U.S. patent application number 11/089158 was filed with the patent office on 2005-10-20 for recombinant plasmid and a method of controlling the effects of yersinia pestis.
This patent application is currently assigned to Michigan State University. Invention is credited to Brubaker, Robert R., Motin, Vladimir L., Smirnov, George B..
Application Number | 20050232940 11/089158 |
Document ID | / |
Family ID | 29250396 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050232940 |
Kind Code |
A1 |
Brubaker, Robert R. ; et
al. |
October 20, 2005 |
Recombinant plasmid and a method of controlling the effects of
yersinia pestis
Abstract
Described is a plasmid prepared by recombinant techniques which
is used to prepare a vaccine against Y. pestis.
Inventors: |
Brubaker, Robert R.;
(Columbus, NC) ; Motin, Vladimir L.; (League City,
TX) ; Smirnov, George B.; (Moscow, RU) |
Correspondence
Address: |
WILMER CUTLER PICKERING HALE AND DORR LLP
60 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Michigan State University
|
Family ID: |
29250396 |
Appl. No.: |
11/089158 |
Filed: |
March 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11089158 |
Mar 24, 2005 |
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10694614 |
Oct 27, 2003 |
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10694614 |
Oct 27, 2003 |
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08302423 |
Sep 8, 1994 |
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6638510 |
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Current U.S.
Class: |
424/190.1 ;
435/252.3; 435/476; 435/69.3; 530/350; 536/23.7 |
Current CPC
Class: |
Y02A 50/407 20180101;
A61K 39/00 20130101; C07K 16/1228 20130101; Y02A 50/30 20180101;
A61K 39/025 20130101; C07K 14/24 20130101 |
Class at
Publication: |
424/190.1 ;
435/069.3; 435/252.3; 435/476; 530/350; 536/023.7 |
International
Class: |
A61K 039/02; C07H
021/04; C12P 021/06; C12N 015/74; C07K 014/24 |
Goverment Interests
[0002] The United States government may have licensing rights to
this application in accordance with U.S. Public Health Service
Grant AI 19353.
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. An immunogenic polypeptide substantially free of antigenic
contaminating proteins, comprising an antigenic polypeptide
sequence of the 259 C-terminal amino acids of V antigen.
11. The immunogenic polypeptide of claim 10, encoded by construct
pAV13 shown in FIG. 1.
12. The immunogenic polypeptide of claim 10, comprising 31.5 kDa of
the 259 C-terminal amino acids of V antigen.
13. The immunogenic polypeptide of claim 10, comprising 19.3 kDa of
the 259 C-terminal amino acids of V antigen.
14. The immunogenic polypeptide of claim 12, comprising a 31.5 kDa
N-terminal portion of the 259 C-terminal amino acids of V
antigen.
15. The immunogenic polypeptide of claim 10, comprising an
antigenic polypeptide sequence of the 31.5 kDa N-terminal portion
of the 259 C-terminal amino acids of V antigen.
16. The immunogenic polypeptide of claim 15, wherein the antigenic
polypeptide sequence does not comprise a portion of the 19.5 kDa
N-terminal portion of the 259 C-terminal amino acids of V
antigen.
17. A V antigen-based Y. pestis vaccine, comprising an antigenic
polypeptide sequence of the 259 C-terminal amino acids of V
antigen.
18. The V antigen-based Y. pestis vaccine of claim 17, wherein the
antigenic polypeptide sequence is encoded by construct pAV13 shown
in FIG. 1.
19. The V antigen-based Y. pestis vaccine of claim 17, comprising
31.5 kDa of the 259 C-terminal amino acids of V antigen.
20. The V antigen-based Y. pestis vaccine of claim 17, comprising
19.3 kDa of the 259 C-terminal amino acids of V antigen.
21. The V antigen-based Y. pestis vaccine of claim 19, comprising a
31.5 kDa N-terminal portion of the 259 C-terminal amino acids of V
antigen.
22. The V antigen-based Y. pestis vaccine of claim 17, comprising
an antigenic polypeptide sequence of the 31.5 kDa N-terminal
portion of the 259 C-terminal amino acids of V antigen.
23. The V antigen-based Y. pestis vaccine of claim 22, comprising
an antigenic polypeptide sequence that is not a part of the 19.5
kDa N-terminal portion of the 259 C-terminal amino acids of V
antigen.
24. A polyclonal antiserum comprising antibodies that specifically
binds to antigenic polypeptide sequences of the 259 C-terminal
amino acids of V antigen.
25. A polyclonal antiserum comprising antibodies that specifically
bind to antigenic polypeptide sequences of the 31.5 kDa N-terminal
portion of the 259 C-terminal amino acids of V antigen.
26. The polyclonal antiserum of claim 25, wherein the antiserum
comprises antibodies that specifically bind to an antigenic
polypeptide sequence that is not a part of the 19.5 kDa N-terminal
portion of the 259 C-terminal amino acids of V antigen.
27. An anti-Yersinia antiserum made by a method which comprises
injecting a mammal with an immunogenic amount of an immunogenic
polypeptide of any of claims 10-16.
28. An anti-bubonic plague antiserum made by a method which
comprises injecting a mammal with an immunogenic amount of an
immunogenic polypeptide of any of claims 10-16.
29. A method of treating or preventing a Yersinia infection in a
mammal comprising administering an immunoprotective amount of an
anti-V antigen antiserum raised against an immunogenic polypeptide
of any of claims 10-16.
30. A method of treating or preventing bubonic plague in a mammal
comprising administering an immunoprotective amount of an anti-V
antigen antiserum raised against an immunogenic polypeptide of any
of claims 10-16.
31. An isolated recombinant V protein antigen truncated at its N
terminus by 67 amino acids and encoded by all but the 201
N-terminal base pairs of a V antigen gene.
32. The truncated V protein of claim 31, encoded by construct pAV13
shown in FIG. 1.
33. A method of controlling Y. pestis in a mammal, comprising: (a)
providing a vaccine comprised of the truncated V protein of claim
31; and (b) administering an effective immunizing amount of the
vaccine to the mammal.
34. A method of controlling Y. pestis in a mammal, comprising: (a)
providing a vaccine comprised of the truncated V protein of claim
32 and (b) administering an effective immunizing amount of the
vaccine to the mammal.
35. A method of treating a mammal infected with Y. pestis,
comprising: (a) providing a vaccine comprised of the truncated V
protein of claim 31; and (b) administering an effective immunizing
amount of the vaccine to the mammal.
36. A method of treating a mammal infected with Y. pestis,
comprising: (a) providing a vaccine comprised of the truncated V
protein of claim 32; and (b) administering an effective immunizing
amount of the vaccine to the mammal.
37. An isolated nucleic acid encoding an immunogenic polypeptide
fragment of a V antigen protein.
38. An isolated nucleic acid encoding the immunogenic polypeptide
of any of claims 10-16.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of 10/694,614, filed Oct.
27, 2003, which is a divisional of 08/302,423, filed Sep. 8, 1994,
now U.S. Pat. No. 6,638,510.
TECHNICAL FIELD
[0003] The present invention is concerned with bubonic plague
caused by Yersinia Pestis and vaccines for treating same.
BACKGROUND ART
[0004] Experimental plague in mice, caused by Yersinia pestis, is
mediated by two distinct types of virulence factors. Members of the
first category serve as whole animal or tissue invasins by
promoting dissemination of the organisms into visceral organs
following infection by peripheral routes of injection (e.g.
intraperitoneal or subcutaneous). Mutants lacking one or more
tissue invasins can exhibit significant reduction in virulent (50%
lethal dose>10.sup.2 to 10.sup.7 bacteria) by peripheral
administration but retain essentially full lethality (50% lethal
dose ca. 10.sup.2 bacteria) upon intravenous injection (4).
Examples of this group (6, 16, 48) include the outer membrane (46)
plasminogen activator (1) mediated by a ca. 10 kb pesticin or Pst
plasmid (12, 41) and a series of iron repressible outer membrane
peptides (10, 40) encoded by a delectable ca. 100 kb chromosomal
segment (11, 22).
[0005] Examples of the second category function to promote
lethality following infection by the intravenous route, known to
facilitate immediate transport of the bacteria to favored niches
within visceral organs (4). Mutational loss of these lethal factors
causes qualitative (intravenous 50% lethal dose>10.sup.7
bacteria) decreases in virulence. Included in this group are
certain ca. 70 kb low calcium response or Lcr plasmid encoded
proteins: V antigen (9, 27), others termed Yops (18, 19, 33, 47):
YopE (23, 35, 43, 44, 47), YopH (3, 34, 44), and probably YpkA
(13), as well as chromosomally encoded antigen 4 or PH 6 antigen
(20) and possibly the murine exotoxin encoded by the ca. 100 kb Tox
plasmid (32.). Considerable effort has been spent in study of the
regulation, processing, and delivery of these proteins to host
cells (2, 15, 26, 28, 30, 31, 35).
[0006] The effectiveness of the immune response directed against
members of the second category of virulence factors has only been
reported for V antigen. Some (17, 24, 27, 39, 49, 50) but not all
(5) antibodies directed against this 37 kDa exported (8, 17, 43,
44) protein provided significant passive protection against
experimental plague in mice. This effect was associated with
release of a potent immunosuppressive block preventing both
synthesis of cytokines (27) and formation of protective granulomas
(50).
[0007] Plague vaccines have been identified in U.S. Pat. No.
3,137,629. The patent describes a process for producing killed
plague vaccines which immunizes mice and guinea pigs by growing
Pasteurella pestis, killing the strain through mechanical action
and solubilizing the extract in strong alkaline solution, and then
preparing parental vaccine by reducing the pH value of the soluble
P. pestis antigenic solution to a neutral pH.
[0008] It is an object of the present invention to prepare a
plasmid by recombinant techniques.
[0009] It is another object of the present invention to prepare an
antigen encoded by the recombinant plasmid.
[0010] It is a further object of the present invention to control
the effect Y. pestis has on mammals by utilizing a vaccine to Y.
pestis constituting the antigen noted above.
SUMMARY OF THE INVENTION
[0011] The present invention is concerned with a plasmid prepared
by recombinant techniques having the construct shown in FIG. 1.
[0012] Also described is a protein encoded by the plasmid shown in
FIG. 1, capable of inducing a protective antibody response.
[0013] The invention is further concerned with a method of
controlling the effects of Y. pestis in mammals comprising the step
of:
[0014] a) providing a vaccine comprised of the protein encoded by
the construct of FIG. 1; and
[0015] b) treating a mammal in need thereof with an effective
anti-Y. pestis amount of the vaccine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1A is a scheme of construction of a recombinant plasmid
formed by joining the DNA encoding the signal sequence and IgG
binding domains of staphylococcal protein A and that of all but the
201 N-terminal base pairs of Y. pestis V antigen; antibody to which
is capable of effecting immunological treatment against Y.
pestis;
[0017] FIG. 1B cites a restriction endonuclease attack on the V
antigen;
[0018] FIG. 2 is a silver-stained extended SDS gel of whole cells
of E. coli BL 21 containing the vector plasmid pKK223-3 (lane 1) or
recombinant plasmid pKVE14 (lane 2);
[0019] FIG. 3 are immunoblots prepared with rabbit polyclonal anti
V antigen (A) mouse monoclonal anti V antigen 15A4.8 (B) and mouse
monoclonal anti V antigen 3A4.1 (C) directed against whole cells of
E. coli containing the vector plasmid pKK223-3 (lane 1) or
recombinant plasmid pKE14 (lane 2);
[0020] FIG. 4 are immunoblots prepared with rabbit anti-native V
antigen purified from Y. pestis KIM (A), anti-recombinant V antigen
(B), anti-protein A-V antigen fusion protein (C), and anti-Protein
A (D) directed against Ca.sup.2+-starved whole Lcr cells of Y.
pestis KIM (lane 1), Lcr.sup.+ cells of Y. pestis KIM (lane 2),
Lcr.sup.- cells of Y. pseudotuberculosis PB1 (lane 3), Lcr.sup.+
cells of Y. pseudotuberculosis PB1 (lane 4), Lcr.sup.- cells of Y.
enterocolitica WA (lane 5), and Lcr.sup.+ cells of Y.
enterocolitica WA (lane 6);
[0021] FIG. 5 is a chart of antiserum for passive immunization
against native V antigen, recombinant V antigen, recombinant
protent A-C antigen fusion and recombinant protein A;
[0022] FIG. 6 are immunoblots prepared with rabbit anti-native V
antigen (A) or mouse monoclonal 17A5.1 anti V antigen (B) directed
against truncated protein A (PA) (lane 1), protein A-V antigen
fusion peptide (PAV) (lane 2), PAV partially hydrolyzed by formic
acid (lane 3), PAV partially hydrolyzed formic acid and passed
through the IgG Sepharose 6FF column (lane 4), whole Lcr.sup.+
cells of Yersinia pestis KIM (lane 5), and whole LCR.sup.-o cells
of Y. pestis KIM (lane 6); A-V.sub.d, V.sub.o, V.sub.d, and A
indicate the positions of PAV, native V antigen (37 Kda), truncated
V antigen (29.5 kDa), and truncated Protein A, respectively. Human
.gamma.-globulin was used to block nonspecific reactions of
monoclonal antibodies against IgG-binding domains of Protein A
(26); and
[0023] FIG. 7 are immunoblots prepared with rabbit anti-protein A-V
antigen fusion peptide (A) and anti-truncated protein A (B)
directed against whole cells of Escherichia coli containing the
vector plasmid pKK223-3 (lane 1) or recombinant plasmid pKE14 (lane
2); Also shown are reactions against disrupted and centrifuged
whole cells of E. coli (pKE14) (lane 3) and further fractionation
of V antigen by chromatography on phenyl-Sepharose CL-4B (lane 4),
DEAE (diethylaminothyl) cellulose (lane 5), Sephacryl S-300SF (lane
6), calcium hydroxylapatite (lane 7), and a second passage on DEAE
cellulose (lane 8).
DESCRIPTION OF THE BEST MODE
[0024] The medically significant yersiniae (Yersinia pestis, Y.
pseudotuberculosis, and Y. enterocolitica) are known to share a ca.
70 kb low calcium response (Lcr) plasmid that mediates restriction
of vegetative growth at 37.degree. C. in Ca.sup.2+-deficient media
while promoting selective synthesis of virulence factors including
V antigen. The latter, encoded by lcrV on the Lcr plasmid, is
established as a 37 kDa protective antigen capable of undergoing
possible autoproteolytic hydrolysis. In this study, lcrV of Y.
pestis was cloned under control of the strong tac promoter into
protease-deficient Escherichia coli BL21. The resulting recombinant
V antigen, like native V antigen, underwent degradation during
purification yielding major peptides of ca. 36,35,34 and 32 to 29
kDa. Rabbit .gamma.-globulin raised against this mixture of
cleavage products provided partial but significant protection
against 10 minimal lethal doses (MLD) of the three species. To
stabilize V antigen and facilitate its purification, plasmid pPAV13
was constructed so as to encode a fusion of lcrV and the structural
gene for staphylococcal protein A (e.g. all but the first 67
N-terminal amino acids of V antigen and the signal sequence plus
IgG binding domains but not cell-wall associated region of protein
A). The resulting protein A-V antigen fusion peptide (PAV) could be
purified to homogeneity in one step IgG affinity chromatography and
was found to be stable thereafter. Rabbit polyclonal
.gamma.-globulin directed against PAV provided substantial passive
immunity against 10 MLD of Y. pestis and Y. pseudotuberculosis but
was ineffective against Y. enterocolitica.
[0025] The vaccine as described herein is generally applied by
parental administration to mammals in need thereof.
[0026] The vaccine of the present invention is generally
administered in the form of pharmaceutical compositions comprising
a pharmaceutically acceptable vehicle or diluent. Such compositions
are generally formulated in a conventional manner utilizing liquid
vehicles or diluents as appropriate to the mode of desired
administration: for parenteral administration (e.g. intramuscular,
intravenous, intradermal), in the form of injectable solutions or
suspensions, and the like. For use as a vaccine in a mammal,
including man, it is given in an amount of about 0.5-100 mg/kg.
[0027] The vaccine described herein is used in conjunction with
normal pharmaceutical excipients to facilitate storage and use.
[0028] The present invention is further illustrated by the
following examples. These examples are provided to aid in
understanding of the invention and not to be construed as a
limitation thereof.
[0029] Materials and Methods
[0030] Bacteria. Escherichia coli K-12 XL1-Blue {recAl endAl gyrA96
thi-1 hsdR17 supE44 relA1 lac [F' proAB lacI.sup.qZ.DELTA.M15Tn10
(tet.sup.r)]} (Stratagene, La Jolla, Calif.) was used as a host for
genetic engineering manipulations. Protease-deficient E. coli BL21
{F.sup.- ompT lon r.sub.B-m.sub.B-56 (Novagen, Madison, Wis.) was
used for expression of cloned genes (17). Mice passively immunized
with the products of cloned genes were challenged with wild type
cells of Y. enterocolitica WA (10) or Y. pseudotuberculosis PB1/+
(9). This purpose was accomplished with Y. pestis KIM by use of a
nonpigmented mutant (20,59) known to lack a spontaneously deletable
ca. 100 kb chromosomal fragment encoding functions of iron
transport and storage (14,28); the isolate in question retained all
other known chromosomally encoded virulence functions as well as
the Tox, Lcr, and Pst plasmids (13,57). Nonpigmented mutants of
this phenotypic background are virulent in mice by the intravenous
(50% lethal dose ca. 10 bacteria, 61) but not by peripheral routes
of infection (50% lethal dose>10.sup.7 bacteria, 21).
[0031] Plasmids. Salient features of plasmids used in this study
are shown in Table 1. The vector pKK223-2 containing the tac
promoter (Pharmacia, Uppsala, Sweden) was used to express a portion
of the lcrGVH-yopBD operon of Y. pestis 358 (22) as described
below. The vector pRIT5 (Pharmacia) encoding the sequence of
Protein A of Staphylococcus aureus was used for preparation of gene
fusions. The recombinant plasmid pBVP5 containing the 1crGVH-YopBD
operon of Y. pseudotuberculosis (38) served as the source of lcrV
in preparing this construction.
1TABLE 1 Characterization of Delectional Variants of HindIII
Fragment From The lcrGVH-yopBD Operon of Yersinia
Pseudotuberculosis 995. Encoding Size Of Designation Of Size of V
Plasmid Fragment Operon V Antigen Antigen (kDa) pBVP5 .about.3,500
lcrgvh-ypoB V.sub.0 37.3 pBVP513D 2,184 lcrGVH V.sub.0 37.3 pBVP53D
1,484 lcrGV.sub.1 V.sub.1 31.5 pBVP514D 1,160 lcrGV.sub.2 V.sub.2
19.3 pBVP515D 878 lcrGV.sub.3 V.sub.3 8.5 pBVP58D 705 lcrGV.sub.4
V.sub.4 2.0 pBVP55D 546 lcrG.sub.1 -- --
[0032] Molecular weights of truncated V antigens were calculated
from the deletion terminus as determined by nucleotide sequencing;
actual values may be slightly greater due to translational overruns
into the vector polylinker region.
[0033] DNA Methods. The preparation of plasmid DNA, digestion with
restriction enzymes, ligation, and transformation of E. coli were
undertaken essentially as described by Maniatis et al (29). The 3.5
kb HindIII fragment of the Lcr plasmid of Y. pestis 358 (22,38) was
introduced into expression vector pKK223-3. The resulting
recombinant plasmid pKVE14 was then selected where the direction of
transcription of the lcrGVH sequence corresponds to the direction
of action of the tac promoter.
[0034] The schema used to construct pPAV13 containing a hybrid gene
encoding a portion of Protein A of S. aureus and lcrV of Y.
pseudotuberculosis is shown in FIG. 1A. The 1.5 kb EcoRV fragment
of recombinant plasmid pBVP5 (38) was introduced into the vector
pRIT5 encoding truncated Protein A (PA). The latter, either alone
or fused with V antigen, maintained its signal sequence and most
IgG-binding domains but lost the region mediating association with
the bacterial cell surface (41,42) (FIG. 1B). PA does not contain
cysteine and is thus unable to form disulfide bridges between
itself and a hybrid domain (60). As a consequence of this fusion,
lcrV lost 201 bp which thus deleted the first 67 amino acids
comprising the N-terminal portion of V antigen. The resulting
Protein A-V antigen fusion peptide (PAV) thus contained 305
N-terminal amino acids from Protein A and 259 C-terminal amino
acids from V antigen (FIG. 1B).
[0035] Purification of Recombinant V Antigen. Cells of E. coli BL21
(pKVE14) were grown in fermenters as described previously (5) in
medium containing 3% Sheffield NZ Amine, Type A (a pancreatic
hydrolysate of casein which contains mixed amino acids and peptides
and is used to facilitate the growth of bacteria) (Kraft, Inc.,
Memphis, Tenn.), 0.5% NaCl, 1% lactose, and ampicillin (100
.mu.g/ml) at 37.degree. C. and harvested by centrifugation
(10,000.times.g for 15 min) at an optical density (620 nm) of about
1.2. After disruption in a French pressure cell (SLM Instruments,
Inc., Urbana, Ill.) and removal of insoluble matter by
centrifugation (10,000.times.g for 30 min), V antigen was subjected
to purification by an established procedure (5). The method
involved use of hydrophobic interaction chromatography with
Phenyl-Sepharose CL-4B (Pharmacia), ion exchange chromatography
with DEAE cellulose (Whatman Inc., Clifton, J.J.), gel filtration
chromatography with Sephacryl S-300SF (trademark of Pharmacia
Biotechnology Group for acrylic resin for chromatograhic separation
of proteins), and Bio-Gel HTP (trademark of Bio-Rad, Richmond,
Calif., for calcium hydroxyapatite (chromatography)). The original
procedure was supplemented by a second chromatographic separation
of DEAE cellulose (linear gradient from 0. 0.35 M NaCl) in order to
remove high molecular weight material peculiar to E. coli.
[0036] Preparation of PA and PAV. Cells of E. coli transformed with
pPAV13 or PRIT5 were grown to late log phase at 37.degree. C. in
Luria broth containing ampicillin (50 .mu.g/ml). Purification of
these recombinant proteins was accomplished by affinity
chromatography on IgG Sepharose 6FF (Pharmacia) according to
directions supplied by the manufacturer. Briefly, the procedure
involved harvesting the organisms by centrifugation (10,000.times.g
for 15 min) with resuspension at a ca. 10-fold increase in number
in 0.01 M Tris.HCl, pH 8.0 (column buffer). Lysis was accomplished
by addition of lysozyme (5 Mg/ml) and, after incubation for 1 h,
further addition of Triton X-100 (trademark of Aahm & Haas Co.
for a nonionic detergent comprised of octyl phenopypolyethoxy
ethanol having an HLB: 13.5.sub.j (0.1%) whereupon incubation was
continued for 3 to 4 h. After clarification by centrifugation
(10,000.times.g for 30 min), samples of 400 ml of the resulting
enriched periplasm were passed through a column (10.times.100 mm)
containing a 10 ml packed volume of affinity resin that selectively
bound PA or PAV. After addition and elution of 10 void volumes of
column buffer to remove contaminating matter, the recombinant
proteins were eluted with 0.2 M acetic acid (ca. pH 3.4),
immediately frozen, and the lyophilized. Resulting purified PA and
PAV were then used directly for qualitative analysis and
immunization.
[0037] Acid Hydrolysis of PAV. Purified PAV was treated with 70%
formic acid for 20 h at 30.degree. C. to cleave the four labile
Asp-Pro peptide bonds within the Protein A domain (60) and the
additional site located at the junction with V antigen (41) (FIG.
1B). After dialysis against column buffer, the partial hydrolysate
was again passed through the IgG Sepharose 6FF column as described
above. In this case, the V antigen moiety plus fragments of PA
lacking IgG binding sites were immediately eluted whereas residual
unhydrolyzed PAV remained bound to the affinity resin.
[0038] Antisera. The same lot of refined rabbit polyclonal anti-V
antigen characterized previously (40) was used as a positive
immunological control. Monoclonal antibodies directed against V
antigen have been defined (3). These reagents consisted of two
groups: monoclonals 3A4.1, 17A5.1, and 17A4.6 that reacted with
nonconformational epitopes located within the last 50 amino acids
comprising the C-terminal part of V antigen (amino acids 276 to
326) and monoclonal 15A4.8 that reacted with an internal
nonconformational epitope located between amino acids 168 to 275
(37).
[0039] Rabbit polyclonal antisera was raised against PA and PAV
with Freund's adjuvant as described previously (62). TiterMax.TM.
adjuvant (Hunger's TiterMax #R-1, CytRx Corp., Norcross, Ga.) was
used to immunize rabbits against recombinant V antigen plus its
degradation products purified from E. coli BL21 (pKVE14). Antisera
prepared against recombinant V antigen or fusion proteins were not
absorbed with material from Lcr.sup.- bacteria although highly
purified .gamma.-globulin was isolated from these reagents as
described previously (62). Antisera raised against V antigen
purified from Y. pestis or E. coli BL21(pKVE14) is termed
anti-native V antigen or anti-recombinant V antigen, respectively.
Immunoblotting. Alkaline phosphatase conjugated with anti-rabbit or
anti-mouse IgG (Sigmal Chemical Co., St. Louis, Mo.) were used as
secondary antibodies in immunoblotting by procedures essentially
identical to those already defined (51,52). In order to prevent
nonspecific reactions of antibodies with PA and PAV, the
nitrocellulose filter was first blocked with 5% fetal calf serum as
usual and then incubated overnight in a solution of 1% normal human
.gamma.-globulin (Calbiochem, San Diego, Calif.). Human
.gamma.-globulin (0.5%) was also added to solutions of primary and
secondary antibodies (26). In addition, Fc-specific anti-mouse IgG
(A-1418, Sigma) was used as a secondary antibody during
immunoblotting of fusion proteins and their derivatives with
monoclonal antibodies.
[0040] Passive Immunity. The ability of highly purified
.UPSILON.-globulin obtained from unabsorbed rabbit polyclonal
antisera raised against recombinant V antigen, PA, and PAV to
provide passive immunity was assayed by defined methods (40,62).
Briefly, this procedure involved intravenous injection of 10
minimum lethal doses (MLD) of Y. pestis (10.sup.2 bacteria), Y.
Pseudotuberculosis (10.sup.2 bacteria), or Y. enterocolitica
(10.sup.3 bacteria) followed by intravenous administration of
either 100 .mu.g or 500 or 500 .mu.g of purified .gamma.-globulins
on postinfection days 1, 3, and 5.
[0041] Miscellaneous. Peptides were located in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gels, prepared as
defined previously (51,52), by silver staining (36). Soluble
protein was determined by the method of Lowry et al. (27).
Results
[0042] Degradation of Recombinant V Antigen. Recombinant plasmid
pKVE14 containing the lcrGVH-yopBD operon of Y. pestis under
control of the tac promoter was transferred into protease-deficient
E. col BL21. After growth in fermenters, the bacteria were
disrupted and the resulting extract was used to prepare nearly
homogenous recombinant V antigen using a method established for
Ca.sup.2+-starved cells of Y. pestis (5). An additional step
involving a second separation with DEAE cellulose was necessary to
eliminate major higher molecular weight proteins present in E. coli
cytoplasm.
[0043] The initial specific activity of recombinant V antigen was
almost 5-fold greater than that obtained from Y. pestis starved for
Ca.sup.2+ (5). Nevertheless, significant loss of precipitin
activity occurred during every step of purification (Table 2). This
phenomenon, as judged by a silver-stained extended lane gel (FIG.
2), reflected gradual loss of the native 37 kDa form with emergence
of ca. 36 kDa, 32 kDa, and possibly smaller peptides. Analysis by
immunoblotting was undertaken to prove that these new peptides
shared epitopes with and thus arose from native V antigen. Use of
rabbit polyclonal anti-native V antigen (FIG. 3A) or mouse
monoclonal antibody 15A4.8, directed against a centrally located
epitope (FIG. 3B), demonstrated emergence of ca. 36, 35, and 34 kDa
degradation products early during the course of purification with
later appearance of a series of smaller fragments ranging from 32
to 29 kDa. The latter were not recognized by mouse monoclonal
antibody 3A4.1 directed against an epitope located near the
C-terminal end (FIG. 3C). These findings indicate that recombinant
V antigen produced in protease-deficient E. coli BL21 undergoes
evidence spontaneous degradation in a manner similar to that
observed for native V. antigen expressed in Y. pestis (5).
Furthermore, patterns observed upon immunoblotting with monoclonal
antibodies indicate that the C-terminal portion of V is involved in
this process.
2TABLE 2 Purification Of Recombinant V Antigen From A Cell-Free
Extract Of Escherichia coli BL21 (pKVE14) Total V Total Vol.
Protein Protein Antigen V-antigen Specific % Preparation (ml)
(mg/ml) (mg) (U/ml) (U) Activity Recovery Crude Extract 200 26
5,200 280 56,000 11 (100) Phenyl- 220 1.6 350 140 30,800 88 55
Sepharose CL- 4B DEAE 40 1.5 60 170 6,800 113 12.1 Cellulose 24 0.7
17 140 3,360 200 6.0 Sephacryl S300SF Ca 35 0.25 8.8 50 1,750 200
3.1 Hydroxylapatite DEAE Cellulose 18 0.1 1.8 15 270 150 0.5
[0044] The unit of V antigen was defined as the reciprocal of the
highest dilution capable of forming a visible precipitate against a
standardized lot of rabbit polyclonal monospecific antiserum by
diffusion in agar under conditions described previously (Lawton et
al, 1963; Brubaker et al., 1987).
[0045] Passive Immunity Mediated By Anti-Recombinant V Antigen. A
portion of the purified lot of recombinant V antigen described
above was used to immunize rabbits. Immunoblots of the resulting
unabsorbed antisera (FIG. 4B) and control absorbed anti-native V
antigen (FIG. 4A) versus Ca.sup.2+-starved whole yersiniae were
identical indicating that the reagent was monospecific. Both
antisera were tested for ability to confer passive immunity against
intravenous infection with yersiniae. As shown in FIG. 5, the
control anti-native V antigen provided complete, partial, and
insignificant protection against y. pestis, Y. pseudotuberculosis,
and Y. enterocolitica, respectively. Anti-recombinant V antigen
promoted a similar degree of passive immunity except that that
directed against Y. pestis was not absolute.
[0046] Characterization of PA and PAV. Additional constructions
encoding truncated staphylococcal Protein A either alone or fused
with V antigen (FIG. 1) were found, after transformation into E.
coli BL21, to promote significant synthesis of PA and PAV,
respectively as judged by intensity of the specific immunoblots
described below. PA and PAV were purified in one step with IgG
Sepharose 6FF and then analyzed by immunoblotting. Anti-native V
antigen reacted nonspecifically with PA (FIG. 6A, lane 1) and both
specifically and nonspecifically with PAV (FIG. 6A, lane 2). Proof
that the salient peptides shown in lanes 2, 3, and 4 of FIG. 6A
reacted specifically rather than nonspecifically with anti-native V
antigen was obtained by blocking PA with human .gamma.-globulin and
then immunoblotting with monoclonal anti V antigen. This process
prevented visualization of PA (FIG. 6B, lane 1) thus demonstrating
that the remaining detectable bands represent a specific
interaction with an epitope of V antigen. Multiple bands appearing
in samples of both PA and PAV (FIG. 6A, lanes 1, 2) reflect
accumulation in the periplasm of E. coli BL21 of the synthesized PA
domain in both native and degraded forms as described by others
(16). To prove that the V antigen domain of the fusion protein was
stable, a sample of purified PAV was hydrolyzed with 70% formic
acid to cleave acid labile Asp-Pro sites defined in FIG. 1B,
neutralized, and then applied to the affinity column. Essentially
pure truncated V antigen (V.sub.d) emerged immediately (FIG. 6,
lane 4); the absence of multiple bands in this sample provides
evidence for the stability of V antigen within PAV.
[0047] Stability of PAV. The number of total units of
near-homogenous PAV recovered after chromatography on IgG Sepharose
6FF was always essentially identical to that present in the crude
extract applied to the affinity column. No significant loss of
purified PAV occurred during storage in 0.01 M Tris. HCl, pH 7.8
for 1 week at 4.degree. C.
[0048] Passive Immunity Mediated By Anti-PAV. Preparations of
homogenous .gamma.-globulin were isolated from unabsorbed rabbit
antisera raised against PA and PAV purified by affinity
chromatography. Control anti-V antigen (FIG. 4A and 4B) and
anti-PAV (FIG. 5C), but not anti-PA (FIG. 5D), reacted with V
antigens of all three Yersinia sp. High molecular weight antigens
(ca. 70 kDa) common to both Lcr.sup.+ and Lcr.sup.- yersiniae were
recognized by both anti-PAV (FIG. 4C) and anti-PA (FIG. 4D).
Anti-PAV (FIG. 7A) but not anti-PA (FIG. 7B) also identified the
same degradation products of V antigen that were detected by the
antisera characterized previously (FIG. 3). These findings verify
that anti-PAV contains antibodies directed against epitopes unique
to V antigen and provide further evidence that its degradation
occurs at the C-terminal end.
[0049] Anti-PA and anti-PAV were tested for ability to confer
passive immunity against intravenous infection with yersiniae. As
shown in FIG. 5, anti-PA failed to provide protection whereas
anti-PAV was highly effective against Y. pestis and Y.
pseudotuberculosis but not Y. enterocolitica.
Discussion
[0050] Since its discovery, V antigen has been ascribed a role as
protective antigen in conferring immunity to plague (8).
Experimental evidence supporting this assumption was initially
limited to the findings that active immunization with V
antigen-rich fractions or passive immunization with antisera raised
against such fractions provided protection against disease (23).
The possibility thus remained that one or more additional antigens
contributed to the immune state described by early workers. This
concern was minimized upon introduction of preparative methods that
permitted recovery of nearly homogenous V antigen (5).
Nevertheless, antisera raised against lots purified by use of these
procedures often contained antibodies directed against highly
antigenic contaminating proteins, especially Yops, present at trace
levels in the final product used for immunization. Although these
antibodies were readily removed by absorption with cross-reacting
material, this process necessitated introduction of bacterial
macromolecules including lipopolysaccharide that might stimulate
nonspecific resistance to infection. To minimize this possibility,
it became necessary to purify the .gamma.-globulin from absorbed
antisera (40,62).
[0051] Concerns that these precautions, undertaken to assure
monospecificity of anti-V antigen, had inadvertently introduced
uncontrolled variables were largely eliminated by use of
.gamma.-globulin purified from antisera raised against highly
purified V antigen cloned in E. coli. However, this process was
also unsatisfactory due to the occurrence of marked degradation
throughout the course of purification. As a result, only a fraction
of the final product consisted of the 37 kDa primary lcrV product.
Although .gamma.-globulin purified from unabsorbed antiserum raised
against this mixture provided satisfactory passive immunity, yields
of antigenic material were insufficient to permit widespread
immunization. The observation that cloned V antigen expressed in
the prototease-deficient background of E. coli BL21, like native V
antigen purified from Y. pestis, underwent marked degradation
during purification further suggests but does not prove that this
process is autocatalytic.
[0052] Problems concerning specificity and degradation were
resolved upon development of the fusion protein PAV that could be
isolated at high yield as a homogenous stable protein in a single
step. Antisera raised against PAV was somewhat more effective in
providing protection against Y. pestis, and especially Y.
pseudotuberculosis than was anti-recombinant V antigen. This
finding emphasizes that passive immunity mediated by anti-V antigen
does not require interaction with N-terminal epitopes because the
latter were absent in PAV. The independent observation that the
N-terminal end of V antigen was poorly antigenic (37) is consistent
with this conclusion.
[0053] Detailed information with respect to the drawing figures is
as follows.
[0054] FIG. 1. Scheme of construction of recombinant plasmid of
pPAV13 encoding staphylococcal protein A-V antigen fusion protein
(PAV) (A) and characterization of PAV (B). Sites of restriction
endonuclease attack are designated; Ap and Cm are locations of
markers of resistance for ampicillin and chloramphenicol,
respectively. Lac designates the position of lacZ which provides
selection of recombinant plasmids in the vector pBluescript SK+.
The genes lcrG, lcrV, and lcrH comprise a portion of the
lcrGVH-yopBD operon of yersinia pseudotuberculosis 995 (38) and the
designation Protein A is the truncated Protein A gene of
Staphylococcus aureus. The dark arrows in A represent the hybrid
gene encoding PAV shown in B to consist of the signal sequence (S),
IgG-binding domains (E to B), the defective domain C' that has lost
the ability to bind IgG, and truncated V antigen that has lost the
first 67 amino acids of its N-terminal portion. Molecular weights
in Kilodaltons are designated for each peptide arising after
hydrolysis of the acid-labile Asp-Pro cleavage sites marked by
arrowheads (60).
[0055] FIG. 2. Silver-stained 12.5% extended sodium dodecyl
sulfate-polyacrylamide electrophoresis gel of whole cells of
Escherichia coli BL21 containing the vector plasmid pKK223-3 (lane
1) or recombinant plasmid pKVE14 (lane 2). Whole cells of E. coli
(pKVE14) were disrupted and centrifugal to prepare a cell-free
extract (lane 3) that was fractionated by chromatography on
phenyl-Sepharose CL-4B (lane 4), DEAE cellulose (lane 5), Sephacryl
S-300SF (lane 6), calcium hydroxylapatite (lane 7), and a second
passage on DEAE cellulose (lane 8). Note the presence of V antigen
in lanes 2 through 8 as a major peptide of 37 kDa.
[0056] FIG. 3. Immunoblots prepared with rabbit polyclonal anti-V
antigen (A), mouse monoclonal anti-V antigen 15A4.8 (B), and mouse
monoclonal anti-V antigen 3A4.1 (C) directed against whole cells of
Escherichia coli containing the vector plasmid pKK223-3 (lane 1) or
recombinant plasmid pKE14 (lane 2). Also shown are reactions
against disrupted and centrifugal whole cells of E. coli (pKE14)
(lane 3) and further fractionation of V antigen by chromatography
on phenyl-Sepharose CL-4B (Lane 4), DEAE cellulose (lane 5),
Sephacryl S-300SF (lane 6), calcium hydroxylapatite (lane 7) and a
second passage on DEAE cellulose (lane 8).
[0057] FIG. 4. Immunoblots prepared with rabbit anti-native V
antigen purified from Y. pestis KIM (A), anti-recombinant V antigen
(B), anti-protein A-V antigen fusion protein (C), and anti-Protein
A (D) directed against Ca.sup.2+-starved whole Lcr.sup.- cells of
Y. pestis KIM (lane 1) , Lcr.sup.+ cells of Y. pestis KIM (lane 2),
Lcr.sup.- cells of Y. pseudotuberculosis PB1 (lane 3), Lcr.sup.+
cells of Y. pseudotuberculosis PB1 (lane 4), Lcr.sup.- cells of Y.
enterocolitica WA (lane 5), and Lcr.sup.+ cells of Y.
enterocolitica WA (lane 6).
[0058] FIG. 5. Ability of 0.033 M potassium phosphate buffer, pH
7.0 (None) or control antiserum raised against native V antigen and
experimental antisera raised against recombinant V antigen, protein
A-V antigen fusion peptide, and recombinant protein A to provide
passive protection in mice against 10 minimum lethal doses of
Lcr.sup.+ cells of Yersinia pestis KIM, Yersinia pseudotuberculosis
PB1, and Yersinia enterocolitica WA. Mice were challenged
intravenously and .gamma.-globulins were then administered
intravenously on postinfection days 1, 3, and 5 at, unless
indicated otherwise, a dose of 100 .mu.g. Lengths of solid bars
show survival in days of individual mice eventually succumbing to
infection; open bars represent survival of independent infected
mice until the experiment was terminated at 21 days. Numbers
adjacent to solid bars show mean survival time in days.
[0059] FIG. 6. Immunoblots prepared with rabbit anti-native V
antigen (A) or mouse monoclonal 17A5.1 anti-V antigen (B) directed
against truncated protein A (PA) (lane 1), protein A-V antigen
fusion peptide (PAV) (lane 2), PAV partially hydrolyzed by formic
acid (lane 3), PAV partially hydrolyzed by formic acid and passed
through the IgG Sepharose 6FF column (lane 4), whole Lcr.sup.+
cells of Yersinia pestis KIM (lane 5), and whole Lcr.sup.- cells of
Y. pestis KIM (lane 6); A-V.sub.d, V.sub.o, V.sub.d, and A indicate
the positions of PAV, native V antigen (37 kDa), truncated V
antigen (29.5 kDa), and truncated Protein A, respectively. Human
.gamma.-globulin was used to block nonspecific reactions of
monoclonal antibodies against IgG binding domains of Protein A
(26).
[0060] FIG. 7. Immunoblots prepared with rabbit anti-protein A-V
antigen fusion peptide (A) and anti-truncated protein A (B)
directed against whole cells of Escherichia coli containing the
vector plasmid pKK223-3 (lane 1) or recombinant plasmid pKE14 (lane
2). Also shown are reactions against disrupted and centrifuged
whole cells of E. coli (pKE14) (lane 3) and further fractionation
of V antigen by chromatography on phenyl-Sepharose CL-4B (lane 4),
DEAE cellulose (lane 5), Sephacryl S-300SF (lane 6), calcium
hydroxylapatite (lane 7), and a second passage on DEAE cellulose
(lane 8).
[0061] While the forms of the invention herein disclosed constitute
presently preferred embodiments, many others are possible. It is
not intended herein to mention all of the possible equivalent forms
or ramifications of the invention. It is understood that the terms
used herein are merely descriptive rather than limiting, and that
various changes may be made without departing from the spirit or
scope of the invention.
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