Patent Grant
7785609
The present invention relates to the field of molecular genetics and, in particular, to the production of recombinant Haemophilus influenzae adhesin (Hia) proteins.
Haemophilus influenzae is the cause of several serious human diseases, such as meningitis, epiglottitis, septicemia and otitis media. There are six serotypes of H. influenzae, designated a to f, that are identified by their capsular polysaccharide. H. influenzae type b (Hib) was a major cause of bacterial meningitis until the introduction of several Hib conjugate vaccines in the 1980' s (ref. 1. Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Vaccines based upon H. influenzae type b capsular polysaccharide conjugated to diphtheria toxoid (ref. 2), tetanus toxoid (ref. 3 and U.S. Pat. No. 4,496,538), or Neisseria meningitidis outer membrane protein (ref. 4) have been effective in reducing H. influenzae type b-induced meningitis. The other serotypes of H. influenzae are associated with invasive disease at low frequencies, although there appears to be an increase in the incidence in disease caused by these strains as the incidence of Hib disease declines (ref. 5; ref. 6). Non-encapsulated or non-typeable H. influenzae (NTHi) are also responsible for a wide range of human diseases including otitis media, epiglottitis, pneumonia, and tracheobronchitis. The incidence of NTHi-induced disease has not been affected by the introduction of the Hib vaccines (ref. 7).
Otitis media is the most common illness of early childhood, with 60 to 70% of all children, of less than 2 years of age, experiencing between one and three ear infections (ref. 8). Chronic otitis media is responsible for hearing, speech and cognitive impairments in children. H. influenzae infections account for about 30% of the cases of acute otitis media and about 60% of chronic otitis media. In the United States alone, treatment of otitis media costs between 1 and 2 billion dollars per year for antibiotics and surgical procedures such as tonsillectomies, adenoidectomies and insertion of tympanostomy tubes. It is estimated that an additional $30 billion is spent per annum on adjunct therapies, such as speech therapy and special education classes. Furthermore, many of the causative organisms of otitis media are becoming resistant to antibiotic treatment. An effective prophylactic vaccine against otitis media is thus desirable.
During natural infection by NTHi, surface-exposed outer membrane proteins that stimulate an antibody response are potentially important targets for bactericidal and/or protective antibodies and, therefore, potential vaccine candidates. A family of high molecular weight proteins (HMW1 and HMW2) that are important in attachment of NTHi to epithelial cells has been identified in about 70 to 75% of NTHi strains (ref. 9; ref. 10). These high molecular weight adhesins have been shown to afford some protection in the chinchilla model of otitis media (ref. 11). A second family of high molecular weight adhesion proteins has been identified in about 25% of NTHi and in encapsulated H. influenzae strains (ref. 12; ref. 13, ref. 14). The NTHi member of this second family is termed Haemophilus influenzae adhesin or Hia and the homologous protein found in encapsulated strains is termed Haemophilus influenzae surface fibril protein or Hsf. The hia gene was originally cloned from an expression library using convalescent sera from an otitis media patient, which indicates that it is an important immunogen during disease. The prototype Hia and Hsf proteins demonstrate about 82% sequence similarity, although the Hsf protein is considerably larger. The proteins are comprised of conserved amino and carboxy termini and several repeat motifs, with Hsf containing more repeat sequences than Hia. A high molecular weight protein (200 kDa) has also been identified from Moraxella catarrhalis that has some sequence homology with the Hsf and Hia proteins (U.S. Pat. No. 5,808,024).
Since Hia or Hsf is conserved amongst encapsulated strains of Haemophilus influenzae and about 20 to 25% of non-encapsulated strains, and has been demonstrated to be an adhesin, the protein has utility in diagnosis of and vaccination against disease caused by H. influenzae or other bacterial pathogens that produce Hia or a protein capable of raising antibodies specifically reactive with Hia.
A disadvantage of Hia for use as an antigen in diagnosis, for the generation of anti-Hia antibodies useful in diagnosis and as an immunogen in vaccination is the low recovery of the native protein from Haemophilus influenzae species.
It would be advantageous to provide recombinant Hia protein for use as antigens, in immunogenic preparations including vaccines, carriers for other immunogens and in the generation of diagnostic reagents.
The present invention is directed towards the provision of recombinant H. influenzae adhesin (rHia) proteins.
In connection with the provision of such recombinant proteins, the present invention provides certain isolated and purified nucleic acid molecules. Accordingly, in one aspect thereof, the present invention provides an isolated and purified nucleic acid molecule encoding a Haemophilus influenzae adhesin (Hia) protein of a strain of Haemophilus influenzae having: (a) a DNA sequence selected from the group consisting of those shown in
Such nucleic acid may be included in a vector, which may be a plasmid vector. In particular, the nucleic acid molecule may encode the Hia protein from strain 11 or 33 of non-typeable Haemophilus.
In another aspect of the present invention, there is provided an isolated and purified nucleic acid molecule encoding an N-truncated Haemophilus influenzae adhesin (Hia) protein of a strain of Haemophilus influenzae which is amplifiable by a pair of nucleotides which are selected from the group consisting of SEQ ID No: 7 and SEQ ID No: 15; SEQ ID No: 9 and SEQ ID No: 15; SEQ ID No: 11 and SEQ ID No: 15; SEQ ID No: 13; SEQ ID No: 15; SEQ ID No: 49; and SEQ ID No: 51.
Such nucleic acid may be included in a vector, which may be a plasmid vector. In particular, the nucleic acid molecule may encode an N-truncated Hia protein from strain 11 or 33 of non-typeable Haemophilus, starting at codon V38 or S44.
The plasmid vector incorporating the isolated and purified nucleic acid provided in accordance with these aspects of the invention may have the identifying characteristics of a plasmid which is selected from the group consisting of:
DS-2008-2-3 as shown in
DS-2186-1-1 as shown in
DS-2201-1 as shown in
DS-2186-2-1 as shown in
DS-2168-2-6 as shown in
1A-191-3-1 as shown in
The vector provided herein may include the cer gene from E. coli. Accordingly, in another aspect of the present invention, there is provided a vector for transforming a host, comprising a nucleic acid molecule encoding a full-length or N-truncated Haemophilus influenzae adhesin (Hia) protein, a promoter for expression of said full-length or truncated Hia protein and, optionally, the cer gene of E. coli. The vector may be a plasmid vector or other non-replicating vector, which may have the identifying characteristics of a plasmid vector which is selected from the group consisting of:
BK-96-2-11 as shown in
DS-2242-1 as shown in
DS-2242-2 as shown in
DS-2340-2-3 as shown in
DS-2447-2 as shown in
DS-2448-17 as shown in
JB-2930-3 as shown in
The vectors provided herein may comprise a replicating vector, including a vector from Salmonella, BCG, adenovirus, poxvirus, vaccinia or poliovirus.
Any of the vectors provided herein may be employed to transform a suitable host cell for expression therein of a protective Haemophilus influenzae adhesin (Hia) protein of a non-typeable strain of Haemophilus, which may be in full-length or truncated form. Such host conveniently may be E. coli. Such expression may be under the control of the T7 promoter and expression of the recombinant Hia from the transformed host may be effected by culturing in an inducing concentration of lactose or other convenient inducing agent.
The present invention further includes, in a further aspect thereof, a recombinant protective Haemophilus influenzae adhesin (Hia) protein of a non-typeable Haemophilus strain producible by the transformed host, particularly E. coli, provided herein. Such Hia protein may be provided in the form of an immunogenic fragment or adhesin-functional analog of the recombinant protein.
The recombinant Hia proteins, full-length or N-truncated, provided herein are useful as antigens in immunogenic compositions, carriers for other immunogens, diagnostic agents and in the generation of diagnostic agents. The nucleic acid molecules which encode the Hia protein, full-length or N-truncated, also are useful as probes for diagnostic use and also in immunogenic compositions.
The present invention, in an additional aspect thereof, provides an immunogenic composition, comprising at least one immunologically active component which is selected from the group consisting of an isolated and purified nucleic acid molecule as provided herein and a recombinant protective Hia protein, full-length or N-truncated, of a strain of Haemophilus, as provided herein, and a pharmaceutically-acceptable carrier therefor.
The immunogenic compositions provided herein may be formulated as a vaccine for in vivo administration to a host to provide protection against disease caused by H. influenzae. For such purpose, the compositions may be formulated as a microparticle, capsule, ISCOM or liposome preparation. The immunogenic composition may be provided in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces.
The immunogenic compositions of the invention (including vaccines) may further comprise at least one other immunogenic or immunostimulating material and the immunostimulating material may be at least one adjuvant or at least one cytokine. Suitable adjuvants for use in the present invention include (but are not limited to) aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein and other adjuvants.
Advantageous combinations of adjuvants are described in copending U.S. patent application Ser. No. 08/261,194 filed Jun. 16, 1994 and 08/483,856 filed Jun. 7, 1995, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference (WO 95/34308 published Nov. 21, 1995).
In accordance with another aspect of the invention, there is provided a method for generating an immune response in a host, comprising the step of administering to a susceptible host an effective amount of the immunogenic composition as recited above. The immune response may be humoral or a cell-mediated immune response. Hosts in which protection against disease may be conferred include primates, including humans.
In accordance with other aspects of the invention, there is provided the immunogenic compositions provided herein when used as a medicament and the use of these components of the immunogenic compositions in the manufacture of an immunogenic composition.
The present invention includes, in a yet additional aspect thereof, a method for the production of a protective Haemophilus influenzae adhesin (Hia) protein of a non-typeable strain of Haemophilus influenzae, which comprises:
transforming a host, such as E. coli, with a vector comprising a nucleic acid molecule encoding an N-truncated form of the Haemophilus influenzae adhesin protein as provided herein,
growing the host to express the encoded truncated Hia, and
isolating and purifying the expressed Hia protein.
The encoded truncated Hia may be expressed in inclusion bodies. The isolation and purification step may be effected by disrupting the grown transformed cells to produce a supernatant and the inclusion bodies containing the Hia, solubilizing the inclusion bodies after separation from the supernatant, to produce a solution of the recombinant Hia, chromatographically purifying the solution of recombinant Hia free from cell debris, and isolating the purified recombinant Hia protein.
The vector transforming the host cell, such as E. coli, may include the T7 promoter and the E. coli or other host cell may be cultured in the presence of an inducing amount of lactose or other convenient inducing agent.
The strain of Haemophilus influenzae herein may be selected from the group of non-typeable strains consisting of strains 11, 33, 32, 29, M4071, K9, K22 and 12. Specific nucleic acid sequences for the genes encoding the respective Hia proteins from such strains are provided herein and are described below.
The nucleic acid molecules provided herein are useful in diagnostic applications. Accordingly, in a further aspect of the invention, there is provided a method of determining the presence, in a sample, of nucleic acid encoding a Haemophilus influenzae adhesin protein, comprising the steps of:
a) contacting the sample with a nucleic acid molecule as provided herein to produce duplexes comprising the nucleic acid molecule provided herein are nucleic acid encoding the Hia protein of a strain of Haemophilus present in the sample and specifically hybridizable therewith; and
b) determining the production of the duplexes.
In addition, the present invention provides a diagnostic kit for determining the presence, in a sample, of nucleic acid encoding a Haemophilus influenzae adhesin protein, comprising:
a) a nucleic acid molecule as provided herein;
b) means for contacting the nucleic acid molecule with the sample to produce duplexes comprising the nucleic acid molecule and any such nucleic acid molecule; and
c) means for determining production of the duplexes.
The recombinantly produced truncated Hia proteins provided herein also are useful in diagnostic applications. Accordingly, in another aspect of the invention, there is provided a method of determining the presence of antibodies specifically reactive with the Hia protein in a sample, comprising the steps of (a) contacting the sample with the recombinant Hia protein provided herein to provide complexes of the recombinant Hia protein and any such antibodies present in the sample specifically reactive therewith; and (b) determining production of the complexes.
Advantages of the present invention include:
an isolated and purified nucleic acid molecule encoding a Haemophilus influenzae adhesin protein or a fragment or an analog of the Hia protein;
recombinantly-produced Hia proteins, free from any other Haemophilus proteins; and
diagnostic kits and immunological reagents for specific identification of Haemophilus.
The present invention will be further understood from the following description with reference to the drawings, in which:
Since H. influenzae strains produce low quantities of the Hia and Hsf proteins, the hia gene from NTHi strains was cloned into an expression vector for overproduction of the recombinant protein in E. coli. When the full-length recombinant Hia (rHia) protein was expressed, it was made in relatively low quantities. In order to confirm that there was expression of the recombinant protein, an immunoblot was performed using antibody raised to a Moraxella catarrhalis high molecular weight adhesin protein identified as 200 kDa in U.S. Pat. No. 5,808,024, assigned to the assignee and the disclosure of which is incorporated herein by reference. Antibody against the gel-purified native 200 kDa protein recognized a specific induced band in the rHia protein sample. The yield of rHia was not significantly improved by increasing the gene copy number of the T7 hia gene cassette.
The E. coli cer gene has been shown to stabilize plasmids containing large inserts (ref. 15), but the yield of rHia was not significantly improved by adding the E. coli cer gene to the expression vector. However, the E. coli cells were observed to clump during culture, suggesting that there was surface expression of the Hia adhesin protein. The apparent toxicity of the rHia protein might be overcome if it were made as inclusion bodies, so truncations were made at the 5′-end of the gene to delete putative signal sequences. This modification resulted in good production and recovery of truncated rHia starting from the V38 position.
The full-length and V38-truncated rHia proteins were immunogenic and the resultant anti-rHia antibodies were protective in passive infant rat models of bacteremia due to H. influenzae type a or type b strains. In addition, the truncated V38 rHia protein was found to be partially protective against nasopharyngeal colonization in an active challenge model in chinchillas. The protection afforded by rHia derived from an NTHi strain against disease caused by NTHi and encapsulated type a or type b strains, indicates that there may be common protective epitopes. The cloning and sequence analysis of additional hia genes may help to identify conserved regions. The full-length or N-terminal truncated rHia proteins may be used as vaccine components to protect against Haemophilus influenzae disease.
Any Haemophilus strains that have hia genes may be conveniently used to provide the purified and isolated nucleic acid molecules (which may be in the form of DNA molecules), comprising at least a portion coding for a Hia protein as typified by embodiments of the present invention. Such strains are generally available from clinical sources and from bacterial culture collections, such as American Type Culture Collection. Appropriate strains of Haemophilus include:
Non-typeable Haemophilus strain 11;
Non-typeable Haemophilus strain 33;
Non-typeable Haemophilus strain 32;
Non-typeable Haemophilus strain 29;
Non-typeable Haemophilus strain M4071;
Non-typeable Haemophilus strain K9;
Non-typeable Haemophilus strain K22;
Non-typeable Haemophilus strain 12;
Type C Haemophilus strain API.
In this application, the term “Hia” protein is used to define a family of Hia proteins that includes those having naturally occurring variations in their amino acid sequences as found in various strains of Haemophilus.
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It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have use in applications in the fields of vaccination, diagnosis, treatment of Haemophilus infection and the generation of immunological agents. A further non-limiting discussion of such uses is further presented below.
Vaccine Preparation and Use
Immunogenic compositions, suitable to be used as vaccines, may be prepared from immunogenic recombinant Haemophilus influenzae adhesin (rHia) proteins of non-typeable Haemophilus strains, immunogenic analogs and fragments thereof and/or immunogenic peptides as disclosed herein. The vaccine elicits an immune response which produces antibodies, including anti-rHia antibodies and antibodies that are opsonizing or bactericidal.
Immunogenic compositions, including vaccines, may be prepared as injectables, as liquid solutions or emulsions. The rHia protein, immunogenic analogs and fragments thereof and/or immunogenic peptides may be mixed with pharmaceutically acceptable excipients which are compatible with the rHia protein, immunogenic fragments analogs or immunogenic peptides. Such excipients may include, water, saline, dextrose, glycerol, ethanol and combinations thereof.
The immunogenic compositions and vaccines may further contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness of the vaccines.
Immunogenic compositions and vaccines may be administered parenterally, by injection subcutaneously or intramuscularly. Alternatively, the immunogenic compositions formed according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces. Thus, the immunogenic composition may be administered to mucosal surfaces by, for example, the nasal or oral (intragastric) routes.
The immunogenic composition may be provided in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces. Some such targeting molecules include vitamin B12 and fragments of bacterial toxins, as described in WO 92/17167 (Biotech Australia Pty. Ltd.), and monoclonal antibodies, as described in U.S. Pat. No. 5,194,254 (Barber et al).
Alternatively, other modes of administration including suppositories and oral formulations may be desirable. For suppositories, binders and carriers may include, for example polyalkalene glycols or triglycerides. Oral formulations may include normally employed incipients such as, for example pharmaceutical grades of saccharine, cellulose and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 1 to 95% of the rHia protein, fragment analogs and/or peptides.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies, and if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms of the rHia, analogs and fragments thereof and/or peptides. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage of the vaccine may also depend on the route of administration and will vary according to the size of the host.
The nucleic acid molecules encoding the rHia proteins of non-typeable Haemophilus may also be used directly for immunization by administration of the DNA directly, for example by injection for genetic immunization or by constructing a live vector, such as Salmonella, BCG, adenovirus, poxvirus, vaccinia or poliovirus, containing the nucleic acid molecule. A discussion of some live vectors that have been used to carry heterologous antigens to the immune system is contained in, for example, O'Hagan (1992) (ref. 16). Processes for the direct injection of DNA into test subjects for genetic immunization are described in, for example, Ulmer et al., 1993 (ref. 17).
Immunogenicity can be significantly improved if the antigens are co-administered with adjuvants, commonly used as an 0.05 to 1.0 percent solution in phosphate—buffered saline. Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.
Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccharides, normally are the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Thus, adjuvants have been identified that enhance the immune response to antigens delivered parenterally. Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established.
A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include the specific adjuvants detailed above as well as saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria and mineral oil, Freund's complete adjuvants, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes.
To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens are emulsified in adjuvants. Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant, FCA), cytolysis (saponins and pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP). Although FCA is an excellent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.
Desirable characteristics of ideal adjuvants include:
U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8, 1989 which is incorporated herein by reference thereto teaches glycolipid analogues including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants. Thus, Lockhoff et al. 1991 (ref. 18) reported that N-glycolipid analogs displaying structural similarities to the naturally-occurring glycolipids, such as glycosphingolipids and glycoglycerolipids, are capable of eliciting strong immune responses in both herpes simplex virus vaccine and pseudorabies virus vaccine. Some glycolipids have been synthesized from long chain-alkylamines and fatty acids that are linked directly with the sugars through the anomeric carbon atom, to mimic the functions of the naturally occurring lipid residues.
U.S. Pat. No. 4,258,029 granted to Moloney, assigned to the assignee hereof and incorporated herein by reference thereto, teaches that octadecyl tyrosine hydrochloride (OTH) functions as an adjuvant when complexed with tetanus toxoid and formalin inactivated type I, II and III poliomyelitis virus vaccine. Also, Nixon-George et al. 1990 (ref. 19), reported that octadecyl esters of aromatic amino acids complexed with a recombinant hepatitis B surface antigen, enhanced the host immune responses against hepatitis B virus.
Immunoassays
The rHia protein of a non-typeable strain of Haemophilus, analogs and fragments thereof produced according to the present invention are useful as immunogens, as antigens in immunoassays including enzyme-linked immunosorbent assay (ELISA), RIAs and other non-enzyme linked antibody binding assays or procedures known in the art for the detection of anti-bacterial, Haemophilus, and/or Hia antibodies. In ELISA assays, the Hia protein, analogs and fragments are immobilized onto a selected surface, for example a surface capable of binding proteins or peptides, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed Hia protein, analogs and/or fragments, a nonspecific protein such as a solution of bovine serum albumin (BSA) or casein that is known to be antigenically neutral with regard to the test sample may be bound to the selected surface. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific bindings of antisera onto the surface.
The immobilizing surface is then contacted with a sample, such as clinical or biological materials, to be tested in a manner conducive to immune complex (antigen/antibody) formation. This may include diluting the sample with diluents, such as BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is then allowed to incubate for from about 2 to about 4 hours, at temperature such as of the order of about 25° to about 37° C. Following incubation, the sample-contacted surface is washed to remove non-immunocomplexed material. The washing procedure may include washing with a solution such as PBS/Tween, or a borate buffer.
Following formation of specific immunocomplexes between the test sample and the bound Hia protein, analogs and/or fragments, and subsequent washing, the occurrence, and even amount, of immunocomplex formation may be determined by subjecting the immunocomplex to a second antibody having specificity for the first antibody. If the test sample is of human origin, the second antibody is an antibody having specificity for human immunoglobulins and in general IgG. To provide detecting means, the second antibody may have an associated activity, such as an enzymatic activity, that will generate, for example, a color development, upon incubating with an appropriate chromogenic substrate. Quantification may then achieved by measuring the degree of color generation using, for example, a visible spectra spectrophotometer.
Use of Sequences as Hybridization Probes
The nucleotide sequences of the present invention, comprising the newly-isolated and characterized sequences of the hia genes, allow for the identification and cloning of the hia genes from other non-typeable strains of Haemophilus.
The nucleotide sequences comprising the sequence of hia genes of the present invention are useful for their ability to selectively form duplex molecules with complementary stretches of other hia genes. Depending on the application, a variety of hybridization conditions may be employed to achieve varying degrees of selectivity of the probe toward the other hia genes in other strains of non-typeable Haemophilus. For a high degree of selectivity, relatively stringent conditions are used to form the duplexes, such as low salt and/or high temperature conditions, such as provided by 0.02 M to 0.15 M NaCl at temperatures of between about 50° C. to 70° C. For some applications, less stringent hybridization conditions are required such as 0.15 M to 0.9 M salt, at temperatures ranging from between 20° C. to 55° C. Hybridization conditions can also be rendered more stringent by the addition of increasing amount of formamide, to destabilize the hybrid duplex. Thus, particular hybridization conditions can be readily manipulated, and will generally be a method of choice depending on the desired results. In general, convenient hybridization temperatures in the presence of 50% formamide and 0.15 M NaCl are: 42° C. for an hia gene which is about 95 to 100% homologous to the target nucleic acid fragment, 37° C. for about 90 to 95 homology and 32° C. for about 85 to 90% homology.
In a clinical diagnostic embodiment, the nucleic acid sequences of the hia genes of the present invention may be used in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of providing a detectable signal. In some diagnostic embodiments, an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of a radioactive tag may be used. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with samples containing Hia genes sequences.
The nucleic acid sequences of Hia genes of the present invention are useful as hybridization probes in solution hybridizations and in embodiments employing solid-phase procedures. In embodiments involving solid-phase procedures the test DNA (or RNA) from samples, such as clinical samples, including exudates, body fluids (e.g., serum, amniotic fluid, middle ear effusion, sputum, bronchoalveolar lavage fluid) or even tissues, is adsorbed or otherwise affixed to a selected matrix or surface. The fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes comprising the nucleic acid sequences of the hia genes or fragments thereof of the present invention under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required depending on, for example, the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe etc. Following washing of the hybridization surface so as to remove non-specifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label. It is preferred to select nucleic acid sequence portions which are conserved among species of Haemophilus. The selected probe may be at least 18 by in length and may be in the range of 30 by to 90 by long.
Expression of the Haemophilus influenzae Adhesin Genes
Plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell may be used for the expression of the hia genes in expression systems. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli may be transformed using pBR322 which contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid or phage, must also contain, or be modified to contain, promoters which can be used by the host cell for expression of its own proteins.
In addition, phage vectors containing replicon and control sequences that are compatible with the host can be used as a transforming vector in connection with these hosts. For example, the phage in lambda GEM™-11 may be utilized in making recombinant phage vectors which can be used to transform host cells, such as E. coli LE392.
Promoters commonly used in recombinant DNA construction include the β-lactamase (penicillinase) and lactose promoter systems and other microbial promoters, such as the T7 promoter system employed herein in preferred embodiments (U.S. Pat. No. 4,952,496). Details concerning the nucleotide sequences of promoters are known, enabling a skilled worker to ligate them functionally with genes. The particular promoter used will generally be a matter of choice depending upon the desired results. Hosts that are appropriate for expression of the Hia protein and immunological fragments or analogs thereof include E. coli, Bordetella species, Bacillus species, Haemophilus, fungi, yeast or the baculovirus expression system may be used. E. coli is the preferred host used herein.
In accordance with this invention, it is preferred to produce the Hia proteins by recombinant methods, particularly when the naturally occurring Hia protein as purified from a culture of a species of Haemophilus may include trace amounts of toxic materials or other contaminants. This problem can be avoided by using recombinantly produced Hia protein in heterologous systems which can be isolated from the host in a manner to minimize contaminants in the purified materials, specifically employing the constructs described herein.
A vector that contains nucleic acid coding for a high molecular weight protein of a non-typeable strain of Haemophilus that is described and referred to herein has been deposited with the America Type Culture Collection (ATCC) located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA, pursuant the Budapest Treaty and prior to the filing of this application. Samples of the deposited vector will become available to the public and all restrictions imposed or access to the deposits will be received upon grant of a patent based on this U.S. patent application. In addition, the deposit will be replaced if viable samples cannot be dispensed by the Depository. The invention described and claimed herein is not limited in scope by the biological materials deposited, since the deposited embodiment is intended only as an illustration of the invention. Any equivalent or similar vectors that contain nucleic acid which encodes equivalent or similar antigens as described in this application are within the scope of the invention.
The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations.
Methods of molecular genetics, protein biochemistry, immunology and fermentation technology used, but not explicitly described in this disclosure and these Examples, are amply reported in the scientific literature and are well within the ability of those skilled in the art.
This Example describes the construction of plasmid DS-2008-2-3 that expresses full-length rHia proteins from NTHi strain 11.
Chromosomal DNA was purified from NTHi strain 11 and the full-length hia gene was PCR amplified using the oligonucleotides (5038.SL and 5039.SL) described in
This Example illustrates the recognition of rHia by anti-native Moraxella catarrhalis high molecular weight adhesin antibody.
There is some sequence conservation observed between the Haemophilus influenzae Hia proteins and a Moraxella catarrhalis high molecular weight adhesin identified as the M. catarrhalis 200 kDa protein in aforementioned U.S. Pat. No. 5,808,024 (FIG. 28). The native M. catarrhalis 200 kDa protein was gel purified as described in U.S. Pat. No. 5,808,024 and guinea pig anti-native 200 kDa antibody was generated. The T7 hia gene was expressed from plasmid DS-2008-2-3 and the cell culture containing the rHia protein was electroblotted to nitrocellulose membrane. Immunoblot analysis using anti-native 200 kDa antibody showed that the antibody recognized the rHia protein, as seen in
This Example describes the construction of plasmids DS-2092-1 and DS-2092-40 that contain tandem copies of T7 hia (11) gene cassettes.
In order to improve the production of full-length recombinant Hia protein, tandem copies of the T7 hia gene cassette containing the strain 11 hia gene (Example 1) were inserted into a single vector. Plasmid DS-2008-2-3 was linearized with Bgl II and dephosphorylated. Plasmid DS-2008-2-3 was also digested with Bgl II and BamH I to excise the T7 hia gene cassette. The T7 hia fragment was ligated into the linearized vector to generate plasmid DS-2092-1 that contains two copies of the T7 hia gene in the anti-clockwise orientation (a,a) and plasmid DS-2092-40 that contains tandem copies in opposite orientations (a,c) (
This Example describes the construction of plasmids expressing truncated strain 11 hia genes.
The production of the rHia protein from single or tandem copies of the T7 hia gene cassette was very low and the protein seemed to be toxic to E. coli (as described below in Example 9). Since H. influenzae Hia is a surface-exposed adhesin molecule, it must either utilize a signal sequence or accessory protein(s) for secretion, but there are no known accessory genes involved. If the signal sequence were removed for expression of the recombinant protein in E. coli, the rHia might be expressed as inclusion bodies and the toxic effect reduced. A putative signal sequence and cleavage sites were identified and four constructs expressing N-terminally truncated rHia proteins were designed (
This Example describes the construction of plasmid BK-96-2-11 that contains the T7 V38 hia (11) cassette, the E. coli cer gene, and the kanamycin resistance gene.
Plasmid DS-1843-2 is a pBR328-based plasmid in which a multiple cloning site and two transcription terminators have been introduced on oligonucleotides, between the EcoR I and Pst I sites, thus destroying both the chloramphenicol and ampicillin resistance genes (
This Example describes the construction of plasmids DS-2242-1 and DS-2242-2 that express the full-length NTHi strain 33 hia gene in the presence of the E. coli cer gene.
Chromosomal DNA was purified from NTHi strain 33 and PCR amplification was performed using oligonucleotides 5039.SL and 5040.SL (
In order to express the full-length strain 33 hia gene, approximately 106 by of the 5′-end of the gene was synthesized from oligonucleotides, from the start codon to an AlwN I site (
In order to include the E. coli cer gene and utilize kanamycin selection, the Bgl II-BamH I fragment containing the T7 hia (33) gene cassette was excised from DS-2103-4 and cloned into BK-2-1-1 that had been digested with Bgl II and dephosphorylated. Plasmids DS-2242-1 and DS-2242-2 contain single copies of the T7 hia (33) gene cassette in opposite orientations, the E. coli cer gene, and the kanamycin resistance gene (
This Example describes the construction of plasmid DS-2340-2-3 that contains a T7 hia gene cassette with a truncated V38 strain 33 hia gene, the E. coli cer gene, and the kanamycin resistance gene.
PCR primers were designed to amplify a 250 by fragment of the 5′-end of the NTHi strain 33 hia gene from a V38 start codon up to an internal SnaB I site. An Nde I site was added at the 5′-end for cloning purposes and the fragment was amplified using plasmid DS-2242-1 as template. The construction scheme is shown in
This Example illustrates the construction of plasmids DS-2447-2 and DS-2448-17 that contain tandem copies of T7 V38 hia (11) or T7 V38 hia (33) gene cassettes, respectively, the E. coli cer gene, and a kanamycin resistance gene.
Plasmid BK-96-2-11, that contains a T7 V38 hia (11) gene cassette, was linearized with Bgl II and dephosphorylated. The Bgl II-BamH I T7 V38 hia (11) gene cassette from DS-2186-2-1 was ligated into BK-96-2-11, generating plasmid DS-2447-2 that contains tandem copies of the T7 V38 hia (11) gene in the same orientation (
Plasmid DS-2340-2-3 was digested with EcoR I and the T7 V38 hia (33) gene cassette was subcloned into pUC-BgXb that had been digested with EcoR I and dephosphorylated. The resultant plasmid, DS-2440-2 was digested with Bgl II and BamH I to release the T7 V38 hia (33) cassette that was ligated with DS-2340-2-3 that had been linearized with Bgl II and dephosphorylated. Plasmid DS-2448-17 contains tandem T7 V38 hia(33) genes in the same orientation (
This Example illustrates the expression of full-length and truncated recombinant hia genes.
DNA from expression plasmids prepared as described in the preceding Examples, was introduced into electrocompetent E. coli BL21 (DE3) cells using a BioRad electroporator. Cells were grown at 37° C. in NZCYM medium using the appropriate antibiotic selection to A578 of 0.3 before the addition of lactose to 1.0% for 4 hours. Samples were adjusted to 0.2 OD/μl with SDS-PAGE lysis+loading buffer and the same amount of each protein sample was loaded onto SDS-PAGE gels (ref. 22).
This Example illustrates the purification of rHia proteins.
All the recombinant Hia proteins were expressed as inclusion bodies in E. coli and were purified by the same procedure (
The resultant pellet (PPT3) obtained after the above extractions contains the inclusion bodies. The pellet was solubilized in 6 ml of 50 mM Tris-HCl, pH 8.0, containing 6 M guanidine and 5 mM DTT. Twelve ml of 50 mM Tris-HCl, pH 8.0 was added to this solution and the mixture was centrifuged at 20,000 g for 30 min. The supernatant (SUP4) was precipitated with polyethylene glycol (PEG) 4000 at a final concentration of 7%. The resultant pellet (PPT5) was removed by centrifugation at 20,000 g for 30 min and the supernatant was precipitated by (NH4)2SO4 at 50% saturation. The (NH4)2SO4 precipitate was collected by centrifugation at 20,000 g for 30 min. The resultant pellet (PPT6) was dissolved in 2 ml of 50 mM Tris-HCl, pH 8.0, containing 6 M guanidine HCl and 5 mM DTT and the clear solution was purified on a Superdex 200 gel filtration column equilibrated in 50 mM Tris-HCl, pH 8.0, containing 2 M guanidine HCl. The fractions were analysed by SDS-PAGE and those containing the purified rHia were pooled and dialysed overnight at 4° C. against PBS, then centrifuged at 20,000 g for 30 min. The protein remained soluble under these conditions and glycerol was added to the rHia preparation at a final concentration of 20% for storage at −20° C. SDS-PAGE analysis of purified V38 rHia (11) and V38 rHia (33) is illustrated in
In order to study the stability of rHia, the purified V38 rHia (11) protein was stored at 4° C. with or without glycerol and at −20° C. with glycerol. The protein was found to be stable under all three conditions and remained intact for at least eight weeks with repeated freezing and thawing (
This Example illustrates the immunogenicity of V38 rHia (11) and V38 rHia (33) proteins.
Hyperimmune antisera against rHia proteins were produced by immunizing two guinea pigs (Charles River) intramuscularly (i.m.) with 5 μg doses of antigen emulsified in complete Freund's adjuvant (CFA, Difco) on day 1. Animals were boosted on days 14 and 28 with 5 μg doses of protein in incomplete Freund's adjuvant (IFA) and sera were collected on day 42. Anti-Hib strain MinnA and anti-Haemophilus type a strain ATCC 9006 antisera were generated using the same protocol, except that a heat-inactivated bacterial preparation was used as the immunogen (1×108 cfu per dose).
To study the immunogenicity of the V38 rHia proteins, groups of five CD-1 mice (Charles River, Quebec) were immunized s.c. on days 1 and 28 with 0.3, 1, 3, and 10 μg of antigen, in the presence of AlPO4 (alum) (1.5 mg per dose). Blood samples were collected on days 1, 28 and 42. Mice generated significant anti-V38 rHia antibody responses even with a single injection of 0.3 μg antigen (
To study the immunogenicity of the V38 rHia (11) protein in BALB/c mice, groups of five animals (Charles River, Quebec) were immunized s.c. on days 1, 28 and 42 with 0.3, 1, 3, and 10 μg of antigen, in the presence of AlPO4 (1.5 mg per dose). Blood samples were collected on days 1, 14, 28, 42 and 56. High antibody titers were observed in all groups, indicating that the protein is very immunogenic even at 0.3 μg per dose (
To study the immunogenicity of the V38 rHia (11) protein in guinea pigs, groups of five animals (Charles River, Quebec) were immunized s.c. on days 1, 28 and 42 with 0.3, 1, 3, and 10 μg of antigen, in the presence of AlPO4 (1.5 mg per dose). Blood samples were collected on days 1, 14, 28, 42 and 56. High antibody titers were observed in all groups, indicating that the protein is also very immunogenic in guinea pigs (
This Example illustrates the analysis of the protection afforded by anti-rHia antibodies in passive infant rat models of bacteremia.
Pregnant Wistar rats were purchased from Charles River. In the H. influenzae type b bacteremia model, groups of 6 to 10 five-day old infant rats were injected s.c. in the dorsal region with 0.1 ml of guinea pig anti-rHia or anti-strain MinnA antiserum. The control animals received injections with pre-immune sera only. Twenty hours later, the animals were challenged intraperitoneally (i.p.) with 200 to 240 colony-forming units (cfu) of freshly grown Hib strain MinnA (0.1 ml). Blood samples were collected 20 h post-challenge, via cardiac puncture under isoflurane anesthesia and plated on chocolate agar plates. Colonies were counted after one day and the results were statistically analyzed by Fisher's Exact test.
In the H. influenzae type a bacteremia model (ref. 23), groups of 9 to 10 five-day old infant rats were injected s.c. in the dorsal region with 0.1 ml of guinea pig anti-rHia or anti-strain ATCC 9006 antiserum. The animals in the control group were injected with guinea pig pre-immune serum. Twenty hours later, the animals were challenged i.p. with 100,000 cfu of freshly grown H. influenzae type a strain ATCC 9006 (0.1 ml). Blood samples were collected 20 h post-challenge and analysed as described above.
As shown in Tables 1 and 2 below, the infant rats that were passively immunized with either guinea pig anti-rHia (11) or anti-V38 rHia (11) antisera, were all significantly protected against type a or type b H. influenzae caused bacteremia. These results demonstrate that antibodies raised to the slightly truncated Hia protein (V38 rHia) are as efficacious as those raised to the full-length protein at protecting animals against bacteremia caused by type a or type b H. influenzae. Such protection afforded by an NTHi-derived recombinant protein against invasive disease caused by encapsulated bacteria, illustrates the utility of the rHia proteins as vaccine antigens.
This Example illustrates the protection afforded by immunization with V38 rHia protein in a chinchilla model of nasopharyngeal colonization.
A nasopharyngeal colonization model has been described by Yang et al (ref. 20). The model works well for those NTHi strains that produce the HMW adhesins, but reproducible colonization could not be established with Hia-producing strains under the same conditions. Repeated attempts to colonize with the prototype Hia-producing NTHi strain 11, were unsuccessful. Colonization was achieved with NTHi strain 33 at 5×108 cfu per inoculum, compared with only 108 cfu required for the prototype HMW-producing NTHi strain 12. Under these conditions, partial protection was observed in animals immunized with 100 μg of V38 rHia (33) and challenged with the homologous NTHi strain 33.
This Example illustrates the cloning and sequence analysis of additional hia genes from H. influenzae strains.
Oligonucleotides (5040.SL and 5039.SL) for PCR amplification were designed based upon the conserved promoter, N-terminal and C-terminal sequences of the hia and hsf genes and proteins (
Chromosomal DNA was prepared from NTHi strains 12, 29, 32, M4071, K9 and, K22 and Haemophilus type c strain API. PCR amplification was performed as follows: each reaction mixture contained 5 to 100 ng of DNA, 1 lag of each primer, 5 units of taq+ or tsg+(Sangon) or taq plus long (Stratagene), 2 mM dNTPs, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, BSA. Cycling conditions were: 95° C. for 1 min, followed by 25 cycles of 95° C. for 30 sec, 45° C. for 1 min, 72° C. for 2 min; then 72° C. for 10 min.
The nucleotide and deduced amino acid sequences of the hia gene from strain 33 are shown in
The nucleotide and deduced amino acid sequences of the hia gene from strain 29 are shown in
The nucleotide and deduced amino acid sequences of the hia gene from strain 4071 are shown in
The nucleotide and deduced amino acid sequences of the hia gene from strain K9 are shown in
The nucleotide and deduced amino acid sequences of the hia gene from strain type c Haemophilus API are shown in
The nucleotide and deduced amino acid sequences of the hia locus from strain 12 are shown in
PCR amplification using primers that would amplify across the putative junction of these two genes in strain 12, confirmed the genetic composition of the locus. Thus it would appear that strain 12 does not produce Hia because it has suffered a deletion of the 5′-end of the hia gene.
An alignment of the deduced Hia sequences from NTHi strains 33, 32, 29, K22, M4071, 11 and K9 and type c strain API compared with H. influenzae type b Hsf, the aidA-like (Hsf/Hia) HI1732 gene from the Rd genome, and the M. catarrhalis 200 kDa protein from strains 4223 and LES-1 is shown in
This Example describes the construction of plasmid JB-2930-3 that contains a T7 hia gene cassette with a truncated S44 strain 11 hia gene, the E. coli cer gene, and the kanamycin antibiotic resistance gene, and expression of S44 Hia proteins.
PCR primers were designed to amplify the S44 Hia N-terminus of the NTHi strain 11 hia gene from the S44 amino acid to an internal Sty I site (
The recombinant S44 hia vector was transformed into E. coli BL21(DE3) for expression studies. The procedure for expression in E. coli was as described in Example 9.
In summary of this disclosure, the present invention provides novel isolated and purified nucleic acid molecules encoding full-length and N-terminal truncated Haemophilus influenzae adhesin (Hia) proteins from Haemophilus which enable protective Hia proteins to be produced recombinantly. Modifications are possible within the scope of this invention.
“This application is a national phase application under 35 U.S.C. 371 of PCT/CA00/00289. This application is a continuation-in-part of U.S. patent application Ser. No. 09/268,347 filed Mar. 16, 1999, now U.S. Pat. No. 6,335,182 issued Jan. 1, 2002.”
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA00/00289 | 3/16/2000 | WO | 00 | 12/19/2001 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO00/55191 | 9/21/2000 | WO | A |
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| Number | Date | Country |
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| 1903055 | Aug 2008 | EP |
| 09170138.3 | Feb 2010 | EP |
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| WO 9534308 | Dec 1995 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| Parent | 09268347 | Mar 1999 | US |
| Child | 09936362 | US |
