KR100216390B1 - Haemophilus outer membrane protein - Google Patents

Abstract

According to the present invention, there is provided an isolated purified nucleic acid from a specific Haemophilus influenzae line that encodes at least part of the D15 outer membrane protein of Haemophilus. These nucleic acids are used to make proteins, peptides, and polypeptides free of contaminants associated with Haemophilus for diagnostic and medical treatment. Moreover, such nucleic acids are used in the diagnosis of Haemophilus infection. Antisera obtained by immunization with nucleic acid D15 outer membrane proteins or peptides can be used for diagnostic medical treatment.

Description

[Name of invention]

Haemophilus outer membrane protein

[Field of Invention]

The present invention relates to molecular genetics, and in particular to the cloning of outer membrane protein D15 of Haemophilus.

[Background of invention]

Haemophilus influenzae type b (Hib) is a major factor causing bacterial meningitis in children under 5 years of age. Antibodies that are protective against these diseases are caused by polysaccharides in the form of encapsulated organisms, and vaccines using purified polyribosyl ribitol sulphate (PRP) as antigens have become gaps. The vaccine provides 90% protection for adults and children over 24 months of age, but not for children under 24 months (Zangwill et al 1993). Like other polysaccharide antigens, PRP does not induce proliferation of T-helper cells. The conjugation of PRP polysaccharides and protein carriers gives the vaccine T-cell related properties to enhance the immune response to the PRP antigen. Recently four PRP-carrier conjugate vaccines are available. They are based on Haemophilus influenzae type b capsular polysaccharide conjugated to diphtheria toxoid, tetanus toxoid, or meningococcal outer membrane protein (Zangwill et al 1993).

However, recent Haemophilus conjugate vaccines are only protective against meningitis caused by Haemophilus influenzae type b. For other invasive strains (types a and c), more importantly, they are not protective against postnatal delivery and non-type (NTHi) strains, a common cause of sepsis, pneumonia and otitis media in newborns. To treat otitis media in the United States alone, it costs between $ 1 to $ 2 billion annually for antibodies and surgical procedures such as tonsillectomy, adenoid extraction, tympanotomy, and the like. In groups of 2 to 6 months and in certain high risk groups, provision of conservative and cross-reactive non-capsular Haemophilus influenzae immunogens is required to achieve universal protection against Haemophilus influenzae related diseases. . Methods of inducing immunity to disease are continually improved and now use the subunits and better defined substances as antigens. This has been undertaken to minimize or eliminate the side effects they can cause, while maintaining their immunogenicity to provide protection against certain natural sources. Therefore, developing a universal vaccine against Haemophilus using cross-reactive outer membrane proteins, fragments, analogs, and / or their corresponding peptides is a very attractive task. Such antigens can be introduced into conventional Haemophilus influenzae type b conjugate vaccines as additional immunogens, or used as carriers of autologous tissue for Haemophilus influenzae encapsulated polysaccharides. High molecular weight outer membrane proteins D15 are known as cross-reactive antigens in both the non-type strains of Haemophilus influenzae and the type b strains (Thomas et al. 1990). D15 is naturally cell surface-exposed and, as determined by SDS-PAGE analysis, has a molecular weight of about 80 kDa. It is necessary to provide a sequence of DNA molecules encoding these D15 outer membrane proteins and peptides corresponding to their portions for diagnosis and immunity, and to generate diagnostic and immunological reagents. Diseases caused by Haemophilus are severe and require improved methods for preventing, detecting and treating diseases such as otitis media, laryngitis, pneumonia and bronchitis.

[Summary of invention]

The present invention is directed to providing purified and isolated nucleic acid molecules comprising at least a portion encoding a D15 outer membrane protein of Haemophilus spp. Nucleic acid molecules comprising at least the portion encoding the D15 outer membrane protein are useful for specific detection of Haemophilus strains and for diagnosis of infection by Haemophilus. Purified and isolated nucleic acid molecules, such as DNA comprising at least a portion encoding a D15 outer membrane protein, are useful for expressing the D15 gene by recombinant DNA methods that provide purified and isolated D15 outer membrane protein in an economical manner. Do.

D15 outer membrane proteins or fragments thereof or analogs thereof are useful for the preparation of vaccines against diseases caused by Haemophilus, immunogenic compositions useful in diagnosing infection by Haemophilus, and as a tool for generating immunogenic reagents. Modulo- or polyclonal antisera (antibodies) raised against the D15 outer membrane protein produced according to the present invention can be used for diagnosis of infection by Haemophilus, specific detection for Haemophilus strains (eg, in vivo and ex vivo). And in diseases caused by infection with Haemophilus.

Peptides corresponding to portions of the D15 outer membrane proteins and their analogs are useful in immunogenic compositions for preparing vaccines against diseases caused by Haemophilus, for diagnosing infection by Haemophilus, and generating immunogenic reagents. It is useful as a tool. Mono- or polyclonal antisera (antibodies) raised against these peptides produced in accordance with the present invention can be used for diagnosis of infection by Haemophilus, specific detection of Haemophilus strains (eg, in vivo and ex vivo verification). And is used for latent immunity as a treatment for diseases caused by infection with Haemophilus.

Therefore, according to one aspect of the present invention, purified and isolated nucleic acid molecules are provided that comprise at least a portion encoding a D15 outer membrane protein. The nucleic acid has a DAN sequence selected from:

(a) a DNA sequence corresponding to any one of the first (a) to the first (e), or their complementary strands, and

(b) a DNA sequence that hybridizes with the DNA sequence defined in (a) under stringent conditions. The DNA sequence defined in (b) is preferably at least 90% identical to the DNA sequence defined in (a). In particular, the DNA sequence defined in (b) may comprise the consensus sequence shown in Figure 1 (f).

Another aspect of the invention is to provide purified and isolated D15 outer membrane proteins or portions thereof. D15 outer membrane protein is Haemophilus D15 outer membrane protein and more particularly Haemophilus influenzae D15 outer membrane protein and Haemophilus influenzae strains are Haemophilus influenzae type b strains such as Haemophilus type b strains, such as Ca Eagan, MinnA, or PAK 12085 It may be a non-type Haemophilus influenzae lineage such as SB33.

The present invention also encompasses recombinant plasmids for the modification of the host in embodiments, recombinant plasmids comprising a plasmid vector in which a DNA fragment comprising the purified and isolated DNA molecules provided herein is inserted into the vector. Such recombinant plasmids comprise a plasmid vector into which a DNA fragment comprising a bp fragment is inserted, at least as shown in Figure 1 from the above-mentioned DNA molecules. The recombinant plasmid could be plasmid DS-712-2-1 with ATCC accession no. 75604 deposited on Nov. 4, 1993 and plasmid JB-1042-5-1 with ATCC accession no. 75006 deposited on Nov. 4, 1993 have.

Plasmids may be adapted for expression of the D15 outer membrane protein encoded in homologous or heterologous host cells by incorporation into a recombinant vector according to another aspect of the present invention. The recombinant vector comprises at least a DNA fragment comprising at least 8 dp fragments selected from at least the aforementioned DNA molecules and expression is effectively a DNA fragment for expression of the gene products encoded by them in a host cell. It means being a couple. The plasmid for the expression of the encoded D15 outer membrane protein could be the plasmid DS-880-1-2 deposited in Nov. 4, 1993 with accession number 75605 adapted for the expression of the D15 outer membrane protein in E. coil. have. The selected DNA fragment encodes polypeptides of at least 6 residues and may be specifically selected from fragments encoding the polypeptides of Table 2 below. Such DNA fragments further comprise a nucleic acid sequence encoding a leader sequence for escape of the gene product from the host. As a host for expression, for example, E. coli, Bacillus, Haemophilus, fungus, yeast or baculovirus system can be used.

Additional aspects of the invention are directed to vaccines, diagnostics, or immunogenic reagents that encode a protein encoded by a DNA molecule comprising at least a D15 outer membrane protein, fragment, or portion encoding a functional analog of that protein. Contains what is used to generate. The invention also includes antisera (antibodies) raised against a D15 outer membrane protein encoded by a DNA protein comprising at least a portion encoding a D15 outer membrane protein and purified peptides corresponding to a portion of the D15 outer membrane protein It is in the treatment and latent immunization of diseases caused by Haemophilus.

Another aspect of the invention is isolated purified peptides comprising at least a portion of the D15 outer membrane protein, or amino acids corresponding to amino acids of their modifications or mutants that maintain immunogenicity. Such peptides can be produced by recombinant methods or prepared by peptide synthesis, which results in pure peptides free of contaminants associated with bacteria, usually containing D15 outer membrane proteins. Such peptide synthesis preferably has amino acids selected from those set forth in Table 2.

According to another aspect of the invention, there is provided an immunogenic composition comprising fragments of D15 outer membrane proteins, their functional analogues, or physiological-carriers for them as described above. Such immunogenic compositions can be made into vaccines, particularly for in vivo infusions that are protective against diseases caused by Haemophilus. For that purpose, immunogenic compositions can be formulated into microparticles, capsules, liposomes. In addition, such immunogenic compositions may be provided in combination with targeting molecules for delivery to the surfaces of specific cells or mucous membranes of the immune system.

According to another aspect of the present invention, Haemophilus comprising the step of providing a protective immunity against Haemophilus by incorporating an effective amount of an immunogenic composition or a nucleic acid as mentioned above in a subject, including a mammal, such as a human. It provides a method of inducing protection against diseases caused.

Still another object of the present invention includes chimeric molecules provided in the form of a D15 outer membrane protein or a peptide or a corresponding polypeptide or other polypeptide or protein or polysaccharide. The linked polypeptide or protein may comprise a surface protein or peptide corresponding to it from a pathogenic bacterium, which may be the P1, P2 or P6 outer membrane protein of Haemophilus influenzae. Linked polysaccharides preferably include PRP molecules from Haemophilus influenzae.

Detailed description of the invention

Any Haemophilus strains with D15 genes are convenient for providing purified nucleic acid molecules (which may be in the form of DNA molecules) that comprise at least a portion encoding a D15 outer membrane protein as typical by embodiments of the invention. Can be used. Such strains are generally available from clinical materials and bacterial culture collections such as the Aerican Type Culture Collection. Haemophilus influenza strains include type a, b and c strains and non-type strains and D15 protein, other bacteria that produce fragments or analogs thereof. Suitable Haemophilus strains include:

Haemophilus influenzae type b strain Ca,

Haemophilus influenzae type b strain MinnA,

Haemophilus influenzae type b strain Eagan,

Haemophilus influenzae type b strain SB33,

Haemophilus influenzae type b strain PAK 12085.

In this application the term D15 outer membrane protein is used to define a group of D15 proteins, including, for example, those having naturally occurring modifications in the amino acid sequences found in the various strains of Haemophilus. Separately defined DNA molecules comprising portions encoding at least the D15 outer membrane protein according to the invention have modifications that occur naturally in their nucleic acid sequences found in, for example, various strains of Haemophilus and DNA molecules encoding functional analogs of membrane proteins. In this application, if the first protein is immunologically related to the second protein and / or has the same function as the second protein, the first protein is said to be a functional analog of the second protein. For example, functional analogs may be fragments of proteins or their substituents, additions or deletions.

In view of the present invention, the D15 gene isolated from Haemophilus influenzae type b strain Ca is shown in Figure 1 (a), Haemophilus influenzae type b strain Eagan, Figure 1 (b), Haemophilus influenzae type b strain MinnA. In Fig. 1 (c), the Haemophilus influenzae type b strain SB33, and in Fig. 1 (d), the Haemophilus influenzae non-type b strain PAK 12085 are shown in Fig. 1 (e). Comparing the D15 gene from the D15 outer membrane protein and the deduced amino acid sequences from these Haemophilus influenza strains, it can be seen that the genes and proteins are very conserved (FIGS. 1 (f) and 3). The consensus sequence (SEQ ID NO: 55) of the D15 genes is shown in Figure 1 (f).

Purified isolated DNA molecules according to the present invention, including portions encoding at least the D15 outer membrane protein of Haemophilus species, have the following advantages.

Haemophilus strains in vivo or ex vivo are used as nucleic acid probes with specific recognition.

Products encoded by DNA molecules are antigens that produce haemophilus-specific antisera as diagnostic reagents, useful as vaccines against diseases caused by Haemophilus spp. And antigens for detecting infection by Haemophilus. .

Peptides corresponding to the portion of the D1 outer membrane protein according to the invention are diagnostic reagents, antigens producing haemophilus-specific antiserum, which are used for vaccines against diseases caused by Haemophilus spp. And infection with Haemophilus. Useful as antigens for detection.

Embodiments illustrating the present invention in detail are as follows.

(i) DNA sequences for encoding D15 outer membrane protein from Haemophilus influenzae type b Ca lineage

Clones that produce D15 outer membrane proteins of Haemophilus influenzae type b (Hib) were prepared using Haemophilus influenzae type b OMP-specific polyclonal antibodies as previously described by Berns and Thomas 1965, Thomas and Rossi 1986. Nomic libraries were screened and separated. The DNA fragment encoding the D15 protein is isolated and subcloned into pUC19 to produce pUC19 / D15 (Figure 2) and used to transform E. coli HB101, as in Example 1. Plasmid DNA was obtained from two individual clones of E. coli HB101 containing the pUC19 / D15 plasmid. End-staining chemistry and oligonucleotide primers synthesized in ABI DNA synthesizer model 380B were sequenced by ABI DNA sequencer model 370A and separated by chromatography. Nucleotide sequence analysis of the D15 gene revealed that it contained an putative promoter and an open reading frame encoding 789 amino acids (FIG. 1A).

The first 19 amino acids of the translated open reading frame form typical leader sequences found in other Haemophilus type b outer membrane proteins such as P1 and P2. The N-terminal sequence of the immuno-affinity purified natural D15 antigen is determined by automated Edman degeneration using an ABI 477A protein sequencer, Ala-Pro-Phe-, which is the D15 gene shown in Figure 1 (a). It is identical to the N-terminal amino acid sequence Ala-Pro-Phe-Val-Ala-Lys- which is expected from the sequencing of.

(ii) sequences of D15 genes from other Haemophilus influenzae strains

As in Examples 2, 3, and 4, other Haemophilus influenzae by screening the chromosomal library of Haemophilus influenzae type b strains Eagan, MinnA and non-type Haemophilus influenzae (NTHi) strains SB33 and PAK 12085. Isolate D15 genes from lines. Positive hybrid clones are spread out on a plate and subjected to secondary screening. The restriction map of the resulting clones is as shown in FIG. The nucleotide sequences of the D15 genes (Figures 1 (b) through 1 (e)) and their derived amino acid sequences were compared for all these clones (Figure 3). The D15 amino acid sequences of the Haemophilus influenzae type b lineages are identical and differ only a few amino acids from the amino acid sequence of the D15 protein from non-type lines (Figure 3).

(iii) expression of D15 protein and its fragments in E. coli

Since D15 is expressed in small amounts by the Haemophilus influenza strains, it may be necessary to express these antigens as recombinant proteins in heterologous systems such as E. coli, or to alter Haemophilus influenzae organisms to enhance natural D15 expression. good. HindIII / EcoR I fragments of Haemophilus influenzae type b Ca lineage DNA are expressed in pUC19 but not pUC18. This is a tightly regulated, inducible system in which the lac promoter is of great use for the expression of heterologous proteins in E. coli despite the presence of the natural D15 gene promoter. The T7 expression system is described in US Pat. No. 4,952,496. Therefore, clones are formed using a T7 system expressing a mature D15 protein containing additional methionine residues at the amino acid terminus. The D15 signal sequence was removed during formation. Full length recombinant D15 (rD15) is expressed in a carrier that allows for easy purification of the D15 protein. The D15 genes from the Haemophilus influenzae type b Ca strain and the Haemophilus influenzae non-type SB33 strain were expressed in high water in E. coli using a T7 system that allowed the mass production of rD15 protein. A clone DS-880-1-2 expressing the SB33 D15 gene is described herein (4th and 5th Examples). rD15 protein is immunogenicly similar to natural protein isolated from Haemophilus influenzae type and non-type lines. Thus, rD15 protein can be used as a cross-reactive antigen in diagnostic tools that detect many, if not all, haemophilus influenzae and other bacteria or analogs that form D15 outer membrane proteins. On the other hand, rD15 can be used as an antigen to specifically detect the presence of Haemophilus influenzae in the sample.

As in Example 6, the truncated D15 fragment was expressed in E. coli in the form of a fusion protein with glutathione S-transferase (GST). The structure was designed to express the N-terminal fragment of the D15 protein. Fusion proteins were expressed at high levels from the pGEX-2T structure and N-terminal fragments were removed by treating GST carrier proteins with thrombin. This process produces molecules called N-maradan rD15 fragments containing amino acids 63-223 of the D15 protein. These N-terminal rD15 fragments are highly immunogenic and induce antibodies that are protective against the challenge of live Haemophilus influenzae.

(iv) Purification of Natural D15 from Haemophilus Influenzae Cell Paste

The present invention also provides a method for obtaining purified natural D15 protein from Haemophilus influenzae. These proteins are affinity-purified and extracted from Haemophilus influenzae type and non-type cell pastes by a process comprising dissolving the protein in an aqueous solution of detergent (third example). D15 protein from non-type Haemophilus influenzae strain 30 is dissolved in 50 mm Tris-HCl / 0.5% Triton X-100 / 100 mM EDTA buffer, pH 8.0 and further purified by D15-specific monoclonal antibody affinity column ( 5a). 80 kDa protein was eluted from the column by 50 mM diethyleneamine, pH 12.0, which reacted with D15-specific monoclonal antibody in immunoblot analysis (Figure 5b). In laboratory animals, the natural D15 protein is very immunogenic. As determined in immunoblot analysis, rabbit anti-D15 antiserum reacted with Haemophilus influenzae isolation mold.

(v) Purification of full length recombinant D15 protein expressed in E. coli

Full length recombinant D15 (rD15) protein was expressed in inclusions in E. coli. As shown in Figure 6, E. coli cells were lysed with 50 mM Tris-HCl / pH 8.0 and purified with 50 mM Tris containing 0.5% Triton X-100 with 100 mM EDTA buffer, pH 8.0 to purify the rD15-containing material. do. After centrifugation, more than 95% of the proteins in the resulting pellets were 80 kDa proteins by SDS-PAGE analysis, which reacted with D15-specific monoclonal antibodies in immunoblot analysis. The N-terminal amino acid sequence of rD15 is Met-Ala-Pro-Phe-Val-Ala-Lys-Asp (SEQ ID NO: 54), which is identical to the expected amino acid sequence.

The rD15 containing can be dissolved in a mixture of PBS, 0.5% Triton X-100, 100 mM EDTA, 8M urea (Example 8). More than 80% of D15 protein is dissolved after dahalisis on PBS to remove urea. Such soluble rD15 antigens are used in immunological studies as described below. From a shake-flask experiment, it was estimated that 10 mg of soluble rD15 protein was obtained from 1 L of E. coli bacterial culture. It is evident that growing recombinant E. coli strains under optimized fermentation conditions significantly increases rD15 production levels.

(vi) immunogenicity of full length recombinant D15 protein (rDNA)

Immunogenicity of full length recombinant D15 protein (rDNA) was studied in guinea pigs and mice. Using the immune protocols described in FIG. 7, high doses of IgG were induced in the guinea pig when 15 μg of rD15 was administered in the presence of Freund's adjuvant or AlPO ₄ . Experiments in rats showed immunogenic proteins in Freund's supplements (Figure 8a), in the presence of AlPO ₄ (Figure 8b), and even in small doses of 5 µg.

The protective ability of rD15 against Haemophilus influenzae type b infection was investigated in a young rat model of bacteremia described essentially by Loeb (1987). Therefore, young mice immunized with the guinea pig anti-rD15 antiserum showed bacteremia much smaller than the control injected with pre-bleed serum. This is consistent with what has already been reported by Thomas et al. (1990).

(vii) Purification and Characterization of N-terminal rD15 fragments

As in Example 6, the truncated rD15 fragment (residues 22-223) corresponding to the N-terminus of the D15 protein was expressed in E. coli as a soluble protein fused to GST. Fusion protein (46 kDa) was easily extracted using phosphate buffered saline (PBS). Purification of GST-D15 fragment fusion protein was by single-step affinity-purification on a glutathione-Sepharose 4B column (lane 3 in FIG. 9). Cutting the 46 kDa fusion protein with thrombin yields two fragments (lane 4 in Figure 9), a 20 kDa polypeptide with the expected size of the 26 kDa protein corresponding to the purified GST standard (lane 2 in Figure 9) and the N-terminus of the rD15 fragment. to be. Separation of these two proteins was performed by the second round of glutathione-Sepharose 4B affinity chromatography. It was estimated from the shake-flask experiment that 1 mg of purified N-terminal rD15 fragment was regenerated from 1 L of E. coli bacterial culture. It is evident that growing recombinant E. coli strains under optimized fermentation conditions significantly increases the level of production of N-terminal rD15 fragments.

Purification of 20kDa polypeptide and 26kDa protein was confirmed by immunoblotting and protein sequencing. The N-terminus of the 20 kDa polypeptide is NH ₂ -Ser-Leu-Phe-Val-Ser-Gly-Arg-Phe-Asp-Asp-Val-Lys-Ala-His-Gln-Glu-Gly-Asp-Val-Leu- Val-Val-Ser- (SEQ ID NO: 12) and corresponds to 63-85 residues of the primary sequence of D15. This result means that there is a pseudo thrombin cleavage site in the D15 sequence, such that the first 42 amino acids of the rD15 fragment are cleaved during thrombin treatment. The final N-terminal rD15 fragment is therefore 161 amino acids, corresponding to 63 to 223 residues of the D15 primary sequence. The N-terminal sequence (NH ₂ -Ser-Pro-Ile-Leu-Gly-Thr-Trp-Lys-SEQ ID NO: 13) of the obtained 26kDa protein was confirmed to be GST.

(viii) immunogenicity of the N-terminal rD15 fragment

Immunogenicity of the N-terminal rD15 fragment was tested in guinea pigs using various adjuvants. Administration of 10 μg of N-terminal rD15 fragment using the immune protocols described in Example 10 resulted in a good palpation response in the guinea pigs for most of the adjuvant tested. The group of guinea pigs immunized with the N-terminal rD15 fragment in Freund's adjuvant showed the highest amount of anti-D15 IgG. The next best aid is Titermax (CytRx Inc.). The other two adjuvants are equally effective with TPAD ₄ (tripalmityl-Cys-Ser-Glu ₄ ) and AlPO ₄ .

(ix) Protective ability of N-terminal rD15 fragments against Haemophilus influenzae type b challenge

An in vitro challenge model to test the protective ability of an antigen against diseases caused by Haemophilus is a young rat model, as described in 1987 by Loeb. The protective ability of the N-terminal rD15 fragment to Haemophilus influenzae type b challenge was investigated in this rat model. As shown in Table 1, young rats latent immunized with rabbit anti-N-terminal rD15 fragment antiserum showed significantly lower bacteremia compared to those injected with sun-bleed.

It was very interesting to find out the protective epitope (s) of these N-terminal rD15 fragments, since we found that antiserum translating to N-terminal rD15 fragments was protective in the young rat model of bacteremia. The first nine overlapping peptides of the D15 protein, shown in Table 2, can be chemically synthesized based on the amino acid sequence (Figure 1) derived from the sequence of the D15 gene from Haemophilus influenzae type b Ca. These synthesized peptides were tested for their reactivity with rabbit or guinea pig antisera raised by ELISAs against purified N-terminal rD15 fragments. As shown in Table 3, both anti-serum of guinea pigs and rabbits reacted with D15 peptides, including D15-P4 to D15-P8 peptides covering 93-209 residues of the D15 primary sequence.

Further studies were conducted to determine if protection against Haemophilus influenzae type b observed using rabbit anti-D15 serum in young rats could be neutralized by D15 peptides. In the first experiment, rabbit anti-N-terminal rD15 fragment antiserum was injected into seven young rats with or without a mixture of nine D15 peptides (D15-P2 to D15-P10). Animals in the positive control group were injected with rabbit anti-N-terminal rD15 fragment antiserum mixed with defined D15 fragments, and the negative control was injected with only nine D15 peptides. As shown in Table 4, young rats that were lately immunized with rabbit anti-N-terminal rD15 fragment antiserum had significantly lower bacteremia levels (3%, compared with the negative control group (group # 4, 64%)). p = 1.2 × 10 ⁻⁷ ), which is consistent with the results obtained earlier. The protection mediated by rabbit anti-N-terminal rD15 fragment antiserum was neutralized significantly by the addition of purified N-terminal rD15 fragment (groups # 3, 64%), between groups # 3 and # 4 (p = 0.09). There is little difference in the level of bacteremia. The addition of a mixture of nine D15 peptides between the two groups, although compared to group # 1 (3%), only slightly neutralized the protection conferred by the antiserum (groups # 2, 13%). The count number of bacteria in is statistically significant (p = 0.0037).

In order to more clearly define the protective epitope (s) of the N-terminal rD15 fragment, the experiment was highly reactive with rabbit anti-N-terminal rD15 fragment antiserum and selected five peptides (D15-P4 to D15-P8). Is repeated with a mixture of The results of this second experiment showed that the protection observed using rabbit anti-N-terminal rD15 fragment antiserum (Table 5, Group # 1) was completely hindered by the addition of a mixture of these five peptides (Table 5). , Group # 2, 106%, p = 0.5310 × ^-8 . These results indicate that mixtures of D15 synthetic peptides can be used as antigens to induce protective antibodies against Haemophilus influenzae.

(xi) Identifying and characterizing immunogenic epitopes of D15 using synthetic peptides

To map the linear B-cell epitopes of D15, overlapping synthetic peptides representing the entirety of D15 are each coated onto ELISA plates and tested with several anti-rD15 antiserum as in Example 19. The results are summarized in Table 6. Rat antiserum raised against rD15 reacted with all D15 peptides, but major epitopes were peptides D15-P8 (180-209 residues-SEQ ID NO: 21), D15-P10 (219-249 residues-SEQ ID NO: 23). ), D15-P11 (241-270 residues-SEQ ID NO: 24), D15-P26 (554-582 residues-SEQ ID NO: 39), respectively. Rabbit anti-D15 antiserum was composed of peptides D15-P4 (93-122 residues-SEQ ID NO: 17), D15-P14 (304-333 residues-SEQ ID NO: 27), D15-P36 (769-798 residues-SEQ ID NO: Number: 49) The antiserum of the guinea pigs raised against rD15 was expressed by peptides D15-P2 (45-72 residues-SEQ ID NO: 15), D15-P4 (93-122 residues-SEQ ID NO: 17), D15-P6 (135-). 164 residues-SEQ ID NO: 19), D15-P8 (180-209 residues-SEQ ID NO: 21), D15-P14 (304-333 residues-SEQ ID NO: 27), D15-P27 (577-602 residues) Field-SEQ ID NO: 40). Since there are only two peptides recognized by rD15-specific antiserum from all three animal species, the immunodominant B-cell epitopes of D15 are therefore peptides D15-P4 (93-122 residues-SEQ ID NO: 17), D15. -P14 (304-333 residues-SEQ ID NO: 27). These results indicate that peptides containing the linear B-cell epitope sequences identified above can be used as target antigens, for example in diagnostic tools that detect the presence of anti-D15 and anti-hemophilus influenza antigens in a sample. it means.

(xii) Identification and characterization of immunodominant T-cell epitopes of D15 using synthetic peptides.

As new elements of the cytokine family have been identified and characterized, the importance of cytokine networks and their replacements in the pathology and immunity within immune and inflammatory responses in new immunity and inflammation are becoming more certain. Mill et al. (1993) recently reported the rapid elimination of pertussis from rat lungs by immunizing twice with a six-week challenge or whole-cell pertussis vaccine following a respiratory infection. Spleen cells from these immunized mice secreted high levels of IL-2 and IFN-γ and low levels of IL-5 in the presence of pertussis antibodies (pertussis toxin, filamentous hemagglutinin (FHA) and pertactin). Turned out. These results suggest that proliferation of Th1 cells (T-cells producing high levels of IL-2 and IFN-γ) is very important for recovery from respiratory infections. The formation of Th1 and Th2 cell subsets is regulated by a balance between other groups of cytokines, mainly IL-12 and IL-4 (TrinCHleri, 1993). IL-12 and IL-4 are responsible for the differentiation of Th1 and Th2 cells, respectively. One of the roles of Th2 cells in the immune system is to provide adjuvant activity to induce high levels of antigen-specific antibodies following immunization. Antigens containing Th1 epitope (s) stimulate antigen-specific T-cells to produce high levels of IL-2 and IFN-γ, while the Th2 epitope (s) produce high levels of IL-4 . Th0 epitope (s) stimulate the synthesis of IFN-γ and IL-4.

Little is known about the immune response of cells against outer membrane proteins of Haemophilus influenzae and their role in protection against Haemophilus influenzae infections and diseases. Eventually, the inventors conducted a study of cellular responses from mice following rD15 immunization. D15-specific T-cell epitopes were determined using D15 peptides and T-cell lines obtained from five BALB / c mice immunized with rD15 (Example 23). Lymphocyte proliferation response of D15-specific T-cell lines to overlapping D15 peptides was determined in conventional cytokine assays, as described in Example 24. The results are summarized in Table 7, which revealed that only stimulation with specific synthetic peptides elicited proliferative responses and secreted specific cytokines. Residues 114-143 (D15-P5-SEQ ID NO: 18), 577-602 (D15-P27-SEQ ID NO: 40) and 219-249 (D15-P10-SEQ ID NO: 23), 262-291 (D15-P12 -SEQ ID NO: 25), 390-416 (D15-P18-SEQ ID NO: 31), 410-435 (D15-P19-SEQ ID NO: 32), 554-582 (D15-P26-SEQ ID NO: 39), 596 Synthetic peptides corresponding to -625 (D15-P28-SEQ ID NO: 41), 725-750 (D15-P34-SEQ ID NO: 47) and 745-771 (D15-P35-SEQ ID NO: 48), respectively; Specificity BALB / c Th0 cells and Th1 cells appeared to be very stimulating. Therefore, these immunogenic T-cell epitopes can be used as germ cell carriers for PRP, and / or OMP B-cell epitopes to enhance their immunogenicity. The Th1 cell epitopes found above are useful for Haemophilus influenzae vaccine formatting that induces Haemophilus influenzae-specific cellular immune responses.

(xiii) immunogenicity of D15 peptides

To determine if synthetic D15 peptides were immunogenic, each of the free peptides was assayed for their immunogenicity. Peptides immunized by ELISA and immunoblotting, as well as natural D15 and rD15 rabbit and guinea pig anti-peptide antiserum were tested. As shown in Table 8, D15-P25 (SEQ ID NO: 39), D15-P29 (SEQ ID NO: 42), D15-P30 (SEQ ID NO: 43), and D15-P31 (SEQ ID NO: 44) were obtained by ELISA. All guinea pig anti-D15 peptide antisera, except those raised for, were immunogenic. Induction of large amounts of peptide-specific IgG antibodies by free peptides means that most peptides contain both functional T-helper determinants and B-cell epitope (s). In addition, these anti-peptide antisera recognized D15 in immunoblot assays. Most peptides contain strong functional T-helper determinants and induce strong IgG antibody responses in mammals, making them suitable for use as immunogens for the preparation of Haemophilus influenzae vaccines. According to immunoblotting, D15 peptide-specific antiserum cross-reacted with D15 from non-type lines of Haemophilus influenzae. These results indicate that immunogenic D15 peptides contain epitopes that are highly conserved in the type and nontype lineages of Haemophilus influenzae. In addition, polyclonal antibodies against these epitopes are useful for detecting Haemophilus influenzae in biological samples.

Therefore, these conservative epitopes of D15 can be used individually or in combination to produce cross-reactive synthetic immunogens against the type and non-type strains of Haemophilus influenzae and other bacteria, fragments or analogs that produce D15 protein. Can be. The peptides described above can be further polymerized, altered into lipopeptides by lipids, or linked to polysaccharides comprising PRP and used to prepare other vaccines as synthetic glucopeptides or lipoglucopeptide conjugates. Such vaccines may be injected into mammals, for example, by intramuscular or non-oral route, or by using antibodies, targeting substances such as microparticles, capsules, liposomes and toxins and their fragments, cells or mucous membranes of the immune system. When delivered to the surface, it can be used to immunize against diseases caused by Haemophilus influenzae.

(xiv) use of D15 as a carrier protein to generate glucoconjugates.

In order to determine whether D15 can be used simultaneously as a carrier and carrier, a D15-PRP conjugation experiment was performed, as in Example 14. D15-PRP conjugates have been found to be very immunogenic in rabbits and, as determined by D15-specific ELISAS and PRP-BSA immunoassay, have been shown to be able to elicit both anti-D15 and anti-PRP IgG antibody responses. . These results demonstrate that D15 protein can be used substantially as a carrier protein for glucoconjugation techniques.

In a preferred embodiment of the present invention, the carrier function of D15 can generally be used to prepare conjugate vaccines and chimeric molecules against pathogenic bacteria, including bacteria in capsules. Therefore, the glucoconjugate according to the present invention is for example Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli, Meningococci, Typhoid fever, Streptococcus mutans, Cryptococcus neoformans, Streptococcus pneumoniae, Staphylococcus aureus It can be applied to vaccines that confer protection against infection by any bacterium with polysaccharide antigens, and the like.

In another embodiment, the carrier function of D15 can be used to generate anti-tumor antibodies, for example, which can induce immunity against abnormal polysaccharides of tumer cells, or be conjugated to chemotherapy or bioactive reagents.

Accordingly, the present invention provides the preparation of the antigen of Haemophilus influenzae (D15) which can be used to diagnose and prevent diseases caused by Haemophilus influenzae and provides a primary sequence. In particular, the inventors of the present invention have found that recombinant D15 or fragments thereof can elicit protective antibody responses against the type b bacterial challenge of live Haemophilus influenzae. The present invention also reveals the nucleotide sequence of the D15 genes isolated from both type b and nontype lineages of Haemophilus influenzae. DNA fragments encoding D15 have been published and reveal that polymorphism is rare for both their nucleotides and derived amino acid sequences (Figures 1 (f) and 3). These fragments of DNA can be used to create immunogens that are essentially free of other Haemophilus influenza antigens (such as PRP and lipoglycosaccharide (LOS)) through the application of recombinant DNA technology. The present invention also presents novel techniques used to prepare essentially pure D15 or fragments thereof, functional analogs. Recombinant D15 proteins, fragments or analogs thereof, can be produced in a suitable expression system such as E. coli, Haemophilus, Bordetella, Bacillus, fungus, yeast, baculovirus, pox virus, vaccinia or mammalian expression system.

One embodiment of the invention is directed to a process for preparing a vaccine comprising purified recombinant D15 protein (rD15) or rD15 fragments that cross-react with a natural D15 immunogen. Specifically, DNA fragments encoding the gene encoding the entire D15 protein and the N-terminal rD15 fragment fused with the glutathione-S-transferase gene were made and expressed in E. coli. The expressed rD15 protein and fragments thereof are immunologically cross-reacted with the natural D15 antigen isolated from both type and nontype Haemophilus influenza isolates and thus are included in the vaccine against diseases caused by Haemophilus influenzae. It was found to be cross-reactive to immunogens. Moreover, as described herein, Haemophilus influenzae recoverer serum recognized D15, rD15, N-terminal rD15 fragments purified from Haemophilus influenzae.

In another embodiment, the present invention provides for the genetic engineering of hybrids or chimeric proteins containing D15 fragments fused to another polypeptide or protein or polysaccharide, such as, for example, P1, P2, P6, PRP. Provided are genes encoding the outer membrane protein D15 from Haemophilus influenzae having the particular nucleotide sequences described in or substantially similar thereto (ie, hybridizing with such sequences under stringent conditions).

Thus, the D15 outer membrane protein functions not only as a protective antigen but also as a carrier in a conjugate vaccine that provides autologous T-cell priming, wherein the hapten portion of the conjugate is a capsular polysaccharide portion (PRP) of Haemophilus influenzae as a carrier. Do it. These D1-hydrocarbon conjugates can modally induce antibodies against PRP and D15, thus increasing the level of protection against Haemophilus influenzae-related diseases, particularly for infants.

In another embodiment, the present invention includes a pure form of at least one peptide or protein, which contains at least one amino acid sequence corresponding to the antigenic determinant of D15, wherein such peptides are polynucleotides against Haemophilus influenzae in mammals. Cronal antibodies can be derived. These D15-specific antibodies are useful in test tools for detecting the presence of Haemophilus influenzae in biological samples. Peptides may have, for example, amino acid sequences corresponding to the following residues of the D15 protein of the Haemophilus influenzae type b Ca lineage as shown in Table 2, or any portion thereof, modification or mutation that maintains immunogenicity There are: 20-49, 45-74, 66-99, 93-122, 114-143, 135-164, 157-187, 180-209, 199-228, 219-249, 241-270, 262-291, 282-312, 304-333, 325-354, 346-375, 367-396, 390-416, 410-435, 430-455, 450-477, 471-497, 491-516, 511-538, 532- 559, 554-582, 577-602, 596-602, 596-625, 619-646, 641-666, 662-688, 681-709, 705-731, 725-750, 745-771, 769-798 ( SEQ ID NOs: 14 to 49).

In another embodiment, the present invention provides pure natural D15 protein, extracted from cultures of Haemophilus influenzae type and non-type isolates and separated by chromatography. Such novel processes include the extraction of D15 protein from cell paste using techniques known for other outer membrane proteins that are treated with aqueous wash solutions and separated by centrifugation and chromatography. Purified natural D15 antigen can be injected, for example, into an intramuscular or non-oral routed mammal, or delivered using antibodies, targeting substances such as microparticles, capsules, liposomes and toxins and fragments thereof. It may be used to immunize mammals against diseases caused by Haemophilus influenzae.

Another aspect of the invention is that the D15 outer membrane protein, fragments or analogs thereof or peptides corresponding to the D15 moiety can be a component of multiple vaccines for otitis media. Such multiple vaccines have the antigenic determinants of D15 described herein, with or without an adjuvant, with at least one protective antigen isolated from pneumococcal streptococci, Branhamella catarrh, Staphylococcus aureus or respiratory synthium virus. At least one.

The D15 peptides (Table 2), or any portion, modification, or mutation thereof, can be readily synthesized manually or using a commercially available peptide synthesizer such as the Applied Biosystem Model 430A synthesizer.

It is evident that various embodiments of the present invention have many applications in the field of vaccination, diagnosis, treatment of immunosuppressive agents, and the preparation of immunogenic reagents for diseases caused by Haemophilus influenzae. Specific non-limiting descriptions of these uses follow.

1. Vaccine Manufacturing and Use

Immunogenic compositions suitable for use as vaccines can be prepared from D15 outer membrane protein, fragments or analogs thereof, and / or peptides corresponding to the D15 moiety, as disclosed herein. Such vaccines have opsonizing action on D15 and include sterile anti-D15 outer membrane protein antibodies to elicit an immune response that produces antibodies. Even if the vaccinated object is challenged by Haemophilus influenzae, bacteria are inactivated by antibodies bound to the D15 outer membrane protein. Antiseptic and bactericidal antibodies represent examples of antibodies useful for protection against disease.

Vaccines containing peptides are generally well known, for example, in US Pat. Nos. 4,601,903, 4,599,231, 4,599,230, 4,596,792, and the like, cited herein. With respect to any further references to these patents, application is made here without particular mention of such effects. Vaccines can be readily prepared for injection as liquid solutions or emulsions. Peptides corresponding to the D15 outer membrane protein, fragments or analogs thereof, or the D15 moiety may be mixed with physiologically acceptable excipients that are compatible with the D15 outer membrane protein, their fragments or analogs or peptides. Excipients are water, saline, dextrose, glycerol, ethanol and combinations thereof. The vaccine may further comprise additional substances such as samples for wetting or emulsifying, pH buffer samples, adjuvants that enhance the effectiveness of the vaccine. Methods of achieving the vaccine adjuvant effect include the use of samples, such as aluminum hydroxide or aluminum phosphate, which are generally used in 0.05 to 0.1% solution in phosphate buffered saline. Vaccines may be injected parenterally by subcutaneous injection or intramuscular injection. Instead, other methods of administration may also be desirable, including left leg or oral formatting. Suppositories include binders and carriers such as, for example, polyalkaline glycols or triglycerides. Oral formalities include commonly used initials such as, for example, pharmaceutical grades of saccharin, cellulose, magnesium carbonate and the like. Such compositions may be in the form of solutions, suspensions, tablets, pills, capsules, supported dosage forms or powders, and correspond to 10% D15 outer membrane protein, fragments or analogs thereof, and / or the D15 moiety. Contains peptides.

Vaccines are injected in therapeutically effective, protective and immunogenic amounts in a manner consistent with the dosage form. This amount injected depends on the object being treated, including, for example, the ability of the individual immune system to form antibodies and, if necessary, to have a cell-mediated immune response. The exact amount of active ingredient required to be injected is at the discretion of the skilled person. However, appropriate dosage ranges can be readily determined by one skilled in the art and are a microgram order of peptides corresponding to the D15 outer membrane protein, fragments or analogs thereof and / or the D15 moiety. Appropriate regimes for initial infusion and adjuvant administrations may also include initial infusions that are important and involve continuous infusions. The dosage of the vaccine is also related to the route of infusion and may vary depending on the size of the host.

Nucleic acid molecules encoding the D15 outer membrane protein according to the present invention may also be directly injected with DNA, for example by injection for genetic immunization or by making a living vector such as Salmonella, BCG, Adenovirus, Poxvirus or Vaccinia. It can also be used directly for immunization. Discussion of several live vectors used to transport heterologous antigens to the immune system has been discussed, for example, by O'Hagen (1992). Processes for injecting DNA directly into a test subject for genetic immunization have been described, for example, by Ulman et al (1993).

The in vitro use of these peptides may not require sufficiently long cerium and / or tissue half-life on their own, and therefore may first require their chemical modification. Such chemically modified peptides are referred to herein as peptide analogs. The term peptide analog extends to any functional chemical equivalent of peptides characterized by increased stability and / or efficiency in vitro or in vivo with respect to the practice of the present invention. The term peptide analog is also used to extend to amino acid derivatives of the peptides described herein. Peptide analogs contemplated herein include methods of altering side branches, adding artificial amino acids and / or derivatives thereof during peptide synthesis and using cross-linkers and other methods of imposing morphological constraints on peptides or their analogs. Generated by them.

Examples of side branch modifications contemplated by the present invention include reductive alkylation with aldehydes and NaBH ₄ , amidation with methyl acetamidate, acetylation with acetic anhydride, carbamylation of amino groups with cyanates, 2, 4 , 6, trinitrovenization of amino groups by trinitrovention sulfoxane (TNBS), alkylation of amino groups by succinic anhydride and tetrahydrophthalic anhydride, pyridoxylation of lysine by pyridoxy-5'-phosphate and NaBH ₄ Includes modifications of amino groups.

Guanidino groups of arginine residues can be altered by forming heterocyclic condensation products with samples such as 2,3-butadiene, phenylglyoxal and glyoxal.

The carboxyl group can be altered by carbodiimide activity through o-acylisourea formation and subsequently by, for example, being derived from the corresponding amide.

Sulfahydryl groups are carboxymethylated with isodoacetic acid or isodoacetamide, perfomic acid oxidation with cysteine acid, formation of mixed disulfides with other thiol compounds, with maleimide, maleic anhydride or other substituted maleimide Reaction, formation of mercuryl derivatives using 4-chloromercurobenzoate, 4-chloromercuryphenylsulfonic acid, phenylmercurylchloride, 2-chloromercuric-4-nitrophenol and other mercuryls, to cyanate at alkaline pH By carbamylation and the like.

Tryptophan residues can be altered by, for example, alkylation of the indole ring with oxidized 2-hydroxy-5-nitrobenzyl bromide or sulfonyl halide with N-bromosuccinimide. Tyrosine residues can be altered by forming 3-nitrotrosine derivatives by retreat with tetranitromethane.

Alteration of the imidazole ring of the histidine residue can be carried out by alkylation with isodoacetic acid derivatives or N-carbetoxylation with diethylpyrocarbonate.

Examples of inserting artificial amino acids or derivatives during peptide synthesis include norleucine, 4-amino butanoic acid, 4-amino3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline , D-isomers of phenylglycine, ornithine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and / or amino acids.

2. Immunoassay

D15 outer membrane proteins, fragments or analogs thereof and / or peptides according to the present invention are known for detecting RIAs, other non-enzyme linked antibody binding assays or anti-bacterial, Haemophilus influenzae, D15 and / or peptide antibodies. Methods are useful as antigens in immunoassays, including enzyme-linked immunosorbent assays (ELISA). In ELISA assays, peptides corresponding to the D15 outer membrane protein, fragments or analogs thereof and / or the D15 outer membrane protein portion are immobilized to selected surfaces that exhibit protein affinity, such as, for example, wells of polystyrene microquantitative plates. After washing to remove incompletely absorbed D15 outer membrane proteins, their fragments or analogs and / or peptides, non-specific proteins antigenically neutral to the test sample, such as bobbin serum albumin (BSA) and casein, May be coupled to the selected surface. This hides nonspecific sites on a fixed surface and thus reduces the background caused by nonspecific binding of antiserum to the surface. Peptides used herein are usually in the range of 12 residues, preferably in the range of 14 to 30 residues.

The immobilized surface then comes into contact with samples of clinical or biological substances to be tested in such a way as to form an immune complex (antigen / antibody). This may include diluting the sample with diluents such as BSA, bobbin gamma globin (BGG) and / or phosphate buffered saline (PBS) / Tween. Samples are incubated for 2 to 4 periods at temperatures of 25 ° C to 37 ° C. After incubation the surfaces in contact with the sample are washed to remove non-immune complexes. The washing process may include washing with a solution such as PBS / Tween, borate buffer.

After formation and subsequent washing of specific immunocomplexes between the bound D15 outer membrane protein, fragments or analogs thereof and / or peptides and the sample, the second antibody having specificity for the first antibody Production and even quantity are determined. If the sample to be tested is human, then the second antibody is an antibody that is specific for human immunoglobulins and generally IgG. In order to provide a means to be sensed, the second antibody may have associated activities such as enzymatic activity that will color, for example when incubated with a suitable pigment substituent. For example, it may be quantified by measuring the degree of color development using a visible light spectrum spectrophotometer.

3. Use as a hybridization probe of sequences

Sequences of nucleotides according to the invention, including the sequence of the D15 outer membrane protein, allow for the identification and cloning of D15 outer membrane protein genes from any species of Haemophilus and other bacteria with genes encoding the D15 outer membrane protein Let's do it.

Sequences of nucleotides comprising sequences encoding the D15 outer membrane protein according to the present invention are useful because they have the ability to selectively form overlapping molecules with complementary strands of other D15 genes. Depending on the application, various hybrid conditions are used to change the degree of selectivity of the probe for other D15 genes. For a high degree of selectivity, low salt and / or high temperature conditions, such as, for example, a salt of 0.02 M to 0.15 M, a temperature of 50 ° C. to 70 ° C., are used as stringent conditions for forming duplicates. . Some applications require less stringent conditions such as salts of 0.15 M to 0.9 M, temperatures of 50 ° C. to 70 ° C. Hybrid conditions may be made more stringent by increasing the amount of framide that destabilizes the hybrid repeaters. Thus, specific hybrid conditions can be easily created and generally selectable according to the desired results.

In clinical diagnostic embodiments, the sequences of the nucleotides of the D15 outer membrane protein gene according to the invention can be used with suitable means, such as a label to determine hybrid. A wide variety of suitable indicator means are known in the art, including other ligands such as avidin / biotin that can provide radioactive, enzymatic or detectable signals. In some diagnostic embodiments, an enzyme tag such as urease, alkaline phosphatase or peroxidase may be used instead of the radioactive tag. In the case of an enzyme tag, colored substituents are known which are used to provide a means that can be seen with the human eye or spectrophotometers in order to transcribe certain hybrids with samples containing the D15 gene sequence. .

The sequences of the nucleotides of the D15 outer membrane protein gene according to the present invention are useful as hybrid probes in solution hybridization and in embodiments using solid-phase processes. In embodiments involving solid phase processes, DNA (or RNA) from samples, such as clinical samples including exudates, body fluids, or even tissues, is absorbed or attached to a selected matrix or surface. The immobilized single strand of nucleic acid is specifically hybridized with selected probes comprising sequences or fragments thereof encoding the D15 outer membrane protein according to the invention, under preferred conditions. The conditions to be selected depend on the particular situation based on the particular measure required, for example depending on the G + C content, the type of target nucleic acid, the material of the nucleic acid, the size of the hybrid probe and the like. Specific hybrids are detected by the label and even quantified after washing the hybrid surface to remove non-specifically bound probe molecules. The pro moiety of choice is at least 11118 bp and may be between 30 and 90 bp in length.

4. Expression of D15 outer membrane protein genes

Control sequences derived from species that are compatible with the replicon and host cell can be used to express the D15 outer membrane protein genes in the expression system. Vectors usually have a site of replication as well as a marker sequence that can provide phenotypic selection in the transformed cells. For example, E. coli can be transformed using pBR322 containing genes of ampicillin and tetracyclone resistance, thus providing an easy means for recognizing the transformed cells. The pBR322 plasmid, or other microbial plasmid or phase, must also be modified to contain or contain a promoter that can be used by microbial organisms to express their own proteins.

In addition, a phi vector containing control sequences and replicons that are compatible with the host microorganism can be used as a modification vector in association with these hosts. For example, phage in lambda GEM ^™ -11 can be used to make recombinant phage vectors that can be used to modify host cells such as E. coli LE392.

Promoters commonly used in recombinant DNA production include promoters of other microorganisms such as β-lactamase (penicillinase) and lactose promoter systems and T7 promoter system. Known in detail with respect to the nucleotide sequence of promoters, this allows them to be functionally linked with plasmid vectors. The particular promoter used is generally a matter of choice depending on the desired results. Suitable hosts for the expression of transferrin receptor genes, fragments or variants thereof, can be used with baculovirus and poxvirus expression systems containing E. coli, Bordetella, Bacillus, fungus, yeast.

According to an aspect of the invention, since naturally occurring D15 protein, specifically purified from the culture of species of Haemophilus, contains unwanted contaminants, including trace amounts of toxic substances, the D15 outer membrane protein, It is desirable to create pieces or analogs. This problem can be solved by using recombinantly generated D15 outer membrane proteins in heterogeneous systems that can be isolated from the host in a manner that minimizes toxins in the purified material. Particularly preferred hosts for expression in this respect include gram positive bacteria that do not have lipopolysaccharide (LPS) and are therefore endotoxin free. Such hosts include species of Bacillus and are particularly useful for the production of non-pyrogenic D15 outer membrane proteins and their fragments and analogs.

Biological Deposits

Certain plasmids containing at least the portion coding for the D15 outer membrane protein from the species of Haemophilus influenzae as described herein are American type culture collections located in Maryland, USA, prior to the filing date under the Budapest Treaty. Was deposited with the Tyep Cluture Collection (ATCC). Samples of deposited plasmids are available in the air if a patent is granted based on a US patent application. The deposited samples are for the purpose of illustrating the invention and are not intended to limit the scope of the invention by the deposited plasmids. Any similar or identical plasmids encoding similar or identical antigens as described herein are within the scope of the present invention.

[Deposit Summary]

The above is a general description of the present invention. With reference to the following specific embodiments, it will be better understood. These embodiments are merely illustrative and are not intended to limit the scope of the invention. Although specific terminology is used, it is for the purpose of description and not of limitation. Immune recombinant DNA methods are not the only ones used in the present invention but are within the scope well known in the art.

[Examples]

Molecular genetic methods, protein biochemistry, immunology are used, but are not the only ones used herein and in the examples, but are fully reported in the literature and are within the scope of those skilled in the art.

Example 1

This example illustrates the cloning and sequencing of the D15 genes.

According to Berns and Thomas (1965), genomic DNA was purified from Haemophilus influenzae type b strain Ca by lysing bacteria with pronase and sodium dedosilsulfate, phenol extraction and isopropane titration. DNA was partially digested with EcoR I and DNA fragments containing 6-10 kb fragments were separated by electrophoresis in low-melting agarose. These fragments are linked to a lambda gt11 Amp1 vector (Thomas Rossi, 1986) and cloned as lysogen into the E. coli strain BTA282. Recombinant clones are selected for their ampicillin resistance conferred by the vector. To recognize clones that produce Haemophilus influenzae type b antigen, clones were replica-plated on nitrocellulose filters and duplicate clones induced expression by varying the temperature to 42 ° C. for 2 hours. The clones are degraded by wetting the filters with 1% sodium dedosilsulfate (SDS). The filters are then left in chloroform-saturated air for about 15 minutes. The filters were then assayed by colony radioimmunoassay using highly immunized rabbit anti-hemophilus influenzae type b serum absorbed by E. coli lysate for antigen production. Autoradiography revealed that the clones produced Haemophilus influenzae type b antigens and their clones were tested for reactivity with the highly immunized anti-hemophilus influenzae type b. Antisera absorbed by complete Haemophilus influenzae type b bacteria (Ca strain) 10 ¹⁰ was used as a negative control.

Many clones that reacted with those that were not absorbed and did not react with the absorbed antiserum were recognized and further analyzed. It was found that one of the clones, D15, was purified and cultured to produce Haemophilus influenzae type b antigen that migrates in dedosyl sulfate polyacrylamide gels having an M _r of about 80 kDa. Lysates from the D15 clone were coupled to Sepharose ^™ 4B gel and used for affinity-tablet-D15 antibodies. This process was explained by Thomas et al. (1990) except that M _r was initially reported at about 103 kDa. Affinity-tablet-D15 antibodies have been shown to react with M _r 80kDa (Sacosyl insoluble portion-Carlone et al. 1986) in the outer membrane protein of Haemophilus influenzae type b. Radiomimuno dot blots and Western blot analyzes of membrane preparations from type b and non-type Haemophilus influenzae strains showed that the affinity-tablet-D15 antibodies reacted with all isolates. These antibodies have been found to potentially protect young mice from bacteremia. The specificity of protection was confirmed by absorbing and removing the protective activity of anti-D15 antibodies by E. coli lysate expressing D15 coupled to Sepharose. Protective studies are described in Thomas et al. It was explained in detail by 1990.

DNA from lambda gt11 Amp1 D15 phage was isolated and 5.7 kb fragments were excreted by EcoR I digestion. This fragment was subcloned into pUC19 and the resulting plasmid was modified into E. coli HB101. When examined by Western blot, recombinant bacteria were found to produce the expected M _r 80kDa Haemophilus influenzae type b antibody. Insert DNA was then analyzed by restriction endonuclease mapping. Exhausted by 2.8 kb Hin III-EcoR I digestion. This fragment was subcloned into pUC19 and the resulting plasmid was modified into E. coli HB101. When examined by Western blot, recombinant bacteria were found to produce the expected M _r 80kDa Haemophilus influenzae type b antibody. DNA was then analyzed by restriction endonuclease mapping. A 2.8 kb HindIII / EcoRl fragment was subcloned into pUC19 to produce the pUC19 / D15 plasmid, which was modified into E. coli HB101. Recombinant bacteria produced an M _r 80kDa protein that was recognized by D15-specific antibodies in the Euster blot of E. coli lysate.

Plasmid DNA was obtained from two clones of recombinant E. coli HB101 containing the pUC19 / D15 plasmid using standard techniques. Oligonucleotide primers of 17-25 bases in length were synthesized by an ABI model 380B DNA synthesizer and purified by chromatography using an OPC cartridge. Samples were sequenced by ABI Model 380B DNA Sequencer. This sequencing shows that the D15 gene contains an open reading frame that encodes 789 amino acids, including similar signal sequences (Figure 1). The derived amino acid sequence was found to contain a sequence of chemically determined, internal peptides obtained by natural trobinbin digestion of D15. The amino acid composition of D15 derived from the D15 gene sequence corresponds to that of natural protein within the limits of experimental error.

Example 2

This example describes the preparation of chromosomal DNA from Haemophilus influenzae strains Eagan, MinnA, SB33, PAK 12085.

Haemophilus influenzae strains were raised in Mueller-Hinton agar, or brain heart infusion broth (Harkness et al., 1992).

[Eagan chromosome DNA]

Bacteria from 50 ml of culture were aggregated by centrifugation at 4 ° C., 5,000 rpm for 20 minutes. The mass was resuspended in 25 ml TE (10 mM Tris, 1 mM EDTA, pH 8.0) and 2 × 5 ml of aricots were used for chromosomal DNA preparation. 0.6 ml of 10% sacosyl and 0.15 ml of 20 mg / ml prokinase K were added to each aricoat, and the samples were incubated at 37 ° C. for one hour. Lysates were extracted once with Tris-saturated phenol (pH 8.0) and three times with chloroform: isoamyl alcohol (24: 1). The aqueous phase was collected in a final 7 ml volume. Then, 0.7 ml of 3M sodium acetate (pH 5.2) and 4.3 ml of isopropanol are added to precipitate the aggregated DNA, rinsed with 70% ethanol, dried and resuspended in 1 ml of water.

[MinnA, SB33, PAK 12085 chromosomal DNA]

Bacteria were agglomerated by centrifugation at 50 ° C. for 15-20 minutes at 5,000 rpm in a Sorvall RC-3B centrifuge in 50 ml of culture. The mass was resuspended in 10 ml TE (10 mM Tris, 1 mM EDTA, pH 7.5), pronase was added at 50 μg / ml and SDS was added 1%. The lysates were extracted once with Tris-saturated phenol, once with Tris-saturated phenol / chloroform (1: 1) and once with chloroform. The final aqueous phase was dialised for 24 hours for 2 × 500 ml NaCl 1M at 4 ° C., buffer was changed once, for 24 hours for 2 × 500 ml TE at 4 ° C., and buffer was changed once. The final dialisate was alicoatted for the next step.

Example 3

This example illustrates the preparation of Haemophilus influenzae chromosome library.

Haemophilus influenzae Eagan and PAK 12085 chromosomal DNA were digested with Sau3A I (0.5 unit / 10 μg DNA) for 15 minutes at 37 ° C. and isolated by size by agarose gel electrophoresis. Gel pieces corresponding to 15-23 kb of DNA were cut off and the DNA was electrophoresed at 14 V overnight in a tubing dialisis containing 3 ml of TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). DNA was precipitated twice and resuspended in water before being linked to EMBL3 BamH I arms (Promega). The ligation mixtures are packed using a lambda ex vivo packing tool and plated over E. coli NM539 cells. The library was titrated, amplified and stored at 4 ° C. or below 0.3% chloroform.

10-30% sucrose gradient in TNE (20 mM Tris-HCl, 5 mM NaCl, 1 mm EDTA, pH 8.0) after MinnA chromosome DNA (10 μg) was digested with Sau3A I (40 units) for 2, 4, 6 minutes. Separated by size from above. Fragments containing more than 5 kb of DNA were collected and precipitated. In the second experiment, chromosomal DNA (2.6 μg) was digested with Sau3A I (4 units) for 1, 2 and 3 minutes and then separated by size by agarose gel electrophoresis. Gel pieces corresponding to 10-20 kb of DNA were cut and DNA was extracted using standard freeze / thaw techniques. Size-separated DNA from both experiments was collected to connect to EMBL3 BamH I arms (Promega). Ligation mixtures are packed using Gigapack II packing tools and plated over E. coli LE392 cells. The library was titrated, amplified and stored at 4 ° C. or below 0.3% chloroform.

10-30% sucrose gradient in TNE (20 mM Tris-HCI, 5 mM NaCl, 1 mM EDTA, pH 8.0) after SB33 chromosomal DNA (20 μg) was digested with Sau3A I (40 units) for 2, 4 and 6 minutes. Separated by size from above. Fragments containing more than 5 kb of DNA were collected and precipitated. In the second experiment, chromosomal DNA (2 μg) was digested with Sau3A I (4 units) for 2, 4 and 6 minutes and then separated by size by agarose gel electrophoresis. Gel pieces corresponding to 10-20 kb of DNA were cut and DNA was extracted using standard freeze / thaw techniques. Size-separated DNA from both experiments was collected to connect to EMBL3 BamH I arms (Promega). Ligation mixtures are packed using Gigapack II packing tools and plated over E. coli LE392 cells. The library was titrated, amplified and stored at 4 ° C. or below 0.3% chloroform.

Example 4

This example describes the screening of DNA libraries.

Eagan, MinnA and SB33 and PAK 12085 DNA libraries are plated onto LE392 cells on NZCYM plates using 0.7% top agarose in NZCYM as overlay. Plaque rises were performed over nitrocellulose filters according to standard procedures, and Peters were processed and hybridized to a digoxigenin-labeled D15 probe. The probe is an EcoR I / Hind III fragment from pUC19 / D15 containing the entire Ca D15 gene (Figure 2). The potential plaques are plated and subjected to a second screening using the same procedures. Phage DNA was digested with Sal I digestion and cloned into pUC to show DS-712-2-1 (Eagan), DS-691-1-5 (MinnA), JB01042-5- as shown in FIG. 1 (SB33), JB-1042-9-4 (PAK12085) Obtained from 500 ml of culture by standard procedure to produce chloros. The nucleotide sequence of the D15 genes from Haemophilus influenzae type b strains Eagan, MinnA non-type Haemophilus influenzae strains SB33, PAK 12085 was determined and compared with that of lineage Ca (1b, 1c, 1d, 1e, 1f also ). The desired amino acid sequence is in degrees 1b, 1c, 1d, 1e and compared with the amino acid sequence of D15 protein of Haemophilus influenzae type b Ca (FIG. 1f).

Example 5

This example illustrates the expression of rD15 protein in E. coli.

A 2.8 kb Hind III / EcoR I fragment is subcloned into pUC19 and this pUC19 / D15 plasmid is modified into E. coli HB101. As soon as induced, positive clones. Western blot expressed 80 kDa protein recognized by D15-specific antiserum. HindIII-Pst I fragments were also shown to be subcloned into pUC19 and express the 67kDa protein. According to the restriction map, this 67kDa protein corresponds to the C-terminal truncated D15 protein. In Western blot analysis, this truncated D15 was recognized by D15-specific antiserum.

Plasmids expressing the D15 gene of the non-type line SB33 were prepared in E. coli. Plasmid JB-1042-5-1, containing the D15 gene of SB33 and its flanks, was digested by EcoRI and HindIII and the 3kb D15 insertion was subcloned into pUC to create plasmid pRY-60-1 (Figure 4). Appropriate oligonucleotides are synthesized to store back the natural D15 sequence between the ATG codon of the expression plasmid pT7-7 and the BsrF I site in the D15 gene. Such oligonucleotides had the following sequence:

Plasmid pRY-60-1 was digested by EcoR I and BsrF I and the DNA fragment containing most of the D15 gene was purified. pUC was digested into purified vector fragments and EcoR I and Nde I. Multi-component linkage between pUC and D15 fragments and oligonucleotides yields plasmid DS-860-1-1 containing the D15 sequence without promoter. pT7-7 was digested with the purified vector fragment EcoR and Nde I. DS-860-1-1 was digested with EcoRI and Nde I and linked with a T7-7 vector where the D15 insert was purified and produced plasmid DS-880-1-2 (Figure 4).

Plasmid formations were performed using E. coli JM109 as a host. For expression, plasmid DS-880-1-2 was altered into E. coli BL21 / DE3, BL21-DE3 / pLysS or JM109 / DE3 cells. Modification of the cells was performed using either calcium chloride-treated competition cells or BioRad electroporator. The modified cells were grown in YT, M9, NZCYM media and induced by IPTG or other induction reagents.

Example 6

This example illustrates the formation and expression of the GST-D15 fragment hybrid gene in E. coli.

The forward primer (primer 1) 5'-GGGGAATTCCAAAAGATGTTCGT (SEQ ID NO: 52) and the reverse primer CACGAATTCCCTGCAAATC-5 '(primer 7-SEQ ID NO: 53) are obtained from 22 to 22 of the primary sequence of the D15 protein by a polymerase chain reaction. It was used to amplify the HindIII-EcoRl 2.8 kb fragment of the D15 gene encoding the 223 N-terminal amino acid residues (Figure 1a). The 609 bp amplified nucleotide sequence was confirmed by DNA sequencing. The amplified gene fragment was linked downward from the GST gene into the pGEX-2T vector and modified into E. coli TG-1. Clones that screened for Haemophilus influenzae type b antigen with rabbit anti-Hemophilus influenzae type b antiserum were cloned and isolated by clone radioassay. The glutathione-S-transferase-D15 fragment fusion protein produced by the modified E. coli was isolated by affinity purification on glutathione agarose.

Example 7

This example illustrates other expression systems for rD15.

The D15 gene or fragments thereof are also expressed in E. coli by other regulated promoters. The D15 gene or fragments thereof are expressed in other crawling systems that lack the leader peptide or that the expression of D15 is not harmful to the host. The genes or their fragments are synthesized by polymerase chain reaction using newly synthesized or suitable primers. These genes are directly subcloned into suitable cloning vectors, bacteriophage vectors in E. coli or other suitable strains in the absence of toxicity. Expression systems are Gram-positive bacteria (such as Bacillus species), pox virus, adenovirus, baculovirus, yeast, fungus, BCG or mammalian expression systems.

Example 8

This example describes a protocol for extracting and purifying rD15 from an E. coli expression system.

As described in Example 5, cell pellets from the resulting 250 ml culture were resuspended in 40 ml of 50 mM Tris, pH 8.0 and broken down by ultrasound (3 × 10 min, 70% duy cycle). The extract was centrifuged at 20,000 × g and the resulting pellet accumulated. The initial pellet was re-extracted with 40 ml 50 mM Tris, 0.5% Triton X-100, 10 mM EDTA, pH 8.0. The suspension was then crushed by ultrasound for 10 minutes at 70% duty cycle. The extract was centrifuged at 300 × g for 5 minutes. The resulting suspension was centrifuged at 20,000 × g for 30 minutes and the resulting pellet accumulated. The pellet was resuspended in 40 ml of 50 mM Tris, pH 8.0. The suspension is then mixed with PBS / 8M urea until the final urea concentration is 6M. The solution was then dialialized against PBS to remove urea. After dialing the solution was centrifuged at 300 x g for 10 minutes and suspended solids accumulated and stored at 4 ° C.

Example 9

This example demonstrates the purification of GST- (D15 fragment) fusion protein using glutathione-Sepharose 4B affinity chromatography.

As explained in Example 6, the obtained 5 mg GST- (D15 fragment) fusion protein initial extract was dissolved in 5 ml of phosphate buffered saline (PBS) containing 1% of Triton X-100. The solution is then loaded onto a glutathione-Sepharose 4B column (2 mL) equilibrated with PBS containing 1% Triton X-100. The flow through of the column was removed. The column is washed with 20 ml PBS and the GST- (D15 fragment) fusion protein is eluted with 50 mM Tris-HCl buffer, pH 8.0, containing 5 mM glutathione. The absorbance of the eluate at 280 nm was monitored. Protein-containing portions (2 mL / portion) were collected and collected. Protein purity was assayed by SDS-PAGE (Figure 9, lane 3). The final volume of purified fusion protein was 6 ml.

Example 10

This example illustrates the protocol used for thrombination of proteins to release truncated D15 molecules.

To remove the protease blocker, the GST- (D15 fragment) fusion protein sample (0.1-0.5 mg protein / ml) from Example 9 was subjected to at least 2 hours at 4 ° C. against 50 mM Tris-HCl buffer, pH 8.5. During three times. After ialisis, the solution was treated with human thrombin (Sigma) at a rate of 1 ml solution per 25 units of thrombin. Cleavage reactions were performed at 37 ° C. for 2 hours and reported on SDS-PAGE (FIG. 9, lane 4). The reaction is stopped by placing the solution on ice.

Example 11

This example illustrates the procedure for purification of N-terminal rD15 fragments from GST using glutathione-Sepharose 4B affinity chromatography.

As described in Example 10, the obtained thrombin-digested GST- (D15 fragment) sample was loaded onto a glutathione-Sepharose 4B column (2 mL) equilibrated with PBS containing 1% of Triton X-100. . Passage of the column containing the N-terminal rD15 fragments accumulated. After washing the column with 20 ml of PBS, the affinity column is regenerated by removing GST using 50 mM Tris-HCl buffer, pH 8.0, containing 5 mM glutathione. The purity of rD15 fragments was analyzed by SDS-PAGE (Figure 9, lane 5). This N-terminal rD15 fragment contains 63-223 amino acids of D15 protein as a result of thrombin cleavage at the site shown in Figure 1a.

Example 12

This example illustrates a protocol for purifying D15-specific polyclonal antibodies by affinity chromatography using recombinant (GST- (D15 fragment) fusion proteins.

As described in Example 9, the resulting recombinant GST- (D15 fragment) fusion protein was conjugated to cyanogen bromide-activated sepharose. Affinity columns were used to purify the eggs from rabbit hyperimmunized anti-Hemophilus influenzae type b antiserum. Affinity purified-antibodies have been shown to react with lysates of E. coli modified by immunoblots to pUC19 / D15 and 80 kDa components present in the lysates of various and non-type Haemophilus influenzae isolates. . These results confirm that the DNA portion encoding the D15 fragment of the fusion protein is part of the open reading frame of the D15 gene. Similarly, antisera raised for recombinant fusion protein (Example 9) or purified N- The terminal rD15 fragment (Example 11) was reacted with D15 protein (Example 13) produced by Haemophilus influenzae strains.

Example 13

This example illustrates the protocol used to purify native D15 from Haemophilus influenzae.

Panezutti et al. As described in 1993, a cell paste of non-type Haemophilus influenzae SB33 strain obtained from cultures grown in brain heart infusion medium supplemented with DNA (2 μg / ml) and HEMIN (2 μg / ml) at 37 ° C. Was resuspended in 50 mM Tris-HCl buffer, pH 8.0, containing 0.5% of Triton X-100 and 10 mM EDTA. The mixture was stirred at room temperature for 2 hours and centrifuged at 20,000 × g for 30 minutes. D15 was placed in the suspension and further purified. Purification of native D15 was performed by affinity chromatography using D15-specific monoclonal antibodies (Example 24). D15 extract (25 mL) was mixed with affinity matrix (1 mL) for 2 hours at room temperature. The mixture was packed into a column and the passage was removed. The column was washed sequentially with the following buffer: 50 mM Tris-HCI buffer containing 0.5% Triton X-100 and 10 mM EDTA, pH 8.0, 1 M HEPES buffer, pH 6.8, 0.5% Triton X-100 and 10 mM EDTA 50 mM Tris-HCI buffer, pH 8.0, 10 mM Persate buffer, pH 8.0. D15 was then eluted with 3 ml of 50 mM diethylamine, pH 12.0, and the protein solution was neutralized by 1 M HEPES, pH 6.8 (1/10 volume). Affinity-purified D15 was analyzed by SDS-PAGE and stored at -20 ° C.

Example 14

This example illustrates the procedure used to prepare the D15-PRP conjugates.

Haemophilus influenzae type b and saccharide (PRP) prepared by controlled acid hydrolysis were subjected to D15 using pyriodate oxidation, as described in US Pat. No. 4,356,170 and more specifically in Example 17. As well as fragments of Example 11 (Example 11), as well as purified natural D15 (Example 13). Or recombinant D15 (Example 8). The average molecular size of the PRP molecules used for conjugation was determined to be approximately 20,000 Daltons. Conjugation was performed without a linking molecule, but the linking molecule may be performed. A PRP / D15 molecular ratio of about 7 was used to provide excess PRP heptene.

PRP / rD15 conjugates were tested for immunogenicity in rabbits according to the protocol in Example 8 and elicited both primary and secondary anti-PRP IgG and anti-D15 antibody responses (Table 9). . Immunoblot analysis showed that rabbit anti-rD15-PRP antiserum also reacted strongly with both natural D15 and rD15. These data indicate that rD15 can be used as a carrier protein in conjugate vaccines. In addition, as a result of the additional homologous protection provided by antibodies to D15, the rD15-PRP conjugate vaccine ensures more lasting protection against Haemophilus influenzae type b disease, especially in infants.

Example 15

This example illustrates the preparation of D15 peptides.

D15 peptides (Table 2) were synthesized using an ABI 430A peptide synthesizer and isolated from resin by HF. Peptides were purified by RP-HPLC on a Vydac C4 column (1 × 30 cm) using 15-555 acetonitrile gradients in 0.1% TFA developed for 40 minutes at a flow rate of 2 ml / min. All synthesized peptides used in biochemical and immunological studies (Table 2) have a purity of at least 95% as determined by analytical HPLC. The amino acid composition analyzes of these peptides, performed in the Water Pico-Tag system, were in good agreement with the theoretical compositions.

Example 16

This example illustrates the protocol used to generate D15 peptide-specific antiserum.

Guinea pigs and rabbits were immunized intramuscularly with each peptide (50-200 μg) emulsified with Freund's complete adjuvant. After two adjuvant administrations with the same amount of peptide in incomplete Freund adjuvant on days +14 and +28, anti-peptide antiserum was collected on day +42 and examined by ELISA and immunoblotting. Peptide-specific ELISA showed a single specificity for the peptides immunized rabbit and guinea pig antisera mode (Table 6). In addition, both the guinea pig and rabbit antiserum raised against D15 peptides in immunoblot analysis reacted with Haemophilus influenzae type b and nontype. Since most D15 peptides induce strong anti-peptide antibody responses in at least one animal species, they are suitable immunogens included in immunogenic compositions including vaccine preparation.

Example 17

This example illustrates the procedure used to prepare PRP-BSA conjugates.

Pyriodate-oxidized PRP (0.1 mg sodium persulfate buffer, 25 mg in 1 mL of pH 6.0) obtained from natural PRP treated with aqueous pyriodic acid (Carlone et al., 1986), 0.2 M sodium perth Pate buffer, pH 8.0 was added to bovine cerium albumin (BSA) in 0.5 ml, (1.32 mg, 0.02 μmol) sodium cyanobor hybrid (14 μg, 0.22 μmol, 10 equivalents to BSA). After incubating at 37 ° C. for 5 days, the reaction mixture was dialyzed against 4 L of 0.1 M Persate Buffer, pH 7.5. The resulting solution was applied over analytical superloss 12 columns (15 × 300 mm, Pharmacia) equilibrated with 0.2 M sodium persate buffer, pH 7.2 and eluted with the same buffer. The parts were monitored for absorbance at 230 m. The first major protein peak was collected and concentrated to 2.2 ml in Centriprep 30. The amount of protein was determined for the Bio Rad protein assay and was 300 μg / ml. The presence of PRP in the protein conjugate was confirmed by Orcinol assay.

Example 18

This example illustrates the protocol used for the production of anti-PRP antiserum in animals using the rD15-PRP conjugate.

Rabbits are immunized intramuscularly (5-50 μg PRP equivalents) with rD15-PRP conjugates (Example 14) mixed with 3 mg AlPO4 per ml (Example 14), two adjuvant doses at two week intervals (half of the same immunogen) Amount). Antisera was collected every two weeks after the first injection, heat-cultured at 56 ° C. and stored at −20 ° C.

Example 19

This example illustrates the reactivity between D15 peptides and anti-peptide and D15-specific antiserum using D15-specific and peptide-specific ELISAs.

Microquantitative wells (Nunc-Immunoplate, Nunc, Denmark) were calibrated with 200 ng of purified rD15 or 500 ng of individual peptides in 50 μg of coating butter (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6) at room temperature. Coded for 16 hours. Plates were blocked for 30 minutes at room temperature with 0.1% (w / v) BSA in phosphate butter saline (PBS). Continuously diluted antiserum was added to the wells and incubated for 1 hour at room temperature. After antisera were removed, plates were washed five times with PBS containing 0.1% (w / v) Tween-20 and from human IgG antibodies conjugated to goat anti-rabbit, guinea pig, rat, or horseradish peroxidase. 0.1% (w / v) of BSA. _Two pieces of F (ab ') were diluted in wash buffer (1 / 8,000) and added onto micro plates. After incubation for 1 hour at room temperature, the plates were washed 5 times with wash buffer. Plates were then developed using the substrate tetramethylbenzidine (TMB) (ADI, Toronto) in H2O2. The reaction was stopped using 1N H ₂ SO ₄ and the optical density was measured at 450 nm using Titretek Multiskan II (Flow Labs., Virginia). As negative controls, two related peptides are used in peptide-specific ELISAs. The assay was performed in triplicate, and the response quantification of each antiserum was defined as a dilution with a consistent 2-fold increase in absorbance values obtained from the negative controls. The reaction results are summarized in Tables 3, 6, 8 and the present description.

Example 20

This example describes a method for measuring anti-PRP IgG in rabbit anti-PRP-D15 conjugated antiserum using PRP-specific ELISA.

Microquantitative wells (Nunc-Immunoplate, Nunc, Denmark) were encoded for about 16 hours with 200 ng of purified PRP-BSA in 200 μl of coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6) at room temperature. . Plates were blocked for 30 minutes at room temperature with 0.1% (w / v) BSA in phosphate buffered saline (PBS). Diluted rabbit antiserum raised against PRP-D15 was added to the wells and incubated for 1 hour at room temperature. After the antisera were removed, the plates were washed 5 times with PBS containing 0.1% (w / v) Tween-20 and 0.1% (w / v) from goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase. BSA. _Two pieces of F (ab ') were diluted in wash buffer (1 / 8,000) and added onto micro plates. After incubation for 1 hour at room temperature, the plates were washed 5 times with wash buffer. Plates were then developed using the substrate tetramethylbenzidine (TMB), (ADI, Toronto) in H ₂ O ₂ . The reaction was stopped using 1N H ₂ SO ₄ and the optical density was measured at 450 nm using Titretek Multiskan II (Flow Labs., Virginia). Known amounts of standard anti-PRP antiserum were included as positive controls. The assay was performed in triplicate, and the response quantification of each antiserum was defined as a dilution that showed a consistent 2-fold increase in OD values obtained from pre-immune serum (Table 9).

Example 21

This example illustrates the protocol used to generate purified D15, rD15 or N-terminal rD15 fragments.

New Zealand white rabbits (Maple Lane) and Guinea Pigs (Charles River) are affinity-purified natural D15 (Example 13) or recombinant D15 (Example 8), emulsified in Freund's complete adjuvant, or 10 μg of the N-terminal rD15 fragment (Example 13) was immunized intramuscularly. Animals were coadministered with 10 μg of affinity-purified D15 or rD15 or rD15 fragments emulsified in Freund's incomplete adjuvant on day 28 and bleeding through the ear margin vein. D15-specific polyclonal antibodies were purified from this material, as described in Example 12.

Example 22

This example illustrates the protective activity of D15-specific antisera against Haemophilus influenzae type b challenge using a young rat model of bacteremia.

Five-day mice were inoculated subcutaneously with 0.15 ml of two different rabbit anti-N-terminal rD15 fragments on the back. Pre-immunized sera were used as negative controls. One day after immunization, young rats were of the Haemophilus influenzae type b MinnA strain grown in brain heart infusion (BHI) medium supplemented with accessory elements and diluted in PBS containing 0.5 mM MgCl ₂ and 0.15 mM CaCl ₂ . 200 cloni-forming units (cfu) (0.1 mL) were injected intraperitoneally. After one day, blood samples were collected through the heart cavity under methoxyflulane anesthesia and spread on a chocolate agar plate. After 24 hours the number of bacteria per ml of blood was measured. By the T test, the levels of bacteremia and the like for the control group were statistically significantly different. The results are summarized in Table 1.

Example 23

This example illustrates the protocol used for the generation of D15-specific T-cell lines.

BALB / c (H-2d) mice purchased from Charles River Animal Farm (Montreal, Canada) were injected subcutaneously with 20 μg of rD15 adsorbed onto 1.5 mg of aluminum phosphate, respectively. At three week intervals animals were dosed with the same amount. Ten days after the last adjuvant administration, spleens of the immunized mice were removed. Lethal cells were 10% heat-inactivated in the presence of different concentrations (1, 10, 100 μg per ml) of each D15 peptide (Table 2) in 96-well plates (Nunc, Denmark). RPMI 1640 medium (Flow Lab.) Supplemented with GiBco, 2 mm L-glutamine (Flow Lab.), 100 U / ml penicillin (Flow Lab.) And 5 × 10 ⁻⁵ M 2-mercaptoethanol (Sigma). Cultured at 5.75 × 10 ⁵ cells per well in 200 μl final volume. Cultures were maintained in a humidified incubator in the presence of 5% CO ₂ / air. Triple cultures were performed for each concentration of each peptide. After 5 days, 150 μl of 10% murine Concanavalin A culture suspension diluted in culture medium was added to microquantitative plate wells as a source of Interleukin-2 (IL-2) to inflate peptide-specific T-cells. After 6 days, 150 μl of suspension was removed and fresh IL-2 containing culture suspension from each microculture was added to maintain and expand the validity of peptide-specific T-cells. After 6 days of culture, the cells are washed three times with 200 μl of culture medium each time.

Each set of cultures was then stimulated with peptides of the corresponding concentrations (1, 10, 100 μl per ml) in the presence of 2 × 10 ⁵ irradiated BALB / c splenocytes in a final volume of 200 μl culture medium. . 60 μl of suspension was removed from each microculture. Floats from each triple culture set were collected. All suspensions were assayed for IL-2, Interleukin-4 and interferon-gamma (IFN-γ). Assays of IL-2 and IL-4 were performed using rat IL-2 and IL-4 ELISA tools purchased from Endogen Inc. (MA, USA). Assays of IFN-γ were performed using mouse INF-γ ELISA tools provided by Genzyme Corporation (MA., USA). The results obtained are shown in Table 7.

Example 25

This example describes the general procedures used to generate murine D15-specific monoclonal antibodies.

BALB / c mice were immunized intraperitoneally with 20-50 μg emulsified, N-terminal rD15 fragment (Example 11) in Freund's complete adjuvant. Two weeks later, mice were injected with the same amount emulsified in Freund's incomplete adjuvant (IFA). Hybridomas were produced by fusion of splenic lymphocytes from immunized mice and non-secreting Sp2 / 0 bone marrow cells previously described by Hamel et al. (1987). D15-specific hybridomas were cloned by sequential restriction dilutions and screened for anti-D15 monoclonal antibody production. Eight D15-specific hybridoma cell lines were found, expanded and cooled in liquid nitrogen. Among the hybridoma cell lines, 6C8-F6-C6 was partially characterized. Monoclonal antibody (MAb 6C8-F6-C6) reacted with peptide D15-P8. This MAb 6C8-F6-C6 was used to make a D15-specific MAb affinity column and to purify natural D15 from Haemophilus influenzae cell paste (Example 13).

TABLE 1

[Protective effect of latent metastasized anti-N-terminal-rD15 fragment antibodies in a young rat model of bacteremia ¹ ]

¹ 5-day-old young mice were immunized with 0.15 mL of rabbit anti-N-terminal rD15 fragments / day afterwards, young mice were challenged with 200 cfu of Haemophilus influenzae type b strain MinnA (0.1 mL, IP). Blood samples were taken from each rat 24 hours after challenge and analyzed for bacterial counts.

² Brackets indicate the number of mice in which bacteremia was found from the total number of mice challenged.

TABLE 2

[Sequences of overlapping synthetic peptides covering the entire D15 antigen sequence]

TABLE 3

[Reactivity of Rabbit and Guinea Pig Anti-N-terminal rD15 Fragment Antiserum with D15 Synthetic Peptides]

TABLE 4

[Interference of protection induced by anti-N-terminal rD15 fragment antibody by D15 peptides in a young rat model of bacteremia]

0.5 ml of rabbit anti-N-terminal rD15 fragment antiserum (anti-rD15 fragment Ab) was mixed with either 9 D15 peptides (100 µg of D15-P2 to D15-P10 peptides in Table 2) or 600 µg of N-terminal rD15 fragment. Mix and leave for 1 hour at room temperature. Antisera and peptides mixed with PBS are used as controls. 0.2 ml of 7-day-old young rats were prepared. After 24 hours the young mice were challenged with 200 cfu of MinnA (0.1 mL, IP) of Haemophilus influenzae type b strains. Blood samples were taken from each rat 24 hours after challenge and analyzed for bacterial counts. The level of bacteremia is expressed as the mean ± standard deviation obtained from each of the seven rats tested.

TABLE 5

[Interference of immunogenicity of rabbit anti-N-terminal rD15 fragment antiserum absorbed with D15 peptides (D15-P4) in a young rat model of bacteremia]

0.5 ml of rabbit anti-N-terminal rD15 fragment antiserum (anti-rD15 fragment Ab) is mixed with five D15 peptides (P8 in peptides P4, 250 μg each) and left for 1 hour at room temperature. Antisera and peptides diluted in PBS are used as controls. Inject 0.2 ml of the indicated substances into young 7-day-old rats. After 24 hours the young mice were challenged with 200 cfu of MinnA (0.1 mL, IP) of Haemophilus influenzae type b strains. Blood samples were taken from each rat 24 hours after challenge and analyzed for bacterial counts. The level of bacteremia is expressed as the mean ± standard deviation obtained from each of the seven rats tested.

TABLE 6

[Reactivity of D15 with Rabbit, Guinea Pig Anti-rD15 Antiserum]

¹ Reactive quantitation is based on peptide-specific ELISAs. +, ++, +++, and ++++ represent the reactive quantification of the antiserum of animals tested at 1/300, 1/1000, 1/2000, and 1/5000 dilutions, respectively, and-is not reactive To indicate that.

² Quantification is the mean for two rabbit antisera raised for rD15.

³ Quantification is the mean of two guinea pig antisera raised for rD15.

⁴ Quantification is the mean for 5 rat antisera raised for rD15.

TABLE 7

[T-Cell Stimulating Activity of D15 Peptides]

¹ Results are the mean from triplicate cultures. In all cases the standard deviation is less than 15%. Immunodominant Th1-cell epitopes are written in bold and Th0-cell epitopes are written in italics.

TABLE 8

Rabbit and Guinea Pig Antibody Responses to D15 Peptides

¹ Reactive quantitation is based on peptide-specific ELISAs. Quantifications below 500 are indicated as not immunogenic.

² Quantification is the mean for two rabbit antisera raised for rD15.

³ Quantification is the mean of two guinea pig antisera raised for rD15.

⁴ NT: Not Tested.

TABLE 9

[Rabbit IgG Antibody Response to D15-PRP Conjugates]

^One rabbit was immunized intramuscularly with rD15-PRP conjugates (equivalent to PRP 5-50 μg) mixed with ₃ mg of AlPO ₄ per ml and administered twice adjuvant (half of the same immunogen) every two weeks do.

² Reactive quantification is based on PRP specificity and D15-specific ELISAs.

[Summary of invention]

The present invention provides nucleic acid molecules comprising genes encoding purified and isolated D15 outer membrane proteins and sequences of these genes and their derived amino acid sequences. The invention also provides peptides corresponding to portions of the D35 outer membrane protein. In addition, it provides antibodies raised against D15 outer membrane proteins, fragments and peptides. Genes, DNA sequences, antibodies, peptides are useful for the preparation of diagnostic, immunological, diagnostic immunological reagents. Vaccines based on expressed recombinant D35, portions thereof, peptides from the provided sequences were prepared to prevent Haemophilus influenzae diseases. Modifications are possible within the scope of the invention.

[references]

O'Hagan (1992)

Ulman et al., 1993.

Berns C. A. and Thomas C. A. (1965) J. Mol. Biol. 11: 476-490.

Thomas W.R. and Rossi A.A. (1986) Infect. Immun. 52: 812-817

Thomas W.R. et al. (1990) Infect. and Immun. 58: 1090-1913.

Carlone G.M. et al. (1986) J. Clin. Microbiol. 24: 330-331.

Smith, D.B. and Johnson K.S. (1988) Gene 67: 31-40.

Harkness, R. et al. (1992) J. Bacteriol. 174: 2425-2430.

Hamel et al. (1987) J. Med. Microbiol. 23-163-170.

Mills et al. (1993) Infect. Immun. 61: 339-410.

Trin CHleri, (1993) Immunol. Today 14: 335-338.

Hope, T.P. (1986) L. Immunol. Methods 88: 1-18.

Zangwill et al., 1993 mm WR 42: 1-15.

Loeb et al. 1987. Infect. Immun. 55: 2612-2618.

Panezutti, 1993. Infect. Immun. 61: 1867-1872.

[Brief Description of Drawings]

The invention can be further understood from the following description of the drawings.

Figure 1 (a) shows the nucleotide sequence (SEQ ID NO: 1) from Haemophilus influenzae type b Ca lineage and the amino acid sequence therefrom (SEQ ID NO: 2),

Figure 1 (b) shows the nucleotide sequence (SEQ ID NO: 3) of the D15 gene from Haemophilus influenzae type b Eagan line (SEQ ID NO: 3) and the amino acid sequence therefrom (SEQ ID NO: 4),

Figure 1 (c) shows the nucleotide sequence (SEQ ID NO: 5) of the D15 gene from Haemophilus influenzae type b MinnA strain (SEQ ID NO: 5) and the amino acid sequence therefrom (SEQ ID NO: 6),

Figure 1 (d) shows the nucleotide sequence of the D15 gene (SEQ ID NO: 7) from the Haemophilus influenzae non-type SB33 strain (SEQ ID NO: 7) and the amino acid sequence therefrom (SEQ ID NO: 8)

Figure 1 (e) shows the nucleotide sequence of the D15 gene (SEQ ID NO: 9) from the Haemophilus influenzae non-type PAK 12085 strain (SEQ ID NO: 9) and the amino acid sequence therefrom (SEQ ID NO: 10),

Figure 1 (f) shows the alignment of the nucleotide sequence of the D15 gene obtained from other Haemophilus influenza isolates (types, Ca, Eagan and MinnA, non-types SB33 and PAK 12085) (SEQ ID NOs: 1, 3, 5, 7, 9),

2 shows clones, pUC19 / D15 (Ca), DS-712-2-1 (Eagan), DS-691-1-5 (MinnA), JB-1042-5-1 (SB33), JB-1042- Restriction Maps of 9-4 (PAK 12085). H = HindIII, R = EcRI, Xb = Xba I,

Figure 3 shows the alignment of amino acid sequences of D15 outer membrane proteins obtained from different Haemophilus influenzae isolates (types, Ca, Eagan and MinnA, non-types SB33 and PAK 12085) (SEQ ID NOs: 2, 4, 6) , 8, 10). Based on the Ca D15 sequence, the dots represent the same amino acid residues as the Ca D15 outer membrane protein,

4 shows the structure of a plasmid (DS-880-1-2) expressing full length SB33 D15 (rD15) from a strong inducible T7 promoter,

5 shows SDS-PAGE analysis of affinity-purified natural D15 from Haemophilus influenzae line 30,

6 shows SDS-PAGE analysis of sequence fragments obtained during purification of full-length rD15 expressed in E. coli containing plasmid DS-880-1-2.

7 shows the response of guinea pig IgG antibodies to full length rD15. Arrows indicate the immunization schedule. Bleeds were taken at 0, 2, 4, 6 and 8 weeks. The first atmospheres represent the standard deviation,

8 shows the response of murine IgG antibodies to full length rD15. Arrows indicate the immunization schedule. The bleeds were taken at 0, 1, 4, 5 and 7 weeks. The first atmospheres represent the standard deviation,

9 shows (SDS-PAGE analysis of N-terminal rD15 fragments isolated from GST- (D15 fragments) fusion protein. Lane 1 shows the low molecular weight indications (14kDa, 21kDa, 31kDa, 45kDa, 68kDa, 97kDa), lanes pre-marked) 2 is GST standard, lane 3 is GST- (D15 fragment) fusion protein, lane 4 is thrombin truncated fusion protein, lane 5 is N-terminal rD15 fragment, lane 6 is GST, lane 7 is low molecular weight,

10 shows the response of guinea pig IgG antibodies to N-terminal rD15 fragments. Arrows indicate the immunization schedule. Bleeds are harvested at 0, 2, 4, 6 and 8 weeks. The first atmospheres represent the standard deviation,

FIG. 11 is a hydrophilicity graph of D15 measured using a window with an average of 7 residues, according to Hope, 1986.

Claims (22)

An isolated and purified nucleic acid molecule having a DNA sequence characterized by at least the portion encoding the D15 outer membrane protein of Haemophilus and selected from: (a) the DNA sequence as set forth in any one of Figures 1a to 1e (SEQ ID NO: : 1, 3, 5, 7, 9), (b) DNA sequence encoding an amino acid having an amino acid sequence (SEQ ID NO: 2, 4, 6, 8, 10) shown in any one of FIGS. 1A to 1E. (c) a DNA sequence comprising the consensus sequence (SEQ ID NO: 55) also set forth in at least 1f and conserved in the sequence defined in (a), (b) above. A recombinant plasmid modified to modify the host's trait characterized by the plasmid vector inserted therein. 34. The recombinant plasmid of claim 33, wherein said recombinant plasmid contains a DNA portion comprising at least 18 bp fragment of the DNA molecule of claim 31 therein. A host characterized by expression means functionally coupled to said at least 18 bp nucleic acid molecule fragment for expression of a genetic product consisting of at least 18 bp fragment of the nucleic acid molecule as claimed in claim 31 and a D15 outer membrane protein of Haemophilus or a fragment thereof. Recombinant vector adapted for transformation of. The recombinant vector according to claim 4, wherein the recombinant vector is plasmid DS-880-1-2 of ATCC No. 75605 deposited on Nov. 4, 1993, and encodes the D15 gene product of Haemophilus influenzae SB33. 5. The recombinant vector of claim 4, wherein said DNA portion encodes a polypeptide of at least six residues. 7. The recombinant vector of claim 6, wherein said polypeptide is selected from those set forth in Table 2. The recombinant vector according to any one of claims 4 to 7, wherein the at least 18 bp nucleic acid molecule consists only of a coding sequence of the D15 outer membrane protein of Haemophilus. The recombinant vector according to claim 8, wherein the DNA portion further comprises a nucleic acid sequence encoding a leader sequence for releasing the genetic product from the host. An isolated purified protein encoded by a DNA fragment contained in the recombinant vector of claim 8 or claim 9. An isolated purified D15 outer membrane protein or part thereof having the amino acid sequence shown in any one of FIGS. 1A to 1E (SEQ ID NOs: 2, 4, 6, 8 or 10). The protein of claim 11, wherein the D15 outer membrane protein is Haemophilus D15 outer membrane protein. The protein of claim 12, wherein the D15 outer membrane protein is Haemophilus influenzae D15 outer membrane protein. The protein of claim 13, wherein the Haemophilus influenzae type b is a Haemophilus influenzae strain or a non-type Haemophilus influenzae strain. 15. The method according to claim 14, wherein the Haemophilus influenzae type b line is Ca MinnA, Eagan line The non-type Haemophilus influenzae strain is selected from * and the PAK 12085 and SB33 strains. A synthetic peptide having an amino acid sequence corresponding to only a portion of an amino acid sequence of a protein claimed in any one of claims 10 to 15 or their modifications or mutants. The synthetic peptide according to claim 16, wherein the synthetic peptide has an amino acid sequence selected from the amino acid sequences shown in Table 2. A chimeric molecule characterized by a polypeptide claimed in any one of claims 10 to 15 or linked to another polypeptide, protein or polysaccharide. 19. The chimeric molecule of claim 18, wherein said other polypeptide or protein comprises a surface protein or peptide corresponding to a pathogenic bacterium. 20. The chimeric molecule of claim 19, wherein said other polypeptide or protein comprises a P1, P2 or P6 outer membrane protein of Haemophilus influenzae, and the constant polysaccharide comprises a PRP molecule from Haemophilus influenzae. A nucleic acid molecule as claimed in claim 1. A protein as claimed in any one of claims 10 to 15 or a synthetic peptide as claimed in claim 16 or 17 or a chimeric molecule as claimed in any one of claims 18 to 20 and An immunogenic composition comprising a physiologically-acceptable carrier. The protein of any one of claims 10-21. Antisera or antibody having specificity for a peptide or immunogenic composition.