Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1996-11-29
2003-08-26
Minnifield, Nita (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S242000, C435S252300, C435S320100, C536S001001, C536S018700, C536S022100, C536S023100, C536S023500, C536S023700, C536S024100, C424S255100
Reexamination Certificate
active
06610506
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel transferrin binding proteins of
Pasteurella haemolytica
, truncations, analogs, homologs and isoforms thereof; nucleic acid molecules encoding the proteins and truncations, analogs, and homologs of the proteins; vaccines containing the proteins; antibodies against the proteins; and, uses of the proteins and nucleic acid molecules.
BACKGROUND OF THE INVENTION
Members of the genus Pasteurella comprise a group of related bacterial species that are important pathogens of ruminants. This group includes the species
Pasteurella haemolytica
which has been classified into two biotypes, A and T, on the basis of sugar utilization, and into 16 serotypes which are recognized on the basis of their somatic antigens (Biberstein, E. L. et al., 1960; Fraser et al., 1982). The T-type strains of
P. haemolytica
, characterized by utilization of trehalose, have been recently reclassified as a new species
P. trehalosi
(Sneath, P. H. A. et al., 1990).
Pneumonic pasteurellosis caused by
Pasteurella haemolytica
is a major economic problem to the cattle, sheep and goat industries world-wide. Shipping fever, a variation of this disease, is a major problem in the cattle industry in North America and is almost exclusively caused by type A1 strains of this species (Babiuk, L. A. and S. D. Acres, 1984). Serotype A2 is the most prevalent disease-causing type in sheep but other serotypes may be important in sheep and goats (Gilmour and Gilmour, 1991). The related species,
Pasteurella trehalosi
(formerly know as T-type
P. haemolytica
) is the causitive agent of septicemia in lambs, a problem plaguing the sheep industry particularly in the United Kingdom. Similarly, strains of the related species
Pasteurella multocida
, are responsible for haemorrhagic septicemia, a serious infection in cattle and water buffalo, which is particularly serious in South East Asia.
Vaccination is a desired method of control for pasteurellosis in ruminants but success has been limited by the lack of immunizing preparations that induce protection against all disease-causing serotypes, particularly if a vaccine effective for all ruminants is considered. Killed whole cell vaccines elicited inconsistent levels of protection and antibody response in calves (Wilkie, B. N., 1980). Homologous vaccines containing sodium salicylate extracts (SSEs) protected sheep against diseases due to serotypes A1, A6 and A9 (Gilmour et al., 1983) but not against the more epidemic serotype A2 (Fraser et al., 1982). An exotoxin produced by
P. haemolytica
which is specifically lethal to leucocytes and alveolar macrophages from ruminants (Benson et al., 1978) has shown a lot of promise as a vaccine candidate in protection experiments in calves and sheep (13,35) but there is limited protection against heterologous serotypes (33). The inclusion of proteins induced under iron-limited growth conditions into a vaccine for pasteurellosis in lambs has been implicated in enhanced protection (15).
Previous studies have established that the ability of pathogenic bacteria to acquire iron in vivo is a critical factor in their pathobiology (7,11). One mechanism of iron retrieval from the host iron-binding glycoprotein, transferrin, involves direct binding of transferrin by surface receptors on the bacteria and the removal of iron from transferrin and uptake into the cell (21). Schryvers (1992) describes the isolation of transferrin receptor proteins from various bacterial pathogens using affinity chromatography. The transferrin receptor has been shown to consist of two proteins, called transferrin binding protein 1 or A (Tbp1 or TbpA) and transferrin binding protein 2 or B (Tbp2 or TbpB). The receptor-mediated type of iron uptake has been demonstrated to operate in serotype A bovine strains of
P. haemolytica
(26). Cells of
P. haemolytica
growing in vitro under iron-limited conditions express a number of iron-repressible outer membrane proteins (IROMPs) identical to those produced by cells recovered in vivo from infected sites in animals with pasteurellosis (9,10). Especially prominent among these proteins were those of molecular sizes 100, 77, 70 and 60 Kda (9,10). The 100 Kda protein has been identified as one of the host specific transferrin receptors in bovine isolates (26) while some of the other IROMPs had been suggested as possibly associated with the 100 Kda protein in an iron acquisition receptor complex (26). The role of the IROMPs expressed by
P. haemolytica
from lambs (10) in iron acquisition has not been elucidated, neither is it known if similar proteins are expressed by goat isolates.
P. haemolytica
acquires iron from bovine host transferrin by a receptor-mediated type of mechanism. The proposal that bacteria with this type of iron acquisition mechanism may be solely dependent upon their surface receptor for iron acquisition in vivo (29) implies that they can only cause disease in those hosts whose transferrin is recognized by their surface receptors.
P. haemolytica
has been reported to cause disease in cattle, sheep and goats and accordingly their surface receptors would be expected to recognize these hosts' transferrins. Therefore it is important to determine whether sheep and goat isolates also possessed transferrin receptors involved in iron acquisition, to evaluate their specificities for different ruminant transferrins and to determine if there is antigenic relatedness amongst the surface receptors from the different strains causing pneumonic pasteurellosis in cattle, sheep and goats.
SUMMARY OF THE INVENTION
Transferrin receptors were identified in a collection of
Pasteurella haemolytica
(and
P. trehalosi
) strains of various serotypes and biotypes (A and T) from cattle, sheep and goats. Growth studies, binding studies and affinity isolation experiments demonstrated that these receptors had identical specificities which recognized transferrins from cattle, sheep and goats. This indicates that there are conserved regions on the receptor proteins, involved in ligand binding, which are accessible at the cell surface.
Antisera prepared against the individual purified receptor proteins (TbpA and TbpB) from a serotype A1 strain of
P. haemolytica
demonstrated considerable crossreactivity against receptor proteins from a representative selection of strains. The cross-reactivity was also observed against intact cells indicating that there are conserved immunological epitopes at the cell surface which could serve as targets for the host's immune effector mechanisms.
The present inventors have cloned, sequenced and expressed tbpA and tbpB genes encoding the transferrin receptor proteins, TbpA and TbpB (also referred to herein as Tbp1 and Tbp2, respectively), from
Pasteurella haemolytica
A1. The genes were organized in an operon arrangement of tbpB- tbpA. The tbpB gene was preceded by putative promoter and regulatory sequences, and followed by a 96 base pair intergenic sequence in which no promoter regions were found, suggesting that the two genes are coordinately transcribed. The deduced amino acid sequences of the TbpA and TbpB proteins had regions of homology with the corresponding
Neisseria meningitidis, N. gonorrhoeae, Haemophilus influenzae
and
Actinobacillus pleuropneumoniae
Lbp and Tbp proteins. The intact tbpB gene was expressed in a T7 expression system and the resulting recombinant TbpB protein retained the functional bovine transferrin binding characteristics. The availability of the recombinant TbpB enabled the inventors to demonstrate its specificity for ruminant transferrin, its ability to bind both the C-and N-terminal lobes of bovine transferrin, and its preference for the iron-loaded form of this protein.
The present inventors also significantly found that vaccination with a formulation containing
P. haemolytica
TbpA and TbpB provided significant protection against experimental bovine pneumonic pasteurellosis. Immunization with two doses of TbpB also provides protection
Broadly stated, the present invention provides a purified and isolated nucleic
Lo Reggie Y. C.
Potter Andrew Allan
Schryvers Anthony Bernard
Baker & Botts L.L.P.
Harris Alana M.
Minnifield Nita
University Technologies International Inc.
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