U.S. patent application number 10/995847 was filed with the patent office on 2005-09-22 for diagnostic device using gp40.
Invention is credited to Andersen, Philip R., O'Connor, Thomas P., Tonelli, Quentin J..
Application Number | 20050208481 10/995847 |
Document ID | / |
Family ID | 26960008 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050208481 |
Kind Code |
A1 |
Andersen, Philip R. ; et
al. |
September 22, 2005 |
Diagnostic device using GP40
Abstract
Assay methods and devices for detecting antibodies to Feline
Immunedeficiency Virus (FIV) using an FIV envelope polypeptide such
as recombinant gp40.
Inventors: |
Andersen, Philip R.;
(Portland, ME) ; O'Connor, Thomas P.; (Westbrook,
ME) ; Tonelli, Quentin J.; (Portland, ME) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
26960008 |
Appl. No.: |
10/995847 |
Filed: |
November 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10995847 |
Nov 23, 2004 |
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09963759 |
Sep 25, 2001 |
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09963759 |
Sep 25, 2001 |
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08852143 |
May 6, 1997 |
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6383765 |
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08852143 |
May 6, 1997 |
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08306539 |
Sep 15, 1994 |
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5656732 |
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08306539 |
Sep 15, 1994 |
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08120311 |
Sep 13, 1993 |
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08120311 |
Sep 13, 1993 |
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07447810 |
Dec 8, 1989 |
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07447810 |
Dec 8, 1989 |
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07348784 |
May 8, 1989 |
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07348784 |
May 8, 1989 |
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07293906 |
Jan 5, 1989 |
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5219725 |
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07293906 |
Jan 5, 1989 |
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07279989 |
Dec 5, 1988 |
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Current U.S.
Class: |
435/5 ; 530/350;
536/23.72 |
Current CPC
Class: |
G01N 33/56983 20130101;
G01N 2333/15 20130101; A61K 39/00 20130101; C12N 2740/15022
20130101; G01N 2469/20 20130101; C07K 16/1054 20130101; C07K 14/005
20130101; C12N 2740/14022 20130101; G01N 33/6878 20130101; G01N
2333/70503 20130101; C07K 16/1063 20130101 |
Class at
Publication: |
435/005 ;
536/023.72; 530/350 |
International
Class: |
C12Q 001/70; C07K
014/15 |
Claims
1-13. (canceled)
14. A method for detecting antibody to FIV in a sample comprising:
providing an isolated Feline Immunodeficiency Virus (FIV) envelope
polypeptide that reacts specifically with a monoclonal antibody
that is specific for the FIV envelope protein gp130, said isolated
polypeptide being the FIV transmembrane envelope protein (gp40) or
a fragment thereof, and detecting a reaction between the
polypeptide and antibody in the sample.
15. The method of claim 14 in which the envelope polypeptide is
gp40.
16. The method of claim 15 in which the envelope polypeptide is
recombinant gp40.
17. The method of claim 15 in which the envelope polypeptide is
synthesized by chemical methods.
18. The method of any one of claims 14-17 in which the reagent
comprises a binding moiety that binds to sample antibody reacted
with the polypeptide.
19. The method of claim 18 in which the binding moiety is attached
to a color-producing moiety.
20. An assay device for detecting antibody to FIV in a sample
comprising: an isolated Feline Immunodeficiency Virus (FIV)
envelope polypeptide that reacts specifically with a monoclonal
antibody that is specific for the FIV envelope protein gp130, said
isolated polypeptide being the FIV transmembrane envelope protein
(gp40) or a fragment thereof; and at least one reagent for
detecting a reaction between the polypeptide and antibody in the
sample.
21. The device of claim 20 in which the envelope polypeptide is
gp40.
22. The device of claim 21 in which the envelope polypeptide is
recombinant gp40.
23. The device of claim 21 in which the envelope polypeptide is
synthesized by chemical methods.
24. The device of any one of claims 20-23 in which the reagent
comprises a binding moiety that binds to sample antibody reacted
with the polypeptide.
25. The device of claim 20 in which the binding moiety is attached
to a color-producing moiety.
26. An ELISA assay device for detecting antibody to FIV in a sample
comprising: a capture protein consisting essentially of recombinant
gp40; and at least one reagent for detecting a reaction between the
capture protein and antibody in the sample.
Description
BACKGROUND OF THE INVENTION
[0001] This application is a continuation-in-part of O'Connor et
al., application Ser. No. 348,784 filed 1989 entitled Polypeptides
of Feline T-Cell Lymphotrophic Lentivirus which is a
continuation-in-part of O'Connor et al., application Ser. No.
293,906, filed Jan. 5, 1989, entitled "Monoclonal Antibodies to
Feline-T-Lymphotropic Lentivirus.", which is a continuation-in-part
of O'Connor et al., application Ser. No. 279,989, filed Dec. 5,
1988, entitled "Monoclonal Antibodies to Feline-T-Lymphotropic
Lentivirus", the whole of which (including drawings) are hereby
incorporated by reference herein.
[0002] This invention relates to purification of polypeptides from
feline T-cell lymphotropic lentivirus (FIV).
[0003] FIV is a retrovirus originally isolated from cats which
exhibit an AIDS-like syndrome. Pederson et al., 235 Science 790,
1987. The virus belongs to the same group as the human
immunodeficiency virus (HIV), the causative agent of human AIDS.
Pederson et al., describe detection of antibody to FIV by an
immunofluorescent assay, and by Western blots, using virus purified
by centrifugation on sucrose gradients in Tris base, pH 7.4,
containing 0.1M NaCl and 1 mM EDTA. A few protein bands were
detected and, although antigenic comparison was not made, the
positions of these bands were tentatively said to correspond to the
major core protein, p24 the gag precursor protean, p55, and the
endonuclease protein, p32, of HIV.
[0004] Pederson et al., U.S. patent application Filed Aug. 26, 1987
entitled "Feline T-Lymphotropic Lentivirus" (which is not admitted
to be prior art to the present application) describes the results
presented by Pederson et al., supra, and states that Western
blotting of FIV infected cell lysates yielded major protein bands
at approximately 22-26 kD, usually about 24 kD; 50-60 kD, usually
about 55 kD; and 28-36 kD, usually about 32 kD.
SUMMARY OF THE INVENTION
[0005] In a first aspect, the invention features a purified
polypeptide having an epitope of an antigenic polypeptide of FIV.
The polypeptide may be glycosylated or unglycosylated. By antigenic
polypeptide is meant a polypeptide which is able to raise (with the
aid of an adjuvant if necessary) an antibody response in cats. The
polypeptide may be a polypeptide fragment of at least 5 amino
acids, or a polypeptide naturally occurring in FIV particles. The
fragment may be obtained from a naturally-occurring polypeptide,
for example, by enzymatic digestion of a naturally occurring
polypeptide, or may be produced by isolation or synthesis of a gene
encoding a desired polypeptide and expression of that polypeptide
within a desired expression system, for example, a bacterial,
yeast, or mammalian expression system.
[0006] By epitope is meant a single antigenic site of an antigenic
polypeptide. Such an epitope is recognized specifically by a
monoclonal antibody to an antigenic polypeptide of FIV.
[0007] By purified is meant that the polypeptide is separated from
other cell components with which it naturally occurs, for example,
FIV polypeptides. Preferably, the polypeptide is sufficiently pure
to permit its use to prepare a monoclonal antibody to the
polypeptide, and even more preferably, pure enough to allow the
amino acid sequence of the polypeptide to be determined by standard
procedure. Generally, the purified polypeptide is biologically
active in that it is suitable for preparation of a monoclonal
antibody, or is suitable for detection of naturally-occurring
antibodies within the serum of a cat.
[0008] In preferred embodiments, the purified polypeptide has at
least 75% amino acid homology to a polypeptide fragment of at least
20 amino acids obtained from an FIV gag or env polypeptide, most
preferably the purified polypeptide includes an amino acid sequence
having at least 75% homology to a whole of a gag or env
polypeptide, even more preferably, the purified polypeptide is an
entire gag or env amino acid sequence. Examples of gag and env
polypeptides include p10, p15, p26, gp40, gp100, and gp130.
[0009] In a second aspect, the invention features a method for
detecting an antibody to FIV within a sample, including the steps
of providing a purified polypeptide as described above, and
contacting that polypeptide under conditions suitable to allow an
antibody/polypeptide complex to form between antibodies within the
sample and the purified polypeptide, and detecting the formation of
such complexes. The presence of antibody/polypeptide complexes is
indicative of antibody to FIV present within the sample.
[0010] In a third aspect, the invention features a purified nucleic
acid including a 50 nucleotide sequence having at least 90%
homology with a 50 nucleotide sequence naturally occurring in an
FIV particle. By purified is meant that the nucleic acid is
substantially separated away from all of the components with which
it naturally occurs, e.g., polypeptides and other nucleic acids.
Preferably, the nucleic acid is completely separated from such
components, and is a pure solution of nucleic acid, or is held
within a cell in which it does not naturally occur, e.g, a
bacterial cell, another viral particle or a non-feline eucaryotic
cell. By 90% homology is meant that the nucleotide sequence is
identical at at least 45 of the 50 nucleotides.
[0011] In preferred embodiments, the nucleic acid encodes a
polypeptide including an epitope of an antigenic polypeptide of
FIV, e.g., an epitope of a gag or env polypeptide of FIV. Most
preferably the nucleic acid is carried in an expression vector and
can be expressed in a bacterial, fungal or other eucaryotic cell,
e.g., a mammalian cell.
[0012] In a related aspect the invention features purified
polypeptide including ten or more contiguous amino acids taken from
the sequence (using standard letters to represent amino acids)
V-Q-S-R-G-S-G-P-V-C-F-N- -C-K-K-P-G-H-L-A-R-Q-S-H or
P-I-Q-T-V-N-G-V-P-Q-Y-V-A-L-D-P-K-M-V-S or
S-V-Q-S-R-G-Q-G-P-V-A-F-N.
[0013] Applicants have provided polypeptides suitable for specific
detection of FIV antibodies and thus have allowed accurate
detection of infection with FIV. Applicants have also provided
polypeptides useful for production of vaccines to prevent disease
caused by FIV in cats.
[0014] Other features and advantages of the invention will be
apparent from the following description of the preferred embodiment
thereof, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] The drawings will first briefly be described.
DRAWINGS
[0016] FIG. 1 is a photograph of the major viral associated
proteins of FIV identified by polyacrylamide gel electrophoresis
(PAGE) and stained with Commassie Blue R250 (lane A); molecular
weight standards are shown in lane B;
[0017] FIG. 2 is a photograph of a Western immunoblot analysis of
antibodies to FIV found in serum from cats identified as positive
by an ELISA assay for FIV antibodies;
[0018] FIG. 3 is a graphical representation of elution of FIV
polypeptides during HPLC purification;
[0019] FIG. 4 is a photograph of an SDS-polyacrylamide gel after
electrophoresis and staining with silver, showing purity of various
FIV polypeptides; and
[0020] FIG. 5 is the nucleic acid sequence, and coresponding amino
acid sequence, of various portions of cloned FIV nucleic acid.
ANTIGENIC FIV POLYPEPTIDES
[0021] FIV polypeptide antigens useful in this invention are
generally described above. Polypeptides useful in this invention
may be purified from virus isolated as described below and
fragments of the purified polypeptides isolated by enzymatic
treatment or other standard procedures. Further, the polypeptides
may be synthesized by standard in vitro expression systems in which
DNA encoding for the FIV polypeptide is cloned and expressed in a
bacterial, yeast, or mammalian cell expression system. Such DNA may
be isolated and expressed as described below. The polypeptides may
also be synthesized by standard chemical methods, for example, the
polypeptide segments of various FIV polypeptides given below can be
synthesized. In the following example FIV polypeptides were
obtained directly from FIV-infected cells and took the form of
polypeptides naturally occurring in an FIV virus particle. This
example is not limiting to the present invention, and those skilled
in the art will recognize the many alternative methods for
obtaining polypeptides of this invention. The polypeptides are
referred to according to molecular weight, thus a polypeptide of 30
kD is termed p30, and a glycoprotein of this weight is termed
gp30.
[0022] Master seed virus producing cultures were obtained in the
form of a continuous feline cell line infected with FIV isolate
#2427 (CRFK-FIV or Petaluma strain) from Dr. Neils Pederson
(University of California, Davis, Calif.). The parent cell line is
Crandell feline kidney cell persistently infected with FIV. The
cell line was deposited with the American Type Culture Collection
on Jul. 13, 1988 and assigned the number CRL9761. Applicants and
their assignees acknowledge their responsibility to replace this
culture should it die before the end of the term of a patent issued
hereon, 5 years after the last request for a culture, or 30 years,
whichever is the longer, and its responsibility to notify the
depository of the issuance of such a patent, at which time the
deposit will be made irrevocably available to the public. Until
that time the deposit will be made available to the Commissioner of
Patents under the terms of 37 C.F.R. .sctn.1-14 and 35 U.S.C.
.sctn.112.
[0023] Other virus cultures can be obtained as described by both
Pederson et al, references, supra, or by Harbour et al., 122 The
Veterinary Record 84, 1988. Seed stocks of virus producing cell
cultures were obtained by freeze-downs of FIV-infected master seed
cell cultures following at least 19 post infection passages in
culture. Additional seed stocks of virus producing cultures were
obtained by either infection of the continuous feline cell culture
with FIV master seed virus or by single cell microwell cloning of
high level FIV producers from the original FIV infected master seed
cell culture. For propogation, master seed virus infected feline
cell cultures were inoculated into tissue cell culture flasks.
Following growth to a confluent monolayer of cells, tissue culture
fluid was harvested at intervals of 2-5 days.
[0024] Working seed virus was produced by propogation by the master
seed cell line permanently infected with FIV. An inoculum was added
to tissue culture flasks, in Dulbecco's Modified Eagles medium
containing 2 mM L-glutamine and 4.5 g per liter/glucose (DME)
containing 100 units per ml. penicillin and streptomycin and 2 mM
glutamine. An inoculum was added to tissue culture flasks,
incubated, and the spent tissue culture fluid harvested when the
cells were grown to confluence. The cells were released from the
culture vessel with trypsin/EDTA and diluted between 1:5 and 1:25
(typically 1:8) in medium. Typically the flasks were incubated at
36.degree. C.-38.degree. C. for a maximum of 7 days (between 3 and
7 days) before fluid and cell harvest. The harvested fluid,
including cell material, was centrifuged in a high speed centrifuge
(Sorval RC-5B or Beckman J2-21) leading to separation of
supernatant and cell pellet material. The cell pellet was
discarded, and the supernatant culture fluid used to prepare
working virus. The clarified supernatant was made 0.5 M in NaCl and
4%-10% (usually 7%) in polyethylene glycol (PEG 8000, Sigma).
Following overnight incubation at 2.degree. C.-7.degree. C., virus
was pelleted (at 13,000.times.g for 30 min.) and resuspended in
buffer (10 mM Tris, pH 7.6 300 mM NaCl, 1 mM EDTA, at 2.degree.
C.-7.degree. C.). After overnight incubation the virus was
centrifuged at 13,000.times.g for 15 min., the pellet discarded and
the supernatant centrifuged on a 50%/80% discontinuous glycerol
step gradient in 10 mM Tris 300 mM NaCl, 1 mm EDTA at pH 7.6.
Centrifugation was at 100,000.times.g for 3 hrs. at 4.degree. C.
and the FIV viral band at the 50%-80% interface collected. The band
was suspended in 10 mM Tris, 0.3 M NaCl and 1 mM EDTA and diluted
1:3 in the buffer and repelleted at 100,000.times.g for 1 hr. The
resulting pellet was purified virus and was resuspended in the
above buffer and stored at -70.degree. C. The resulting virus was
substantially free from FIV host cell proteins and was composed of
at least 5% p26 (the major nucleocapsid protein, as measured by
densitometric scans of Commassie Blue 250 stained SDS/PAGE as total
protein).
[0025] Such purified virus may be obtained by other techniques,
however, applicants have found that high molecular weight
contaminants present in virus preparations may be eliminated by use
of the high salt (i.e., greater than physiological range salt
concentration) used in the gradient centrifugation procedure.
[0026] Referring to FIG. 1, polypeptides associated with purified
FIV were analyzed by SDS/PAGE and compared with polypeptides
isolated in an identical manner from the scent culture medium of
uninfected cells. Analysis of the Commassie Blue stained gels
revealed three major polypeptides with molecular weights of about
10, 15, and 26 kD, named p10, p15 and p26, respectively.
[0027] When an ELISA test was performed using disrupted FIV to
identify cats possessing polyclonal antibody to FIV polypeptides,
and Western blot analyses then performed on feline sera determined
to be positive by ELISA, each of the cats had antibodies which
reacted with one or more polypeptides of molecular weight p10, 15,
26, 40 and 65 kD under the conditions used.
[0028] Referring to FIG. 2, a standard Western immuno blot was
performed as described by Towbin et al., 76 Proc. Natl. Acad. Sci
USA 4350, 1979. Briefly, FIV was disrupted with SDS and proteins
transferred to a sheet of nitrocellulose. The nitrocellulose sheet
was blocked with 30% calf serum, 1% bovine serum albumin (BSA), and
0.05% Tween 20 in Dulbecco's phosphate buffer saline. The sheets
were cut into 0.5 cm strips and incubated with a 1:100 dilution of
serum sample in blocking buffer for 2 hrs. for 20-22.degree. C.
Strips were repeatedly washed with washing buffer (0.05% Tween 20
in Dulbecco's phosphate buffer saline) and then incubated with a
second antibody (specific For feline heavy and light chain Ig)
horseradish peroxidase conjugate (obtained from Kirkguard and Perry
Laboratories Inc. Gaithersburg, Md.). After 1 hr. incubation, the
strips were repeatedly washed with washing buffer and incubated
with the precipitating substrate 4-chloronaphthol for 10 min. The
strips were partially dried and the results interpreted
immediately. The serum in each of the lanes A-G was obtained from
various cats infected with FIV. Predominant cross-reactivity is
detected with p26 and p15 and to a lesser extent with p10. Other
proteins of 32, 40, 47 and 65 kD molecular weight are also
detected.
[0029] Certain viral polypeptides, such as the gag (e.g., p26)
antigenic polypeptides, are abundant in purified viral
preparations, others such as the viral envelope polypeptides (e.g.,
gp130) tend to be lost during viral purification, and
electrotransfer less efficiently for Western blot analysis than the
gag antigens. Therefore, in order to more readily detect the viral
envelope (env) and the gag precurser polypeptides, FIV cell
extracts were labeled with .sup.35S-methionine and cysteine and
examined by immunoprecipitation (RIPA). Confluent cultures of cells
infected with FIV were incubated for 30 min. in methionine and
cysteine-free Dulbecco's modified Eagle's medium. The cell cultures
were then incubated for 4 hrs. in 8 ml of the same medium
containing 100 microCuries per ml of .sup.35S-methionine and
.sup.35S-cysteine (specific activity 1200 Curies per mM, New
England Nuclear Corporation, Boston, Mass.). The radioactive tissue
culture fluids were removed and the cells lysed with 5 ml of 10 mM
sodium phosphate buffer pH 7.5 containing 100 mM NaCl, 1% Triton X
100, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mM
phenylmethylsulfonylfluoride, and 100 Kallikren inactivator units
of aprotenin per ml. (Sigma Chemical Co., St. Louis, Mo.). Before
use, the cell lysates were clarified by centrifugation
100,000.times.g for 30 min. and the pellet discarded. Aliquots of
the labelled cell lysates (0.1 ml) and 5 .mu.l of serum being
tested were mixed in a microcentrifuge tube and incubated for 1 hr.
at 37.degree. C. and then overnight at 4.degree. C. The next day,
0.2 ml of a 5% suspension of protein A Sepharose CL-4B beads
(Pharmacia, Piscataway, N.J.) in 10 mM of phosphate buffer, pH 7.5
containing 100 mM NaCl, 1% Triton X-100 and 0.1% SDS was added to
each tube and mixed for 30 min. at 4.degree. C. The
antibody/antigen complexes bound to the protein A Sepharose beads
were collected by centrifugation (2 min. at 20,000.times.g) and
washed 3 times in lysing buffer. The final pellet was resuspended
in 25 .mu.l SDS/PAGE loading buffer and heated and 100.degree. C.
for 3 minutes. The Sepharose beads were removed by centrifugation
and the supernatant applied to a PAGE. Gels were processed for
fluorography using enlightening.TM. (New England Nuclear
Corporation, Boston, Mass.) and exposed at -70.degree. to Kodak
XR-5 film. Sera from experimentally infected cats recognize
polypeptides of 15, 22, 36, 40, 47, 110 and 130 kD. Although there
were some quantitative and qualititative differences all cats
appear to mount a response to p22, gp40, gp47 and gp130.
[0030] In order to determine which of the polypeptides identified
by RIPA-PAGE analysis were related to the major internal structural
protein, p26, RIPA-PAGE analysis was carried out using monoclonal
antibodies which reacted with p26 as determined by Western
blotting. This monoclonal immunoprecipited proteins p47, p36, p22
and p15. High molecular weight polypeptides (130 kD) of FIV which
were not detected by the p26 specific monoclonal antibody, were
identified by RIPA-PAGE. A protein of molecular weight 100 kD was
also detectable utilizing serum antibodies obtained from some
infected cats.
[0031] In another example, antigenic glycopeptides of FIV can be
obtained as follows. Actively growing CRFK FTLV infected cells were
scraped from roller bottles, gently washed with phosphate-buffered
saline (PBS), and pelleted. The cell pellet was gently resuspended
in 10 mM sodium phosphate, pH 7.2, at a ratio of 1 ml buffer to 0.1
ml of cell pellet. This suspension was incubated on ice or
refrigerated for 5-10 min., vigorously vortex mixed for 30 seconds,
and four volumes of PBS with 1 mm PMSF added. The mixture was then
vigorously homogenized for 90-120 seconds with a Brinkmann
Homogenizer PT10/35 with a PTA 20 generator.
[0032] The resulting homogenate was clarified for 20 minutes at
5,000.times.g. The supernatant fraction was discarded and the cell
membrane pellet resuspended in PBS+0.2% Triton X-100 at a ratio of
2.5 ml buffer to 0.1 ml original cell pellet. The mixture was then
vigorously homogenized for 90-120 seconds with a Brinkmann
Homogenizer PT 10/35 with a PTA 20 generator. The resulting
homogenate was clarified at 100,000.times.g for 1 hr, the
supernatant decanted off and batch bound overnight at 20-23.degree.
C. on Pharmacia Lentil Lectin Sepharose 4B at a ratio of 6 ml of
resin to 5 ml of original cell pellet.
[0033] The Lentil Lectin Suspension was poured through a column,
the resin collected, and washed with 15 column volumes of PBS+0.2%
Triton X-100. The glycoproteins were then eluted from the resin by
subjecting the resin to 5-10 column volumes of PBS+0.2% Triton
X-100+200 mm methyl .alpha.-D mannopyranoside, collecting fractions
of 1 column volume/tube.
[0034] The isolation of glycoproteins was verified by 9% SDS-PAGE
electrophorisis, and checked using .sup.35S-radiolabeled cell
preparations in conjunction with RIPA data.
[0035] Further purification of viral glycoprotein from host cell
glycoprotein includes use of HPLC, or of a polyclonal or monoclonal
antibody or affinity chromatography.
EXAMPLE 1
gag Polypeptide Purification
[0036] Isolated virus (250-500 microliters) was combined with two
volumes of 6M guanidine hydrochloride, pH 3 (adjusted with 20%
trifluoro acidic acid in water). The mixture was vortexed and
incubated at 37.degree. C.-40.degree. C. in a water bath for 20-25
minutes. The incubated solution was filtered through a pre-wetted
(100 microliters of 6M guanidine hydrochloride, pH 3) 0.45 micron
gel aquadisk filter (No. 4184) and the filter rinsed with 100
microliters of 6M guanidine hydrochloride pH 3. The filtered sample
was loaded onto an HPLC column for purification.
[0037] The HPLC system consisted of a Beckman HPLC having three
110V pumps, a 421 controller, a 166 variable wavelength detector, a
427 integrator, a 210A injector with dynamic mixer, and a 1000
microliter sample. The column was a Waters radial compression
cartridge held in an RCM-100 column holder with a modified inlet
connector (Waters MicroBond-A-Pak Fenile 10 MU 8 mm.times.10 cm
cartridge No. 85722 with a guard pak resolve CN cartridge, No.
85826). The system was set such that two levers were compressed and
the pressure was in the mid-yellow zone.
[0038] Purification was by a multi-step gradient from aqueous 0.1%
trifluoroacetic acid (v/v solvent A) to aceto nitrol containing
0.1% trifluoroacetic acid (v/v, solvent B).
[0039] Fractions were collected and condensed by a Savant Speed Vac
to remove all solvents to a final volume ranging from 50-100
microliters. The condensed fractions where neutralized by addition
of 200-300 microliters of 50 mM or 100 mM sodium phosphate buffer,
pH 7.2. The buffered fraction pHs were then checked with pH paper
and, if the pH was still below 6, 1N NaOH added until the pH was
brought within the range of 6.5-7.5. The neutralized fractions were
frozen at -20.degree. C. until use.
[0040] Referring to FIG. 3, the printout from an HPLC column
described above is shown. The flow rate was at 1 ml per minute
starting with 100% of solvent A. After 15 min. the solvent was
changed to contain 26% solvent B, 10 min. later to 31% solvent B,
12 min. later to 37.5% solvent B, 5 min. later to 40% solvent B, 6
min. later to 43% solvent B, 8 min. later to 45% solvent B, 15 min.
later to 60% solvent, and 20 min. later to 100% solvent B. Peaks
corresponding to p10, p15, and p26 are indicated in FIG. 3.
Fractions containing these peaks were collected.
[0041] Referring to FIG. 4, fractions corresponding to p10, p15 and
p26 were run in an SDS-polyacrylamide gel containing 15%
bis-acrylamide at 70V. The gel was stained with silver using a
Biorad Silver Staining Kit 161-0443. The isolated fractions
corresponding to p15, p10, and p26 were essentially homogeneous
solutions of the FIV polypeptides.
[0042] The isolated FIV polypeptides were analyzed by standard
technique for their amino acid sequence, yielding the following
results (? indicates uncertainty in the actual amino acid, or no
knowledge at all).
1 p26 Number 1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-- 17-18-19-20
Amino Acid P-I-Q-T-V-N-G-V-P-Q-Y-V-A-L-- D-P-K-M-V-S p10 Number
1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19- Amino Acid
V-Q-S-R-G-S-G-P-V-C-F-N-C-K-K-P-G-H-L- Number 20-21-22-23-24 Amino
Acid A- R- Q- S- H P15 Number 0-1-2-3-4-5-6-7-8-9-10-11-12-13 Amino
Acid S-V-Q-S-R-G-Q-G-P-V- A- F- N-?
[0043] In order to determine if the purified polypeptide is useful
in this invention, that is, whether the polypeptide is antigenic,
any standard procedure can be used. For example an ELISA test can
be performed using a polyclonal antibody from cat serum to
determine whether the polypeptide is cross-reactive. Alternatively,
the polypeptide can be injected with or without an adjuvant, into
an animal, e.g., a mouse, to determine if it causes antibodies to
be raised to it. These polypeptides are useful for production of
vaccines to prevent FIV-caused disease symptoms and FIV infection
in cats. These vaccines are produced by standard procedure.
Preferably the gag or env polypeptides are provided in a standard
inoculation medium and injected intravenously, intrarterially or
otherwise into a cat at a level of 1-100 .mu.g/kg animal at
intervals of 3-4 weeks until immunity to FIV is produced.
[0044] FIV Monoclonal Antibodies
[0045] Antibodies to FIV polypeptides are useful aids for
identification of a purified polypeptide (as described above), and
for purifying polypeptides. They are also useful to determine the
antigenicity of any polypeptide. An example of preparation of
useful antibodies follows. These antibodies are monoclonal
antibodies which allow specific detection and purification of
either individual or a small number of FIV polypeptides.
[0046] Balb/CJ (Jackson Labs) mice were immunized with an initial
injection of 50 micrograms of FIV virus (prepared as described
above) per mouse mixed 1:1 with Difco Bacto adjuvant complete.
After two weeks a booster injection of 100 micrograms of FIV virus
was injected into each mouse intravenously without adjuvant. Three
days after the booster injection a fusion was performed with mouse
myeloma cell lines FO or p3X63-Ag8.653. Mid log phase myeloma lines
were harvested on the day of fusion and checked for viability. The
cells were spun at 300.times.g for 8 min., separated from the
growth medium, and resuspended in serum free DME.
[0047] For fusion, an FIV-inoculated mouse was killed by cervical
dislocation and the spleen aseptically removed. The spleen was
washed three times in serum free DME and placed in a sterile Petri
dish containing 20 mls of complete medium (DME containing 20%
bovine fetal serum, 100 units per ml. of penicillin and
streptomycin, and 1 mM sodium pyruvate). To release cells, the
spleen was perfused with a 23 gauge needle.
[0048] Cells were placed in a 50 ml conical centrifuge tube and
pelleted at 300.times.g for 8 min. The pellet resuspended in 5 ml
of 0.17M ammonium chloride and placed on ice for 8 min. 5 ml of
bovine fetal serum (20%) was added and the cells pelleted again at
330.times.g for 8 min. After resuspension in 10 ml DME the cells
were counted and the spleen and myeloma cells mixed in a ratio of
3:1. The cell mixture was pelleted at 200.times.g for 10 minutes,
the supernatant decanted, and the pellet allowed to stand for 5
min. Over a period of 1 min., 1 ml of 50% PEG (PEG 1500 mixed 1:1
with Hepes pH 8.1) at 37.degree. C. was added. After 1 min.
incubation at 37.degree. C., 1 ml of DME was added over a period of
another 1 min. and then a second 1 ml of serum free medium added
over a period of 1 min. Finally, 10 mls of DME was added over a
period of 2 min., the cells pelleted at 200.times.g for 8 min., and
the pellet resuspended in complete medium containing 0.016 mM
thymidine, 0.1 mM hypoxanthine, 0.5 micromolar aminopterin, and 10%
hybridoma cloning factor (1.times.HAT). The cells were plated into
96-well plates.
[0049] After 3, 5 and 7 days half of the medium in the fusion
plates was removed and replaced with fresh 1.times.HAT. After 11
days the hybridoma cell supernatant was screened by an ELISA test.
In this test, 96 well plates were coated with FIV virus by standard
technique. One hundred microliters of supernatant from each well
was added to a corresponding well on a screening plate and
incubated for 1 hr. at 20-22.degree. C. After incubation, each well
was washed three times with distilled water and 100 microliters of
a horseradish peroxidide conjugate of goat anti-mouse IgG (H+ L),
A, M (1:1500 dilution) was added to each well and incubated for 1
hr. at 20-22.degree. C. After three washes with distilled water,
the substrate OPD/hydrogen peroxide was added and incubation
continued for five to fifteen minutes. One hundred microliters of a
stop solution (1 M hydrochloric acid) was then added and the
absorbance at 490 nm read. Cultures which had an optical density
reading greater than the control wells were removed to 2 cm.sup.2
culture dishes, with the addition of normal mouse spleen cells in
1.times.HT medium. After a further three days all of the 2 cm.sup.2
cultures were rescreened for antibody and those testing positive
again were cloned by limiting dilution. The cells in each 2
cm.sup.2 culture were counted and cell concentration adjusted to
1.times.10.sup.5 cells per ml. The cells were diluted in complete
medium and normal mouse spleen cells at concentrations of hybridoma
cells of 5, 10 and 50 cells per ml added. The cells were plated
into 96-well plates for each dilution. After 10 days the cloning
plates were screened for growth. About 37% of all wells showed
growth. The growth-positive wells were screened for antibody and
those testing positive expanded to 2 cm.sup.2 cultures and provided
with normal mouse spleen cells. The cloning procedure was repeated
2 times until a stable antibody-producing hybridoma was obtained.
At this point the cell culture was expanded from 2 to 9 to 75 to
150 cm.sup.2 culture vessels, at which point ascite production
could be commenced.
[0050] For ascites production, pristane primed IRCF1 female mice
were used. 0.5 ml of pristane was injected intraperitoneally (IP)
to each mouse, and the mouse allowed to rest for 10-60 days. At
this time 4.5.times.10.sup.6 cells were injected IP into each mouse
and ascites formed in 7-14 days. Ascites fluid was harvested with a
pasteur pipette through a hole in the peritoneum.
[0051] Antibodies to glycoproteins can also be isolated and
detected. In particular, antibodies to two glycoproteins of
molecular weight 40 kD (gp40) and 130 kP (gp130) which are detected
using PAGE and RIPA respectively.
[0052] Monoclonals useful in this invention for purification and
identification of specific polypeptides of FIV include those which
are specific for FIV and form a sufficiently strong interation with
an FIV epitope, and an FIV antigen, to be useful in an immunoassay,
for example, an ELISA, to detect an FIV antigen. In order to
determine which of the above monoclonal antibodies are useful in
this invention one main test was used. This entailed determination
of whether the monoclonal antibody can bind FIV antigen and be
detected with a conjugate of polyclonal antibody to FIV (an ELISA
test, described above).
[0053] Another test is to perform a Western blot to determine
whether the monoclonal antibody has good reactivity with one or
more FIV antigens. Generally, those monoclonals which show poor
reactivity, that is, produce faint bands on the Western blot, are
not suitable in this invention. Yet another test involves
radioimmunoprecipitation assay (RIPA) where FIV virus labeled with
.sup.35S-methionine is reacted with a monoclonal antibody to form
within immunoprecipitate, and the immunoprecipitate run in a
SDS-PAGE and autoradiographed to detect the labelled proteins. This
analysis determines which of the monoclonal antibodies is able to
detect precursor FIV antigenic polypeptides and not just mature
polypeptides.
[0054] Antibody Detection
[0055] The above antigenic polypeptides can be used to detect
naturally occurring antibodies produced by cats. Such detection can
be any standard immunoassay procedure, for example, by an ELISA
test, as described above. One example of such a test follows. This
example is not limiting to the present invention and those skilled
in the art will recognize that any of many other standard
procedures can be used.
EXAMPLE 2
Antibody Assay
[0056] Materials required to perform this assay include 96 well
flat bottom microtiter strips coated with a solution containing the
appropriate test antigen (e.g., p26, p15, or p10). The test wells
were coated with 100 .mu.l of a solution containing 0.15 micrograms
antigen in 0.25 molar sodium citrate, pH 7.5. The wells were
covered with parafilm, incubated overnight at 4.degree. C., and
tapped until dry. The antigen was then overcoated by adding 200
.mu.l 1% BSA in 0.25 molar sodium citrate, incubating at room
temperature (20-25.degree. C.) for 1 hour, and tapping the wells
dry. 200 microliters of 7.5% sucrose in 0.25 molar sodium citrate
was then added to each well and incubated at room temperature for
30 minutes. The resulting strips were used immediately, or dried
under vacuum for 6 hours at room temperature for later use.
[0057] Assays were performed by adding 100 .mu.l of feline serum
sample (positive control, negative control, or test sera) diluted 1
to 100 in Dulbecco's PBS containing 0.1% Bovine serum albumin, 30%
calf serum, and 0.05% Tween-20 (Sigma Chemical, St. Louis, Mo.) to
a well, incubating at room temperature for 30 minutes, and tapping
the well dry. The wells were washed immediately two times with
Dulbecco's PBS containing 0.05% Tween-20, tapping the wells dry
after the second wash.
[0058] 100 .mu.l of a solution containing antibodies to feline
immunoglobulin was then added. These antibodies were conjugated to
an indicator enzyme (e.g., alkaline phosphatase) and then dissolved
in a solution of 50% fetal calf serum, 0.05% Tween-20 in 0.05M
Tris-HCl, pH 7.6. The wells were incubated at room temperature for
30 minutes, tapped dry, and then washed two times with Dulbecco's
PBS containing 0.05% Tween-20.
[0059] A solution containing 0.1% 3,3',5,5' Tetramethylbenzidene in
40% Glycerol and 60% methanol was mixed with an equal volume of a
solution containing 22.82 grams dibasic potassium phosphate, 19.2
grams citric acid and 1.34 milliliters 30% hydrogen peroxide
solution per liter. One hundred microliters of this solution was
added to each well, and then incubated at room temperature for 15
minutes. At the end of the incubation period, 100 .mu.l of 0.25%
hydrofluoric acid was added to each well. The optical density at
650 nanometer of the solution in each well was then determined with
a microtiter plate reader. An immune response to p10, p15 and p26
was detectable either following experimental infection of a cat
with FIV, or in feline sera possessing antibodies to FIV.
[0060] FIV Nucleic Acid
[0061] FIV nucleic acid is useful for production of large amounts
of FIV polypeptides, or fragments thereof, and also for detection
of homologous nucleic acid in vivo, using standard techniques.
There follows an example of cloning of FIV viral DNA. The specific
FIV strain chosen is not meant to be limiting in this invention and
those skilled in the art will recognize that equivalent nucleic
acid may be isolated by use of the cloned sequences which are
provided as specific deposits, or by techniques similar to those
described in this example.
[0062] A Crandal feline kidney cell line productively infected with
FIV strain 2428 (Pentaluma isolate) was used as a source of
unintegrated viral DNA. The unintegrated viral DNA was prepared by
Hirt extraction and CsCl-ethidium bromide centrifugation to resolve
linear and supercoiled viral DNA. (Hirt, 26 J. Mol. Biol. 365,
1967; Canaani et al., 282 Nature 378, 1979).
[0063] The supercoiled viral DNA was used to construct libraries
which contain overlapping viral DNA sequences. The procedures used
to construct these libraries were similar to those described by
Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Lab, Cold Spring Harbor, N.Y.) and Glover, DNA
Cloning, Vol. 1, A Practical Approach, IRL Press, Washington, D.C.)
and are familiar to those of ordinary skill in the art.
[0064] Two viral DNA libraries were generated by cleavage of
supercoiled viral DNA by one of two restriction endonucleases.
Partial cleavage by the restriction endonuclease RsaI, which
recognizes the DNA sequence 5'GTAC3', or cleavage by the
restriction endonuclease HaeIII, which recognizes the DNA sequence
5'GGCC3', of the supercoiled viral DNA generates two sets of
blunt-ended DNA molecules whose sequences overlap. The blunt-ended
DNA molecules were then treated with EcoRI methylase, according to
the manufacturer's directions, to modify the 3' adenine residue of
the EcoRI recognition sequence 5'-GAATCC-3'. Methylation at this
site inhibits the cleavage of treated DNA by the restriction
endonuclease EcoRI. The methylated DNA molecules were then ligated
to linker DNA molecules which contained a cleavage site for the
restriction endonuclease EcoRI. The linker containing DNA molecules
were then treated with the restriction endonuclease EcoRI to
generate molecules whose termini were compatible with the EcoRI
cloning site in the recombinant DNA phage vector .lambda. ZAP.TM.
(Stratagene, La Jolla, Calif.). Linker fragments resulting from
this cleavage were separated from the large DNA molecules by size
separation on a quickspin column (Boehringer, Mannheim,
Indianapolis, Ind.). The DNA molecules were then ligated into the
EcoRI cleaved .lambda. ZAP vector using T4 DNA ligase (New England
Biolabs, Beverly, Mass.). Ligated DNA molecules were then packaged
into phage using Gigapack gold (Stratagene, La Jolla, Calif.).
Viable phage from the packaging reaction were then amplified by
infecting BB4 cells (Stratagene) and harvesting plate lysates of
those infected cells in order to obtain a stock of recombinant
.lambda.ZAP clones.
[0065] Because the insert DNA of the recombinant .lambda.ZAP clones
contain host cellular DNA as well as FIV proviral DNA sequences, it
was necessary to screen each library with an FIV DNA probe that
contains a readily detectable label. Such a probe was made from RNA
isolated from FIV. A radioactive complementary DNA was synthesized
from total viral RNA essentially as described by Maniatis et al.
1982, supra except that selection of poly A-containing RNA was not
performed, and methymercuric hydroxide was omitted from the
protocol.
[0066] The bacteriophage libraries were plated at a density of
10,000 bacteriophage per 150 mm dish. They were then screened by
hybridization of the radioactively labeled probe to phage DNA which
was immobilized on nitrocellulose filters (Maniatis, et al., 1982
supra). Each hybridizing bacteriophage plaque was then picked,
replated, and hybridized as described, until a single well was
isolated which contained a .lambda.ZAP recombinant clone. XL-1-Blue
cells (Stratagene) were then infected with recombinant .lambda.ZAP
phage, and plasmids containing the insert DNA were obtained
following superinfection with R408 helper phage according to the
manufacturer's directions (Stratagene, La Jolla, Calif.). This
procedure also provides both recombinant plasmids (which can be
isolated from the cell) and single stranded phage stock which can
be isolated from the medium for DNA sequence analysis.
[0067] The above recombinant plasmids were analyzed for inserts by
preparing plasmid DNAs from overnight culture of bacteria
replicating these plasmids as follows. One and one half ml. of an
overnight culture was placed in a microcentri-uge tube and spun for
four minutes at 4000.times.g. The supernatant was removed and the
tube respun for four minutes at 4000.times.g. The supernatant was
removed and the tube respun for a few seconds, and residual liquid
removed carefully with a pasteur pipet. The bacterial pellet was
then thoroughtly resuspended in 200 microliters of a solution
containing 8% sucrose, 50 mm EDTA, 5% Triton X-100 and 50 MM
Tris/HCl, pH 8.05. 20 microliters of a lysozome solution at a
concentration of 10 milligrams lysozyme per milliliter in 10
millimolar Tris/HCl, pH 8 and one millimolar EDTA was then added,
mixed, and the mixture was incubated at 4.degree. C. for 15
minutes. The solution containing bacteria was then placed in a
boiling water bath for 90 seconds. The mixture was chilled on ice,
and spun in a microfuge in the cold for 10 minutes at
11,000.times.g. The pellet was carefully removed with a glass
pipet. Ice cold isopropanol (200 microliters) was then added, the
solution thoroughly mixed, and incubated at -20.degree. C. for 5
minutes. The chilled solution was centrifuged at -20.degree. C. at
11,000.times.g for 10 minutes to pellet the plasmid DNA. The
supernatant was carefully removed and the pelleted DNA briefly air
dried. The DNA pellet was then dissolved in 100 microliters of
sterile double distilled water. Plasmid DNAs thus isolated were
anlayzed for inserts by restriction enconuclease cleavage and
electrophoresis in 0.8% agarose gels (Maniatis et al., 1982
supra).
[0068] Standard dideoxy sequence analysis was performed on the
recombinant DNA containing clones. Single stranded phage were
isolated from the media used to propagate cells containing the
bluescript plasmid using the method generally described in the M13
dideoxy sequencing manual published by Bethesda. Research
Laboratories (Gaithersburg, Md.). A number of clones were sequenced
and analyzed by this method. Sequence information for clones termed
10CX, 2BY, and R5X is presented in FIGS. 5a, b, and c along with
the putative translated amino acid sequence for each clone. These
amino acid sequences show homology with the amino acid sequence of
the envelope gene of equine infectious anemia virus, a lentivirus,
immunologically closely related to FIV.
[0069] Nucleic acid probes derived from the 2BY DNA sequence
hybridize to DNA isolated from FIV infected but not uninfected
cells. These probes can be used to isolate other FIV genes from
other strains and can be expressed by standard procedures to
provide the purified polypeptides described above.
[0070] Deposit
[0071] Strains 10CX, 2B4 and R5X have been deposited with the ATCC
and assigned numbers 67937, 67938, and 67939, respectively.
[0072] Applicants' and their assignees acknowledge their
responsibility to replace these cultures should they die before the
end of the term of a patent issued hereon, 5 years after the last
request for a culture, or 30 years, whichever is the longer, and
its responsibility to notify the depository of the issuance of such
a patent, at which time the deposits will be made irrevocably
available to the public. Until that time the deposits will be made
available to the Commissioner of Patents under the terms of 37 CFR
Section 1-14 and 35 USC Section 112.
[0073] Other embodiments are within the following claims.
* * * * *