Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-03-07
2004-05-18
Minnifield, Nita (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C424S192100, C424S200100, C424S184100, C424S244100, C536S023700, C435S173300, C435S252300, C435S253400, C435S320100
Reexamination Certificate
active
06737521
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to products and processes useful to deliver proteins, including protein antigens, to the surface of gram-positive bacteria and firmly attach them to the cell. More specifically, it relates to the production of a fusion protein containing at least the anchor region of the cell associated region of a gram-positive surface protein and any protein, peptide or polypeptide that may be usefully administered to an animal host. The products of the invention which comprise proteins, peptides or polypeptides, as further discussed below, placed on the surface of a gram positive bacteria may be used to deliver such materials to the animal host to elicit an immungenic response, for example the production of protective antibodies or for another useful purpose. The protein, peptide or polypeptide may, for example, be an enzyme or other functional protein necessary for a specific purpose. It may be an antigenic determinant from a bacteria, virus, parasite or fungus. It may be a surface antigen from a mammalian tumor cell or of male sperm. It may be an allergen such as a vespid venom. The invention also relates to novel plasmids, genes, chromosomes and transformed bacteria employed in the production of such fusion proteins. The invention, additionally, provides novel vaccines which employ gram-positive bacteria designed to deliver to an animal host a foreign antigen normally present on a pathogenic microorganism which is associated with the virulence of that pathogen and which will elicit antigens to protect the host against infection or disease caused by the pathogenic microorganism.
The products of the invention are also useful as diagnostic agents.
The invention also provides a means to deliver enzymes, placed on the bacterial surface, to specific areas of interest.
The term “animal” as used herein refers to living beings including mammals such as man; bovines, especially beef cattle; sheep and goats; poultry, especially chickens, ducks and turkeys; as well as fish, especially those raised in fish farms such as salmon, trout and catfish. This invention is of special importance to mammals.
BACKGROUND OF THE INVENTION
The essence of this invention is that it provides a method for the production of novel non-pathogenic gram positive bacteria expressing a hybrid surface protein which may be a hybrid surface antigen, comprising two principal parts, an anchor segment comprised of amino acid residues and an N-terminal active polypeptide segment, both of which will be defined and discussed in more detail below.
This invention will be better understood by consideration of the M protein and its structure. The M protein is a coiled coil surface antigen which is the virulence factor of group A streptococci, a gram positive bacteria. The M protein of
Streptococcus pyogenes
of M type 6 contains 441 amino acid residues.
Its structure will be discussed in more detail below, but it will be useful to discuss the cell associated region at this point. Using the standard one letter representation of amino acids, the structure of the anchor region of the cell associated region of the M6 protein from amino acid residue 407 to residue 441 may be represented as:
LPSTGETANPFFTAAALTVMATAGVAAVVKRKEEN (SEQ ID NO:1)
Reading from the first leucine residue (L) at the N-terminal of the anchor region, the region includes the LPSTGE segment (SEQ ID NO:36); a spacer segment containing three amino acid residues TAN (SEQ ID NO:60); a hydrophobic segment of twenty amino acids, PFTAAALTVMATAGVAVV (SEQ ID NO:2); followed by a highly charged tail segment, KRKEEN (SEQ ID NO:61).
It has been observed as a result of structural studies of a large number of surface proteins of gram positive bacteria that the above described anchor region of the M6 protein is highly conserved among all known surface proteins of gram positive bacteria. Many of them are shown by Fischetti et al (Reference 1). Generally, the hydrophobic segment contains about 15 to 20 amino acid residues, the charged tail segment about 4 to 6 amino acid residues, and the spacer segment from about 3 to 6 amino acid residues. Most remarkable however, is the high degree of homology, practically 100% in the LPSTGE (SEQ ID NO:36) segment of the known surface proteins. The variations that occur are almost exclusively at the 3 and 6 positions. Therefore, the region may be generally represented as LPXTGX (SEQ ID NO:37).
The following Table 1 shows the remarkable extent of this homology thus far established amongst forty different surface proteins of gram positive bacteria.
TABLE 1
[SEQ ID NOS: 36, 38-55, AND 59]
SEQUENCED SURFACE PROTEINS FROM
GRAM-POSITIVE BACTERIA
SEQ
NAME/
SURFACE
ID
GENE
PROTEIN
ORGANISM
LPSTGE
REF
NO.
1.
M6
M protein
S. pyogenes
LPSTGE
(2)
36
2.
M5
M protein
S. pyogenes
LPSTGE
(3)
36
3.
M12
M protein
S. pyogenes
LPSTGE
(4)
36
4.
M24
M protein
S. pyogenes
LPSTGE
(5)
36
5.
M49
M protein
S. pyogenes
LPSTGE
(6)
36
6.
M57
M protein
S. pyogenes
LPSTGE
(7)
36
7.
M2
M protein
S. pyogenes
LPSTGE
(8)
36
8.
ARP2
IgA binding
S. pyogenes
LPSTGE
(8)
36
protein
9.
ARP4
IgA binding
S. pyogenes
LPSTGE
(9)
36
protein
10.
FcRA
Fc binding
S. pyogenes
LPSTGE
(10)
36
protein
11.
Prot H
Human IgG
S. pyogenes
LPSTGE
(11)
36
Fc binding
12.
SCP
C5a peptidase
S. pyogenes
LPTTND
(12)
38
13.
T6
Protease
S. pyogenes
LPSTGS
(13)
39
resistant
protein
14.
bac
IgA binding
Gr. B strep
LPYTGV
(14)
40
protein
15.
Prot G
IgG binding
Gr. G strep
LPTTGE
(15)
41
protein
16.
PAc
Surface protein
S. mutans
LPNTGE
(16)
42
17.
spaP
Surface protein
S. mutans
LPNTGE
(17)
42
18.
spaA
Surface protein
S. sobrinus
LPATGD
(18)
43
19.
wapA
Wall-associated
S. mutans
LPSTGE
(19)
36
protein A
20.
Sec10
Surface protein
E. fecalis
LPQTGE
(20)
44
21.
Asc10
Surface protein
E. fecalis
LPKTGE
(20)
59
22.
asa1
Aggregation
E. fecalis
LPQTGE
(21)
44
substance
23.
Prot A
IgG binding
S. aureus
LPETGV
(22)
45
protein
24.
FnBP
Fibronectin
S. aureus
LPETGG
(23)
46
binding
protein
25.
wg2
Cell wall
S. cremoris
LPKTGE
(24)
59
protease
26.
In1A
Internalization
L. monocyto
-
LPTTGE
(25)
47
protein
genes
27.
Fim-
Type 1
A. viscosis
LPLTGA
(26)
48
briae
fimbriae
28.
Fim-
Type 2
A. naeslundii
LPLTGA
(27)
48
briae
fimbriae
29.
Mrp4
IgG/
S. pyogenes
LPSTGE
(10)
36
Fibrinogen
binding
30.
sof22
Serum opacity
S. pyogenes
LPASGD
(54)
49
factor
31.
Sfb
Fibronectin
S. pyogenes
LPATGD
(55)
43
binding
32.
Prot L
Light chain
P. magnus
LPKAGS
(56)
50
binding
33.
bca
alpha antigen
Gr. B strep
LPATGE
(57)
51
34.
fnbA
Fibronectin
S. dysgalactiae
LPQTGT
(58)
52
binding
35.
fnbB
Fibronectin
S. dysgalactiae
LPAAGE
(59)
53
binding
36.
EmmG1
Mprotein
Gr. G Strep
LPSTGE
(60)
36
37.
DG12
Albumin
Gr. G strep
LPSTGE
(61)
36
binding
protein
38.
MRP
Surface protein
S. suis
LPNTGE
(62)
44
39.
FnBp
Fibronectin
S.aureus
LPETGG
(63)
54
binding
protein
40.
cna
Collagen
S. aureus
LPKTGM
(64)
55
binding
protein
It is apparent that this highly homologous region of the surface proteins of gram-positive bacteria is essential to anchoring bacterial surface proteins to the cell (28). This segment, which is referred to herein as the LPXTGX segment (SEQ ID NO: 37), is the crucial segment of the cell associated region of surface protein for anchoring the proteins to the surface of gram positive bacteria.
This discovery has been confirmed by Schneewind et al (65). Using the Protein A molecule of Staphylococcus aureus, these investigators have established that the complete complex (LPXTGX motif (SEQ ID NO:37), hydrophobic domain and charged tail) are necessary to deliver the Protein A molecule to the cell surface and that changes in the LPXTGX motif (SEQ ID NO:37) or deletion thereof will not inhibit expression of the molecule but will prevent it from anchoring to the surface.
REFERENCES:
patent: 4722840 (1988-02-01), Valenzuela et al.
patent: 4859465 (1989-08-01), Rutter
patent: 5124153 (1992-06-01), Beachey et al.
patent: 5149532 (1992-09-01), Brunnel
patent: 5500353 (1996-03-01), Smit et al.
patent: 5616686 (1997-04-01), Fischetti et al.
patent: 5707822
Fischetti Vincent A.
Pozzi Gianni
Schneewind Olaf
Burns Doane , Swecker, Mathis LLP
Minnifield Nita
LandOfFree
Delivery and expression of a hybrid surface protein on the... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Delivery and expression of a hybrid surface protein on the..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Delivery and expression of a hybrid surface protein on the... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3221227