The present invention relates generally to bacterial antigens and genes encoding the same. More particularly, the present invention pertains to the cloning, expression and characterization of transferrin-binding proteins from Haemophilus somnus (H. somnus) and the use of the same in vaccine compositions.
Haemophilus somnus is a Gram-negative bacterium which causes a number of disease syndromes in cattle, collectively referred to as bovine hemophilosis. The bacterium is commonly associated with thromboembolic meningoencephalitis (ITEME), myocarditis, septicemia, arthritis, and pneumonia (Corbeil, L. B. (1990) Can. J. Vet. Res. 54: S57-S62; Harris and Janzen (1990) Can. Vet. J. 30:816-822; Humphrey and Stephens (1983) Vet. Bull. 53:987-1004). These diseases cause significant economic losses to the farm industry annually.
Conventional vaccines against H. somnus infection are either based on killed whole cells or on a protein fraction enriched in outer membrane proteins (OMPs). However, whole cell bacterins and surface protein extracts often contain immunosuppressive components which can render animals more susceptible to infection. Recombinant vaccines containing H. somnus lipoproteins, LppA, LppB and LppC, have been described. See, e.g., International Publication No. WO 93/21323, published Oct. 28, 1993. However, there remains a need for efficacious subunit vaccines against H. somnus infection.
Iron is an essential element for growth of most microbes. Weinberg, E. D. (1978) Microbiol. Rev. 42:45-66. Even though iron is abundant within mammalian tissues, virtually all iron within the mammalian body is held intracellularly as ferritin or as heme compounds, pools which are generally inaccessible to invading microorganisms. Additionally, the small amount of iron present in extracellular spaces is effectively chelated by high-affinity iron-binding host glycoproteins such as transferrin, present in serum and lymph, and lactoferrin, present in secretory fluids and milk. Otto et al. (1992) Crit. Rev. Microbiol. 18:217-233.
Hence, bacterial pathogens have developed specific iron-uptake mechanisms. In many bacterial species, these mechanisms involve the synthesis and secretion of small compounds called siderophores which display high affinity for ferric iron (FeIII). Siderophores are capable of removing transferrin-bound iron to form ferrisiderophore complexes which in turn are recognized by specific iron-repressible membrane receptors and internalized into the bacterium where the iron is released. Crosa, J. H. (1989) Microbiol. Rev. 53:517-530. Some gram-negative bacteria do not secrete detectable siderophores when grown in an iron-deficient environment but produce outer membrane proteins that bind directly and specifically to transferrin, thereby allowing iron transport into the bacterial cell. Transferrin binding proteins tend to be highly specific for the transferrin of their natural host. The ability of microorganisms to bind and utilize transferrin as a sole iron source, as well as the correlation between virulence and the ability to scavenge iron from the host, has been shown (Archibald and DeVoe (1979) FEMS Microbiol. Lett. 6:159-162; Archibald and DeVoe (1980) Infect Immun. 27:322-334; Herrington and Sparling (1955) Infect. Immun. 48:248-251; Weinberg, E. D. (1978) Microbiol. Rev. 42:45-66).
Two transferrin-binding proteins, termed a transferrin-binding protein 1 and 2 (Tbp1 and Tbp2), respectively, have been identified in bacterial outer membranes. For example, Gonzalez et al. (1990) Mol. Microbiol. 4:1173-1179, describes 105 and 56 kDa proteins from Actinobacillus pleuropneumoniae, designated porcine transferrin binding protein 1 (pTfBP1) and porcine transferrin binding protein 2 (pTfBP2), respectively. U.S. Pat. Nos. 5,417,971, 5,521,072 and 5,801,018 describe the cloning and expression of two transferrin binding proteins from A. pleuropneumoniae, as well as the use of the proteins in vaccine compositions. Schryvers, A. B. (1989) J. Med. Microbiol. 29:121-130, describes two putative transferrin-binding proteins isolated from Haemophilus influenzae. U.S. Pat. No. 5,708,149 and International Publication No. WO 95/13370, published May 18, 1995, describe the recombinant production of H. influenzae Tbp1 and Tbp2. U.S. Pat. Nos. 5,141,743 and 5,292,869 and International Publication No. WO 90/12591 describe the isolation of transferrin-receptor proteins from Neisseria meningitidis and the use of the isolated proteins in vaccine compositions. International Publication No. WO 95/33049, published Dec. 7, 1995, and European Publication No, EP 586,266, describe DNA encoding N. meningitidis transferrin binding proteins. Finally, Ogunnariwo et al. (1990) Microbiol. Path. 9:397-406, describe the isolation of two transferrin-binding proteins from H. somnus. 
However, to date, the transferrin binding proteins from H. somnus have not been recombinantly produced.
The present invention is based on the discovery of genes encoding H. somnus transferrin-binding proteins and the characterization thereof. The proteins encoded by the genes have been recombinantly produced and these proteins, immunogenic fragments and analogs thereof, and/or chimeric proteins including the same, can be used, either alone or in combination with other H. somnus antigens, in novel subunit vaccines to provide protection from bacterial infection in mammalian subjects.
Accordingly, in one embodiment, the subject invention is directed to an isolated nucleic acid molecule comprising a coding sequence for an immunogenic H. somnus transferrin-binding protein selected from the group consisting of (a) an H. somnus transferrin-binding protein 1 and (b) an H. somnus transferrin-binding protein 2, or a fragment of the nucleic acid molecule comprising at least 15 nucleotides.
In additional embodiments, the invention is directed to recombinant vectors including the nucleic acid molecules, host cells transformed with these vectors, and methods of recombinantly producing H. somnus transferrin-binding proteins.
In still further embodiments, the subject invention is directed to vaccine compositions comprising a pharmaceutically acceptable vehicle and an immunogenic H. somnus transferrin-binding protein selected from the group consisting of (a) an H. somnus transferrin-binding protein 1, (b) an H. somnus transferrin-binding protein 2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, as well as methods of preparing the vaccine compositions.
In yet other embodiments, the present invention is directed to methods of treating or preventing H. somnus infections in a mammalian subject. The method comprises administering to the subject a therapeutically effective amount of the above vaccine compositions.
In additional embodiments, the invention is directed to methods of detecting H. somnus antibodies in a biological sample comprising:
(a) providing a biological sample;
(b) reacting the biological sample with an immunogenic H. somnus transferrin binding protein selected from the group consisting of (a) an H. somnus transferrin-binding protein 1, (b) an H. somnus transferrin-binding protein 2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, under conditions which allow H. somnus antibodies, when present in the biological sample, to bind to the H. somnus transferrin-binding protein to form an antibody/antigen complex; and
(c) detecting the presence or absence of the complex,
thereby detecting the presence or absence of H. somnus antibodies in the sample.
In yet further embodiments, the invention is directed to an immunodiagnostic test kit for detecting H. somnus infection. The test kit comprises an H. somnus transferrin-binding protein selected from the group consisting of (a) an H. somnus transferrin-binding protein 1, (b) an H. somnus transferrin-binding protein 2 and (c) an immunogenic fragment of (a) or (b) comprising at least 5 amino acids, and instructions for conducting the immunodiagnogtic test.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.