MXPA01003571A - Protective recombinant haemophilus influenzae - Google Patents

Abstract

Protective high molecular weight (HMW) proteins are produced recombinantly by expression from E. coli by using a promoter effective in E. coli and a nucleic acid molecule which contains a modified operon of a non-typeable strain of Haemophilus. The modified operon contains the portion only of the A region which encodes the mature HMW protein and the complete B and C regions of the operon. Enhanced levels of expression of the HMW proteins can be achieved by including the E. coli cer gene, a further copy of the portion of the A region of the operon encoding the mature protein or both, in the expression vector. Nucleotide and deduced amino acid sequences of the hmw1 and hmw2 genes and HMW1 and HMW2 proteins, respectively, of several non-typeable Haemophilus influenzae strain have been identified.

Description

PROTEXEA8 RECOMBXMANTE8 PROTECTIVE HIGH MOLECULAR WEIGHT OF HAEMOPHILUS INFLUENZAE ?????????? m and g.iciT.ro BIACICMADA This application is a continuation in part of application 09 / 167,568, filed on October 7, 1998.

FIELD PE LA IMVEHCl6H The present invention relates to the field of molecular genetics and, in particular, to the production of high molecular weight recombinant proteins of Haemophilus influenzae and nucleic acid molecules and of the vectors used therein.

AMTBCBDKMTISS DB IMVmCldW Encapsulated strains of Haemophilus influenzae type b are the leading cause of bacterial meningitis as well as other invasive infections in young children. However, unencapsulated or non-typeable H. influenzae (NTHi) is responsible for a wide range of human diseases that include: otitis media, epiglottitis, pneumonia and tracheobronguitis. Vaccines based on capsular polysaccharide of * H. influenzae type b conjugated with diphtheria toxoid (reference 1). Throughout this application, the various references will be mentioned in parentheses to more fully describe the state of the art to which this invention pertains. The complete bibliographic information of each citation is found at the end of the specification, immediately after the claims. The findings of these references are incorporated in this manner as a reference in the present discovery), tetanus toxoid (reference 2 and U.S. Patent No. 4,496,538) or outer membrane protein of Neisseria meningititis (reference "^ 10 3) have been effective to reduce meningitis induced by H. influenzae type b, but not the disease induced by NTHi (reference 4). Otitis media is the most common disease of young children, where 60% to 70% of all 15 children under 2 years of age suffer between one and three otic infections (reference 5) Chronic otitis media is responsible for auditory, speech and cognitive impairment in children.Infections with JET.influenzae are the main cause Approximately 30% of the cases of acute otitis media and approximately 60% of those of chronic otitis media, only in the United States, treatment of otitis media costs between one thousand and two thousand million dollars a year, in regard to antibiotics and surgical procedures, such as 25 tonsilectomies, adenoidectomies, and the insertion of tympanostomy tubes. It is estimated that an additional $ 30 billion is spent annually on additional therapies, such as speech therapies and special education classes. In addition, many of the organisms that cause otitis are becoming resistant to antibiotic treatment. Thus, an effective prophylactic vaccine against otitis media is desirable. During the natural infection with NTHi, the exposed surface outer membrane proteins that stimulate an antibody response are potentially important targets for bactericidal and / or protective antibodies and, therefore, potential vaccine candidates. Barenkamp and Borod (reference 6) showed that the serum of convalescent children suffering otitis media due to antibodies containing NTHi with high molecular weight proteins (HMW) approximately 60 to 75% of the f- N NTHi strains express HMW proteins and most of these strains contain two groups of genes called hnnrlABC and hmw2ABC. The hmtrA genes code for the structural HMWA proteins and the hmwS and hnarC genes are accessory genes responsible for the processing and secretion of the HMWA proteins (references 7, 8, 9, U.S. Patent No. 5,603,938, WO 97/36914 ). It has been demonstrated that the HMWA proteins are adhesins that mediate binding with human epithelial cells (reference 10) and it seems that only the HMWA proteins processed in an appropriate manner are effective adhesins (reference 8). Immunization with a mixture of native HHW1A and HHW2A proteins resulted in protection against otitis media in the chinchilla intra-ampule inoculation model (reference 11).; WO 97/36914). The prototype of the hnnr1A gene of NTHi strain 12 codes for a 160 kDa HMWIA protein that is processed by cleaving a 35 kDa amino terminal fragment that generates the mature 125 kDa HMWIA protein. Similarly, the hitar 2A gene of NTHi strain 12 codes for a 155 kDa HMW2A protein that is processed by cleavage of a nearly identical 35 kDa amino terminal fragment to produce the mature 120 kDa HMW2A protein. Plasmid pHMWI-15 (reference 8) has a backbone of pT7-7 (reference 12) and contains the hmwlABC operon of NTHi strain 12 with 5 'and 3' flanking regions. There are approximately 400 bp of 5 * flanking sequences located between the T7 promoter and the start of the hnnr1A structural gene. Plasmid pHMW2-21 (reference 10) has a backbone of pT7-7 and contains the complete hmr2ABC operon with 5 'and 31 flanking sequences. There are approximately 800 bp of 5' flanking sequences located between the T7 promoter and P1269 the start of the structural gene Hmr2A. The rHMWlA and rHW2A proteins are produced with a relatively low yield ^ from the plasmids pHMWl-15 and pHMW2-21. The Hmtrl ABC or hmr2 ABC genes of H. influenzae 5 can be genetically engineered to produce the mature recombinant HMWIA or HMW2A proteins by deleting the sequence encoding the 35 kDa guide sequence, which is normally removed by processing in the H. Influenzae. Since this sequence If the gula has been deleted, the hmrlBC or hmr2BC genes that serve to process and secrete the mature HMWIA and HMW2A structural proteins of H. influenzae should not be needed (reference 9). The performance of the rHMWlA or rHMW2A protein can be significantly increased by the deletion of the leader sequence and the processing of the genes, however, the purified recombinant proteins are not protective. As set forth herein, the, -. genes hmrlBC and hmr2BC or their protein products apparently contribute to the protective capacity of proteins of rHMWlA and rHMW2A. This requirement is not expected for accessory genes that are rwise redundant. The E. coli gene is thought to stabilize the plasmids by preventing multimerization (reference 13). For expression vectors with large inserts, you can be used to the cer gene to stabilize the plasmids.

The present invention is directed to the delivery of high molecular weight recombinant proteins of H.

V non-typeable influences that are protective by providing certain nucleic acid molecules and vectors that contain them. It has now been found that in order to obtain high-molecular-weight recombinant proteins (HW) of non-typeable Haemophilus that are protective, it is V necessary to provide a vector containing only the segment of the A portion of the operon encoding the mature HMW protein, i.e., lacking the segment of the A gene coding for the glula sequence and the B and C portions of the operon. It has also been found that the expression level of the mature protein can be improved by including in the vector at least one additional segment coding for the mature protein, the cer gene of E. coll, or b In accordance with the above, in one aspect of the present invention, there is provided a nucleic acid molecule comprising a functional promoter in E. coll and which is operatively coupled to a modified operon of a non-typeable strain of Haemophilus comprising the genes A, B and C, wherein the A gene of the operon contains only a nucleic acid sequence that codes for a mature, high molecular weight protein of the tipificable strain of Haemophllus y, dé aguí, from which the portion of gene A that codes for the leader sequence. To effect the expression of the mature HMW protein in E. coli, any suitable promoter can be used. However, it is preferred to use the T7 promoter. The high molecular weight mature protein encoded may be the HMW1 or HMW2 protein of the non-typeable Haemophilus strain. The nontypable Haemophillus strain can be selected from the group consisting of strains 12, Joyc, K21, LCDC2, PNH1 and 15 of non-typeable Haemophilus influenzae. The present invention also provides the nucleotide sequences of the hmwlA and / or hmw2A genes of certain non-typeable strains of Haemophilus influenzae, which have not previously been isolated, purified or expressed, together with the deduced amino acid sequences of the corresponding HMW1 proteins. and HMW2 of the non-typeable Haemophilus strains. In accordance with the foregoing, in anr aspect of the invention there is provided an isolated and purified nucleic acid molecule which codes for a high molecular weight protein (HMW) of a non-typeable strain of Haemophilus Influences, which has: (a) a DNA sequence selected from the group consisting of those shown in Figures 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 (SEQ ID NOS: 25, 27, 29, 32, 33 , 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64) or a complementary sequence thereof; or (b) a DNA sequence encoding a high molecular weight protein having an amino acid sequence selected from the group consisting of those shown in Figures 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 (SEQ ID NOS: 26, 28, 30, 32, 34, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65), or a complementary sequence thereof. The modified operon in the first aspect of the invention may include the sequences coding for the mature protein (SEQ ID NOS: 27, 31, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72) or a DNA molecule that codes for the mature protein having the amino acid sequences (SEQ ID NOS: 28, 32, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73) shown in Figures 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29. The nucleic acid molecule provided according to the first aspect of the invention can additionally comprise a sequence containing at least one copy additional of the coding region for the mature one only of the operon of a non-typeable strain of Haemophllus, the cer gene of E. coli or these two segments. The nucleic acid molecules provided in accordance with the first aspect of the invention can be incorporated into a vector, typically a plasmid vector, for the transformation of E. coli in order to express the mature high molecular protective protein of a strain non-typeable of Haemop ílua. Plasmid vectors for the latter purpose can have the identification characteristics of a plasmid that is selected from the group consisting of: DS-1046-1-1 JB-2507-7 BK-86-1-1 BK-35-4 BK-76-1-1 DS-2334-5 DS-2400-13 The details of the structures and preparation of these plasmids are given in the figures and in the examples. The present invention extends, in a further aspect thereof, to a strain of E. coli transformed by the vectors provided herein and expressing a high molecular weight protective protein of a non-typeable strain of Haemophilus. The present invention further includes a recombinant protein of high molecular weight isolated and purified from a non-typeable strain of Haemophilus, an immunogenic segment or an analogue thereof, which can be produced by the transformed E. coli. The present invention further includes, in a further aspect thereof, a recombinant method for the production of a high molecular weight protective protein of a non-typeable strain of Haemophilus, comprising: transforming E. coli with a vector comprising the nucleic acid molecule provided in the first aspect of the invention; develop E. coli to express the mature protein of high molecular weight (HMW) encoded and isolate and purify the HMW protein expressed. The non-typeable Haemophilus strain can be any of the above-mentioned strains and the high molecular weight protein can be the HMW1 protein or the HMW2 protein, which is provided in a free form from the contamination of the other protein. The purification steps may include separating the HMW A protein from protein B and C.

The present invention, in a further aspect thereof, provides an isolated and purified HMW 1 protective protein from a non-typable Haemophilus strain, which is free from contamination with the HMW2 protein of the same non-typeable Haemophilus strain. In still another aspect, the present invention provides an isolated and purified protective HMW2 protein from a non-typable Haemophilus strain, which is free from contamination with the HMW1 protein of the same non-typable Haemophilus strain. The HMW1 or HMW2 protein may come from any of the non-typeable strains of Haemophilus mentioned above and may be one that has SEQ 10 NO: 28, 32, 37, 41, 45, 49, 53, 57, 61, 65, 69673 In accordance with another aspect of the invention, there is provided an immunogenic composition comprising at least one immunologically active component, selected from the group consisting of at least one nucleic acid molecule as provided herein, at least one recombinant protein of the invention. HMW as provided herein, or at least one novel HMW protein as provided herein and a pharmaceutically acceptable carrier therefor. The at least one active component produces an immune response when administered to a host. The immunogenic compositions provided herein can be formulated as vaccines for in vivo administration to a host to provide protection against the disease caused by H. influenzae. For such a purpose, the compositions can be formulated as a microparticle, ISCOM or liposome preparation. The immunogenic composition can be provided in combination with a target or target molecule for distribution to specific cells of the immune system or mucosal surfaces. The immunogenic compositions of the invention (including vaccines) may further comprise at least one other immunogenic or immunostimulatory material and the immunostimulatory material may be at least one adjuvant or at least one cytosine. Adjuvants suitable for use in the present invention include (but are not limited to): aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide , zinc hydroxide, a glycolipid analog, an octadecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOP EP, DC-chol, DDBA and a lipoprotein and other adjuvants. Advantageous combinations of adjuvants are described in copending US Patent Applications No. 08 / 261,194 filed June 16, 1994 and 08 / 483,856 filed June 7, 1995, assigned to the assignee hereof, and whose descriptions are incorporated herein by reference (O 95/34308, published November 21, 1995). According to still another aspect of the invention, there is provided a method for the generation of an immune response in a host, comprising the step of administering to an susceptible host, such as a human, an effective amount of the immunogenic composition as mentioned earlier. The immune response may be a humoral or cell-mediated immune response and may provide protection against the disease caused by Haemophllus. Hosts that can be protected against disease include primates, including humans. It has been found that the nucleic acid sequences of portions B and C of the operon encoding the HMW1 and HMW2 proteins are highly conserved in the nucleic acid sequence between non-typeable Haemophilus species, which enables them to be supplied in a universal plasmid vector for the reception of the nucleic acid sequence coding for the mature HMWIA or HHW2A protein from a variety of non-typeable Haemop ilus strains for the purpose of expression of the HMWIA or HMW2A from a transformed host, such as E. coli. In accordance with the above, in yet another aspect of the invention, a plasmid vector is provided for the expression of a high molecular weight protein of a non-typeable strain of Haemophilus and comprising the T7f promoter a cloning site and portions thereof. B and C of the hnnr operon of a non-typeable Haemophilus strain. The plasmid can also contain the cer gene of E. coli. The plasmid vector can be plasmid JB-2646-1. The present invention, in its various aspects, allows the production of high-molecular-weight protective proteins of non-typeable Haemophilus that are useful to provide immunogenic compositions that confer protection against diseases caused by infection with non-typeable Haemophilus strains.

BBBYB PfffffiRTFCrtff PB K > B PlffWQS The present invention will be further understood from the following description with reference to the drawings, in which: Figure 1A shows the construction scheme for generating the plasmid DS-1091-2 which expresses the genes P1269 hmwlABC coding for the HMWIA protein of 160 kOa full length. The enzyme restriction sites are: B, BamH I; Bg, Bgl II; Bsy Bsm I; Hf Hind III; R, EcoR I; Xb, Xba I. Other abbreviations are: T7p, T7 promoter; HMWlp, promoter hmwlj ApRf ampicillin resistance gene; CAP, calf alkaline phosphatase. Figure IB shows the sequence of the nucleotides used in the construction scheme of Figure 1A (SEQ ID NOS: 1, 2 and 3). Figure 2 shows the construction scheme for generating the plasmid DS-1094-2 which expresses the hmw2ABC genes coding for the full-length 155 kDa HMW2A protein. The restriction enzyme sites are: Bg, Bgl II; Bs, Bsm I; H, Hind III, R, EcoR I; Xb, Xba I. Other abbreviations are: T7p, T7 promoter; HM 2p, hmw2 promoter; ApR, ampicillin resistance gene; CAP, calf alkaline phosphatase. Figure 3A shows the construction scheme for generating the plasmid DS-1046-1-1 expressing the hmwlABC genes coding for the mature HMWIA 125 kOa protein. The enzyme restriction sites are: B, BamH I; Bg, Bgl II; H, Hind III; R, EcoR I; Xb, Xba I. Other abbreviations are: T7p, T7 promoter; HMWlp, promoter hmwl; ApR, ampicillin resistance gene. Figure 3B shows the sequence of the P1269 ollgonucleotides used in the construction scheme of Figure 3A (SEQ ID NOS: 4, 5 and 6). Figure 4A shows the construction scheme for generating the plasmid DS-1200-3 expressing the hmw2AB genes encoding the mature 120 kDa HMW2A protein. The enzyme restriction sites are: Bg, Bgl II; H, Hind III; R, EcoR I; S, salt I; Xb, Xba I; Xho, Xho I. Other abbreviations are: T7p, T7 promoter; HMW2p, lumr2j ApR promoter, ampicillin resistance gene; CAP, calf alkaline phosphatase. Figure 4B shows the sequence of ollgonucleotides used in the construction scheme of Figure 4A (SEQ ID NOS: 7, 8 and 9). Figure 5 shows the construction scheme for generating the plasmid DS-1122-2 which contains the hmtrlA gene coding for the mature 125 kDa HMW1A protein and part of the hmrlB gene. The enzyme restriction sites are: B, BamH I; Bg, Bgl II; H, Hind III; R, EcoR I; Xb, Xba I. Other abbreviations are: T7p, T7 promoter; ApR, ampicillin resistance gene. Figure 6A shows the construction scheme for generating plasmid JB-2330-7 expressing the hmtrlA gene coding for the mature 125 kDa HMW1A protein. The enzyme restriction sites are: B, BamH I; Bgl Bgl II; H, Hind III; K, Kpn I; R, EcoR I; Xho, Xho I. Other P1269 abbreviations are: T7p, T7 promoter; Ap, ampicillin resistance gene; KanR, kanamycin resistance gene, CAP. calf alkaline phosphatase. Figure 6B shows the oligonucleotides used to PCR amplify the 3 'end of the hmwlA in the construction scheme of Figure 6A (SEQ ID NOS: 10, 11, 12, 13 and 14). Figure 7 shows the construction scheme for generating the plasmid JB-2369-6 which expresses tr series copies of the T7 hmwlA gene cassette encoding the mature 125 kDa HMW1A protein. The restriction enzyme sites are: B, BamH I; Bgl Bgl II; Hr Hind 111; K, Kpn I; Rf EcoR I; S, Sal I; Xbf Xba I; Xho, xho I. Other abbreviations are: T7p, T7 promoter; ApR, 15 ampicillin resistance gene; TcR, tetracycline resistance gene; CAP, calf alkaline phosphatase; ttl, transcription terminator 1; tt2, transcription terminator 2; MCS, ^ multiple cloning site. Figure 8A shows the construction scheme to generate the plasmids DS-2084-3 and DS-2084-1 containing one or two copies, respectively, of the cassette of the T7 gene hmw2A coding for the mature protein of HMW2A of 120 kDa. The restriction enzyme sites are: B, BamH i; Bg, Bgl II; H, Hind 1 III; M, Mlu I; R, EcoR I; 25 Xb, Xba I. Other abbreviations are: T7p, T7 promoter; ApR, P1269 ampicillin resistance gene; TcR, tetracycline resistance gene; CAP, calf alkaline phosphatase; ttl, transcription terminator 1; tt2f transcription terminator 2; MCSf multiple cloning site. Figure 8B shows the oligonucleotides used to PCR-amplify the 3 * end of hnar2A in the construction scheme of Figure 8A (SEQ ID NOS: 15, 16, 17, 18 and 19). Figure 9 shows the construction scheme for generating the plasmids JB-2507-7 and BK-86-1-1 containing the T7 hnnrlA / T7 hnarlABC series genes coding for the mature 125 kDa HMWIA protein, with selection by ampicillin or kanamycin, respectively. The restriction enzyme sites are: B, BamH I; Bg, Bgl II; H, ffind III; K, Kpn I; R, EcoR I; S, Sal I; Xb, Xba I; Xho, Xho I. Other abbreviations are: T7p, T7 promoter; ApR, ampicillin resistance gene; KanR, kanamycin resistance gene; TcR, tetracycline resistance gene; CAP, calf alkaline phosphatase. Figure 10 shows the construction scheme for generating plasmids BK-35-4 and BK-76-1-1 containing the G7 hnarlABC / cer series genes coding for the mature 125 kDa HMWIA protein, using the selection by ampicillin or kanamycin, respectively. The restriction enzyme sites are: B, BamH I; Bg, Bgl P1269 II; ?, Hínd III; ?, ??? I; R, EcoR I; S, Sal I; Xb, Xba I. Other abbreviations are: T7pf T7 promoter; ApR, ampicillin resistance gene; anRf kanamycin resistance gene; CAP, calf alkaline phosphatase. Figure 11 shows the SDS-PAGE analysis of the expression of recombinant HMW1 proteins from various constructs. Band 1, indicators with a wide range of molecular weight; band 2, DS-1046-1-1 [p7 hmrlABC (125)], without induction; lane 3, DS-1091-2 [p 7 hmrlABC (160)]; lane 4, DS-1046-1-1 [pT7 hmrlABC (125)]; band 5, DS-1122-2 [p 7 hmrlA partial B (125)], band 6, JB-2330-7 [p 7 hmwlA (125)]; band 7, JB-2369-6 [pBr328 T7 hmrlA (125) / T7 hmrlA (125)]; band 8, BK-86-1-1 [pT7 hnnrlA (125) / T7 hmrlABC (125) / kanR] band 9, BK-76-1-1 [pT7 hnnrlABC (125) / cer / kanR]; band 10, indicators with wide molecular weight range. Figure 12 shows a purification scheme for recombinant proteins of HMW1 and HNW2. The abbreviations are PPT, pellet; SUP, supernatant; OG, octylglucoside; PEG, polyethylene glycol. Figure 13 shows the SDS-PAGE analysis of rHMWl extractions. Band 1, pre-stained protein molecular weight indicators; band 2, whole cell lysates of E. coli, band 3, proteins soluble in the extraction with Tris-HCl / NaCl; band 4, proteins P1269 soluble in the extraction with Tris-HCl / Triton? -100 / EDTA; 5r band soluble proteins in the extraction of Tris-HCl / octylglucosido; band 6, pellets after extraction of Tris-HCl / octylglucoside; band 7, guanidine insoluble proteins HC12M; band 8, supernatant of precipitation with 7% PE6; band 9, pellet of precipitation with 7% PEG; band 10, interphase pellet of precipitation with 50% ammonium sulfate; band 11, proteins recovered in the lower phase; band 12, proteins recovered in the upper phase. Figure 14, comprising panels A and B, shows an SDS-PAGE analysis of purified rHMW1 and rHMW2. Figure 15 contains an SDS-PAGE analysis showing the stability of rHMW1 from the construction of T7 hnnrABC / cer / kanR stored at -20 ° C in the presence of 20% glycerol. Figure 16, comprising panels A and B, shows the immunogenicity of the rHMWlA protein produced from various constructions. Figure 17 shows the sequences of oligonucleotides used to PCR amplify additional »ha genes from chromosomal DNA of H. Influences not typable (SEQ ID NOS: 20, 21, 22, 23 and 24).

P1269 Figure 18 shows the nucleotide sequence (SEQ ID NO: 25) and the deduced amino acid sequence (SEQ ID NO: 26) of the hawlA gene of the NTHi Joyc strain. The arrow marks the predicted start of the mature protein (mature protein: coding for the sequence SEQ ID NO: 27; amino acid sequence SEQ ID NO: 28). Figure 19 shows the nucleotide sequence (SEQ ID NO: 29) and the deduced amino acid sequence (SEQ ID NO: 30) of the hnnr2A gene of the NTHi Joyc strain. The arrow marks the predicted start of the mature protein (mature protein: coding for the sequence SEQ ID NO: 31, amino acid sequence SEQ ID NO: 32). Figure 20 shows the nucleotide sequence (SEQ ID NO: 33) and the deduced amino acid sequence (SEQ ID NO: 34, 35) of the defective hnnRlA gene of the strain NTHi Kl. The arrow marks the predicted start of the mature protein (mature protein: coding for the sequence SEQ ID NO: 36, amino acid sequence SEQ ID NO: 37, 35). Figure 21 shows the nucleotide sequence (SEQ ID NO: 38) and the deduced amino acid sequence (SEQ ID NO: 39) of the hmtr2A gene of strain NTHi K21. The arrow marks the predicted start of the mature protein (mature protein: coding for the sequence SEQ ID NO: 40, amino acid sequence SEQ ID NO: 41). Figure 22 shows the nucleotide sequence P1269 (SEQ ID NO: 42) and the deduced amino acid sequence (SEQ ID NO: 43) of the h wlA gene of the NTHi strain LCDC2. The arrow marks the predicted start of the mature protein (mature protein: coding for the sequence SEQ ID NO: 44, amino acid sequence SEQ ID NO: 45). Figure 23 shows the nucleotide sequence (SEQ ID NO: 46) and the deduced amino acid sequence (SEQ ID NO: 47) of the hnm2A gene of the NTHi strain LCDC2. The arrow marks the predicted start of the mature protein (mature protein: coding for the sequence SEQ ID NO: 48, amino acid sequence SEQ ID NO: 49). Figure 24 shows the nucleotide sequence (SEQ ID NO: 50) and the deduced amino acid sequence (SEQ ID NO: 51) of the hmtrlA gene of the NTHi PMH1 strain. The arrow marks the predicted start of the mature protein (mature protein: coding for the sequence SEQ ID NO: 52, amino acid sequence SEQ ID NO: 53). Figure 25 shows the nucleotide sequence (SEQ ID NO: 54) and the deduced amino acid sequence (SEQ ID NO: 55) of the hnar2A gene of the NTHi PMH1 strain. The arrow marks the predicted start of the mature protein (mature protein: coding for the sequence SEQ ID NO: 56, amino acid sequence SEQ ID NO: 57). Figure 26 shows the nucleotide sequence (SEQ ID NO: 58) and the deduced amino acid sequence (SEQ.

P1269 ID NO: 59) of the iwrlA gene of strain NTHi 15. The arrow marks the predicted start of the mature protein (mature protein: coding for the sequence SEQ ID NO: 60, amino acid sequence SEQ ID NO: 61). Figure 27 shows the nucleotide sequence (SEQ ID NO: 62) and the deduced amino acid sequence (SEQ ID NO: 63) of the hmw2A gene of the NTHi 15 strain. The arrow marks the predicted start of the mature protein (protein mature: coding for the sequence SEQ ID NO: 64; amino acid sequence SEQ ID NO: 65). Figure 28 shows the nucleotide sequence (SEQ ID NO: 66) and the deduced amino acid sequence (SEQ ID NO: 67) of the hmlA gene of strain NTHi 12. The arrow marks the predicted start of the mature protein (mature protein : coding for the sequence SEQ ID NO: 68, amino acid sequence SEQ ID NO: 69). Figure 29 shows the nucleotide sequence (SEQ ID NO: 70) and the deduced amino acid sequence (SEQ ID NO: 71) of the hmw2A gene of strain NTHi 12. The arrow marks the predicted start of the mature protein (mature protein : coding for the sequence SEQ ID NO: 72, amino acid sequence SEQ ID NO: 73). Figure 30 shows the alignment of the deduced HMW1A and HMW2A protein sequences (SEQ ID NOS: 26, 30, 34, 35, 39, 43, 47, 51, 55, 59, 63) with the P1269 published sequences of the HMHIA and HMW2A protein of strain 12 (U.S. Patent No. 5,603,938) (SEQ ID NOS: 67, 71). Figure 31 shows the oligonucleotides (SEQ ID NOS: 74, 75, 76, 77, 78, 79, 80, 81) used to determine whether the himrABC genes amplified by PCR were hmwl or hsar2. Figure 32A shows the construction scheme for generating the T7 expression plasmid himrABC JB-2646-1 in which any hmeA gene can be inserted. The enzyme restriction sites are: B, BaH i; Bg, Bgl II; H, Hind III; K, Kpn I; R, EcoR I; S, Sal I; Xb, Xba I. Other abbreviations are: T7p, T7 promoter; ApR, ampicillin resistance gene; KanR, kanamycin resistance gene; CAP, calf alkaline phosphatase. Figure 32B illustrates the oligonucleotides (SEQ ID NOS: 82, 83, 84, 85, 86, 87, 88, 89, 90) Used to PCR amplify the 3 'end of himrlA and the 5' end of hmtrlB in the scheme of construction of figure 32A. Figure 33A shows the construction of DS-2334-5 containing a chimeric hmrABC T7 gene of the LCDC2 gene hw2A genes hmtrBC of NTHI 12. The restriction enzyme sites are: B, BamH I; Bg, Bgl II; H, Hind III; K, Kpn 1; N, Nde I; R, EcoR I; S, Sal I; Xb, Xba I, Xho, Xho I. Other abbreviations are: T7p, T7 promoter; ApR, gene of P1269 resistance to ampicillin; KanR, kanamycin resistance gene; CAP, calf alkaline phosphatase. Figure 33B shows the oligonucleotides (SEQ ID NOS: 91, 92, 93, 94, 95) used to PCR amplify the hmr2A gene of LCDC2 for expression in the generic expression vector constructed as shown in Figure 33A. Figure 34 shows the construction of DS-2400-13 containing the T7 genes hmrA / T7 hmtrABC and the cer gene of E. coll. The enzyme restriction sites are: B, BamH I; Bg, Bgl II; H, Hind III; R, EcoR I; S, Sal I; Xb, Xba I, Xho, Xho I. Other abbreviations are: T7p, T7 promoter; ApR, ampicillin resistance gene; KanR, kanamycin resistance gene; TetR, tetracycline resistance gene; CAP, calf alkaline phosphatase.

Any strain of Haemophilus having hann genes can conveniently be used to provide purified and isolated nucleic acid molecules (which may be in the form of DNA molecules), comprising at least a portion encoding a protein of HMW1A, HMW1B, HMW1C, HMW2A, HMW2B or HMW2C, as typified by the embodiments of the present invention. Such strains are generally available from clinical sources and from bacterial culture collections, such as the American Type Culture Collection. Appropriate strains of non-typeable tfaemophilus include: H. influenzae non-typeable strain 12; H. influenzae not typeable strain Joyc; H. influenzae non-typeable strain Kl; H. influenzae non-typeable strain 21; H. influenzae nontypeable strain LCDC2 H. influenzae nontypeable strain PMH1; or H. influenzae nontypeable strain 15. In this application, the term "HMW protein" is used to define a family of HNW proteins that includes those that have natural variants in their amino acid sequences, as they are found in diverse non-typeable Haemophilus strains and characterized by an apparent molecular weight of between about 100 to about 150. Reference will now be made in detail to the presently preferred embodiments of the invention, which, together with the following examples, serve to explain the principle of the invention. For reasons of clarity of the discovery and not in a limiting manner, the detailed description of the invention is divided into the following sections: 1. Improved production of recombinant HMW proteins from E.coli. The production of native HMW1A or HMW2A proteins of H. influenzae is very low. The plasmids pHMWI-15 and pHMW2-21 (references 8 and 10) contain the complete operons hmtrlABC and hmtr2ABC of the strain NTHI 12 cloned in the expression vector pT7-7. The production of rHMWlA or rHMW2A recombinant proteins is low from these plasmids, possibly due to the 5 'flanking and the hnar promoter sequences inserted between the T7 promoter and the start codon of the hmwA genes. By removing the flanking sequences 5 * and the hnar promoter, the production of rHMWlA and rHMW2A proteins produced from the plasmids DS-1091-2 and DS-1094-2 (Figures 1 and 2) is marginally improved. When produced in H. Influenzas, native HMWA proteins are processed and secreted with the removal of the 35 kDa N-terminal fragment. Instead of relying on the correct processing and secretion of the rHMWA proteins by E, coli, the gene sequences encoding the N-terminal 35 kDa fragments were genetically deleted from the hnnrlABC and hmtf2ABC genes. In the resulting new constructions, DS-1046-1-1 and DS-1174-4 (Figures 3 and 4) the production of the mature proteins rHMWlA and rHMW2A was increased. The rHMW BC proteins were also produced in excess. Inserts of the hmvlABC and hmr2ABC gene in the pT7-7 vector were still approximately 8.6 kb and approximately 8.3 kb, respectively. Since HMWA proteins are the structural and protective proteins, it was thought that the size of the gene insert could be reduced by removing some or all of the hmtfBC genes. Expression vectors with smaller inserts are generally more efficient to produce recombinant proteins and it is thought that overproduction of rHNWBC proteins is undesirable. The production of rHMWA proteins was marginally improved when the tumrBC genes were suppressed in the vectors DS-1200-3, DS-1122-2, JB-2330-7 and DS-2084-3 (figures 4, 5, 6 and 8). . However, the production of rHMWB and rHMWC proteins was eliminated, which simplified the protein purification process. When serial copies of the T7 hwA gene cassettes were used to express the rHMWA proteins of vectors JB-2369-6 and DS-2084-1 (Figures 7 and 8), production was marginally improved. The construction of this series of expression vectors demonstrated that it was possible to increase the production of rHMW1 and rHMW2 proteins from E. coli. However, when tested for protection in a nasopharyngeal colonization model, the rHMWA proteins produced from the improved vectors were not P1269 protective. Only mixtures of native HMW1A + HM 2A proteins or rHMWA proteins produced from lower yielding vectors containing complete hmwABC genes were protective. 2. Modification of expression vectors to produce protective proteins that are protective of HMW. The expression vectors containing the hwABC genes encoding the full-length HMW1A (DS-1091-2) or HNW2A (DS-1094-2) proteins that were dependent on E. coli for processing did not produce enough protein for the test in animal models. Expression vectors containing hmwABC genes encoding mature HMW1A (DS-1046-1-1) or HMW2A (DS-1174-4) proteins expressed protective rHMWA proteins with moderate performance. The vectors that over produced rHMWA proteins alone did not produce protective antigens. Two approaches were attempted to increase the yield of the rHMWA protective protein. To the vector containing the cassette of the T7 gene hmwABC expressing the mature protein rHMWlA, the cer gene of E. coli was introduced. The gene is thought to stabilize the plasmids by preventing multimerization and their presence can stabilize expression vectors containing large cassettes of the hmwABC gene. We have also found that sometimes the P1269 presence of car also increased the production of recombinant proteins. The rHMWlA antigen that was over produced from the T7 constructions himrlABC / cer (Figure 10) was protective in the nasopharyngeal colonization model (Table 2). The second approach to the overproduction of rHNWA protective protein was to construct a vector in which the rHMWA protein was overproduced in the presence of rHMWBC proteins. To the vector containing the cassette of the T7 gene hmwABC expressing the mature protein rHMWlA, an additional cassette of the T7 hmrA gene was added. The rHMWlA antigen that was produced from the T7 constructions hmrlA / T7 hwlABC (Figure 9) was protective in the nasopharyngeal colonization model (Table 2). The two approaches can be combined, so that serial copies of the T7 hmrA / T7 hmrABC genes are coexpressed with the cer gene of E. col! in the same plasmid, DS-2400-13 (figure 34). 3. Cloning and analysis of the sequence of the additional hwA genes. The hmrA genes and the encoded proteins have variable sequences. In order to produce a fully effective vaccine, it may be necessary to use rHMWA proteins generated from multiple strains of P1269 Hae ophllUB not typifloables. The hmtrlA and / or hnnr2A genes were amplified by PCR and sequenced from different strains of Haemophllus nontypeable influences. Figures 18 to 26 illustrate the nucleotide and deduced amino acid sequences of the hmtrlA gene of the Joyc strain, the hmr2A gene of the Joyc strain, the defective hmwlA gene of strain K, the himr2A gene of strain K21, the bmrlA gene of strain LCDC2, the hmtr2A gene of strain LCDC2, the hmwlA gene of strain PMH1, the mr2A gene of strain PMH1, the hmwlA gene of strain 15, and the hmr2A gene of strain 15, respectively. The alignment of the protein sequences deduced with the protein sequences HNW1A and HMW2A of strain 12 previously described (Figures 28 and 29) identifies both regions of sequence conservation and divergence (Figure 30). This information can be useful for the identification of potential epitopes to generate peptides for vaccination or diagnostic purposes. The molecular weights of the mature HMW proteins of the various strains of non-typeable Haemophllus are contained in Table 34. Construction of a generic expression vector for the production of protective reeombinating proteins of HMW. New hmwA genes can be amplified by PCR from non-typeable Haemophllus strains and P1269 sequenced as described above. However, to produce protective rHMWA antigens, the hmvA genes must be expressed in the presence of hnarBC genes. It was found that the deduced sequences of the accessory proteins HMW1B and HMW2B of the prototype strain 12 were 99% identical, while the HMW1C and HMW2C proteins deduced from the same strain were 96% identical (reference 8, U.S. Patent No. 5,603,938). The very well-conserved nature of the hnarBC genes led to the possibility of constructing a generic expression vector using the hnarBC genes of a prototype strain and introducing any gene for its expression in it. Figure 32 illustrates the construction of a generic expression vector (JB-2646-1) containing the T7 promoter, an Xba I cloning site for the introduction of hmtrA genes, the hnnrlBC genes of strain 12 and the cer gene of E. coli. Figure 33 illustrates the construction of a cassette of the chimeric hmvABC G7 gene in the generic expression vector, wherein a hmw2A gene of LCDC2 amplified by PCR is combined with the hmrlBC genes of strain 12 to produce the plasmid DS-2334-5 . The expression of the genes from the chimeric construct was as observed for the T7 constructs h wlABC or T7 hnnr2ABC based on the hmrlA genes of the strain of NTHi 12. It is clearly evident for some of experience P1269 in the art, that the various embodiments of the present invention are used in applications in the fields of vaccination, diagnosis, treatment of Haemophilus infections and the generation of immunological agents. Next, a non-limiting additional description of said use is presented.

. Preparation and Use of the Vaccine The immunogenic compositions, suitable for use as vaccines, can be prepared from immunogenic high molecular weight (HMW) proteins of non-typeable Haemophilus strains, analogs and immunogenic fragments thereof and / or peptides. immunogenic, as described herein. The vaccine elicits an immune response that produces antibodies, including anti-HMW antibodies and antibodies that are opsonizing or bactericidal. Immunogenic compositions, including vaccines, can be prepared as injectable, liquid or emulsion solutions. The HMW protein, the immunogenic analogs and fragments thereof and / or the immunogenic peptides can be mixed with pharmaceutically acceptable excipients that are compatible with the HMW protein, the immunogenic fragments, analogs or immunogenic peptides. These excipients may include water, saline, dextrose, P1269 glycerol, ethanol, and combinations thereof. The immunogenic compositions and vaccines may also contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents or adjuvants, to improve the effectiveness of the vaccines. The immunogenic compositions and vaccines can be administered parenterally, by subcutaneous or intramuscular injection. Alternatively, the immunogenic compositions formed according to the present injection can be formulated and distributed in such a way as to elicit an immune response on the mucosal surfaces. In this way, the immunogenic composition can be administered to the mucosal surfaces by, for example, nasal or oral (intragastric) routes. The immunogenic composition can be provided in combination with a target molecule for distribution in specific cells of the immune system, or to the mucosal surfaces. Some of these target molecules include vitamin B12 and fragments of bacterial toxins, as described in International Patent O 92/17167 (Biotech Australia Pty. Ltd.), and monoclonal antibodies, as described in US Patent No. 5,194,254 ( Barber et al.). Alternatively, other modes of administration may be desirable, including suppositories and P1269 oral formulations For suppositories, binders and carriers can be included, for example, polyalkylene glycols or triglycerides. Oral formulations may include excipients normally employed such as, for example, saccharin, cellulose and magnesium carbonate in their pharmaceutical grades. These compositions may take the forms of solutions, suspensions, tablets, pills, capsules, sustained-release formulations or powders and may contain from about 1 to 95% of the HNW protein, fragments, analogues and / or peptides. The vaccines are administered in a manner compatible with the dosage formulation and in such an amount as to be therapeutically effective, protective and immunogenic. The amount to be administered depends on the subject to be treated, including, for example, the ability of the individual's immune system to synthesize antibodies and, if needed, to produce a cell-mediated immune response. The precise amounts of the active ingredient required to be administered depend on the judgment of the attending physician. However, suitable dose ranges are easily determined by one of skill in the art, and may be of the order of micrograms of the high molecular weight protein, analogs P1269 and fragments thereof, and / or peptides. Suitable regimens for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dose of the vaccine may also depend on the route of administration and will vary according to the size of the host. The nucleic acid molecules encoding the non-typeable Haemophilua HMW proteins can also be used directly for immunization by direct administration of the DNA, for example, by injection for genetic immunization or by the construction of a living vector, such as Salmonella. , BCG, adenovirus, poxvirus, vaccinia or poliovirus, which contains the nucleic acid molecules. A description of some live vectors that have been used to transport heterologous antigens to the immune system is contained, for example, in O'Hagan (1992) (ref 17). The processes for the direct injection of DNA in test subjects for genetic immunization are described, for example, in Ulmer et al 1993 (ref 18). Immunogenicity can be significantly improved if the antigens are coadministered together with adjuvants, commonly used as a solution to the P1269 0.05-1.0 percent in phosphate buffered saline. The adjuvants improve the immunogenicity of an antigen, but are not necessarily immunogenic themselves. The adjuvants can act by retaining the antigen locally near the site of administration, to produce the deposition effect that facilitates the prolonged and slow release of the antigen to the cells of the immune system. The adjuvants can also attract the cells of the immune system to an antigen deposit and stimulate said cells to elicit immune responses. Agents or immunostimulatory adjuvants have been used for many years to improve host immune responses, for example, to vaccines. Intrinsic adjuvants, such as lipopolysaccharides, are usually the components of killed or attenuated bacteria used as vaccines. The extrinsic adjuvants are immunomodulators that are typically non-covalently bound to the antigens, and are formulated to improve host immune responses. Thus, adjuvants have been identified that improve the immune response to antigens administered parenterally. However, some of these adjuvants are toxic and can cause undesirable side effects, making them unsuitable for P1269 use in humans and in many animals. Of course, only aluminum hydroxide and aluminum phosphate (collectively and commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The effectiveness of alum to increase the antibody responses in diphtheria and tetanus toxoids is well established. A wide range of extrinsic adjuvants can elicit potent immune responses to antigens. These include saponins complexed with membranal protein antigens (immune stimulation complexes), pluronic polymers with mineral oil, killed mycobacteria and mineral oil, complete Freund's adjuvants, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A and liposomes. To efficiently induce humoral immune responses (HZR) and cell-mediated immunity (CMI), immunogens are frequently emulsified in adjuvants. Many adjuvants are toxic and induce granulomas, acute and chronic inflammations (complete Freund's adjuvant, FCA for its acronym in English), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveltis (LPS and MDP).

P1269 Although FCA is an excellent adjuvant and is widely used in research, it is not authorized for use in human and veterinary vaccines, due to its toxicity. The desirable characteristics of ideal adjuvants include: 1) lack of toxicity; 2) ability to stimulate a long-lasting immune response; 3) manufacturing simplicity and stability in long-term storage; 4) ability to provoke CMI and HIR against antigens administered through various routes, if required; 5) synergy with other adjuvants; 6) ability to selectively interact with populations of antigen presenting cells (APC); 7) ability to specifically elicit appropriate immune responses specific to TH1 or TH2 cells; and 8) ability to selectively increase the appropriate levels of antibody isotype (e.g., IgA) against antigens. U.S. Patent No. 4,855,283 issued to Lockhoff and collaborators of August 8, 1989, which P1269 is incorporated herein by reference, teaches glycolipid analogues including N-glycosylamides, N-glucosylureas and N-glucosylcarbamates, each of which is substituted on the sugar residue by a amino acids, as immunomodulators or adjuvants. Thus, Lockhoff et al., 1991, (ref.19) reported that N-glycolipid analogs that show structural similarities to naturally occurring glycolipids, such as glycosphingollipids and glucoglycerolipids, are capable of eliciting immune responses. strong both in the herpes simplex virus vaccine and in the pseudorabies virus vaccine Some glycolipids have been synthesized from long chain alkylamines and fatty acids that are directly linked to the sugars through the anomeric carbon atom , to mimic the functions of lipid residues of natural origin US Pat. No. 4,258,029 issued N. to Noloney, assigned to the assignee hereof and incorporated by reference thereto, teaches that octadecyl-tyrosine hydrochloride ( OTH) works as an adjuvant when it forms a complex with tetanus toxoid and poliomyelitis virus vaccine type I, II and III ina ctivated with formalin. Also, Nixon-George et al, 1990 (ref 20), reported that the octadecyl esters of aromatic amino acids complexed P1269 with a surface recombinant antigen of hepatitis B, improved host immune responses against hepatitis B virus. 6. Immunoassays The HMW protein of a non-typeable strain of Haemophilus, the analogues and fragments thereof and / or peptides of the present invention, are useful as immunogens, as antigens in immunoassays, including the enzyme-linked immunosorbent assay (ELISA), RIAs and other antibody binding assays, not linked to enzymes, or methods known in the art for the detection of anti-bacterial, Haemophilus, HHW and / or peptide antibodies. In ELISA tests, the HMW protein, analogues, fragments and / or peptides corresponding to portions of the HMW protein, are immobilized on a selected surface, for example, a surface capable of binding to proteins or peptides. , such as the cavities of a polystyrene microtiter plate. After washing to remove HMW protein, incompletely adsorbed analogs, fragments and / or peptides, a non-specific protein such as a solution of bovine serum albumin (BSA) or casein, which is known to be antigenically neutral with respect to The test sample can be P1269 linked to the selected surface. This allows the blocking of non-specific adsorption sites on the immobilization surface and, thus, reduces the noise or background caused by the non-specific bonds of the antisera on the surface. The immobilization surface is then placed in contact with a sample, such as clinical or biological materials, to be tested in a manner conducive to the formation of the immune complex (antigen / antibody). This may include dilution of the sample with diluents, such as BSA, bovine gamma-globulin (BGG) and / or phosphate buffered saline (PBS) / Tween. The sample is then allowed to incubate for about 2 to 4 hours, at temperatures such as between about 25 ° and 37 ° C. After incubation, the surface put in contact with the sample is washed to remove the material that did not form the immune complex. The washing process may include washing with a solution such as PBS / Tween or a borate buffer. After the formation of the specific immunocomplexes between the test sample and the HMW protein, linked analogs, fragments and / or peptides, and the subsequent washing, the presence, and even the amount, of the immunocomplex formed, can be determined by subjecting the immunocomplex before a second antibody. If the P1269 test sample is of human origin, the second antibody is an antibody that has specificity for human immunoglobulins and in general, for IgG. To provide the detection means, the second antibody can have an associated activity, such as enzymatic activity, which will generate, for example, the development of coloration, after incubation with an appropriate chromogenic substrate. Quantification can then be achieved by measuring the degree of color generation using, for example, a visible spectrum spectrophotometer. 7. Sequence sequences as hybridization probes The nucleotide sequences of the present invention comprising newly isolated and characterized sequences of the hmtr genes allow the identification and cloning of the genes to "from other non-typeable strains of Haemophilus. The nucleotide sequences comprising the sequence of the hmr genes of the present invention are useful for their ability to selectively form duplex, ie, double, molecules with complementary stretches of other hmw genes. Depending on the application, a variety of hybridization conditions may be employed to achieve the various grades of P1269 selectivity of the probe towards the other hnar genes. For a high degree of selectivity, relatively stringent conditions are used to form the duplexes or doubles, such as conditions of low salt concentration and / or high temperature, such as those that provide NaCl from 0.02 N to 0.15 N at room temperature. approximately 50BC and 70BC. For some applications, less stringent hybridization conditions, such as between 0.15 N and 0.9 N of salt, are required at temperatures in the range between 20BC and 55ac. Hybridization conditions can also be made more stringent by the addition of increasing amounts of formamide, to destabilize the hybrid duplex. In this way, the particular hybridization conditions can be easily manipulated and will generally be a method of choice, depending on the desired results. In general, suitable hybridization temperatures in the presence of 50% formamide and 0.15 M NaCl are: 42BC for \ a hnar gene that is 95 to 100% homologous with the fragment of white nucleic acid, 37 & C for 90 to 95% homology and 32BC for 85 to 90% homology. In a clinical diagnostic modality, the nucleic acid sequences of the hnar genes of the present invention can be used in combination with an appropriate means, such as a marker, for the P1269 determination of hybridization. A wide variety of suitable indicator means are known in the art, including radioactive, enzymatic or other ligands, such as avidin / biotin, which are capable of providing a detectable signal. In some diagnostic modalities, an enzymatic label, such as urease, alkaline phosphatase or peroxidase, may be used instead of a radioactive label. In the case of enzymatic labels, it is known that colorimetric indicator substrates can be used to provide a visible medium to the human eye or visible spectrophotometrically, to identify the specific hybridization with samples containing the hnnr gene sequences. The nucleic acid sequences of the hw genes of the present invention are useful as hybridization probes in solution hybridizations and in modalities employing solid phase methods. In modalities involving solid-phase procedures, test DNA (or RNA) from samples, such as clinical samples, including exudates, body fluids (eg, serum, amniotic fluid, middle ear effusion, sputum, fluid from bronchoalveolar lavage) or even tissues, is adsorbed or fixed in some way to a selected matrix or surface. The fixed single-stranded nucleic acid is then subjected to hybridization P1269 specifies with selected probes comprising the nucleic acid sequences of the hanr genes or fragments thereof of the present invention, under the desired conditions. The selected conditions will depend on the particular circumstances that are based on the particular criteria required, which depend, for example, on the content of G + c, the type of target nucleic acid, the source of the nucleic acid, the size of the probe Hybridization, etc. After washing the hybridization surface to remove probe molecules, not specifically bound, specific hybridization is detected, or even quantified, by means of the label. It is preferred to select portions of the nucleic acid sequence that are conserved among Haemophilus species. The selected probe may be at least 18 bp long and may be in the range of about 30 to 90 bp in length. 8. Expression of high molecular weight protein genes The plasmid vectors containing the replicon and the control sequences that are derived from species compatible with the hcell, can be used for the expression of the high molecular weight protein genes in the expression systems. He P1269 vector ordinarily possesses a replication site, as well as tagging sequences, which are capable of providing phenotypic selection in transformed cells. For example, E. coli can be transformed using pBR322 which contains the genes for ampicillin and tetracycline resistance and thus provides simple means to identify the transformed cells. Plasmid pBR322, or another microbial plasmid or phage, must also contain, or be modified to contain, promoters that can be used by the hcell for the expression of their own proteins. In addition, phage vectors containing the replicon and control sequences that are compatible with the hcan be used as the transformation vector in connection with these h. For example, phage in Lambda GEMMR-11 can be used in the preparation of recombinant phage vectors, which can be used to transform hcells, such as LE392 from E. coli. Promoters commonly used in the construction of recombinant DNA include β-lactamase (penicillinase) and lactose promoter systems, and other microbial promoters, such as the T7 promoter system used herein in the preferred embodiments (Patent P1269 North American No. 4,952,496). The details related to the nucleotide sequences of the promoters are known, which makes it possible for anyone skilled in the art to link them functionally with the genes. The particular promoter used will generally be a matter of choice depending on the desired results. H which are suitable for the expression of the HMW protein and the fragments or immunological analogues thereof include E. coli, Bordet & lla species, Bacillus species, Haemophilus, fungi, yeasts or the expression system of baculovirus. In the present, the preferred his E. coli. In accordance with the present invention, it is preferred to produce the HMW proteins, by recombinant methods, particularly when the HMW protein of natural origin, such as purified from a culture of a Haemophilus species, can include trace amounts of materials toxic or other pollutants. This problem can be avoided by using the HMW protein, produced by recombinant techniques, in heterologous systems that can be isolated from the h in order to minimize the contaminants in the purified materials, specifically using the constructions described herein. In addition, recombinant production methods allow the manufacture of HMW1 P1269 or HMW2 or immunogenic fragments and analogs thereof, separated from each other and in a highly purified form, which is distinct from the normal combined proteins present in the Haemophllus strains.

Biological Deposits Certain vectors containing nucleic acid encoding a high molecular weight protein of a non-typeable strain of Haemophilus that are described and referenced herein have been deposited with the American Type Culture Collection (ATCC) located at 10801 University Boulevard, Hanassas, Virginia 20110-2209, United States of America, in accordance with the Budapest Treaty, and prior to the filing of this application. Samples of the deposited vectors will be available to the public and all restrictions imposed on access to the deposits will be removed after the granting of a patent based on this North American patent application. In addition, deposits will be replaced if the Depositing Agency can not provide viable samples. The invention described and claimed herein is not limited in scope by the biological materials deposited, since it is intended that the deposited form be only as an illustration of the invention. Any equivalent or similar vectors P1269 containing nucleic acid encoding similar antigens and equivalents, as described in this application, are within the scope of the invention.

Deposit Summary ATCC Plasma Deposita Peat DS-1046-1-1 (pT7 hmwlABC (125)) 203263 September 25, 1998 (pT7 hmwlA (125) / JB-2507-7 203262 T7 hmwlABC (125)) September 25, 1998 ( pT7 hmwlA (125) / BK-86-1-1 203258 September 25, 1998 T7 hmwlABC (125) / KanR) BK-35-4 (pT7 hmwlABC (125) / cer) 203259 September 25, 1998 (pT7 BK -76-1-1 203261 September 25, 1998 hmwlABC (125) / cer / KanR) (pT7 hmw2A (LCDC2) / DS-2334-5 203260 September 25, 1998 hmwlBC (12) / cer / KanR) (pT7 JB-2646-1 203256 September 25, 1998 hmwlBC (12) / cer / KanR) (pBRT7 hm lA / T7 DS-2400-13 203257 September 25, 1998 hmwlABC / cer / KanR) The above disclosure generally describes the present invention. Further understanding can be obtained P1269 complete by reference to the following specific examples. These examples are described for illustrative purposes only and are not intended to limit the scope of the invention. Both changes in the form and substitution of equivalents are contemplated, depending on the circumstances, they may suggest or make it convenient. Although specific terms have been used herein, it is intended that such terms have a descriptive and not limiting sense. The methods of molecular genetics, of protein biochemistry, of immunology and of fermentation technology used, although not explicitly described in this description and in these Examples, are widely reported in the scientific literature and are within the capacity of those experienced in the art.

Example 1 This Example describes the construction of the plasmid DS-1091-2 which expresses the JumrlABC genes encoding the full length 160 kDa HHW1A protein. Plasmid pHMWI-15 (ref.8) contains approximately 400 bp of 5'-flanking region, including the hnnr1 promoter, inserted between the T7 promoter and the start of the hmwlABC coding region (Figure 1).

P1269 There is a unique Bgl II site in the multiple cloning site of pHMWl-15 and a unique BamH I site in the hmwlA coding region. The 2.2 kb Bgl II-BamH i fragment was subcloned for subsequent manipulation, which generated the plasmid DS-1035-12. A 400 bp Xba I-Bsm I fragment containing the 5'-flanting region was replaced by oligonucleotides of approximately 86 bp (Figure IB) that bound the T7 promoter directly with the initial ATG codon of the hmwlk gene in the plasmid DS-1055R -2. The Xba fragment I-BamH I of 1.5 kb of the DS-1055R-2 was inserted into pHMWI-15 which had been digested with the same enzymes to generate the plasmid DS-1091-2.

Example 2 This Example describes the construction of plasmid DS-1094-2 expressing the hmw2KBC genes encoding the full-length 1055 kDA HMW2A protein. Plasmid pHMW2-21 (reference 10) contains approximately 800 bp of 5 · -flanking sequence, including the hmw2 promoter, between the T7 promoter and the start of the smttABC coding sequence (Figure 2). Plasmid pHMW2-21 has two EcoR I sites, one at the multiple cloning site and the other within the coding sequence of the hmw2 gene. The 2.5 kb EcoR I fragment is P1269 subcloned for subsequent manipulation, which generates the plasmid DS-1036-9. The approximately 800 bp Xba I-BamH I fragment containing the 5'-flanking sequences was replaced by the same oligonucleotides of approximately 86 bp that were used for hwl (Figure IB), to bind the T7 promoter directly to the initial AT6 codon of hnnr2A, which generates the plasmid DS-1056R-1-1. An intermediate plasmid (DS-1078-4) was needed to introduce convenient sites of restriction enzymes and the Xba I insert was removed, then religated to change the orientation of the plasmid DS-1085-8. Plasmid DS-1085-8 was linearized with EcoR I, dephosphorylated and ligated with the 8 kb EcoR I fragment of pH W2-21, to generate the plasmid DS-1094-2 containing the T7 promoter directly linked to the coding sequence of Ajimr2ABC.

Example 3 This Example illustrates the construction of the plasmid DS-1046-1-1 expressing the unrlABC genes encoding the mature 125 kDa HMW1A protein. Plasmid pHNWI-15 (reference 8) contains the Xba I site within the sequence of the T7 promoter and a unique BamH I site within the coding sequence of the mature HMW1A protein (Figure 3A). The fragment Xba I- P1269 BamH I of 1.8 kb of pHMWI-15 was removed and replaced by an approximately 114 bp Xba I-BamH I fragment generated from oligonucleotides (Figure 3B). The resulting 11.3 kb plasmid, DS-1046-I-1, thus contains the T7 promoter bound in frame with the AwlABC operon encoding the mature 125 kDa HMW1A protein.

EXAMPLE 4 This Example illustrates the construction of plasmid DS-1200-3 expressing the Aw2AB partial C genes encoding the mature 120 kDa HMW2A protein. Plasmid pHMW2-21 (reference 10) contains an EcoR I site within the coding sequence of the mature HMW2a protein. However, this is not unique (Figure 4A). A multistep construction process involved first recreating part of the T7 promoter and the start of the hmtr2h gene encoding the mature HMW2A protein, from oligonucleotides of 105 bp (Figure 4B). Plasmid DS-1134-2 was linearized with EcoR I, dephosphorylated and the 8 kb EcoR I fragment of pHMW2-21, which contains most of the hmtr2 gene and the full hmw2B and hmw2C genes, was inserted. The plasmid DS-1147-4 is a pUC-based plasmid containing the T7 gene cassette 2ABC. The whole cassette was removed in a 6.5 kb Bgl II-Xho I fragment and P1269 was inserted into pT7-7 which had undergone digestion with Bgl II and Sal I, to generate the plasmid DS-1200-3. Part of the mw2C gene was deleted in this construction.

Example 5 This Example illustrates the construction of the plasmid DS-1122-2 which contains the hnnrA gene encoding the mature 125 kDa HMWIA protein and part of the mvlB gene. Plasmid DS-1046-1-1 (Figure 3A, Example 3) contains three Hind III sites, one with the umrlB gene, another with the hnmrlC gene and another in the 3 'region of the multiple cloning site (Figure 5). When the plasmid DS-1046-1-1 was digested with Hind III and then religated, plasmid DS-1122-2 containing a complete himrlh gene encoding the mature 125 kDa HMWIA protein was generated. of the Hunnal gene and does not contain Junarle gene.

EXAMPLE 6 This example illustrates the construction of plasmid JB-2330-7 containing the lA gene encoding the mature 125 kDa HMWIA protein, without other hmr genes. PCR amplification was performed on ADM of plasmid DS-1122-2 (Figure 5, Example 5) to generate a 500 kb fragment of the Kpn I site near the 3 'end of imlA, through the terminator and introduce sites of P1269 restriction enzymes for Xho I and Hind III at the 3 'end. The fragment was cloned into pCR II, whereby the plasmid DS-2056-1-1 is generated (Figure 6A) and the oligonucleotides that were used are shown in Figure 6B. The plasmid DS-1122-2 was digested with Kpn I and Hind IIIf which removed most of the hsarlA gene and the entire fragment of the ñnvlB gene. The 2.6 kb Kpn 1-Hind III vector fragment of DS-1122-2 was ligated with the 0.5 kb Kpn 1-Hind III fragment of DS-2056-1-1 to generate plasmid JB-2321-1, which contains approximately 0.2 kb of the 5 * -hmtrlK sequence and approximately 0.5 kb of the 31-hmtrlK sequence, joined at the Kpn I site. The plasmid JB-2321-1 was linearized with Kpn 1, dephosphorylated and the Kpn I internal fragment 2.7 kb of DS-1122-2 was inserted to generate plasmid JB-2330-7. the plasmid contains a T7 hnarl gene cassette without additional hmrl gene sequences.

Example 7 This Example illustrates the construction of plasmid JB-2369-6 containing tandem copies of the T7 hmtrlA gene cassette encoding the mature 125 kDa HMW1A protein. Plasmid JB-2330-7 (Figure 6A, Example 6) was digested with Bgl II and Hind III and the gene cassette T7 wlA was subcloned into pUC-BgXb which had been digested with Bgl II and Hind III, generating he P1269 plasmid JB-2337-1 (Figure 7). Plasmid JB-2337-1 was digested with Sal I and Xho I which released the T7 hnnrlk cassette in a fragment with compatible ends. The vector DS-1843-2 is a plasmid based on pBR328 that contains transcription terminators and a multiple cloning site with a unique Xho I site. The vector DS-1843-2 was digested with Xho I, dephosphorylated and then ligated with the gene casse Sal I-Xho I T7 hmwlA. 3.5 kb to generate the plasmid JB-2347-5. Because the Sal I and Xho I sites are sealed, this plasmid contains a unique Xho I site at the 3 'end of the T7 honrlA gene that can be used to insert genetic cassettes Sal 1-Xho I T7 additional himrlA derived from JB- 2337-1. Plasmid JB-2369-6 contains two T7 himlA genes in tandem introduced in this manner.

This Example illustrates the construction of the plasmids DS-2084-3 and DS-2084-1 which contain one or two tandem copies of the T7 gene cassette hnnr2 which encodes the mature HMW2A. Plasmid DS-1200-3 (Figure 4A, Example 4) contains the partial T7 w2AB C gene cassette. There are two lu I sites in DS-1200-3, one located near the 3 'end of hm »2K and the other located near the 5' end of P1269 w2B (Figure 8A). Oligonucleotide primers were used to PCR amplify a 247 bp fragment from the 3 'end of the hmw2h gene from the Mlu I site and introduce a unique BamH I site after the hmw2A stop codon (Figure 8B). The 247 bp PCR fragment was subcloned into pCRII and the plasmid DS-2056-3-1 was generated. Plasmid DS-1200-3 was digested with Bgl II and Mlu I and the 3.2 kb fragment containing the T7 promoter and most of the hmw A gene was purified. Plasmid pUC-BgXb was digested with Bgl II and BamH I and dephosphorylated. The Bgl II-Mlu I hav2A gene fragment and the Mlu 1-BamH i PCR fragment of DS-2056-3-1 were ligated into the pUC vector to generate the plasmid DS-2073-3, which thus contains a T7 gene cassette hmw2A of 3.4 kb in a Bgl II-BamH lr fragment without additional hmw2 genes. Plasmid DS-1843-2 was linearized with Bgl II and the 3.4 kb Bgl II-BamH I cassette was inserted, which generated the plasmid DS-2084-3 containing a single T7 hmv2A gene cassette and the plasmid DS-2084- 1 containing two T7 hmw2 genetic cassettes in tandem.

Example 9 This Example illustrates the construction of plasmids JB-2507-7 and BK-86-1-1 containing tandem T7 genes hm * rlA / T7 hmwlKBC encoding the HMWIA protein P1269 matures at 125 kDa and are resistant to ampicillin or kanamycin, respectively. Plasmid DS-1046-1-1 (Figure 3A, Example 3) contains the T7 hmtrl &BC gene cassette and has a unique BamH I site within the coding region of the mature HMW1A protein. Plasmid JB-2369-6 (Figure 7; Example 7) contains genetic cassettes in tandem T7 hmwlK, each of which contains an internal BamH I site within the coding sequence for HMW1A. When plasmid JB-2369-6 was digested with BamH I, a 3.5 kb fragment containing the 3 * end of the first hmwlh gene and the T7 promoter and the 5 'end of the second hwlA gene was generated. This fragment was ligated into the BamH I site of DS-1046-1-1 to generate plasmid JB-2507-7 containing genetic cassettes in tandem T7 hmwlh / ?? hmwlKBC (Figure 9). The only Sal I site found in the multiple cloning site of the pT7-7f vector backbone was used to linearize JB-2507-7. The cassette with kanamycin resistance was separated from pUC-4K by digestion with Sal I and ligated with vector JB-2507-7 to generate plasmid BK-86-1-1. Example 1Q This Example illustrates the construction of plasmids BK-35-4 and BK-76-1-1 containing the T7 gene cassette jmrlABC and a cer gene of E. coli and are resistant P1269 to ampicillin or kanamycin, respectively. Plasmid DS-2224-1-4 (Figure 10) contains a cer gene from E. coli (reference 13) that was generated from oligonucleotides of approximately 290 bp, cloned in the BamH I site of pUC-BgXb. Plasmid DS-1046-1-1 (Figure 3, Example 3) contains a unique Bgl II site towards the 5 'end of the T7 promoter. Plasmid DS-1046-1-1 was linearized with Bgl II, dephosphorylated and ligated with 290 bp BamH I fragment containing the cer gene of DS-2224-1-4, to generate the plasmid BK-35- Four. The cassette with kanamycin resistance was separated from pUC-4K by digestion with Sal I and inserted into the unique Sal I site of BK-35-4 to generate the plasmid BK-76-1-1.

Example 11 This Example illustrates the analysis of the production of rHMW1 and rHMW2 proteins of the different constructions produced in the preceding Examples. The plasmids were introduced into BL21 (DE3) cells of E. coli by electroporation using a BioRad apparatus. Strains were cultured at 37 ° C in NZCYM medium to an optical density of A57 e = 0.3, then induced by the addition of lactose at 1.0% for 4 hours. The samples were adjusted to 0.2 OD / μ? with lysis in SDS-PAGE + charge absorber and the same amount of P1269 sample of SDS-PAGE gel protein. Figure 11 illustrates the relative production of rHMW proteins from various constructs as analyzed on SDS-PAGE gels. The identification of the bands in relation to the specific constructions is given in the description of the previous figure, "a" indicates the band for the HMWA proteins, nb "indicates the band for the HMWB proteins and" c "indicates the band for the proteins. HMWC proteins As can be seen in the Figure, the production of proteins HMW1A, B and C from the construction T7 hmwlABC (l60) (band 3) is negligible.The production of the three proteins is improved in the T7 construction hmwlABC (125) (band 4) In band 5, there is no production of the HMW1C protein and the HMW1B protein is slightly reduced in size due to the truncation of its gene in the T7 construction hmwlA partial B. In band 6, no there is production of HMW1B or HMW1C from the construction T7 hmwlA (125). Band 7 shows that the improvement was marginal, if any, in the production of HMW1A from the construction T7 hmwlA / T7 hmwlA. 8, the production of HMW1A, HMW1B and H is evident MW1C when expressed from the construction T7 hmvlA / T7 hmwlABC. In band 9, proteins HMW1A, HMW1B and HMW1C are produced from the T7 construct hmwlABC / cer.

P1269 Example 12 This Example illustrates the purification of recombinant HMW1 and HMW2 proteins. All the recombinant HHW proteins were expressed as inclusion bodies in E. coli, without taking into account whether there were partial or complete deletions of genes B and C in the various constructions, they were purified by the same procedure (Figure 12). Cellular agglomerates of E. coli from 500 ml of culture were resuspended in 50 ml of 50 mM Tris-HCl, pH 8.0, with a content of 0.1 N NaCl and broken by sonic energy. The extract was centrifuged at 20,000 g for 30 minutes and the supernatant that formed was discarded. The agglomerate (PPTi) was then extracted in 50 ml of 50 mM Tris-HCl, pH 8.0, containing 0.5% Triton X-100 and 10 mM EDTA, then centrifuged at 20,000 g for 30 minutes and the supernatant was added. discarded. The pellet (PPT2) was then extracted in 50 ml of 50 mM Tris-HCl, pH 8.0, containing 1% octylglucoside, then centrifuged at 20,000 g for 30 minutes and the supernatant discarded. The resulting agglomerate (PPT3), obtained after the previous extractions, contains the inclusion bodies. The agglomerate was solubilized in 6 ml of Tris-HCl 50mM P1269, H 8.0, containing 6M guanidine and 5mM DTT. 12 ml of 50 mM Tris-HCl, pH f N 8.0 were added to this solution and the mixture was centrifuged at 20 ° C., 000 g for 30 - minutes. The supernatant (SUPA) was precipitated with polyethylene 5 glycol (PE6) 4000 at a final concentration of 7%. The resulting agglomerate (PPT5) was removed by centrifugation at 20,000 g for 30 minutes and the supernatant was precipitated with (NHA) 2 SO at 50% saturation. After the addition of (NH) 2SO, 10-phase separation was given in the solution and the protein went to the upper phase, which was then subjected to centrifugation at 20,000 g for 30 minutes. The resulting agglomerate (PPT6) was dissolved in 2 ml of 50 mM Tris-HCl, pH 8.0, containing 6M guanidine hydrochloride and 5M DTT and the clear solution was purified in a Superdex 200 gel filtration column equilibrated in 50 mM Tris-HCl, pH 8.0, containing 2M guanidine hydrochloride. The fractions were analyzed by SDS-PAGE and (those containing the purified rHMWl were mixed and dialyzed overnight at 4 ° C against PBS, then centrifuged at 20,000 g for 30 minutes. The protein remained soluble under these conditions and glycerol was added to the rHMW1 preparation at a final concentration of 20% for storage at -20 * C. The SDS-PAGE analysis of a rHMWl protein representative (abc / cer) in various stages of purification P1269 is shown in Figure 13. The identification of the bands is given above in the description of the figures. Three major protein bands at approximately 110, 80 and 60 kDa (lane 6) became apparent after the three initial extractions with 50 mN Tris-HCl / 0.1M NaCl, pH 8.0 (lane 3); 50 mN Tris-HCl / 0.5% Triton X-100, pH 8.0 (band 4); and 50 mH Tris-HCl / 1% octylglucoside, pH 8.0 (band 5). These three proteins represent the products of the hnwlA, C and B genes, respectively, as confirmed by N-terminal amino acid sequencing. The products of genes B and C were less soluble in the guanidine hydrochloride solution (band 7) and were more easily separated from the gene A product (HM 1, band 8) by diluting the concentration of guanidine hydrochloride of 6M to 2M. Precipitation with 7% polyethylene glycol (PE6) 4000 removed other contaminating proteins (band 9) from the rHMW1 preparation. A final precipitation with ammonium sulfate not only concentrated the rHMW1 of the PE6 soluble fraction (band 10), but also effectively eliminated the residual PE6 (band 11) and the salt (H4> 2S0 (band 12) through of a separation phase that occurred when mixing the PE6 solution with a high concentration of (NH4) 2S04 The rHMW1 agglomerate was then dissolved in 50 mM Tris-HCl, pH 8.0, containing 6M guanidine hydrochloride and 5M DTT and it was purified P1269 on a Superdex 200 gel filtration column pre-equilibrated with 50 mMf Tris-HCl pH 8.0, containing 2M guanidine hydrochloride (Figure 1, block A). The average yield of the purified rHMWl is approximately 10 mg IT1 per culture. The SDS-PAGE analysis of the purification of rHMW2A from the T7 construction imr2A / T7 hnsw2 is shown in Figure 14, block B.

Example ¾? This Example illustrates the stability of the rHMWlA protein. To study the stability of rHMWlA, the purified rHMWlA protein produced according to Example 12 was stored at 4 ° C or -20 ° C, with or without glycerol. in the absence of glycerol, it was found that the protein degraded when stored at 4 ° C and tended to precipitate when stored at -20 ° C. The addition of glycerol to a final concentration of 20% not only significantly increased the solubility of rHMWlA, but also increased the stability of the protein when stored at -20 ° C. The protein remained intact for at least eight weeks even after repeated freezing and thawing (Figure 15).

Example 14 This Example illustrates the immunogenicity of P1269 proteins rHM lA and rHMW2A produced from different constructions. To study the immunogenicity of the rHMW1 protein produced from T7 constructs nomrlABC (pDS-1046-1-1; Figure 3A, Example 3) or T7 AjtmrlABC / cer (pBK-76-1-1); Figure 10, Example 10) and purified by the procedure of Example 12, s.c. (subcutaneously) groups of five BALB / c mice (Charles River, Quebec) on days 1, 29 and 43 with 0.3, 1 and 3 antigen, in the presence of AIPO (1.5 mg per dose). Blood samples were collected on days 0, 14, 28, 42 and 56. Mice immunized with purified rHMWl derived from the T7 constructs hmtrlABC or T7 JunvlABC / cer (0.3 to 3 per dose) generated anti-rHMWlA antibody responses dose-dependent (Figure 16), suggesting that the two proteins remain immunogenic after removal of inclusion bodies and solubilization. No statistically significant differences were found in the antibody titers induced by the protein derived from these two constructs. In order to compare the immunogenicity of rHMW1 and rHMW2 proteins produced from several different and purified constructs according to Example P1269 12, groups of 9 chinchillas (Rancho de Chinchillas Moulton) were immunized via i.m. (intramuscular) on days 1, 14 and 28, with 30 ig of rHMW protein in the presence of AIPO4 (1.5 per dose). Blood samples were collected on day 42. Chinchilla anti-HMW antibody responses induced by various forms of rHMW are summarized in Table 1. It was found that the rHMW1 protein prepared from the T7 constructions hmwlABC (abe) ( pDS-1046-1-l; Figure 3A, Example 3), T7 hmwlK / t? hmvlABC (a / abe) (pBK-86-1-1, Figure 9, Example 9), T7 hamrlABC / cer (abe / cer) (pBK-76-1-1, Figure 10, Example 10) and T7 h vlK / n hmrlK (2xa) (pJB-2369-6; Figure 7, Example 7), but not the T7 construct hmtrlKB (125) (abé) (pDS-1122-2; Figure 5 Example 5), induced in the chinchillas Significant antibody titers after three immunizations. Similarly, the rHMW2 prepared from the T7 constructions JumttABC (abe) (pDS-1147-4; Figure 4A, Example 4) or T7 himv2A / T7 hmv2A (2xa) (pDS-2084-1; Figure 8A, Example 8) and purified according to the procedure of Example 12 elicited significant antibody titres in the chinchillas after three immunizations. Anti-rHMW IgG titers were determined by antigen-specific enzyme-linked immunoabsorbent assay (EIAs).

P1269 Microtiter wells (Nunc-MAXISORP, Nunc, Denmark) were coated with 50 μ? of protein antigen (0.5 ig mi-1). The reagents used in the assays are the following: F (ab *) 2 fragments affinity purified from goat anti-mouse IgG (Fc-specific) conjugated with horseradish peroxidase (Jackson immunoResearch Labs., Missiseauga, Ontario); anti-guinea-pig IgG antibody purified by affinity (1 iq mi'1) (prepared in this laboratory); and F (ab ') 2 fragment affinity of goat anti-guinea pig IgG (H + L) antibodies conjugated to horseradish peroxidase (HRP) (Jackson ZmmunoResearch Laboratories) used as a reporter. Chinchilla IgG was purified from chinchilla serum according to Barenkamp (reference 14). The generation and purification of chinchilla IgG anti-guinea pig antibodies, have already been described previously (reference 15). Reactions were developed using tetramethylbenzidine (TMB / H202, ADI, Mississauga, Ontario) and absorbances were measured at 450 nm (using 540 nm as a reference wavelength) on a Flow Multiskan MCC microplate reader (ICN Biomedicals, Mississauga , Ontario). The reactive titre of an antiserum was defined as the reciprocal of the dilution that consistently shows an increase in absorbance corresponding to twice the amount obtained in the serum sample prior to blood extraction.

Example 15 This Example illustrates the protective ability of rHMWlA and rHMW2A proteins produced from different constructs. Immunization and intranasal stimulation with streptomycin-resistant freshly cultured NTHi strain 12 in chinchillas has been described previously (reference 15). Briefly, groups of 8 to 9 animals were immunized three times i.m. with one of the following preparations: 30 μg of purified rHMW1 or rHMW2, 2 x 109 ufe (colony forming units) of complete NTHi cells inactivated by heat (56 eC, 10 minutes) in alum; or in alum alone, on days 0, 14 and 28. On day 42 serum and nasal lavage samples were taken for the measurement of anti-HMW1 or anti-rHMW2 antibody titers by EIAs. On day 44, the animals were lightly anaesthetized by means of xylazine / ketamine hydrochloride by intramuscular injection (0.06 mg of xylazine and 0.3 mg of ketamine hydrochloride per kg body weight). Intranasal inoculations were performed via passive inhalation (50 μm per nostril, total 0.1 ml per animal) of freshly cultivated streptomycin-resistant strain 12 NHTi in BHI medium supplemented with hemin and NAD both at 2 μg mi-1. The dose of bacterial stimulation was 1 x 108"s per animal. Nasopharyngeal washes were performed after 4 days postinoculation in anesthetized chinchillas 5 (xylazine / ketamine hydrochloride, in the same route and in the same dose as on day 44). Secretions were obtained by nasopharyngeal irrigation with 1 ml of sterile saline and the fluid was collected out of the contralateral nasal passages. In general, they were collected from each animal approximately 500 μ? of fluid and 25 μ? of the sample were deposited on a chocolate agar plate in the presence of 50 μ? of streptomycin (20 mg ml -1). The protective effect of parenteral immunization with several preparations of rHMW1 and rHMW2 in colonization PN of chinchilla nasopharyngeal fluid with strain 12 NTHi are summarized in Table 2. From 67 to 88% of the control animals that were immunized only with alum, had positive nasal lavage fluids in culture. In contrast, from 67 to 80% of the animals immunized with the protein purified rHMWl derived from the constructions abe (pDS-1046-1-1), a / abe (pBK-86-1-1) or abe / cer (pBK-76-1-1) were sufficiently protected. Animals immunized with the rHMWl protein derived either from the abA construct (pDS-1122-2) or from the 2xa construct (pJB-2369-6), were infected by 70 to 90%. These results clearly indicate that in order to achieve significant protection against the colonization of strain 12 NTHi in the chinchilla model, the rHMW1 protein must be derived from a construct with intact ABC genes. Similar results were also observed with the rHMW2 protein. As shown in Table 2, animals immunized with purified rHMW2 protein from the abe construct (pDS-1147-4), but not from the 2xa construct (pDS-2084-1), were protected against colonization of the strain 12 NTHi in the chinchilla model. In all cases, significant protection was observed in the chinchillas immunized with the preparations of NTHi 12 whole cells inactivated with heat, prepared according to the US Patent NO. 5,603,938.

E-iemolo 16 This Example illustrates cloning and sequence analysis of himrh genes from additional NTHi strains. Chromosomal DNA was prepared from several NTHi strains and PCR was carried out using the oligonucleotide primers shown in Figure 17. The sense primer (5522.SL, SEQ ID NO: 21) corresponds to the P1269 region conserved in genes h wK encoding the immediate residues in the direction of the 3 'end of the processing site for mature HMW proteins The antisense primer (5523.SL, SEQ ID NO: 24) corresponds to the start of the hm / B gene which is also preserved. The PCR amplification was performed as follows: each reaction mixture contained 5 to 100 ng of DNA, 1 of each primer, 5 units of taq + or tsg + (Sangon) or longer taq (Stratagene), 2mM dNTPs, Tris- 20 mM HCl (pH 8.8), 10 mM KC1, 10 mM (NH4) 2 SO4, 2 mM MgSO4, 0.1% Triton X-100, BSA. Cycling conditions were: 95 ° C for 1 min, then 25 cycles of 95 ° C for 30 seconds, 45 ° C for 1 min, 72 ° C for 2 min; then 72 ° C for 10 min. The nucleotide sequences (SEQ ID NO: 25) and deduced amino acid sequences (SEQ ID NO: 26) of the mvlA gene of the Joyc strain are shown in Figure 18. The mature HMW1A protein predicted from the Joyc strain (coding for the sequence SEQ ID NO: 27, amino acid sequence SEQ ID NO: 28) has a molecular weight of 125.9 kDa and a pl of 8.21. No RGD motifs were found in HMW1A Joyc. The nucleotide sequences (SEQ ID NO: 29) and deduced amino acids (SEQ ID NO: 30) of the hmw2 gene of the Joyc strain are shown in Figure 19. The mature HMW2A protein predicted from the Joyc strain (coding for the P1269 sequence SEQ ID NO: 31, amino acid sequence SEQ ID NO: 32) has a molecular weight of 100.9 kDa and a pl of 6.91. No R6D motifs were found in Joyc HMW2A. The nucleotide sequences (SEQ ID NO: 33) and deduced amino acids (SEQ ID NOS: 34 and 35) of the defective avlA gene of the Kl strain are shown in Figure 20. Although there is a complete hwlA gene in the Klr strain there is a change in the reading pattern immediately after a poly G tract, which results in the premature termination of the HMW1A protein after 326 amino acids. The nucleotide sequences (SEQ ID NO: 38) and deduced amino acid sequences (SEQ ID NO: 39) of the hWlA gene of strain K21 are also shown in Figure 21. The mature HMW1A protein predicted from strain K21 (coding for the sequence SEQ ID NO: 40, amino acid sequence SEQ ID NO: 41) has a molecular weight of 104.4 kDa and a pl of 8.71. There is a single RGD motif located at residues 20 to 22 in HMW1A of K21. The nucleotide sequences (SEQ ID NO: 42) and deduced amino acids (SEQ ID NO: 43) of the JimwA gene of the LCDC2 strain are shown in Figure 22. The mature HMW1A protein predicted from the LCDC2 strain (which encodes the sequence SEQ ID NO: 44, amino acid sequence SEQ ID NO: 45) has a molecular weight of 114.0 kDa and a pl of 8.72. No RGD motifs were found in the HMW1A of LCDC2.

P1269 The nucleotide sequences (SEQ ID NO: 46) and deduced amino acids (SEQ ID NO: 47) of the hnor h gene. of the strain LCDC2 are shown in Figure 23. The mature HMW2A protein predicted from the strain LCDC2 (which encodes the sequence SEQ ID NO: 48, amino acid sequence SEQ ID NO: 49) has a molecular weight of 111.7 kDa and a pl of 8.22. No RGD motifs were found in the HMW2A of LCDC2. The nucleotide sequences (SEQ ID NO: 50) and deduced amino acids (SEQ ID NO: 51) of the hmtrlA gene of the PMH1 strain are shown in Figure 24. The mature HMW1A protein predicted from the FMH1 strain (coding for the sequence SEQ ID N0: 52f amino acid sequence SEQ ID NO: 53) has a molecular weight of 102.4 kDa and a pl of 6.73. Two RGD motifs were found in the HMW1A of PMH1, the first in residues 19 to 21 and the second in residues 505 to 507. The nucleotide sequences (SEQ ID NO: 54) and deduced amino acids (SEQ ID NO: 55) of the hnmr2A gene of the PMH1 strain are shown in Figure 25. The mature HMW2A protein predicted from the PMH1 strain (which encodes the sequence SEQ ID NO: 56, amino acid sequence SEQ ID NO: 57) has a molecular weight of 103.9 kDa and a pl of 9.07. Two RGD motifs were found in the HMW2A of PMH1, the first in residues 26 to 28 and the second in residues 532 to 534. The nucleotide sequences (SEQ ID NO: 58) and P1269 deduced amino acids (SEQ ID NO: 59) of the hmwlA gene of strain 15 are shown in Figure 26. The mature HMW1A protein predicted from strain 15 (coding for the sequence SEQ ID NO: 60, amino acid sequence SEQ ID NO: 61) has a molecular weight of 103.5 kDa and a pl of 8.06. No R6D motifs were found in the HMW1A protein of strain 15. The nucleotide sequences (SEQ ID NO: 62) and deduced amino acid sequences (SEQ ID NO: 63) of the hmw2A gene of strain 15 are shown in Figure 27. Mature HMW2A protein predicted from strain 15 (coding for the sequence SEQ ID NO: 64, amino acid sequence SEQ ID NO: 65) has a molecular weight of 121.9 kDa and a pl of 8.22. No RGD motifs were found in the HMW2A protein of strain 15. The nucleotide sequences (SEQ ID NOS: 66, 70) and deduced amino acids (SEQ ID NOS: 67, 71) of the genes iwlA and hr2K, from strain 12 , as described in U.S. Patent No. 5,603,938, are shown in Figures 28 and 29, respectively. An alignment of the protein sequences of HMW1A and HMW2A deduced with the protein sequences of HMW1A and HMW2A published from strain 12 (SEQ ID NOS: 67, 71) is shown in Figure 30. The cleavage site for mature proteins it is shown by means of the arrow. The regions of similarity can be P1269 identify in particular approximately between residues 980 and 1168, at the carboxyl terminus, approximately between residue 1360 and the end. There appear to be repeat sequences in some proteins inserted around residue 1219, most notably in Joyc HMW1A and Kl HMW1A, which appear to have two repeat sequences in tandem, while K21 HMW1A and LCDC2 HMW2A contain single copies of the repeat. The HMW2A of strain 15 contains a different repeat segment located in the same area. There is a short segment of semiconserved sequence inserted in residue 583 that is found in all HMW2A proteins, except in the HMW2A of strain 15. However, this is found in the HMW1A protein of strain 15.

Example 17 This Example illustrates the PCR amplification used to determine whether the jmvlA or hnnr2K genes have been amplified or not. The hmwX genes were amplified by PCR using primers based on conserved sequences between hmvl operons and hmw2 and in this way the amplified genes could be either h l or hm »2. Although the hnar genes do not occur in the encapsulated strains, the 3'- and 5'-flanking sequences can be found in the sequence P1269 genomic strain Rd of H. influenzas (reference 16). Oligonucleotide sense primers were generated based on the flanking 51-hxmrl sequence from the HI1679 gene of the Rd strain (primer 5672.SL, SEQ ID NO: 74) and the flanking 5 · -hmtr2 region of the HI1598 gene of strain Rd (primer 5676. SL (SEQ ID NO: 75)). The antisense primers were generated based on the internal sequences of the amplified hmrh genes. The oligonucleotide primers are shown in Figure 31. Primer 57 2.SL (SEQ ID NO: 78) was used to amplify the hmwA genes of strains Kl, K21, PMH1 and 15, while primer 5743.SL (SEQ ID NO: 81) was used for the PCR of the hnor genes of the Joyc and LCDC2 strains. The amplified fragments were sequenced directly by means of specific primers of tvmrA (5742.SL and 5743.SL) and the sequence was compared to the sequence of the genes that were cloned in Example 16. After the h wA genes amplified by PCR were identified as himrlA or hmr2At specific PCR primers were used to PCR amplify a second copy of the gene with a start codon manipulated at the beginning of the mature protein. A pair of representative PCR primers, which was used to amplify the hmr2h gene of LCDC2 for expression, are illustrated in Figure 33B (5972.S, SEQ ID NO: 92; 5973. SL, SEQ ID NO: 95).

P1269 Example 18 This Example illustrates the construction of a generic plasmid for the expression of hmtrABC genes in E. coli. As shown in Example 16, the hvnr1K and mw2A genes can be amplified by PCR from any strain that contains non-typeable H. influenzae hmr, but to produce protective recombinant antigen, they should be expressed in the presence of humrBC genes. A generic expression plasmid containing the T7 promoter, unrlBC genes of strain 12, the E. coli cer gene, a gene with kanamycin resistance and a cloning site to insert any hmrh gene was constructed. Plasmid BK-76-1-1 (Figure 10, Example 10) was digested with EcoR I and religated to generate plasmid JB-2581-2-1, which has the 2 kb EcoR I fragment that present deletion at the 3 'end of jmrlA and the 5' end of hnnrlB (Figure 32A). The EcoR I 2kb fragment of BK-76-1-1 was subcloned into pUC-BgXb for further manipulation, which generates the plasmid JB-2581-ll, Figure 32B shows the oligonucleotide primers used to amplify the 3 * end of hmtrlh (5947.SL, SEQ ID NO: 83; 5948.SL, SEQ ID NO: 86) and the 5 * end of mvlB (5949.SL, SEQ ID NO: 87; 5950.SL, SEQ ID NO: 90), that introduce an Xba I site at the junction of the two genes. He P1269 plasmid JB-2603-1-1 contains a fragment 3'- EcoR 1-Xba I of 1.5 kb of the hmtrlK gene and the plasmid JB-2603-2-1 contains the Xba 1-EcoR I fragment of approximately 550 bp of the intergenic AB hmr sequence and the 5 'end of nmriB. Plasmid JB-2581-2-1 was linearized with EcoR I, dephosphorylated and ligated with the EcoR 1-Xba I inserts of JB-2603-1-1 and JB-2603-2-1, which generates the JB plasmid -2641-1. This plasmid is identical to B-76-1-1, with the exception that it contains an additional Xba I site between the genes hmwlh and JunvlB. Plasmid JB-2641-1 was digested with Xba I which removed the complete JimvlA gene, but left the BC bmrl genes intact. Re-ligating the vector fragment generated the plasmid JB-2646-1 which is the generic expression vector to which the hmvh genes can be cloned into the Xba I site (Figure 32A). In order to demonstrate the usefulness of the generic expression vector, a chimeric HjmrABC T7 gene cassette containing the hw2 gene of LCDC2 combined with the JumrlBC genes of strain 12 was generated. The hnmr2A gene of LCDC2 was amplified by PC using the primers that they are illustrated in Figure 33B and cloned into pCR II, which generated the plasmid BK-137-3-10, which contains the bmZ gene with a counter-clockwise orientation. In order to change the orientation of the hnnrlh gene. for cloning purposes, the plasmid was digested with BamH I P1269 to release the hmr2 insert, then the two fragments were re-ligated. Plasmid BK-177-3-2 contains the hmw2K gene of LCDC2 with a clockwise orientation. Plasmid BK-177-3-2 was digested with Nde I and Xho I and the hw2A fragment was ligated to pT7-7 which had been digested with Nde I and Sal I, to generate plasmid BK-189- 2-5. The generic expression plasmid JB-2646-1 (Figure 32A) was linearized with XJa I and dephosphorylated. Plasmid BK-189-2-5 was digested with XJba I which released the hnnr2 gene ready to be inserted into the expression vector. Plasmid DS2334-5 thus contains a T7 JumrABC gene cassette consisting of the hmr2h gene of LCDC2 and the unrlBC genes of strain 12.

Example 19 This Example illustrates the construction of plasmid DS-2400-13, which contains a T7 nmrA / T7 / uinrABC cassette, the E. coli cer gene and a kanamycin-resistant gene. Plasmid DS-1843-2 is a vector based on tetracycline resistant pBR328 containing a multiple cloning site inserted between the EcoR I and Pst I sites. DS-1843-2 was linearized with Xho I and dephosphorylated and the gene Kanamycin-resistant protein from pUC-4K was inserted into a Sal I fragment, which generated the plasmid DS-2372-31 which is resistant to both kanamycin and P1269 tetracycline. Plasmid DS-2372-31 was linearized with Bgl II and dephosphorylated and the synthetic E. coli cer gene from N DS-2224-1-4 was inserted into a BamR I fragment, which generated the plasmid DS-2379-2 -6. Plasmid DS-1046-1-1 (Figure 5 3A, Example 3) was digested with Bgl II and Sal I and the T7 gene fragment hmwABC was inserted into DS-2379-2-6 which had been digested with BamH I and Sal I. The resulting plasmid (DS-2391-1) is a vector based on kanamycin resistant pBR and sensitive to tetracycline, which contains the T7 genes umvABC and the cer gene of E. coli. JB-2369-6 (Figure 7f Example 7) was digested with BamH I to release an internal 3 'hmwA / T7 fragment 5' hmwh that was inserted into the unique BamH I site of the vector pBR T7 hmnrABC / cer / kanR . The resulting plasmid pBR T7 anrA / n 15 hmrhBC / cer / kanR (DS-2400-13) thus contains multiple hmwh genes (See Figure 34).

RBanMBM PB IA EXPOSURE In summary, the present invention provides 20 nucleic acid molecules and constructs that incorporate it, which allow the recombinant production of high molecular weight proteins of Haemophilus Influenzae nontypeable, which are protective. Modifications are possible within the scope of the invention.

P1269 Table l. Immunogenicity of various forms of HMW1 and HMW2 in chinchillas.

Groups of 9 chinchillas (i.m.) were immunized on day 1, 14 and 28 with 30 of the indicated antigens adsorbed on alum. Blood samples were collected on day 42. The reactive titre of an antiserum was defined as the reciprocal of the dilution that consistently shows an increase in absorbance corresponding to the P1269 double with respect to that obtained with the serum sample prior to blood extraction. Two groups of numbers indicate two sets of experiments.

Table 2. Protective capacity of various forms of HHW1 and HNW2 against NP colonization with strain 12 of NTHi in chinchillas.

Groups of 9 chinchillas (i.m.) were immunized on days 1, 14 and 28 with 30 ig of indicated antigens adsorbed on alum. The blood samples are P1269 collected on day 42. On day 44, animals were challenged by intranasal inoculations with freshly cultured Streptomycin-resistant strains 12 of NTHi. The dose of bacterial stimulation was 1 x 108 ufe per animal. Nasopharyngeal washes were performed 4 days after inoculation and 25 μ? of nasal lavage were placed on chocolate agar plates. An animal was considered infected if they recovered > 50 ufe of bacteria from 25 μ? of nasal lavage fluid. * Statistical significance was found when compared to control animals using the Nann-Whitney Rank Sum test (p <0.5).

TABLE 3 Molecular weights of mature HMW proteins from several non-typeable strains of H. influenzae Molecular Peeo (kDa) Non-lipophilic strain of H. influenza * 12 Joyc X21 LCDC2 PMH1 15 Protelna Madurat HMW1 125 125.9 104.4 114.0 102.4 103.5 HMH2 120 100.9 111.7 103.9 121.9 P1269 1. Berkowitz et al. 1987. J. Pedlatr. 110: 509. 2. Claesson et al. 1989. J. Pediatr. 114: 97. 3. Black, S.B., H.R. Shinefield, B. Fireman, R. Hiatt, N. Polen, E. Vittinghoff, The Northern California Kaiser Permanent Vaccine Study Center Pediatrics Group. Efficacy in infancy of oligosaccharide conjugate Haemophilus influenzae type b (HbOC) vaccine in a United States population of 61,080 children. 1991. Pediatr. Infect. Dis. J. 10: 97-104. 4. Madore, D.V. 1996. Impact of inununization on Haemophilus influenzae type b disease. Infectious Agents and Disease 5: 8-20.

. Bluestone, C.D. 1982. Current concepta in otolaryngology. Otitis media in children: to treat or not to treat? N. Engl. J. Med. 306: 1399-1404. 6. Barenkamp, S.J. , and F.F. Bodor. 1990. Development of bactericidal serum activity following nontypable Haemophilus influenzae acute otitis media. Pediatr. Infect. Dis. 9: 333-339. 91269 7. Barenkamp, S.J., and E. Leininger. 1992. Cloning, expression, and DNA seguence analysis of genes encoding nontypeable Haemophilus influenzae high-molecular-weight ^ surface-exposed proteins related to filamentous hemagglutinin of Bordetella pertussis. Infect. Immun. 60: 1302-1313. 8. Barenkamp, S.J., and J.W. St. Geme III. 1994. Genes encoding high-molecular-weight adhesion proteins of ^ 10 nontypeable Haemophilus influenzae are part of gene clusters. Infect. Iatmun 62: 3320-3328. 9. St. Geme III, J.W. and S. Grass. 1998. Secretion of the Haemophilus influenzae HMW1 and HMW2 adhesins involves a periplasmic intermediate and requires the HMWB and HMWC proteins. Molec. Hicrobiol. 27: 617-630. ^ 10. St. Geme III, J.W. , S. Falkow, and S.J. Barenkamp. v 1993. High-molecular-weight proteins of nontypeable Haemophilus influenzae mediate attachment to human epithelial cells. Proc. Nati Acad. Sci. USA 90: 2875-2879. 11. Barenkamp, S.J. 1996. Immunization with high-molecular-weight adhesion proteins of nontypeable 25 Haemophilus influenzae modifies experimental otitis media P1269 in chinchillas. Infect. Immun. 64: 1246-1251. 12. Tabor, S., and C.C. Richardson. 1985. A bacteriophage T7 RNA polymerase / promoter system for controlled exclusive expression of specific genes. Proc. Nati Acad. Sci. USA 82: 1074-1078. 13. Patient, M.E. , and D.K. Summers. 1993. ColEl multimer formation triggers inhibition of Escherichia coli cell division. Holec. Microbiol. 9: 1089-1095. 14. Barenkamp, S. 1986. Protection by serum antibodies in experimental nontypeable Haenophílus influenzas otitis media. Infect. Immun. 52: 572-578.

. Yang, Y.-P., S.M. Loosmore, B. Underdown, and M.H. Klein. 1998. Nasopharyngeal colonization with nontypeable H. influenzae in chinchillas. Infect. Immun. 66: 1973-1980. 16. Fleischmann et al. 1995. Whole-genome random seguencing and assembly of Haemophilus influenzae Rd. Science 269: 496-512. 17. O'Hagan, DT. 1992. Oral delivery of vaccines. Formulation and clinical pharmaco kinetic considerations.

P1269 Clin. Pharmacokinet 22 (t): 1-10. 18. Ulmer et al. 1993. Curr. Opinion Invest. Drugs 2: 983-989. 19. Lockhoff, 0., 1991. Glycolipids as immunomodulators: Synthesis and properties.

. Nixon-George A., et al., 1990. The adjuvant effect of stearyl tyrosine on a recombinant subunit hepatitis B surface antigen. J. Immunol 144 (12): 4798-4802.

P1269

Claims (21)

1. wsivnroK-AciowEsi 1. A nucleic acid molecule characterized by a functional promoter in E. coli and functionally coupled to a modified operon of a non-typeable strain of Haemophilus comprising genes A, B and C, wherein the A gene of the operon contains only a nucleic acid sequence encoding a mature, high molecular weight protein of the non-typeable strain of Haemophilus.
2. 2. The nucleic acid molecule according to claim 1, wherein the promoter is the T7 promoter.
3. 3. The nucleic acid molecule according to claims 1 6 2, wherein the operon encodes the high molecular weight protein (HMW1) of the non-typeable strain of Haemophilus or the high molecular weight protein 2 (HMW2) of the strain not typifiable of Haemophilus.
4. 4. The nucleic acid molecule according to any of claims 1 to 3, wherein the non-typeable strain of Haemophilus is selected from the group consisting of strains 12, Joyc, K21, PMH1 and 15 of non-typeable Haemophilus influenzae.
5. 5. The nucleic acid molecule according to any of claims 1 to 4, wherein the nucleic acid sequence encoding a protein P1269 high mature molecular weight has a nucleic acid sequence selected from those with SEQ ID NOS: 27,, 31, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 or encodes a "protein HMW1 or HMW2 that has an amino acid sequence 5 selected from those having SEQ ID NOS: 28, 32, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73.
6. 6. The nucleic acid molecule according to any of claims 1 to 5 which it is also characterized by an additional nucleic acid sequence . { '- 10 which encodes the mature high molecular weight protein of a non-typeable strain of Haemophilus.
7. 7. The nucleic acid molecule according to any of claims 1 to 6, further characterized by the cer gene of E. coli.
8. 8. An isolated and purified nucleic acid molecule encoding a high molecular weight protein (HMW) of a non-typeable strain of Haemophilus influenzas, which is characterized by: (a) a DNA sequence selected from group 20 consisting of of those shown in Figures 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 (SEQ ID NOS: 25, 27, 29, 31, 33, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64); Or (b) a DNA sequence encoding a high molecular weight protein having a sequence of P1269 amino acids selected from the group consisting of those shown in Figures 18, 19, 20, 21, 22, 22, 24, 25, 26 and 27 (SEQ ID NOS: 26, 28, 30, 32, 34, 35 , 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65).
9. 9. A vector adapted for transformation of a host, characterized by the nucleic acid molecule according to any of claims 1 to 8.
10. The vector according to claim 9 which is a plasmid vector.
11. The vector according to claim 10, wherein the plasmid has the identifying characteristic of a plasmid that is selected from the group consisting of: DS-1046-1-1 (ATCC No: 203263) JB-2507-7 (ATCC No: 203262) BK-86-1-1 (ATCC No: 203258) BK-35-4 (ATCC No: 203259) BK-76-1-1 (ATCC No: 203261) DS-2334-5 (ATCC No: 203260) DS-2400-13 (ATCC No: 203257)
12. 12. A strain of E. coli transformed by an expression vector according to any of claims 9 to 11 and expressing a high molecular weight protective protein of a non-typeable strain. of Haemophllus.
13. 13. A high-weight protective protein Recombinant molecular p1269 of a non-typeable strain of Ha &amphibole or an immunogenic fragment or an analogue thereof capable of being produced by the transformed E.coli strain according to claim 12.
14. 14. A plasmid vector for the expression of a high protein molecular weight of a non-typeable strain of Haemophllus and characterized by the T7 promoter, a cloning site for the insertion of a nucleic acid molecule in the plasmid vector and portions B and C of the operon of a non-typeable strain of Haemophilus.
15. 15. The plasmid vector according to claim 14, which is also characterized by the gene cer of?. coli
16. 16. The plasmid vector according to claim 14, having the identification characteristics of a plasmid which is the plasmid JB-2646-1 (ATCC No: 203256).
17. 17. An isolated and purified protective HMW1 protein from a non-typeable strain of Haemophil s that is free from contamination by the HMW2 protein of the same non-typeable strain of Haemophilus or a protective HMW2 protein isolated and purified from a non-typeable strain of Haemophilus that it is free from contamination by the HM 1 protein of the same non-typeable strain of Haemophilus.
18. 18. The HMW1 or HMW2 protein according to claim 17, wherein the non-typeable strain ofP1269 Haemophllus is selected from the group consisting of Joyc strains, K21, LCDC2, PMH1 and 15 Haemophilus influenzas not typable.
19. 19. The HMW1 or HMW2 protein according to any of claims 17 or 18 which is characterized by an amino acid sequence selected from the group consisting of SEQ ID NOS. 28, 32, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73.
20. 20. A method for the production of a high molecular weight protective protein of a non-typeable strain of Haemophllus, comprising: transforming E. coli with a vector according to any of claims 9 to 12, cultivating Z. coli to express the mature high molecular weight encoded protein (HNW), and isolating and purifying the expressed HNW protein. The method according to claim 20, wherein the isolation and purification process includes separating the HMWA protein from proteins B and C. P1269 REffTMfflf? ' IMVGHCIÓ High molecular weight protective proteins (HMW) that are produced in recombinant form by the expression of E. coli using an efficient promoter in E. coli and a nucleic acid molecule containing a modified operon from a non-typeable Haemophilus strain. The modified operon contains only the portion of the A region encoding the mature HMW protein and the entire B and C regions of the operon. Reinforced levels of expression of the HMW proteins can be achieved by including the E. coli cer gene, an additional copy of the A region of the operon encoding the mature protein, or both, in the expression vector. The nucleotide and deduced amino acid sequences of the hnorl and hmw2 genes and the HMW1 and HMW2 proteins, respectively, of several strains of non-typifiable Haemophilus influenzas have been identified. P1269