgtt gataaaaata   1440 

ataatcctgt aacctataaa ttagaaaatc gttctgtaac atactattct tacattgatg   1500 

aatctatctt taatcgatac agtaacttta aagaagaagt tcctgtagaa ctcgctaagg   1560 

aatggaaact taaagaatat ggtggaaaat attatattga ctcgcctcgc tgctttaaaa   1620 

atcacggtga tgctaatcac gaggggatgt gtagactaag atctgatgta aaagaggaaa   1680 

aggaaacatt agtagctaat aacatcactt atgatttaaa aaaggagtat tttattaact   1740 

caaggctcac gaatagtgat aatttattat cttgtgatgg aattaactgt gataaaggta   1800 

caattcaagg tttcgaagct gatggaacgc ctaaggattt accaataaaa ataatcccaa   1860 

aagaaggtaa aaaatttgca cttattgaaa aaatttcaga tcaaaatggc tacaatattg   1920 

gcccagagaa agcatctcgt tttctagtac ctaattcacc tggttataat agaaacattt   1980 

ggaaaaaacg tgaccttgat actcgtactc aacaaattaa tttggattta acaaaacatt   2040 

ttgaactagg aaaaagccaa catgatttat cttatggttt agtttggagt aaaacaacaa   2100 

aatcaatgat aaataaagaa gggttaaaag ttaatagtgg aaaatggtgg attgattatc   2160 

caaaagactg tgaatctagt acatcagatt tatgtacaaa aaatagtaca gcatcatttc   2220 

ttattcctgt agaaacaaaa gatggttctc tctattttaa agatgaattt agagtaaatg   2280 

atcgtcttgg cttagatatc ggttatcgat atgacaaagt caaatacaaa accaattatc   2340 

aaccgggtat aacgccaaaa atccctgatg atatgttagt taatttattt ataaaagaac   2400 

cttttgtaaa aaacacacgg agtctaaacc ctaatgatcc aaatgaaatt aatcgacgaa   2460 

aaaatgcaga agctaatatt aattatattt ctcaacccaa aaaatttaat gcgagttctt   2520 

atgctttaag cacaaaattc gatccattgg attggttaca agttcaagca aaatatagca   2580 

aaggtttcag agcaccaaca gctgatgaat tatacttcac attcaagcac ccagaattta   2640 

ctgttcttcc aggctctaaa ttaaaacctg aaattgcaaa aactaaggaa ttatcattaa   2700 

ctttacatga tgatgaaatc ggttttattt ctggtggata tttcatcaca aattataata   2760 

attttattga ttttagttat ctaggaacaa aatcatttgg ttctcaagca actaagcatg   2820 

aattatatca atctgttaat ttagataatg ctaaagtgac aggatttgaa ttgaaaacca   2880 

aatttacatt aggaaaatgg atatcatggt tgaagaatgt tgattttggt taccaattaa   2940 

ctaaacaaaa aggtaaagca agcgataacc gcccacttaa tgctattcag ccaatgacac   3000 

aagtgatgag tttagcctat acgcatcctg ataatctgtt tggggcaaat ttatatctta   3060 

ctcatgtttc ccaaaaagaa gcgagtgaca catataacat ttattcaaaa gatgctacag   3120 

caggagataa agaatatgtt caaaataaac atattaaatg gcgtagtaaa gcttacacag   3180 

tgacagattt tactttcttc gtgaaaccta tgaagaattt aactttacga gcgggtgttt   3240 

acaatttatt tgacaaaaaa tatagacttg ggatggggat nctatagagt ncgacctggc   3300 

aggcatgcat agtctggc                                                 3318 

 
           
             19  
             3494  
             DNA  
             H. influenzae  
           
            19 

ggcattcttg ctgcctgcag ttcactctat gagaccccct tcttatggtg gtcaataatg     60 

tcagtaatat aagccattaa aagtaagctc ttcaaaacaa tttgtaagag atttaactga    120 

tttagctaaa cgctccccca atgtgttaat tggctcaaaa tacattactg caatatactg    180 

tttattaaat cgtttacctt tacccgaaaa ctaccaagat cacgcattag taggagagtg    240 

gaaaggttat cgagattgcc atattcaagg caatttggta ctgatttacc aatacgttat    300 

acacgatgaa tttgatgaat tgaaattttc tcgtttaaat acacactcac aaaccgcttt    360 

aaaatagaag taatcttaaa taataatccg cacgtaaaat gtgcggatta tttttacata    420 

ggattaaaat tcaaactgaa cagacattct gtaatttctg ccaggtgcat aaaagcggtt    480 

taagccttca ccagtttctg tttttactcg gttaattgtg ccaagatgtc gaacagagcg    540 

agcagagtcc caagtgagat attttttgtt ggttaaatta tatacaccag cggtaaatgt    600 

aagattttta attggtttcc aataggcaat cgtatcgatc actgtatagc gattattacg    660 

ccataatccg cgagaatctt ttacatcttt gccgtttgct tttgtagcag gtatatctgt    720 

tgcattgtca gtatattgct tttcttttct ggcgaccata cttgtccatt gagaattaaa    780 

gctatctttt gctttttttg ctgcaacatt agtgatatac atatccacgc cccatttttg    840 

gcttggtgca tcatagccaa tattgtatac cgatgtggtt ggttgtaagg cattcattgg    900 

ttgaggttta cgagcaatgg cttcgtattc tggatgttca tctttattca attccaaaaa    960 

ttctttatat tttggatgta atccattgtc tttgattctg cctttttgat aggtaaattt   1020 

atagcctaaa tggaaacctt gtagtttttc aaataaatcg cccatctcaa gacgtgaagc   1080 

aatttcgata cctctgactc ttgctcgatc tcgattttgg ttttgatgga acggatattt   1140 

tattgcagag ccttcttcaa taggacgttc gcccacttct actaagtcaa taaaattacg   1200 

gtaatcgttt tgaaacgcat ttaacgtaat ataactactg tttttataaa aagtgaacgc   1260 

gacttctttt gttttggagg tttcagcttt taaatctgtg ttaggctgaa tggaaaattg   1320 

tggatgttta aatgtcatat aaatttcatc agaggttggc gcacgaaaac cattggcgta   1380 

tttaagctgt acacgaagcc aatttgtggg atcaagattt aaccctaaat tgtaagaatg   1440 

atgtttatag tcagttttgc gtaataacag ggcaagattg tcttcaaaat tttttttata   1500 

acacggtgtg tcgtaagtgc aattttcata tcctggaggt attgagtatt tactaccata   1560 

aacatattcc tttgagctaa atttcttaaa tagcccagta attaatccat taggaacagg   1620 

aatgtttttg tcataactag gcaaatattt tacgtggtca taacgataat ttaaatctag   1680 

tcctagccaa gaggtaagtt gcacattatc tccaaaatac aacacattat ttttagtggt   1740 

aacagggatt aaataggtat ctttaccttt attggaattc attaagctac atctgtaagc   1800 

actatgatca ggtgcaggag tatgctccac aggatatgtg ccatctacag gtttgttaca   1860 

gaaaaaattg ccagcccacc attgcacatt ggcaacagtg tagtattgat gattcaccat   1920 

actttttagt gttttttcat aaagtccacc atattttagt tggtgttggg tatgccatag   1980 

gtgaaattct ttgtctaaat caagtttaat ttgttgggta tgggtgttta aatcacggtc   2040 

attaacgaag tctgtgctgt agccatggct ttttggaaat aaaaatttag cactttcata   2100 

ttgggttaat ccataattac cttctgctga ttttagtgag atttcaccat attttttgcc   2160 

atttaattct ttaatggtaa tatttcggtc ttcataatta tatttgtcat tcccattttc   2220 

atcttttcca acaaaaacct gaaatttttt attacaattt aatttttcac aattaattaa   2280 

gaccgaatca agtgagcctc cttctgtatc tacatcatta ctaacatctt cacccttttt   2340 

gttttgtagt tctaagccat aattatattt tcctgtaaat tcttgattgt ctttatcttt   2400 

aatcttataa attccacctt cttctactaa atgtagccct tgtggattac gtacaccagc   2460 

acaagtggat tgatgacaat actcatcaga gcgtgcttta ttggtaattt tttgtgagga   2520 

ataacttagt ttaatatgat cccaaaaagg ggtttgactg aaattttcat aactaaattg   2580 

aatatttttt cttttagatt gatcattaat aactcgctcg ccatattttt cctcacattt   2640 

agtatttttg cattgattga aaatataaga caaatccata ccctttgttt ctaaagtgga   2700 

atcatctaat gccacgctca agcgatggtt ttcattaggc tgaaagccca attttattaa   2760 

tgtgctttgg cgggtaattt gataaggatc ggctttttca cgggtaggac caaccgcact   2820 

taaatccgcc tgtttattgg gataaatttt ataatcgtag ttttctattt cgtgcccatc   2880 

acgttttgta tcaacgacta aaatatcaaa ttttttagaa cgtcctgcta atgtaagtgt   2940 

cttaaggttt tgattattca ttgtttgata gccacgttta taggaaagat aataatcttt   3000 

atctatcaga taatctcgag catctttagt ttcaaatata acagagccac ccaatgcacc   3060 

actaccggat tttaaggagt cagcaccttt ggtaatagtt gctgttttaa cattttcaat   3120 

ttcaatgcta ttacgagtat tattaaaatt gccatagcct tcaaataatt ctttaaatcc   3180 

ttgagagctt aaggtttcag cttgacgaag cccatcaacc ataataccta cacggttttc   3240 

atcaacccct cgaacagcat aaccacttgc gcccgttcta cctgtttcaa ccaccgtaat   3300 

acctgtttca tagcgaacga gatcacgaga atcagacgcc tgctgtttcg ctaatttttt   3360 

ggcagaaatt tgggtttcac ctaccttttt ctctttcaca ttaattgttt ctgtacttcc   3420 

tgaaacatta atttgttcta gttgttcaga aacattacta ttttggttgg ttggttggtt   3480 

ggttggttgg ttgg                                                     3494 

 
           
             20  
             5002  
             DNA  
             H. influenzae  
             
               misc_feature  
               (4906)..(4906)  
               n = a, c, g, or t  
             
           
            20 

tataagccca accaggcgcc aacgcagtaa cagtttcctt gagagaaaac ataacaaaag     60 

atggggtatt tctggaagag aaaccaatgc caccaaacca gaaatgaggg cttttaggta    120 

agtaaaatag tgttaataaa tggaaataat agttgccatc taaagttttc gatttaataa    180 

cgagattctc catactcaag tgcggtagaa tttgccaagt tttacttagc cagtctacgt    240 

tttctaaacg aaatacccaa tttatccgca tattgcttca ttgtattcgt tccagagcta    300 

ccgcctgcat accaacgctt ttcttgaaaa ggaaccaacg agacttcaac taaggcagtt    360 

tgataactta accccccacc tatacgcaaa gtagcctcat tgtatttttt attatcccaa    420 

taatattttc cattcccatt aaacatagtt ttactaaaaa aatgatctgc ccatggccat    480 

ttttttctac tgataaagaa taccctaccc cccttcccac tttctttttc ccaagcggtc    540 

caactaccaa tttttgtgcc actttttgga gcgttattca aattatcatc atttaaaaaa    600 

tttaagccta cttgccatat ccattgattc cgctgattta gtgttaaaag atactgatca    660 

ataacaccta aaaatttttc atcatctacc tctgtacgta atttttcaaa ttgaattttg    720 

gcagattcat tttcatagtt aaaaaatagg gcttgagcta attgataacg caaaggtagt    780 

aaagatgcgt ctagagcgaa taattcacga taataagcaa tagattgagt taaatcacct    840 

tgttcacgcg cgtcaatagc ctttgcccaa gtgagtaaga aattatcgtg ctgaggaaat    900 

tgtttatata gtggtaataa cagttgaact gcctgagtgt tattttgata taaagcaaga    960 

attaacccac gcaaaacaag tcttggatgt tgtgctaatt ggcttttgga aagagaaata   1020 

atatgcctat ttggtatttc tttcttaggc atagaggaaa aagaggaggt ttttaattcc   1080 

gcactttgaa tcgtatttgt cagtgtatca tttttaggac gtgcaacttt tgcccaagct   1140 

atattcgtta atgataagcc tattaatgat aagcctatta atgataagaa agaaatttgt   1200 

tttacgccat ttttcatatt ttatccatat tcttaaaaaa ctctaacttg acattattac   1260 

aaaaaaagaa caataatgcg aattattatc aattttgtat aagtattaat tctatgaaat   1320 

ctgtacctct tatcactggt ggactttcct ttttattaag cgcttgtagc gggggaggtg   1380 

gttcttttga tgtagatgac gtctctaatc cctcctcttc taaaccacgt tatcaagacg   1440 

atacttcaag ttcaagaaca aaatctaatt tggaaaagtt gtccattcct tctttaggag   1500 

gagggatgaa gttagtggct cagaatctga gtggtaataa agaacctagt ttcttaaatg   1560 

aaaatggcta tatatcatat ttttcctcac cttctacgat tgaagatgat gttaaaaatg   1620 

ttaaaacaga aaataaaata catacaaatc caattgggct tgaacctaat agagcattac   1680 

aagaccccaa tttacaaaaa tacgtttatt ctggtttgta ctatattgag aattggaaag   1740 

acttttccaa attagcaaca gaaaaaaaag cctatagtgg ccattatggt tatgcgtttt   1800 

attatggtaa taaaactgca acagacttgc cagtaagcgg tgtagcaacg tataaaggaa   1860 

cttgggattt catcactgca actaaatatg gccaaaatta ttctttgttc agtaatgcta   1920 

gaggtcaagc ttattttcga cgtagtgcta ctcgaggaga tattgattta gaaaataatt   1980 

caaagaatgg tgatataggc ttaataagtg aatttagtgc agattttggg actaaaaaac   2040 

tgacaggaca actgtcttac accaaaagaa aaactgatat tcaacaatat gaaaaggaaa   2100 

aactctatga tatagatgcc catatttata gtaatagatt caggggtaaa gttactccta   2160 

cgaaatccac atcggatgaa catcccttta ccagcgaggg aacattagaa ggtggttttt   2220 

atggacctaa tgctgaagaa ctagggggta aattcttagc tagggataaa cgagtttttg   2280 

gggtatttag tgccaaagaa acgccagaaa cagaaaagga aaaattatcc aaagaaacct   2340 

taattgatgg caagctaatt actttctcta ctaaaacagc cgatgcaaca accagtacaa   2400 

cagccagtac aacagccgat gtaaaaaccg atgaaaaaaa ctttacgaca aaagatatat   2460 

caagttttgg tgaagctgat taccttttaa ttgataatta ccctgttcct cttttccctg   2520 

aaggggatac tgatgacttc gtaacgagta aacatcacga tattggaaat aaaacctata   2580 

aagtagaagc atgttgcaag aatctaagct atgtaaaatt tggtatgtat tatgaggata   2640 

aagagaagaa aaacacaaat caaacaggac aataccacca atttttgtta ggtctccgta   2700 

ctcccagttc tcaaattcct gtaacgggaa acgtgaaata tctcggtagt tggtttggtt   2760 

atattggtga tgacaagaca tcttactcca ctacaggaaa taaacaacaa gataaaaatg   2820 

ctcccgccga gtttgatgtc aattttgaca ataaaacatt aacaggcaaa ttaaaacgag   2880 

ccgactcaca aaataccgtg tttaacattg aggcaacctt taaaaatggt agtaatgcct   2940 

tcgaaggtaa agcaaccgca aatgtagtga ttgatcccaa aaatacacaa gccacatcta   3000 

aagtcaattt cacgacaaca gtaaacgggg cattttatgg tccgcacgct acagaattag   3060 

gcggttattt cacctataac ggaaacaatc ctacagctac aaattctgaa agttcctcaa   3120 

ccgtaccttc accacccaat tcaccaaatg caagagctgc agttgtcttt ggagctaaaa   3180 

gacaagtaga aaaaaccaac aagtagaaac aaccaacaag tagaaaaaaa caaataatgg   3240 

aatactaaaa atgactaaaa aaccctattt tcgcctaagt attatttctt gtcttttaat   3300 

ttcatgctat gtaaaagcag aaactcaaag tataaaagat acaaaagaag ctatatcatc   3360 

tgaagtggac actcaaagta cagaagattc agaattagaa actatctcag tcactgcaga   3420 

aaaagtaaga gatcgtaaag ataatgaagt aactggactt ggcaaaatta tcaaaactag   3480 

tgaaagtatc agccgagaac aagtattaaa tattcgtgat ctaacacgct atgatccagg   3540 

gatttcagtt gtagaacaag gtcgcggtgc aagttctgga tattctattc gtggtatgga   3600 

cagaaataga gttgctttat tagtagatgg tttacctcaa acgcaatctt atgtagtgca   3660 

aagcccttta gttgctcgtt caggatattc tggcactggt gcaattaatg aaattgaata   3720 

tgaaaatgta aaggccgtcg aaataagcaa gggggggagt tcttctgagt atggtaatgg   3780 

agcactagct ggttctgtaa catttcaaag caaatccgca gccgatatct tagaaggaga   3840 

caaatcatgg ggaattcaaa ctaaaaatgc ttattcaagc aaaaataaag gctttaccca   3900 

ttctttagct gtagcaggaa aacaaggtgg atttgaagga cttgctattt acactcaacg   3960 

aaattcaatt gaaacccaag tccataaaga tgcattaaaa ggcgtgcaaa gttataatcg   4020 

attaatcgcc aaagaagatg gatctaatgc atactttgtg atggaagatg agtgtccaaa   4080 

ggattataac agttgtatac cttcagccaa accacctgcg aagttatcct cccaaagaga   4140 

aaccgtaagc gtttcagatt atacgggggc taaccgtatc aaacctaatc caatgaaata   4200 

tgaaagccag tcttggtttt taagaggagg ctatcatttt tctgaacaac attatattgg   4260 

tggtattttt gaattcacac aacaaaaatt tgatatccgt gatatgacat ttcccgctta   4320 

tttaagatca acagaaaaac cggatttaga aaatagttct ttttatccaa agcaagatta   4380 

tggtgcatat caacgtattg aggatggccg aggcgttaaa tatgcaagtg ggctttattt   4440 

cgatgaacac catagaaaac agcgtgtagg tattgaatat atttacgaaa ataagaacaa   4500 

agcgggaatc attgacaaag cagtgttaag tgctaatcaa caaaacatta tacttgacag   4560 

ttatatgcaa catacacatt gcagtcttta tcctaatcca agtaagaatt gccgcccaac   4620 

acttgataaa ccttattcat actatcattc tgatagaaat gtttataaag aaaaacataa   4680 

tatgttgcaa ttgaatttag agaaaaaaat tcaacaaaat tggcttactc atcaaattgt   4740 

cttcaatctt tgggttttga tgactttact tcagcgcttc agcataaaga ttatttacct   4800 

cgacggtgtt accgctacgg caaagagtat ttcagagaaa cctggtgaaa caccaagaag   4860 

aaatggtttc aaattacaac cttacttata cccaaaacca aatgcnatct ttgnnaggac   4920 

gagatcattg taattatcaa ggtagctcct ctattatagt gactgtaaag gggcggtaat   4980 

ttaagggaaa aattattatt ca                                            5002 

 
           
             21  
             6  
             PRT  
             H. influenzae  
           
            21 

Phe Tyr Ala Pro Gly Arg 
1               5 

 
           
             22  
             5  
             PRT  
             H. influenzae  
           
            22 

Leu Trp Gln Glu Arg 
1               5 

 
           
             23  
             9  
             PRT  
             H. influenzae  
           
            23 

Phe Gly Gln Ser Gly Phe Ala Ile Arg 
1               5 

 
           
             24  
             9  
             PRT  
             H. influenzae  
           
            24 

Ala Gly Val Tyr Asn Leu Thr Asn Arg 
1               5 

 
           
             25  
             8  
             PRT  
             H. influenzae  
           
            25 

Tyr Ile Thr Trp Asp Ser Ala Arg 
1               5 

 
           
             26  
             9  
             PRT  
             H. influenzae  
           
            26 

Lys Tyr Ile Thr Trp Asp Ser Ala Arg 
1               5 

 
           
             27  
             10  
             PRT  
             H. influenzae  
           
            27 

Glu Phe Ala Arg Ile Asn Asn Gly Thr Arg 
1               5                   10 

 
           
             28  
             9  
             PRT  
             H. influenzae  
           
            28 

Tyr Asp Asn Ile His Tyr Gln Pro Lys 
1               5 

 
           
             29  
             10  
             PRT  
             H. influenzae  
           
            29 

Leu Ser Phe Asn Pro Thr Glu Asn His Arg 
1               5                   10 

 
           
             30  
             11  
             PRT  
             H. influenzae  
           
            30 

Ser Arg Gly Gln Asp Leu Ser Tyr Thr Leu Lys 
1               5                   10 

 
           
             31  
             12  
             PRT  
             H. influenzae  
           
            31 

Tyr Glu Thr Gly Val Thr Val Val Glu Ala Gly Arg 
1               5                   10 

 
           
             32  
             11  
             PRT  
             H. influenzae  
           
            32 

Asn Pro Glu Asp Thr Tyr Asp Ile Tyr Ala Lys 
1               5                   10 

 
           
             33  
             11  
             PRT  
             H. influenzae  
           
            33 

Phe Thr Leu Ala Ala Asp Leu Tyr Glu His Arg 
1               5                   10 

 
           
             34  
             13  
             PRT  
             H. influenzae  
           
            34 

Glu Leu Phe Glu Gly Tyr Gly Asn Phe Asn Asn Thr Arg 
1               5                   10 

 
           
             35  
             14  
             PRT  
             H. influenzae  
           
            35 

Thr Met Val Tyr Gly Leu Gly Tyr Asp His Pro Ser Gln Lys 
1               5                   10 

 
           
             36  
             16  
             PRT  
             H. influenzae  
           
            36 

Val Glu His Asn Leu Gln Tyr Gly Ser Ser Tyr Asn Thr Thr Met Lys 
1               5                   10                  15 

 
           
             37  
             17  
             PRT  
             H. influenzae  
           
            37 

Gly Tyr Ala Thr Glu Asn Asn Gln Ser Phe Asn Thr Leu Thr Ala Gly 
1               5                   10                  15 

Arg 

 
           
             38  
             19  
             PRT  
             H. influenzae  
           
            38 

Lys Gly Tyr Ala Thr Glu Asn Asn Gln Ser Phe Asn Thr Leu Thr Leu 
1               5                   10                  15 

Ala Gly Arg 

 
           
             39  
             901  
             DNA  
             H. influenzae  
             
               misc_feature  
               (1)..(2)  
               n = a, c, g, or t  
             
           
            39 

nntttcatgn cactatccca ctatgtgncc tgcagaacaa tcttatagga ccccttcata     60 

gtttttatgg gaattaaaat gaccgatttt agattaaaca aacatcccta ttccgttatg    120 

cttgggctaa cggcaggtgt tgcttatgca gctcaaccaa ccaaccaacc aaccaaccaa    180 

ccaaccaacc aaccaaccaa ccaaaatggt aatgtttctg aacaactaga gcaaattaat    240 

gtatctggtt ctaccgaaga tagtgataca aaaacaccac caaaaattgc tgaaacggta    300 

aaaacggcca aaacgccccc cccagaacaa gcaaacaata ttaaagacat cgccaaatac    360 

catacgggtg ttattgtccc tgaagctggg ctttttcgtc caaccgctcc cccattcgtg    420 

ttgtccataa caccccccca tttattacta ccgcccgctt acgttcacat ctttcctttt    480 

cttcgccgcg ctttcatcat ttttctccgg catttttaca taagtagtcc cttcccgctt    540 

ccctcctctc ctcttcctcc ttatttttat tatgatgttt ataagaatct cctctcttac    600 

ctattccagc ctcgttgttc tactcgcctt ctgctaaccc tttctccctt ttccatcctc    660 

tctaccccgc cccccctttc tctttttttt ccccctttct tttttccccc cacccctcac    720 

ttttccccgc tttatttttt acacaccccc cgacacaaca ttcatctccc tttgtatccg    780 

ctcatctttc cccccccccc cccaccatcc tccgcactct atctttccat tctatacccc    840 

cccttccctt ttcccccccc cccccctttc cgactgcaat tttttttctt ctccccctcc    900 

g                                                                    901 

 
           
             40  
             2979  
             DNA  
             H. influenzae  
           
            40 

atgggaatta aaatgaccga ttttagatta aacaaacatc cctattccgt tatgcttggg     60 

ctaacggcag gtgttgctta tgcagctcaa ccaaccaacc aaccaaccaa ccaaccaacc    120 

aaccaaccaa ccaaccaaaa tggtaatgtt tctgaacaac tagagcaaat taatgtatct    180 

ggttctaccg aaaatagtga tacaaaaaca ccaccaaaaa ttgctgaaac ggtaaaaacg    240 

gctaaaacgc tggaaagaga acaagcaaac aatattaaag acatcgttaa atacgagacg    300 

ggcgttactg ttgttgaagc tgggcgtttt gggcaaagcg gttttgccat tcgtggtgta    360 

gatgaaaacc gtgtagcgat taatattgat ggattacgtc aagctgaaac cctatcttct    420 

caaggcttta aagagctttt tgagggttat ggtaacttca ataatacgcg taatggtgca    480 

gaaattgaaa ctttaaaaga agtaaatatt acaaaagggg caaattcaat caaaagtggt    540 

agtggttcct taggtggatc tgtaatttat aaaacaaaag atgcgagaga ttatctcctt    600 

aacaaggatt actatgtaag ctacaaaaag ggatacgcta cagaaaataa tcaatcattc    660 

aataccctta ctcttgcagg acgttataaa aagtttgatg ccttagtggt tacaacaagc    720 

agaaatggac acgaacttga aaactatgat tacaaaaatg caaatagcct tactcaaggt    780 

aaaaaaagag aaaaagcaga cccatacaaa attgaacaag atagtacatt attaaaatta    840 

tcttttaacc ctacagaaaa tcatcgtttt acccttgcag cagatttata tgaacatcgt    900 

tctcgtgggc aagatttatc ctatacacta aaatatcaaa aaacagatcc taatttactc    960 

gaggttgatt ctagacacac caatgataaa acaaagagac gtaatatttc ctttagctat   1020 

gaaaatttct ctcaaacgcc attttgggat acattaaaaa tcacttattc agatcaacgt   1080 

attaaaactc gcgcacgtac agatgactat tgtgatgcag gtgtaagata ttgtgaggga   1140 

actgcaaatc ctgcgggact aaaattaaca gatgggaaaa taacacgtcg agatggttca   1200 

gaacttcaat ttgaaaaaaa agataaaaat attgataaca acatctatga cttcgataaa   1260 

tttattgata ctgatgatcg agtaatagaa ggtaaactag gcctaaggag atcctctgga   1320 

acttggtatg attgttcaat atttgattgt aaagataaaa caaaaatgaa aatttttgaa   1380 

actgaacatc cttatggcta tggcactact ggaacgtgga aaaaagactt tgaacttgaa   1440 

ataaaaaaat taaatgacaa aaatttcgct agagtaaagg atgcgaataa taaaacatac   1500 

tctattcttc cctcatctcc aggttattta gaacgcctct ggcaagagcg agatttagac   1560 

accaacaccc aacaattaaa tttagattta accaaagact tcaaaacttg gcgtgttgaa   1620 

cataatctac aatatggtag ctcatataat acaacgatga agcgaatggt taatcgtgct   1680 

ggtaatgatg cttctgatgt gcaatggtgg gcagagccta cacttggtta cagtttgttg   1740 

tatgataaac ctcacacttg taaaaccgct tatggggggt ggaaagctaa cctttgccct   1800 

cgagttgatc ctaaattttc attcttatta cccattaaaa caaaagaaaa atcagtctat   1860 

ctctttgata atgttgttat aactgattat ttatcttttg atttgggtta tcgttatgac   1920 

aatatccatt atcaaccaaa atataaacac ggcgttacac cgaaattacc tgatgatatt   1980 

gtgaaaggat tgtttattcc attaccaagt ggtaaaaata ataatgatga tcctgaagtt   2040 

aagaaaaacg tacaacaaaa tattgactat atcgctaaac aaaacaaaaa atataaagca   2100 

cattcttaca gttttgtttc aacgattgat ccaacgagtt ttcttcgttt acaactaaaa   2160 

tattcaaaag gttttagagc accaacttca gatgaaatgt atttcacctt taaacaccct   2220 

gatttcacta ttttgccaaa tactaatcta aaaccagaga tagcaaaaac aaaagaaatt   2280 

gcttttacat tacataatga tgattggggt tttatctcca caagtctatt taaaaccaac   2340 

tataaaaact ttatcgacct aatatttaaa ggagaaaaag attttaaatt agttagcgga   2400 

ggtagcacat taccattttc tctttatcaa aatattaata gagatagtgc ggtagtaaaa   2460 

ggaatagaaa ttaattcaaa agtattcctt ggtaaaatgg caaaatttat ggatggattt   2520 

aacctaagct ataaatatac ctatcaaaaa ggaagaatgg atggcaatat tcctatgaat   2580 

gcaattcagc ctaaaacgat ggtgtatggc ttaggctatg accacccaag ccaaaaattt   2640 

ggatttaatt tctacactac ccacgtagca agtaaaaatc cagaagatac ttatgatatt   2700 

tatgcgaaag ataaaaacca aaccaacact agcataaaat ggcgcagtaa atcttatact   2760 

attctagatt taattggata tgtgcaacca attaaaaatt taaccataag agccggcgta   2820 

tataatctta caaaccgtaa atacatcact tgggattctg cgcgttcaat tcgttcattt   2880 

ggtacaagta atgttataga tcaaaagaca ggtcaaggca ttaaccgctt ctacgcacca   2940 

ggtagaaatt ataagatgtc agttcaattt gagttttaa                          2979 

 
           
             41  
             992  
             PRT  
             H. influenzae  
           
            41 

Met Gly Ile Lys Met Thr Asp Phe Arg Leu Asn Lys His Pro Tyr Ser 
1               5                   10                  15 

Val Met Leu Gly Leu Thr Ala Gly Val Ala Tyr Ala Ala Gln Pro Thr 
            20                  25                  30 

Asn Gln Pro Thr Asn Gln Pro Thr Asn Gln Pro Thr Asn Gln Asn Gly 
        35                  40                  45 

Asn Val Ser Glu Gln Leu Glu Gln Ile Asn Val Ser Gly Ser Thr Glu 
    50                  55                  60 

Asn Ser Asp Thr Lys Thr Pro Pro Lys Ile Ala Glu Thr Val Lys Thr 
65                  70                  75                  80 

Ala Lys Thr Leu Glu Arg Glu Gln Ala Asn Asn Ile Lys Asp Ile Val 
                85                  90                  95 

Lys Tyr Glu Thr Gly Val Thr Val Val Glu Ala Gly Arg Phe Gly Gln 
            100                 105                 110 

Ser Gly Phe Ala Ile Arg Gly Val Asp Glu Asn Arg Val Ala Ile Asn 
        115                 120                 125 

Ile Asp Gly Leu Arg Gln Ala Glu Thr Leu Ser Ser Gln Gly Phe Lys 
    130                 135                 140 

Glu Leu Phe Glu Gly Tyr Gly Asn Phe Asn Asn Thr Arg Asn Gly Ala 
145                 150                 155                 160 

Glu Ile Glu Thr Leu Lys Glu Val Asn Ile Thr Lys Gly Ala Asn Ser 
                165                 170                 175 

Ile Lys Ser Gly Ser Gly Ser Leu Gly Gly Ser Val Ile Tyr Lys Thr 
            180                 185                 190 

Lys Asp Ala Arg Asp Tyr Leu Leu Asn Lys Asp Tyr Tyr Val Ser Tyr 
        195                 200                 205 

Lys Lys Gly Tyr Ala Thr Glu Asn Asn Gln Ser Phe Asn Thr Leu Thr 
    210                 215                 220 

Leu Ala Gly Arg Tyr Lys Lys Phe Asp Ala Leu Val Val Thr Thr Ser 
225                 230                 235                 240 

Arg Asn Gly His Glu Leu Glu Asn Tyr Asp Tyr Lys Asn Ala Asn Ser 
                245                 250                 255 

Leu Thr Gln Gly Lys Lys Arg Glu Lys Ala Asp Pro Tyr Lys Ile Glu 
            260                 265                 270 

Gln Asp Ser Thr Leu Leu Lys Leu Ser Phe Asn Pro Thr Glu Asn His 
        275                 280                 285 

Arg Phe Thr Leu Ala Ala Asp Leu Tyr Glu His Arg Ser Arg Gly Gln 
    290                 295                 300 

Asp Leu Ser Tyr Thr Leu Lys Tyr Gln Lys Thr Asp Pro Asn Leu Leu 
305                 310                 315                 320 

Glu Val Asp Ser Arg His Thr Asn Asp Lys Thr Lys Arg Arg Asn Ile 
                325                 330                 335 

Ser Phe Ser Tyr Glu Asn Phe Ser Gln Thr Pro Phe Trp Asp Thr Leu 
            340                 345                 350 

Lys Ile Thr Tyr Ser Asp Gln Arg Ile Lys Thr Arg Ala Arg Thr Asp 
        355                 360                 365 

Asp Tyr Cys Asp Ala Gly Val Arg Tyr Cys Glu Gly Thr Ala Asn Pro 
    370                 375                 380 

Ala Gly Leu Lys Leu Thr Asp Gly Lys Ile Thr Arg Arg Asp Gly Ser 
385                 390                 395                 400 

Glu Leu Gln Phe Glu Lys Lys Asp Lys Asn Ile Asp Asn Asn Ile Tyr 
                405                 410                 415 

Asp Phe Asp Lys Phe Ile Asp Thr Asp Asp Arg Val Ile Glu Gly Lys 
            420                 425                 430 

Leu Gly Leu Arg Arg Ser Ser Gly Thr Trp Tyr Asp Cys Ser Ile Phe 
        435                 440                 445 

Asp Cys Lys Asp Lys Thr Lys Met Lys Ile Phe Glu Thr Glu His Pro 
    450                 455                 460 

Tyr Gly Tyr Gly Thr Thr Gly Thr Trp Lys Lys Asp Phe Glu Leu Glu 
465                 470                 475                 480 

Ile Lys Lys Leu Asn Asp Lys Asn Phe Ala Arg Val Lys Asp Ala Asn 
                485                 490                 495 

Asn Lys Thr Tyr Ser Ile Leu Pro Ser Ser Pro Gly Tyr Leu Glu Arg 
            500                 505                 510 

Leu Trp Gln Glu Arg Asp Leu Asp Thr Asn Thr Gln Gln Leu Asn Leu 
        515                 520                 525 

Asp Leu Thr Lys Asp Phe Lys Thr Trp Arg Val Glu His Asn Leu Gln 
    530                 535                 540 

Tyr Gly Ser Ser Tyr Asn Thr Thr Met Lys Arg Met Val Asn Arg Ala 
545                 550                 555                 560 

Gly Asn Asp Ala Ser Asp Val Gln Trp Trp Ala Glu Pro Thr Leu Gly 
                565                 570                 575 

Tyr Ser Leu Leu Tyr Asp Lys Pro His Thr Cys Lys Thr Ala Tyr Gly 
            580                 585                 590 

Gly Trp Lys Ala Asn Leu Cys Pro Arg Val Asp Pro Lys Phe Ser Phe 
        595                 600                 605 

Leu Leu Pro Ile Lys Thr Lys Glu Lys Ser Val Tyr Leu Phe Asp Asn 
    610                 615                 620 

Val Val Ile Thr Asp Tyr Leu Ser Phe Asp Leu Gly Tyr Arg Tyr Asp 
625                 630                 635                 640 

Asn Ile His Tyr Gln Pro Lys Tyr Lys His Gly Val Thr Pro Lys Leu 
                645                 650                 655 

Pro Asp Asp Ile Val Lys Gly Leu Phe Ile Pro Leu Pro Ser Gly Lys 
            660                 665                 670 

Asn Asn Asn Asp Asp Pro Glu Val Lys Lys Asn Val Gln Gln Asn Ile 
        675                 680                 685 

Asp Tyr Ile Ala Lys Gln Asn Lys Lys Tyr Lys Ala His Ser Tyr Ser 
    690                 695                 700 

Phe Val Ser Thr Ile Asp Pro Thr Ser Phe Leu Arg Leu Gln Leu Lys 
705                 710                 715                 720 

Tyr Ser Lys Gly Phe Arg Ala Pro Thr Ser Asp Glu Met Tyr Phe Thr 
                725                 730                 735 

Phe Lys His Pro Asp Phe Thr Ile Leu Pro Asn Thr Asn Leu Lys Pro 
            740                 745                 750 

Glu Ile Ala Lys Thr Lys Glu Ile Ala Phe Thr Leu His Asn Asp Asp 
        755                 760                 765 

Trp Gly Phe Ile Ser Thr Ser Leu Phe Lys Thr Asn Tyr Lys Asn Phe 
    770                 775                 780 

Ile Asp Leu Ile Phe Lys Gly Glu Lys Asp Phe Lys Leu Val Ser Gly 
785                 790                 795                 800 

Gly Ser Thr Leu Pro Phe Ser Leu Tyr Gln Asn Ile Asn Arg Asp Ser 
                805                 810                 815 

Ala Val Val Lys Gly Ile Glu Ile Asn Ser Lys Val Phe Leu Gly Lys 
            820                 825                 830 

Met Ala Lys Phe Met Asp Gly Phe Asn Leu Ser Tyr Lys Tyr Thr Tyr 
        835                 840                 845 

Gln Lys Gly Arg Met Asp Gly Asn Ile Pro Met Asn Ala Ile Gln Pro 
    850                 855                 860 

Lys Thr Met Val Tyr Gly Leu Gly Tyr Asp His Pro Ser Gln Lys Phe 
865                 870                 875                 880 

Gly Phe Asn Phe Tyr Thr Thr His Val Ala Ser Lys Asn Pro Glu Asp 
                885                 890                 895 

Thr Tyr Asp Ile Tyr Ala Lys Asp Lys Asn Gln Thr Asn Thr Ser Ile 
            900                 905                 910 

Lys Trp Arg Ser Lys Ser Tyr Thr Ile Leu Asp Leu Ile Gly Tyr Val 
        915                 920                 925 

Gln Pro Ile Lys Asn Leu Thr Ile Arg Ala Gly Val Tyr Asn Leu Thr 
    930                 935                 940 

Asn Arg Lys Tyr Ile Thr Trp Asp Ser Ala Arg Ser Ile Arg Ser Phe 
945                 950                 955                 960 

Gly Thr Ser Asn Val Ile Asp Gln Lys Thr Gly Gln Gly Ile Asn Arg 
                965                 970                 975 

Phe Tyr Ala Pro Gly Arg Asn Tyr Lys Met Ser Val Gln Phe Glu Phe 
            980                 985                 990