U.S. patent application number 12/761239 was filed with the patent office on 2011-08-04 for method and device for detecting feline immunodeficiency virus.
This patent application is currently assigned to IDEXX Laboratories, Inc.. Invention is credited to Randall G. Groat, Quentin J. Tonelli.
Application Number | 20110189651 12/761239 |
Document ID | / |
Family ID | 34316504 |
Filed Date | 2011-08-04 |
United States Patent
Application |
20110189651 |
Kind Code |
A1 |
Groat; Randall G. ; et
al. |
August 4, 2011 |
Method and Device for Detecting Feline Immunodeficiency Virus
Abstract
A method and device for determining a feline immunodeficiency
virus infection or vaccination in an animal. The method includes
contacting a biological sample from a felid with various FIV
polypeptides and determining the binding of antibodies in the
sample to the polypeptides. The determination of whether an animal
is infected with FIV or has been vaccinated against FIV can be
determined by measuring the animal's immune response to an FIV env
polypeptide. A device for detecting FIV antibodies is provided.
Inventors: |
Groat; Randall G.;
(Freeport, ME) ; Tonelli; Quentin J.; (Portland,
ME) |
Assignee: |
IDEXX Laboratories, Inc.
Westbrook
ME
|
Family ID: |
34316504 |
Appl. No.: |
12/761239 |
Filed: |
April 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10938097 |
Sep 10, 2004 |
7776546 |
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12761239 |
|
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60584106 |
Jun 30, 2004 |
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60501982 |
Sep 11, 2003 |
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Current U.S.
Class: |
435/5 ;
436/501 |
Current CPC
Class: |
C07K 14/005 20130101;
G01N 2469/20 20130101; G01N 33/56988 20130101; G01N 33/56983
20130101; G01N 2333/155 20130101; C12N 2740/15022 20130101 |
Class at
Publication: |
435/5 ;
436/501 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; G01N 33/566 20060101 G01N033/566 |
Claims
1-38. (canceled)
39. A method for detecting whether a felid has received a whole
killed FIV vaccine that comprises an inactivated FIV Env protein
("FIV vaccine") or whether the felid is infected with FIV, the
method comprising: (a) obtaining a biological sample from a felid;
(b) contacting the sample with a first antigen consisting of SEQ ID
NO:9, consisting of SEQ ID NO:20 or comprising an FIV Env peptide
selected from the group consisting of SEQ ID NO:1-8 and 10 under
conditions that permit antibody-antigen binding, wherein said first
antigen is attached to a solid phase; (c) detecting for
antibody-antigen binding, wherein: (i) a positive result is
indicative of an FIV-infected felid or an uninfected felid that has
received the FIV vaccine within about 12 weeks before obtaining the
sample; (ii) a negative result is indicative of an unvaccinated and
uninfected felid, or a felid that has not received the FIV vaccine
within about 12 weeks before obtaining the sample.
40. The method of claim 39, further comprising obtaining a second
biological sample from the felid detected positive in step (c)(i)
at about 10 to 12 weeks after the first biological sample is taken,
and repeating step (b), wherein a positive result is indicative of
an FIV-infected felid, and a negative result is indicative of an
uninfected felid that has received the FIV vaccine.
41. The method of claim 39 wherein the first antigen consists of
SEQ ID NO:9.
42. The method of claim 39 wherein the first antigen consists of
SEQ ID NO:20.
43. The method of claim 39 wherein the first antigen comprises SEQ
ID NO:1.
44. The method of claim 39 wherein the first antigen comprises SEQ
ID NO:2.
45. The method of claim 39 wherein the first antigen comprises SEQ
ID NO:3.
46. The method of claim 39 wherein the first antigen comprises SEQ
ID NO:4.
47. The method of claim 39 wherein the first antigen comprises SEQ
ID NO:5.
48. The method of claim 39 wherein the first antigen comprises SEQ
ID NO:6.
49. The method of claim 39 wherein the first antigen comprises SEQ
ID NO:7.
50. The method of claim 39 wherein the first antigen comprises SEQ
ID NO:8.
51. The method of claim 39 wherein the first antigen comprises SEQ
ID NO:10.
52. A method for detecting vaccination and/or infection of FIV in
felids comprising: (a) obtaining a first biological sample from a
felid; (b) contacting the sample with a first antigen consisting of
SEQ ID NO:9, consisting of SEQ ID NO:20 or comprising an FIV Env
peptide selected from the group consisting of SEQ ID NO:1-8 and 10
under conditions that permit antibody-antigen binding, wherein said
first antigen is attached to a solid phase; (c) contacting the
biological sample with a second antigen comprising an FIV Gag
polypeptide, wherein the second antigen is attached to a solid
phase under conditions that permit antibody-antigen binding, (d)
detecting for antibody-antigen binding, wherein (i) a positive
result in both step (b) and step (c) is indicative of an
FIV-infected felid or a felid that has been vaccinated with a
whole-killed FIV vaccine that comprises an inactivated FIV Env
protein ("FIV vaccine") within about 12 weeks before obtaining the
sample; (ii) a negative result in both step (b) and step (c) is
indicative of an unvaccinated and uninfected felid; (iii) a
negative result in step (c) and a positive result in step (b) is
indicative of an uninfected felid that has been vaccinated with the
FIV vaccine.
53. The method of claim 52 further comprising obtaining a second
biological sample from the felid detected positive in step (d)(i)
at about 10 to 12 weeks after the first biological sample is taken,
and repeating step (b), wherein a positive result is indicative of
an FIV-infected felid, and a negative result is indicative of an
uninfected felid that has been vaccinated with the FIV vaccine.
54. The method of claim 52, wherein the FIV Gag polypeptide is FIV
p15 or FIV p24.
55. The method of claim 52 wherein the first antigen consists of
SEQ ID NO:9.
56. The method of claim 52 wherein the first antigen consists of
SEQ ID NO:20.
57. The method of claim 52 wherein the first antigen comprises SEQ
ID NO: 1.
58. The method of claim 52 wherein the first antigen comprises SEQ
ID NO:2.
59. The method of claim 52 wherein the first antigen comprises SEQ
ID NO:3.
60. The method of claim 52 wherein the first antigen comprises SEQ
ID NO:4.
61. The method of claim 52 wherein the first antigen comprises SEQ
ID NO:5.
62. The method of claim 52 wherein the first antigen comprises SEQ
ID NO:6.
63. The method of claim 52 wherein the first antigen comprises SEQ
ID NO:7.
64. The method of claim 52 wherein the first antigen comprises SEQ
ID NO:8.
65. The method of claim 52 wherein the first antigen comprises SEQ
ID NO:10.
66. A method for determining an FIV infection of FIV in felids
comprising: (a) obtaining a biological sample from a felid; (b)
contacting the sample with an antigen consisting of SEQ ID NO:9,
consisting of SEQ ID NO:20 or comprising an FIV Env peptide
selected from the group consisting of SEQ ID NO:1-8 and 10 under
conditions that permit antibody-antigen binding; (c) detecting
antibody-antigen binding, thereby determining that the felid has
been infected with FIV of has received a whole killed FIV vaccine
that comprises an inactivated FIV Env protein within about 12 weeks
before obtaining the sample.
67. The method of claim 66 wherein the antigen consists of SEQ ID
NO:9.
68. The method of claim 66 wherein the antigen consists of SEQ ID
NO:20.
69. The method of claim 66 wherein the antigen comprises SEQ ID
NO:1.
70. The method of claim 66 wherein the antigen comprises SEQ ID
NO:2.
71. The method of claim 66 wherein the antigen comprises SEQ ID
NO:3.
72. The method of claim 66 wherein the antigen comprises SEQ ID
NO:4.
73. The method of claim 66 wherein the antigen comprises SEQ ID
NO:5.
74. The method of claim 66 wherein the antigen comprises SEQ ID
NO:6.
75. The method of claim 66 wherein the antigen comprises SEQ ID
NO:7.
76. The method of claim 66 wherein the antigen comprises SEQ ID
NO:8.
77. The method of claim 66 wherein the antigen comprises SEQ ID
NO:10.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/938,097 filed on Sep. 10, 2004, now U.S. Pat. No. 7,776,546,
which claims the benefit of U.S. Provisional Patent Application
Ser. No. 60/584,106, filed Jun. 30, 2004, and U.S. Provisional
Patent Application Ser. No. 60/501,982 filed Sep. 11, 2003.
FIELD OF THE INVENTION
[0002] The invention is related to the detection of antibodies
directed to Feline Immunodeficiency Virus.
BACKGROUND OF THE INVENTION
[0003] Feline immunodeficiency virus (FIV), formerly called feline
T-lymphotrophic lentivirus, was first isolated in 1986 from a large
multiple cat household in Petaluma, Calif. (Pederson et al.,
Science (1987) 235:790). FIV infects cats to produce an AIDS-like
syndrome. Although FIV is morphologically and pathologically
similar to the human immunodeficiency virus (HIV), it has been
shown to be antigenically distinct from HIV. Like HIV, once a cat
becomes infected with FIV, the disease progresses from a primary
infection (viraemia, fever, general lymphadenitis) to a lengthy
asymptomatic phase, followed by severe impairment in immune
function caused by a reduction in CD4 lymphocytes, and resulting in
heightened susceptibility to secondary infections and ultimately
death.
[0004] FIV has been classified as a member of the subfamily
Lentiviridae in the family Retroviridae, the family that includes
human and simian immunodeficiency viruses, equine infectious
anaemia, maedi visna of sheep and caprinearthritis encephalitis
viruses (CAEV). The genome of FIV is organized like other
lentiviruses with three long open reading frames corresponding to
gag, pol and env (Talbott et al., Proc. Natl. Acad. Sci. (1989)
86:5743; Olmsted et al., Proc. Natl. Acad. Sci. (1989) 86:2448).
The gag gene codes for the major structural components of the
virus, the env gene codes for the envelope glycoprotein, and the
pol gene codes for the polymerase protein.
[0005] The gag gene is expressed as a 55 kD polyprotein that is
processed into three subunits: a p15 matrix protein, a p24 capsid
protein, and a p10 nucleocapsid protein. The pol gene encodes three
proteins: the protease, reverse transcriptase and a p14.6 protein
of unknown function. Autoprocessing by the protease portion of the
gene gives rise to all three proteins of the pol region.
Additionally, the protease is responsible for the processing of the
gag precursor. The pol gene is expressed as a gag pol fusion
protein. The envelope gene is expressed as a 160 kD glycoprotein,
gp160. The antigenicity of the FIV core proteins is similar to
other lentiviruses.
[0006] Several independent viral isolates have been prepared across
the world, and a certain number of studies have been carried out in
order to demonstrate the structure of the isolated strains: the
American strain Petaluma, Talbott et al. Natl. Acad. Sci. USA,
1989, 86, 5743-5747; Philipps et al., J. Virol., 1990, 64, 10,
4605-4613), the Japanese strains (the TM1 and TM2 strains),
Miyazawa et al., Arch. Virol., 1989, 108, 59-68, and the Swiss
isolates (FIVZ1 and FIVZ2), Morikawa et al., Virus Research, 1991,
21, 53-63.
[0007] The nucleotide sequences of three proviral clones derived
from American FIV isolates (Petaluma strain) have been described
(clones FIV34TF10, FIV14 and isolate PPR) (Olmsted, et al. 1989;
Philipps et al., 1990; Talbott et al., 1989) and compared with two
Swiss isolates (Morikawa et al. 1991). This comparison led Morikawa
et al. to specify the presence of certain conserved regions and
certain variable regions within the env gene of FIV. French strains
have also been isolated (strains Wo and Me) (Moraillon et al.,
1992, Vet. Mic., 31, 41-45).
[0008] The virus replicates optimally in blood mononuclear cells
and has a tropism for T-lymphocytes, peritoneal macrophage, brain
macrophage and astrocytes. In common with other retroviruses, the
genetic material of FIV is composed of RNA and the production of a
DNA copy of the viral RNA is an essential step in the replication
of FIV in the host. This step requires the enzyme reverse
transcriptase that is carried into the host by the invading virus.
The DNA version of the viral genome is inserted into the genetic
material of infected host cells in which it continues to reside as
a provirus. This provirus is replicated every time the cell divides
and can code for the production of new virus particles. Cells
infected with FIV remain infected for the duration of their
lifespan.
[0009] The virus appears to be spread naturally by horizontal
transmission, predominantly by bite wounds from an infected cat as
these animals shed appreciable amounts of virus in saliva (Yamamoto
et al., Am. J. Vet. Res. 1988, 8:1246). Vertical transmission has
been reported, but is rare.
[0010] Current diagnostic screening tests for FIV infection detect
serum antibody (Ab) to FIV. Virus detection kits are also available
but not as prevalent. A number of diagnostic tests are available to
determine the presence of FIV antibody in infected animals. For
example, PetChek.RTM. FIV Ab test kit and the SNAP.RTM. Combo FeLV
Ag/FIV Ab test kit (IDEXX Laboratories, Westbrook, Me.) are
immunoassay based diagnostic tests for FIV infection.
[0011] Detecting FIV infection is becoming increasingly important
as studies reveal FIV infection is widespread worldwide. As
vaccines have been developed in attempt to combat the disease, it
is even more important to be able to detect the effectiveness of a
vaccine and to discriminate between vaccinated cats versus
naturally infected cats.
SUMMARY OF THE INVENTION
[0012] In one aspect, the present invention provides a method for
determining whether a felid has been vaccinated against FIV or is
naturally infected with FIV by determining the felid's immune
response to an FIV polypeptide, such as an FIV env polypeptide.
[0013] In another aspect, the invention is directed to a method of
distinguishing among animals that have been naturally infected with
FIV versus animals that have not been infected or have been
vaccinated against an FIV infection. The method includes contacting
a biological sample from an animal with a polypeptide that does not
substantially bind to an FIV antibody that is a significant
component of the animal's immune response to an FIV vaccine. FIV
antibodies in the sample that substantially bind to the polypeptide
are detected. It is determined that the animal is naturally
infected by correlating a positive result in the detecting step to
a natural infection and it is determined that the animal has been
vaccinated or not infected by correlating a negative result to a
vaccination or no infection. The polypeptide may be derived from
FIV env.
[0014] In a further aspect, the invention is directed to a method
of determining whether a cat has been vaccinated against FIV or is
naturally infected with FIV. The method includes (a) detecting,
before a period a time following vaccination sufficient for certain
FIV antigen-specific antibodies raised in response to the vaccine
to be not detected, whether the cat has antibodies against an FIV
peptide; (b) detecting, after a period of time following
vaccination sufficient for certain FIV antigen-specific antibodies
raised in response to the vaccine to be not detected, whether the
cat has antibodies against an FIV polypeptide; (c) determining that
the animal has been vaccinated by detecting antibodies in step a
but not in step b, and (d) determining that the animal is naturally
infected by detecting antibodies in steps a and b.
[0015] The invention also provides for a method of determining
whether a cat has not been infected by FIV or has been vaccinated
against FIV. The method includes analyzing a biological sample from
the cat to detect antibodies against a polypeptide derived from
FIV, and determining that the animal has not been infected or has
been or vaccinated by not detecting such antibodies.
[0016] In yet another aspect, the invention provides a method of
determining whether or not a cat has been vaccinated for FIV or
naturally infected with FIV. The method includes providing a test
device comprising a polypeptide, obtaining a blood sample from a
cat, running the blood sample on the test device, and reading the
test device. A positive result indicates the cat has been naturally
infected with FIV or vaccinated against FIV and a negative result
indicates the cat has not been naturally infected with FIV and not
vaccinated against FIV.
[0017] Still further, the invention is directed to a diagnostic
device having a dry porous carrier, a first detection reagent
immobilized on the porous carrier where the first detection reagent
includes a protein that captures FIV antibodies generated by a host
in response to either a natural FIV infection or an FIV
vaccination, and a second detection reagent immobilized on the
porous carrier wherein the second detection reagent includes a
protein that captures FIV antibodies generated by a host in
response to a natural FIV infection but does not substantially
capture antibodies generated by the host in response to an FIV
vaccination. The first detection reagent may be FIV p15 or p24
antigen, and the second detection reagent may be an FIV env
protein.
[0018] In another aspect, the invention is directed to novel FIV
polypeptides.
DETAILED DESCRIPTION
[0019] Before describing the present invention in detail, a number
of terms will be defined. As used herein, the singular forms "a,"
"an", and "the" include plural referents unless the context clearly
dictates otherwise.
[0020] As used herein, the term "polypeptide" refers to a compound
of a single chain or a complex of two or more chains of amino acid
residues linked by peptide bonds. The chain(s) may be of any
length. A protein is a polypeptide and the terms are used
synonymously. Also included within the scope of the invention are
functionally equivalent variants and fragments of FIV polypeptides.
The polypeptide is capable of binding one or more antibodies
specific for the polypeptide.
[0021] Polypeptides derived from FIV include any region of the of
the FIV proteome including for example, portions of the gag and env
regions and mimitopes thereof. U.S. Pat. Nos. 5,648,209, 5,591,572,
and 6,458,528, which are incorporated by reference herein in their
entirety, describe FIV polypeptides derived from the FIV env and
gag proteins. These peptides, and others like them, from the env
and gag proteins, are suitable for use in the methods of the
present invention. Examples of a suitable env polypeptides include
the following:
TABLE-US-00001 [SEQ ID NO: 1]
ELGSNQNQFFSKVPPELWKRYNKSKSKSKSKNRWEWRPDFESEKC [SEQ ID NO: 2]
CNRWEWRPDFESEKSKSKSKSMQELGSNQNQFFSKVPPELWKRYN
[0022] SEQ ID NO:1 is a trimeric sequence; the first monomer is the
native FIV env sequence, amino acids 696-717 with C to S, I to V, L
to P, and T to K substitutions; the second monomer (underline) is a
KS linker; the third monomer is the native FIV surface env protein,
amino acids 396-408 with a C-terminal C addition. In one aspect of
the invention, the length of the KS linker is varied from 2-20
amino acids.
[0023] SEQ ID NO:2 is a trimeric sequence; first monomer is the
native FIV env protein, amino acids 396-408 with an N-terminal C
addition; the second monomer (underline) is a KS linker; the third
monomer is native FIV env protein, amino acids 694-717, with C to
S, I to V, L to P, and T to K substitutions. In one aspect of the
invention, the length of the KS linker is varied from 2-20 amino
acids.
[0024] "Binding specificity" or "specific binding" refers to the
substantial recognition of a first molecule for a second molecule,
for example a polypeptide and a polyclonal or monoclonal antibody,
or an antibody fragment (e.g. a Fv, single chain Fv, Fab', or
F(ab')2 fragment) specific for the polypeptide.
[0025] "Substantial binding" or "substantially bind" refer to an
amount of specific binding or recognizing between molecules in an
assay mixture under particular assay conditions. In its broadest
aspect, substantial binding relates to the difference between a
first molecule's incapability of binding or recognizing a second
molecule, and the first molecules capability of binding or
recognizing a third molecule, such that the difference is
sufficient to allow a meaningful assay to be conducted
distinguishing specific binding under a particular set of assay
conditions, which includes the relative concentrations of the
molecules, and the time and temperature of an incubation. In
another aspect, one molecule is substantially incapable of binding
or recognizing another molecule in a cross-reactivity sense where
the first molecule exhibits a reactivity for a second molecule that
is less than 25%, preferably less than 10%, more preferably less
than 5% of the reactivity exhibited toward a third molecule under a
particular set of assay conditions, which includes the relative
concentration and incubation of the molecules. Specific binding can
be tested using a number of widely known methods, e.g, an
immunohistochemical assay, an enzyme-linked immunosorbent assay
(ELISA), a radioimmunoassay (RIA), or a western blot assay.
[0026] Animals infected with FIV are felids, which is to be
understood to include all members of the order Felidae, including
domestic cats, lions, tigers, jaguars, leopards, puma, ocelots,
etc. As used herein, the terms "felid," "cat" or "animal" is a
reference to all felids.
[0027] A "biological sample" refers to a sample from an animal
subject including saliva, whole blood, serum, plasma or other
sample known to contain FIV antibodies
[0028] An "antibody that is a significant component of an animal's
immune response to a FIV vaccine" refers to an antibody that is
elicited as the result of a vaccination with a FIV vaccine. These
antibodies may be identical to or similar to antibodies elicited as
the result of a natural FIV infection. A successful vaccination
produces a measurable level of the antibody that is a significant
component of the FIV vaccine.
[0029] Vaccines for FIV are described, for example, in U.S. Pat.
Nos. 6,667,295, 5,833,993, 6,447,993, 6,254,872 and 6,544,528, and
published U.S. Patent Application 20040096460, each of which is
incorporated herein by reference in their entirety. U.S. Pat. Nos.
6,447,993 and 6,254,872 describe vaccines that are prepared from
cell free-viral isolates of different FIV subtypes or a combination
of cell lines each infected with different prototype FIV virus from
a different subtype. U.S. Pat. 5,833,933 describes vaccines
containing DNA sequences encoding FIV gag protein and FIV env
protein. These vaccines include an expression system for expressing
the sequences. One available vaccine is FEL-O-VAX.RTM. FIV vaccine
(Fort Dodge Animal Health, Overland Park, Kans.).
[0030] Biological samples from animals that have been vaccinated
against an FIV infection have the potential for producing a
positive result in a test for an FIV infection due to the presence
of antibodies produced by the animal in response to the vaccine. In
one aspect, the invention provides for a method of distinguishing
between animals that have been naturally infected with FIV and
animals that have not been infected or have been vaccinated against
an FIV infection. The method includes contacting a biological
sample from the animal with a polypeptide derived from an FIV that
does not substantially bind to an antibody that is a significant
component of the animal's antibody response to an FIV vaccine.
[0031] In another aspect, the invention includes a method of
determining whether a cat has not been infected by FIV and has not
been vaccinated against FIV. A biological sample from a cat is
analyzed to detect antibodies against a polypeptide, derived from
FIV env and/or gag. It is then determined that the animal has not
been infected and has not been or vaccinated by determining the
absence of such antibodies.
[0032] In some instances, during an initial phase following a
vaccination, an animal may temporarily (transiently) produce lower
levels of certain antibodies to specific FIV polypeptides that are
elements of a vaccine, as compared to those produced in response to
a natural infection. These antibody levels taper off after a period
of time to the point that antibody to these polypeptides is not
detected after the initial phase. Generally, this amount of time is
about ten to twelve weeks, but will vary between species and
individual subject animals. Transient antibodies are not a
significant component of the animal's immune response to the
vaccine.
[0033] For example, the development of FIV antibodies in an animal
against a vaccine is dependent upon the vaccine. For example, it
has been found that animals test seropositive for FIV antibodies
against p24 (gag) about two to four weeks after vaccination with
the FEL-O-VAX.RTM. vaccine. However, animals so vaccinated do not
generate persistent levels of antibodies against one or more
regions of the env protein, even though this protein was included
as an element of the vaccine. In contrast, naturally infected
animals typically generate persistent levels of antibodies to both
FIV gag and env proteins.
[0034] The differences in the immune response between animals that
are vaccinated and animals that are naturally infected provide a
means for determining whether an animal has been vaccinated or is
naturally infected. Using the method of the invention, animals that
have been naturally infected with FIV can be distinguished from
animals that have not been infected or have been vaccinated against
an FIV infection. Accordingly, the detection of the substantial
binding between a polypeptide derived from FIV and an antibody that
is not a significant component of an animal's immune response to a
vaccine can indicate a natural infection. The relative absence of
such binding can indicate vaccination or no infection. In addition,
a second, separate antibody capture reagent can be included in the
test that substantially binds to antibodies produced in response to
vaccination and/or natural infection, such as p15 or p24 proteins.
As such, various combinations of separate capture reagents can lead
to a determination of the vaccination and/or infection status of
the test subject.
[0035] For example, FIV gag proteins p15 and p24 may be immunogenic
components of a killed whole virus FIV vaccine. It is expected that
these components elicit a persistent antibody response when
administered to an animal. On the other hand, some vaccines may not
include immunologically significant quantities of FIV env protein
or, this protein has been altered in the process of virus
inactivation, or presentation of this protein by vaccination
differs from that for natural infection to a point where antibodies
produced thereto, if any, are detected for a period of time less
than antibodies to p15 and p24. Thus, while during the initial
phase following vaccination, an animal may transiently produce low
levels of such antibodies that bind to env proteins, any such
antibody production declines over a period of time and is not
detected after about 12 weeks. In this example, the transiently
produced antibodies are not a significant component of the animal's
immune response to the vaccine after a period of time.
[0036] Given that the production of detectable antibodies that are
directed toward specific FIV env polypeptides usually drops off
after about 12 weeks from completion of vaccination, in one aspect
of the invention the biological sample is obtained from the animal
that has not received an FIV vaccine within about the prior 12
weeks. If the vaccination status is unknown and the test indicates
infection (based on a reaction with the antibody capture protein),
a retest after an additional 12 weeks can be recommended.
[0037] Differences in the immune response between vaccinated
animals and naturally infected animals, such as specific antibody
levels and/or kinetic parameters for antibody-antigen binding
reactions (e.g., affinities, avidities), should be considered in
the design of an assay for distinguishing between vaccinated and
infected animals. Differences in immune response can be significant
such that even after the initial phase following a vaccination, an
animal may persistently produce lower levels of antibodies to
specific FIV polypeptides, and/or antibodies with different binding
properties as compared to those antibodies produced in response to
a natural infection.
[0038] The method of the invention can be optimized in many ways
and one of skill in the art could simultaneously adjust the sample
dilutions, reagent concentrations, incubation temperatures and
times used in the method to accomplish a differential detection of
serum having antibodies to an FIV infection or vaccination. For
instance, at optimized sample dilution and other conditions for an
immunoassay for antibodies to specific polypeptides, samples from
vaccinated animals may, for one specific FIV polypeptide, give a
negative assay result and samples from infected animals will give a
positive assay result. For a second FIV polypeptide, both samples
may give a positive result.
[0039] In one aspect of the invention, the proteins are immobilized
on a suitable solid support. The biological sample is brought into
contact with the protein, to which the anti-FIV antibodies bind, if
such antibodies are present in the sample. The binding may be
detected by any suitable means, e.g., enzymes, radionuclides,
particulates or fluorescent labels. In a suitable embodiment, the
detection reagent can be associated with a protein that is the same
or similar to that which is used to capture anti-FIV antibodies (if
present).
[0040] In another aspect, the method is directed to a test device
to determine whether a cat has been vaccinated for FIV or naturally
infected with FIV. A method of using the test device includes
providing a test device having an FIV env protein and a separate
FIV gag protein. The device can be used to test a biological sample
from a cat by contacting the device with the biological sample.
Upon reading the device, the detection of the binding of an
antibody to the gag protein (a positive result on the gag protein)
indicates the cat has been naturally infected with FIV or
vaccinated against FIV. A concurrent positive result on the env
protein indicates natural infection (or, perhaps a transient post
vaccination response), while a concurrent negative result on the
env protein indicates vaccination. The following table summarizes
the above:
TABLE-US-00002 gag protein env protein No vaccination or infection
- - Vaccination + - Potential Recent Vaccination + + Infection + +
Infection and Vaccination + +
[0041] The polypeptides used in the invention contain at least six
amino acids, usually at least nine amino acids, and more usually
twelve or more amino acids found within one of the natural FIV
proteins and mimitopes and functionally equivalent variants
thereof.
[0042] "Functional equivalent" or "Functionally equivalent" refers
to polypeptides related to or derived from the native FIV envelope
(env) and viral core (gag) polypeptide sequences where the amino
acid sequence has been modified by a single or multiple amino acid
substitution, insertion, deletion, and also sequences where the
amino acids have been chemically modified, such as amino acid
analogs, but which nonetheless retain substantially equivalent
function. Functionally-equivalent variants may occur as natural
biological variations or may be prepared using known techniques
such as chemical synthesis, site-directed mutagenesis, random
mutagenesis, or enzymatic cleavage and/or ligation of amino acids.
Thus, modification of the amino-acid sequence to obtain variant
sequences may occur so long as the function of the polypeptide is
not affected.
[0043] FIV functionally-equivalent variants within the scope of the
invention may comprise conservatively substituted sequences,
meaning that one or more amino acid residues of the FIV polypeptide
are replaced by different residues that do not alter the secondary
and/or tertiary structure of the FIV polypeptide. Such
substitutions may include the replacement of an amino acid by a
residue having similar physicochemical properties, such as charge
density, size, configuration, or hypdrophilicity/hydrophobicity.
For purposes of example only, such substitutions could include
substituting one aliphatic residue (Ile, Val, Leu, or Ala) for
another, or substitution of basic residues Lys and Arg, acidic
residues Glu and Asp, amide residues Gln and Asn, hydroxyl residues
Ser and Tyr, or aromatic residues Phe and Tyr. Conservative
variants can generally be identified by modifying a polypeptide
sequence of the invention and evaluating the antigenic activity of
the modified polypeptide using, for example, an immunohistochemical
assay, an enzyme-linked immunosorbent assay (ELISA), a
radioimmunoassay (RIA), or a western blot assay. Further
information regarding the making of phenotypically silent amino
acid exchanges may be found in Bowie et al., Science 247:1306-1310
(1990).
[0044] Examples of functional equivalents of SEQ ID NOS: 1 and 2
are shown here with a description of the various modifications to
the peptides.
TABLE-US-00003 SEQ ID NO: Sequence Description 3 CWEWRPDFESER
Multimer sequence; ELGSNQNQFFSK first monomer comprising SFFQNQNSGL
native FIV env protein, ELGSNQNQFFSK amino acids 398-408,
N-terminal C addition, K to R substitution; second monomer
(underline) comprising native FIV env protein, amino acids 696-707,
C to S substitutions; third monomer comprising inverted native FIV
env protein, amino acids 706-697, C to S substitutions; fourth
monomer (underline) comprising native FIV env protein, amino acids
696-706, C to S substitutions 4 CNRWDWRPDFES Dimeric sequence;
KKSKTAFAMQEL first monomer comprising GSNQNQFFSKIP native FIV env
protein, LELWTR amino acids 396-408, N-terminal C addition, E to W,
E to K substitution, SK addition; second monomer (underline)
comprising native FIV env protein, amino acids 690-715, C to S, K
to T substitutions 5 CNRWEWRPDFES Dimeric sequence; first
EKMQELGSNQNQ monomer comprising native FFSKVPPELWKR FIV env
protein, amino YN acids 396-408, N-terminal C addition; second
monomer (underline) comprising native FIV env protein, amino acids
694-717, C to S, Ito V, L to P, T to K substitutions
[0045] Additional variants are also contemplated within the scope
of the invention, and such variants include amino and/or carboxyl
terminal fusions, for example achieved by addition of amino acid
sequences of any number of residues, as well as intrasequence
insertion of one or more amino acids. For example, amino acid
sequences added may be those derived from the whole or parts of
other polypeptides or proteins, or may be those provided in the
corresponding positions in the FIV envelope or viral protein.
Longer peptides may comprise multiple copies of one or more of the
polypeptide sequences. Moreover, multiple copies of the
polypeptides may be coupled to a polyamino acid backbone, such as a
polylysine backbone to form multiple antigen peptides (MAPs).
[0046] Deletional amino acid sequence variants are those in which
one or more amino acid residues are removed from the sequence.
Insertional variants exist when one or more amino acids are
integrated into a predetermined site in the protein, although
random insertion is an option with suitable screening of the
resulting product. In all cases, these and other FIV variants used
retain substantially the same antigenicity of the FIV polypeptides.
Other variants are also contemplated, including those where the
amino acid substitutions are made in the area outside the antibody
recognition regions of the protein. Fusion proteins comprising two
or more polypeptide sequences of FIV are also within the scope of
the invention provided the sequences provide the appropriate
antigenicity. Such polypeptides will generally correspond to at
least one epitope or mimitope that is characteristic of FIV. By
characteristic, it is meant that the epitope or mimitope will allow
immunologic detection of antibody directed to FIV in a
physiological sample with reasonable assurance. Usually, it will be
desirable that the epitope or mimitope, variant or fusion protein
be immunologically distinct from (i.e., not cross-reactive with
antibodies which recognize) viruses other than FIV.
[0047] An antigenically active variant differs by about, for
example, 1, 2, 3, 5, 6, 10, 15 or 20 amino acid residues from SEQ
ID NOS: 1 and 2, such as those shown in SEQ ID NOS: 3-10, or a
fragment thereof Where this comparison requires alignment the
sequences are aligned for maximum homology. Deletions, insertions,
substitutions, repeats, inversions or mismatches are considered
differences. The differences are, preferably, differences or
changes at a non-essential residue or a conservative substitution.
The site of variation can occur anywhere in the polypeptide, as
long as the resulting variant polypeptide is antigenically
substantially similar to SEQ ID NOS: 1 and 2, such as, for example,
the variations shown in SEQ ID NOS: 3-10 (see Tables 3 and 4).
Exemplary functionally-equivalent variants include those displaying
50% or more amino acid homology. Preferably, such homology is 60%,
70%, or greater than 80%. However, such variants may display a
smaller percentage of homology overall and still fall within the
scope of the invention where they have conserved regions of
homology.
[0048] In some cases, one or more cysteine residues may be added to
the termini of the polypeptides in order to facilitate specific
carrier linkage or to permit disulphide bonding to mimic antigenic
loops and thus increase the antigenicity. Moreover, a fatty acid or
hydrophobic tail may be added to the peptides to facilitate
incorporation into delivery vehicles and to increase
antigenicity.
[0049] Some examples of monomers that can be used to produce
variants of the polypeptides of SEQ ID NOS: 1-5 are as follows:
TABLE-US-00004 SEQ ID NO Sequence Description 6 TAFAMQELG Native
FIV env protein, SNQNQFFSK amino acids 690-707, C to S
substitutions 7 TAFAMQELG Native FIV env protein, CNQQQFFCA amino
acids 690-707, N to Q, K to A substitutions 8 YTAFAMQEIG Native FIV
env protein, CNQNQFFCA amino acids 689-707, L to I, K to A
substitutions 9 ELGCNQNQF Native FIV env protein, FCK 7amino acids
696-07 10 CEGSNQNQF Native FIV env protein, FSK amino acids
696-707, N-terminal C addition, L deletion, C to Ssubstitutions
[0050] In yet another aspect, the invention provides novel
polypeptides. These polypeptides may be used, for example, as
detection reagents in kits or in vaccines. One such polypeptide has
the following formula
[(P.sup.1).sub.a-(L.sup.1).sub.b-(P.sup.2).sub.c].sub.n, wherein
P.sup.1 is a polypeptide that is the native, or an antigenic
fragment and functionally-equivalent variant of native FIV env
peptides 696-717, and P.sup.2 is a polypeptide that is the native,
or an antigenic fragment and functionally-equivalent variant of
native FIV env protein, amino acids 396-408. Either P.sup.1 or
P.sup.2 can be inverted. For example, P.sup.1 can be, for example,
any of SEQ ID NOs. 6-10, or one of
TABLE-US-00005 ELGSNQNQFFSKVPPELWKRYN, [SEQ ID NO: 11]
MQELGSNQNQFFSPPELWKRYN, [SEQ ID NO: 12] ELGSNQNQFFSK, [SEQ ID NO:
13] LGSNQNQFFS, [SEQ ID NO: 14] and TAFAMQELGSNQNQFFSKIPLELWTR,
[SEQ ID NO: 15]
and P.sup.2 can be, for example, one of
TABLE-US-00006 NRWEWRPDFESEKC, [SEQ ID NO: 16] CNRWEWRPDFESEK, [SEQ
ID NO: NO: 17] CWEWRPDFESER, [SEQ ID NO: 18] and CNRWDWRPDFESKK,
[SEQ ID NO: 19]
where either P.sup.1 and/or P.sup.2 is inverted. L.sup.1 is a
linker polypeptide consisting of 2-20 alternatively repeating S and
K peptides, beginning and ending with either S or K, and where a, c
and n may independently be an integer from 1 to 3, and b may be an
integer from 0 to 1.
[0051] The FIV polypeptides used as detection reagents may be
natural, i.e., including the entire FIV protein or fragments
thereof isolated from a natural source, or may be synthetic. The
natural proteins may be isolated from the whole FIV virus by
conventional techniques, such as affinity chromatography.
Polyclonal or monoclonal antibodies may be used to prepare a
suitable affinity column by well-known techniques.
[0052] Proteins that are immunologically cross-reactive with a
natural FIV protein can be chemically synthesized. For example,
polypeptides having fewer than about 100 amino acids, more usually
fewer than about 80 amino acids, and typically fewer than about 50
amino acids, may be synthesized by the well-known Merrifield
solid-phase synthesis method where amino acids are sequentially
added to a growing chain. Merrifield, 1963, J. Am. Chem. Soc.,
85:2149-2156). Recombinant proteins can also be used. These
proteins may be produced by expression in cultured cells of
recombinant DNA molecules encoding a desired portion of the FIV
genome. The portion of the FIV genome may itself be natural or
synthetic, with natural genes obtainable from the isolated virus by
conventional techniques. Of course, the genome of FIV is RNA, and
it will be necessary to transcribe the natural RNA into DNA by
conventional techniques employing reverse transcriptase.
Polynucleotides may also be synthesized by well-known techniques.
For example, short single-stranded DNA fragments may be prepared by
the phosphoramidite method described by Beaucage and Carruthers,
1981, Tett. Letters 22:1859-1862. Double-stranded fragments may
then be obtained either by synthesizing the complementary strand
and then annealing the strands together under appropriate
conditions, or by adding the complementary strand using DNA
polymerase with an appropriate primer sequence.
[0053] The natural or synthetic DNA fragments coding for the
desired FIV protein or fragment may be incorporated in a DNA
construct capable of introduction to and expression in in vitro
cell culture. Usually, the DNA constructs will be suitable for
replication in a unicellular host, such as yeast or bacteria. They
may also be intended for introduction and integration within the
genome of cultured mammalian or other eukaryotic cells. DNA
constructs prepared for introduction into bacteria or yeast will
include a replication system recognized by the host, the FIV DNA
fragment encoding the desired polypeptide product, transcriptional
and translational initiation regulatory sequences joined to the
5'-end of the FIV DNA termination regulatory sequences joined to
the 3'-end of the fragment. The transcriptional regulatory
sequences will include a heterologous promoter that is recognized
by the host. Conveniently, a variety of suitable expression vectors
are commercially available for a number of hosts.
[0054] To be useful in the detection methods of the present
invention, the polypeptides are obtained in a substantially pure
form, that is, typically from about 50% w/w or more purity,
substantially free of interfering proteins and contaminants.
Preferably, the FIV polypeptides are isolated or synthesized in a
purity of at least 80% w/w, and more preferably, in at least about
95% w/w purity. Using conventional protein purification techniques,
homogeneous polypeptide compositions of at least about 99% w/w
purity can be obtained. For example, the proteins may be purified
by use of the antibodies described hereinafter using the
immunoabsorbant affinity columns described hereinabove.
[0055] The method of the invention may be accomplished using
immunoassay techniques well known to those of skill in the art,
including, but not limited to, using microplates and lateral flow
devices. In one embodiment, an FIV protein is immobilized on a
solid support at a distinct location. Detection of protein-antibody
complexes on the solid support can be by any means known in the
art. For example, U.S. Pat. No. 5,726,010, which is incorporated
herein by reference in its entirety, describes an example of a
lateral flow device, the SNAP.RTM. immunoassay device (IDEXX
Laboratories), useful in the present invention. Colloidal particle
based tests can also be used, such as the commercially available
WITNESS.RTM. FIV diagnostic test (Synbiotics Corporation, Lyon,
France).
[0056] Immobilization of one or more analyte capture reagents,
e.g., FIV proteins, onto a device or solid support is performed so
that an analyte capture reagent will not be washed away by the
sample, diluent and/or wash procedures. One or more analyte capture
reagents can be attached to a surface by physical adsorption (i.e.,
without the use of chemical linkers) or by chemical binding (i.e.,
with the use of chemical linkers). Chemical binding can generate
stronger attachment of specific binding substances on a surface and
provide defined orientation and conformation of the surface-bound
molecules.
[0057] Another embodiment of the invention provides a device that
is suitable for a lateral flow assay. For example, a test sample is
added to a flow matrix at a first region (a sample application
zone). The test sample is carried in a fluid flow path by capillary
action to a second region of the flow matrix where a label capable
of binding and forming a first complex with an analyte in the test
sample. The first complex is carried to a third region of the flow
matrix where an FIV protein is immobilized at a distinct location.
A second complex is formed between an immobilized protein and the
first complex including the antibody from the sample. For example,
a first complex comprising a gold sol particle and an FIV protein
bound to an FIV antibody will specifically bind and form a second
complex with a second immobilized FIV protein or with a second
antibody directed to feline antibodies. The label that is part of
the second complex can be directly visualized.
[0058] In another aspect, the invention includes one or more
labeled specific binding reagents that can be mixed with a test
sample prior to application to a device for of the invention. In
this case it is not necessary to have labeled specific binding
reagents deposited and dried on a specific binding reagent pad in
the device. A labeled specific binding reagent, whether added to a
test sample or pre-deposited on the device, can be for example, a
labeled FIV protein that specifically binds an antibody for
FIV.
[0059] Any or all of the above embodiments can be provided as a
kit. In one particular example, such a kit would include a device
complete with specific binding reagents (e.g., a non-immobilized
labeled specific binding reagent and an immobilized analyte capture
reagent) and wash reagent, as well as detector reagent and positive
and negative control reagents, if desired or appropriate. In
addition, other additives can be included, such as stabilizers,
buffers, and the like. The relative amounts of the various reagents
can be varied, to provide for concentrations in solution of the
reagents that substantially optimize the sensitivity of the assay.
Particularly, the reagents can be provided as dry powders, usually
lyophilized, which on dissolution will provide for a reagent
solution having the appropriate concentrations for combining with a
sample.
[0060] An FIV protein can be an immobilized analyte capture reagent
in a reaction zone (solid phase). A second analyte capture reagent,
i.e. a second FIV protein, that has been conjugated to a label, can
either be added to the sample before the sample is added to the
device, or the second analyte capture reagent can be incorporated
into the device. For example the labeled specific binding reagent
can be deposited and dried on a fluid flow path that provides fluid
communication between the sample application zone and the solid
phase. Contact of the labeled specific binding reagent with the
fluid sample results in dissolution of the labeled specific binging
reagent.
[0061] The device may also include a liquid reagent that transports
unbound material (e.g., unreacted fluid sample and unbound specific
binding reagents) away from the reaction zone (solid phase). A
liquid reagent can be a wash reagent and serve only to remove
unbound material from the reaction zone, or it can include a
detector reagent and serve to both remove unbound material and
facilitate analyte detection. For example, in the case of a
specific binding reagent conjugated to an enzyme, the detector
reagent includes a substrate that produces a detectable signal upon
reaction with the enzyme-antibody conjugate at the reactive zone.
In the case of a labeled specific binding reagent conjugated to a
radioactive, fluorescent, or light-absorbing molecule, the detector
reagent acts merely as a wash solution facilitating detection of
complex formation at the reactive zone by washing away unbound
labeled reagent.
[0062] Two or more liquid reagents can be present in a device, for
example, a device can comprise a liquid reagent that acts as a wash
reagent and a liquid reagent that acts as a detector reagent and
facilitates analyte detection.
[0063] A liquid reagent can further include a limited quantity of
an "inhibitor", i.e., a substance that blocks the development of
the detectable end product. A limited quantity is an amount of
inhibitor sufficient to block end product development until most or
all excess, unbound material is transported away from the second
region, at which time detectable end product is produced.
[0064] The following are provided for exemplification purposes only
and are not intended to limit the scope of the invention described
in broad terms above. All references cited in this disclosure are
incorporated herein by reference.
EXAMPLES
Example 1
[0065] Eight cats testing negative for FIV with the SNAP.RTM. FeLV
Ag/FIV Ab test kits were vaccinated with FEL-O-VAX.RTM. FIV
vaccine, Fort Dodge Animal Health, Fort Dodge Iowa. This vaccine is
produced from multiple strains of the whole killed FIV virus. The
cats were vaccinated following the manufacturer's directions at day
0, 14, and 28. Two cats testing negative for FIV were not
vaccinated and were included as controls for this study.
[0066] Blood samples were obtained from each of the ten cats in the
vaccination study at day zero and every seven days for 12 weeks and
stored frozen until testing. In addition, blood samples from FIV
negative cats and cats naturally infected with FIV, confirmed FIV
Ab negative or positive by a western immunoblot confirmatory test,
were also tested.
[0067] Sample testing was accomplished using a SNAP.RTM.
immunoassay device, which provides a solid phase with reversible,
chromatographic flow of sample, and automatic, sequential flow of
wash and enzyme substrate solutions as described in U.S. Pat. No.
5,726,010.
[0068] For the SNAP.RTM. device, FIV gag p24 (recombinant) and an
FIV env 696-707 with additional N-terminal cysteine--CELGCNQNQFFCK
[SEQ ID NO:20]--proteins were deposited to form a single antibody
capture spot on the solid phase. A negative control reagent was
deposited to form a negative control spot and a positive control
reagent was deposited to form a positive control spot on the solid
phase of the SNAP.RTM. device. The gag or env proteins were
chemically conjugated to the enzyme horseradish peroxidase and
provided in a solution consisting of a buffer, detergent, and
animal serum components.
[0069] Serum samples were combined with either gag or env
protein-enzyme conjugate solution, and applied to the SNAP.RTM.
device. Following a short incubation period, the device was
activated. Color development on the positive control spot indicated
the test was valid. Color development on the sample spot greater
than color development on the negative control spot indicated the
presence of antigen in the sample and was scored as a positive test
result. Test results were determined visually and are shown in
Table 1.
TABLE-US-00007 TABLE 1 gag Ab env Ab test result test result Animal
ID Status Day (visual) (visual) NV1 not vaccinated, not infected 0
NEG NEG NV1 not vaccinated, not infected 7 NEG NEG NV1 not
vaccinated, not infected 14 NEG NEG NV1 not vaccinated, not
infected 21 NEG NEG NV1 not vaccinated, not infected 28 NEG NEG NV1
not vaccinated, not infected 35 NEG NEG NV1 not vaccinated, not
infected 42 NEG NEG NV1 not vaccinated, not infected 49 NEG NEG NV1
not vaccinated, not infected 56 NEG NEG NV1 not vaccinated, not
infected 63 NEG NEG NV1 not vaccinated, not infected 70 NEG NEG NV1
not vaccinated, not infected 77 NEG NEG NV1 not vaccinated, not
infected 84 NEG NEG NV2 not vaccinated, not infected 0 NEG NEG NV2
not vaccinated, not infected 7 NEG NEG NV2 not vaccinated, not
infected 14 NEG NEG NV2 not vaccinated, not infected 21 NEG NEG NV2
not vaccinated, not infected 28 NEG NEG NV2 not vaccinated, not
infected 35 NEG NEG NV2 not vaccinated, not infected 42 NEG NEG NV2
not vaccinated, not infected 49 NEG NEG NV2 not vaccinated, not
infected 56 NEG NEG NV2 not vaccinated, not infected 63 NEG NEG NV2
not vaccinated, not infected 70 NEG NEG NV2 not vaccinated, not
infected 77 NEG NEG NV2 not vaccinated, not infected 84 NEG NEG V1
vaccinated, not infected 0 NEG NEG V1 vaccinated, not infected 7
NEG NEG V1 vaccinated, not infected 14 NEG NEG V1 vaccinated, not
infected 21 POS NEG V1 vaccinated, not infected 28 POS NEG V1
vaccinated, not infected 35 POS POS V1 vaccinated, not infected 42
POS NEG V1 vaccinated, not infected 49 POS NEG V1 vaccinated, not
infected 56 POS NEG V1 vaccinated, not infected 63 POS NEG V1
vaccinated, not infected 70 POS NEG V1 vaccinated, not infected 77
POS NEG V1 vaccinated, not infected 84 POS NEG V2 vaccinated, not
infected 0 NEG NEG V2 vaccinated, not infected 7 NEG NEG V2
vaccinated, not infected 14 NEG NEG V2 vaccinated, not infected 21
NEG NEG V2 vaccinated, not infected 28 NEG NEG V2 vaccinated, not
infected 35 POS NEG V2 vaccinated, not infected 42 POS NEG V2
vaccinated, not infected 49 POS NEG V2 vaccinated, not infected 56
POS NEG V2 vaccinated, not infected 63 POS NEG V2 vaccinated, not
infected 70 POS NEG V2 vaccinated, not infected 77 POS NEG V2
vaccinated, not infected 84 POS NEG V3 vaccinated, not infected 0
NEG NEG V3 vaccinated, not infected 7 NEG NEG V3 vaccinated, not
infected 14 NEG NEG V3 vaccinated, not infected 21 NEG NEG V3
vaccinated, not infected 28 NEG NEG V3 vaccinated, not infected 35
POS NEG V3 vaccinated, not infected 42 POS NEG V3 vaccinated, not
infected 49 POS NEG V3 vaccinated, not infected 56 POS NEG V3
vaccinated, not infected 63 POS NEG V3 vaccinated, not infected 70
POS NEG V3 vaccinated, not infected 77 POS NEG V3 vaccinated, not
infected 84 POS NEG V4 vaccinated, not infected 0 NEG NEG V4
vaccinated, not infected 7 NEG NEG V4 vaccinated, not infected 14
POS NEG V4 vaccinated, not infected 21 POS NEG V4 vaccinated, not
infected 28 POS NEG V4 vaccinated, not infected 35 POS NEG V4
vaccinated, not infected 42 POS NEG V4 vaccinated, not infected 49
POS NEG V4 vaccinated, not infected 56 POS NEG V4 vaccinated, not
infected 63 POS NEG V4 vaccinated, not infected 70 POS NEG V4
vaccinated, not infected 77 POS NEG V4 vaccinated, not infected 84
POS NEG V5 vaccinated, not infected 0 NEG NEG V5 vaccinated, not
infected 7 NEG NEG V5 vaccinated, not infected 14 NEG NEG V5
vaccinated, not infected 21 POS POS V5 vaccinated, not infected 28
POS NEG V5 vaccinated, not infected 35 POS NEG V5 vaccinated, not
infected 42 POS NEG V5 vaccinated, not infected 49 POS NEG V5
vaccinated, not infected 56 POS NEG V5 vaccinated, not infected 63
POS NEG V5 vaccinated, not infected 70 POS NEG V5 vaccinated, not
infected 77 POS NEG V5 vaccinated, not infected 84 POS NEG V6
vaccinated, not infected 0 NEG NEG V6 vaccinated, not infected 7
NEG NEG V6 vaccinated, not infected 14 NEG NEG V6 vaccinated, not
infected 21 POS NEG V6 vaccinated, not infected 28 POS NEG V6
vaccinated, not infected 35 POS POS V6 vaccinated, not infected 42
POS NEG V6 vaccinated, not infected 49 POS NEG V6 vaccinated, not
infected 56 POS NEG V6 vaccinated, not infected 63 POS NEG V6
vaccinated, not infected 70 POS NEG V6 vaccinated, not infected 77
POS NEG V6 vaccinated, not infected 84 POS NEG V7 vaccinated, not
infected 0 NEG NEG V7 vaccinated, not infected 7 NEG NEG V7
vaccinated, not infected 14 NEG NEG V7 vaccinated, not infected 21
POS NEG V7 vaccinated, not infected 28 POS NEG V7 vaccinated, not
infected 35 POS NEG V7 vaccinated, not infected 42 POS POS V7
vaccinated, not infected 49 POS POS V7 vaccinated, not infected 56
POS NEG V7 vaccinated, not infected 63 POS NEG V7 vaccinated, not
infected 70 POS NEG V7 vaccinated, not infected 77 POS NEG V7
vaccinated, not infected 84 POS NEG V8 vaccinated, not infected 0
NEG NEG V8 vaccinated, not infected 7 NEG NEG V8 vaccinated, not
infected 14 POS NEG V8 vaccinated, not infected 21 POS NEG V8
Vaccinated, not infected 28 POS NEG V8 Vaccinated, not infected 35
POS NEG V8 Vaccinated, not infected 42 POS NEG V8 Vaccinated, not
infected 49 POS NEG V8 Vaccinated, not infected 56 POS NEG V8
Vaccinated, not infected 63 POS NEG V8 Vaccinated, not infected 70
POS NEG V8 Vaccinated, not infected 77 POS NEG V8 Vaccinated, not
infected 84 POS NEG Inf1 Not vaccinated, infected ND POS POS Inf2
Not vaccinated, infected ND POS POS Inf3 Not vaccinated, infected
ND POS POS Inf4 Not vaccinated, infected ND POS POS Inf5 Not
vaccinated, infected ND POS POS Inf6 Not vaccinated, infected ND
POS POS Inf7 Not vaccinated, infected ND POS POS Inf8 Not
vaccinated, infected ND POS POS Inf9 Not vaccinated, infected ND
POS POS inf10 Not vaccinated, infected ND POS POS
Example 2
[0070] Microplate ELISA analysis was performed on serum samples
collected from confirmed FIV negative and infected cats, and cats
vaccinated with the FEL-O-VAX.RTM. FIV vaccine in an indirect assay
format with individual FIV polypeptides on the solid phase and
anti-(feline IgG) peroxidase conjugate. Antibodies to FIV env were
detected using these peptides as antigen reagents:
TABLE-US-00008 [SEQ ID NO: 1]
ELGSNQNQFFSKVPPELWKRYNKSKSKSKSKNRWEWRPDFESEKC [SEQ ID NO: 2]
CNRWEWRPDFESEKSKSKSKSMQELGSNQNQFFSKVPPELWKRYN [SEQ ID NO: 3]
CWEWRPDFESERELGSNQNQFFSKSFFQNQNSGLELGSNQNQFFSK [SEQ ID NO: 4]
CNRWDWRPDFESKKSKTAFAMQELGSNQNQFFSKIPLELWTR [SEQ ID NO: 5]
CNRWEWRPDFESEKMQELGSNQNQFFSKVPPELWKRYN [SEQ ID NO: 10]
CEGSNQNQFFSK
[0071] The polypeptides were synthesized using a commercial
instrument and following the manufacturer's instructions.
Polypeptide stocks were prepared at 5 mg/ml in DMSO. The
polypeptides were then coated on microplate wells (peptide @ 10
ug/ml in 50 mM Tris-HCl pH 7.4, 100 ul/well). The plates were then
blocked/overcoated with 2% Tween-20/2.5% sucrose, allowed to dry in
mylar bags with desiccant.
[0072] For the assays, feline serum samples (100 ul/well, diluted
1/1000 in 50% fetal bovine serum) were added to the wells and the
plates were incubated for ten minutes at room temperature.
Following incubation, the microplates were washed with PBS/Tween.
Goat Anti-(cat IgG):peroxidase conjugate was added to the wells
(100 ul/well anti-catIgG:peroxidase diluted in 50% fetal bovine
serum). The plates were incubated for another fifteen minutes at
room temperature and washed a second time with PBS/Tween.
Peroxidase substrate was added (100 ul/well, tetramethyl benzidine
peroxidase substrate) and the plates were incubated a third time
for ten minutes at room temperature. A hydrofluoric acid stop
solution (50 ul/well) was added to the plates. Sample antibody
binding was measured by determining peroxidase activity (colored
product) with a spectrophotometer (A650 nm). Significant,
substantial antibody binding for a sample is considered to be A650
nm greater than 0.200. The IDEXX PETCHEK.RTM. Anti-FIV antibody
test kit was also run on these samples as a reference test. The
results are shown in Table 2.
Table 2
TABLE-US-00009 [0073] TABLE 2 seq ID 1 seq ID 2 seq ID 3 seq ID 10
seq ID 4 seq ID 5 PetChek sample A (650 nm) A (650 nm) A (650 nm) A
(650 nm) A (650 nm) A (650 nm) result FIV infected, not vaccinated:
58376-274 1.400 1.944 1.838 1.370 1.910 2.109 positive JL-60 1.139
1.906 1.433 2.127 2.014 1.639 positive 21636 1.301 1.838 1.944
1.918 1.986 2.080 positive Gonzalez 0.951 1.775 1.281 1.920 1.520
1.407 positive 2605 0.500 1.593 0.746 0.965 1.152 0.871 positive
Stanley 0.972 1.590 0.834 0.442 1.124 1.044 positive 2614 0.328
1.029 0.382 1.095 0.527 0.945 positive mean 0.942 1.668 1.208 1.405
1.462 1.442 std. deviation 0.398 0.314 .582 .615 .558 .520 FIV
vaccinated, not infected: Vx 3520 D84 0.032 0.042 0.035 0.040 0.043
0.045 positive Vx 3519 D84 0.032 0.084 0.036 0.041 0.040 0.038
positive Vx 3532 D84 0.026 0.038 0.036 0.042 0.038 0.040 positive
Vx SK4 D84 0.031 0.047 0.033 0.048 0.042 0.046 positive Vx G1 wk5
0.032 0.111 0.100 0.101 0.114 0.116 positive Vx G1 wk7 0.035 0.114
0.105 0.103 0.121 0.137 positive Vx G1 wk8 0.035 0.095 0.088 0.096
0.095 0.108 positive Vx G1 wk12 0.036 0.100 0.087 0.085 0.101 0.111
positive mean 0.032 0.079 0.065 0.070 0.074 0.080 std. deviation
0.003 0.032 0.033 0.029 0.037 0.041 FIV negative, not vaccinated:
2151-05H 0.032 0.064 0.050 0.049 0.039 0.047 negative F6263E 0.039
0.045 0.038 0.058 0.039 0.037 negative Abraham 0.032 0.045 0.035
0.035 0.037 0.035 negative AWL 2002 0.033 0.041 0.033 0.072 0.039
0.051 negative 14834 0.030 0.035 0.024 0.036 0.038 0.034 negative
D1606315 0.032 0.035 0.033 0.035 0.041 0.038 negative 2483-83-33
0.029 0.034 0.033 0.035 0.036 0.036 negative 2483-83-23 0.032 0.033
0.033 0.035 0.038 0.039 negative 2483-83-30 0.030 0.033 0.033 0.035
0.038 0.035 negative 2377-1-38 0.030 0.033 0.033 0.034 0.037 0.035
negative 769703 0.029 0.032 0.035 0.035 0.042 0.036 negative
2377-23-3 0.026 0.032 0.032 0.033 0.036 0.035 negative 14151 0.032
0.032 0.034 0.034 0.037 0.035 negative 768547 0.030 0.031 0.034
0.034 0.037 0.035 negative 768513 0.032 0.030 0.034 0.036 0.037
0.042 negative mean 0.031 0.037 0.034 0.040 0.038 0.038 std.
deviation 0.003 0.009 0.005 0.011 0.002 0.005
Example 3
[0074] Microplate ELISA analysis was performed as in Example 2 on
serum samples collected from confirmed FIV negative and infected
cats, and cats vaccinated with the FEL-O-VAX.RTM. FIV vaccine.
Antibodies to FIV env were detected using these peptides as antigen
reagents:
TABLE-US-00010 [SEQ ID NO: 2]
CNRWEWRPDFESEKSKSKSKSMQELGSNQNQFFSKVPPELWKRYN [SEQ ID NO: 6]
TAFAMQELGSNQNQFFSK [SEQ ID NO: 7] TAFAMQELGCNQQQFFCA [SEQ ID NO: 8]
YTAFAMQEIGCNQNQFFCA [SEQ ID NO: 9] ELGCNQNQFFCK
Significant, substantial antibody binding for a sample is
considered to be A650 nm greater than 0.200). The IDEXX
PETCHEK.RTM. Anti-FIV antibody test kit was also run on these
samples as a reference test. The results are reported in Table
3.
TABLE-US-00011 TABLE 3 seq ID 2 seq ID 6 Seq ID 7 seq ID 8 seq ID 9
PetChek sample A (650 nm) A (650 nm) A (650 nm) A (650 nm) A (650
nm) result FIV infected, not vaccinated: 2689:44 7 2.212 0.908
1.696 0.893 1.153 positive 197 2.123 0.687 1.623 1.590 1.027
positive 22488 2.121 0.802 1.162 1.663 1.296 positive 23804 255
2.065 0.991 0.829 1.078 1.036 positive 24034 283 1.973 0.663 1.436
1.318 1.202 positive F0-138 1/23/00 1.951 1.409 1.835 1.412 1.548
positive 56360 60 1.881 1.391 1.553 1.270 1.298 positive 57561 181
1.875 1.782 1.747 1.000 1.293 positive 56897 187 1.864 0.813 1.492
0.929 1.102 positive 21518 1.849 1.000 1.419 1.032 0.955 positive
58178 232 1.839 1.386 1.338 1.392 1.237 positive 58376 274 8/17
1.826 0.617 0.448 0.314 0.471 positive 23805 253 1.796 1.005 0.562
0.721 0.599 positive 21636 1.795 1.726 1.225 1.811 1.238 positive
58232 242 1.724 0.454 1.580 0.686 1.297 positive 23119 1.721 0.888
1.154 0.447 1.156 positive 57601 215 1.659 0.725 0.987 0.722 0.601
positive 22373 275 1.646 0.641 1.853 0.695 1.584 positive F9-881
1.574 0.926 0.587 1.272 0.589 positive 23938 323 1.554 0.635 1.475
0.999 1.333 positive 58036 224 1.418 1.387 1.218 0.828 1.102
positive 22879 1.332 0.468 1.117 0.752 1.055 positive F0-162
2/13/00 1.273 1.068 1.284 0.702 1.046 positive 23321 211 1.214
1.183 0.751 1.308 0.886 positive mean 1.762 0.981 1.265 1.035 1.088
std. deviation 0.267 0.371 0.404 0.385 0.290 FIV negative,
vaccinated: Vx C3520 D35 0.211 0.058 0.142 0.065 0.116 positive Vx
C3511 D49 0.173 0.155 0.209 0.111 0.180 positive Vx C3519 D35 0.110
0.053 0.091 0.058 0.055 positive Vx C3517 D35 0.110 0.082 0.093
0.063 0.074 positive Vx SK4 D21 0.066 0.035 0.082 0.038 0.044
positive mean 0.134 0.077 0.123 0.067 0.094 std. deviation 0.058
0.047 0.054 0.027 0.055 FIV negative, not vaccinated: F9-1164 0.061
0.038 0.054 0.039 0.036 negative 57435 272 8/17 0.058 0.039 0.046
0.035 0.039 negative 57975 236 0.048 0.035 0.040 0.043 0.036
negative 57323 174 0.046 0.046 0.032 0.042 0.034 negative 56728 200
0.045 0.039 0.049 0.044 0.042 negative 58238 251 8/14 0.044 0.031
0.032 0.033 0.033 negative 56956 209 0.044 0.035 0.049 0.038 0.036
negative F9-1638 143119 0.042 0.035 0.037 0.036 0.039 negative
F9-1191 0.041 0.027 0.040 0.037 0.037 negative 57528 197 0.041
0.033 0.039 0.035 0.035 negative 57095 125 0.041 0.034 0.038 0.035
0.035 negative 56704 154 0.039 0.033 0.040 0.027 0.035 negative
57911 235 0.039 0.040 0.056 0.050 0.038 negative F9-1455 11/14/99
0.038 0.034 0.037 0.037 0.033 negative F0-53 1/23/00 0.038 0.035
0.038 0.037 0.036 negative 57222 153 0.038 0.036 0.041 0.037 0.036
negative 56746 226 0.038 0.036 0.038 0.036 0.034 negative F9-1278
0.037 0.024 0.038 0.036 0.034 negative 57238 0.037 0.035 0.037
0.036 0.035 negative 2873 0.037 0.034 0.036 0.033 0.034 negative
22151 80 0.036 0.026 0.039 0.036 0.047 negative 57611 216 0.036
0.035 0.067 0.043 0.044 negative 57211 147 0.036 0.035 0.039 0.037
0.034 negative F9-1211 0.035 0.033 0.036 0.035 0.031 negative mean
0.041 0.035 0.042 0.037 0.036 std. deviation 0.007 0.005 0.008
0.005 0.004
[0075] Although various specific embodiments of the present
invention have been described herein, it is to be understood that
the invention is not limited to those precise embodiments and that
various changes or modifications can be affected therein by one
skilled in the art without departing from the scope and spirit of
the invention.
Sequence CWU 1
1
20145PRTArtificial sequenceSynthetic polypeptide 1Glu Leu Gly Ser
Asn Gln Asn Gln Phe Phe Ser Lys Val Pro Pro Glu1 5 10 15Leu Trp Lys
Arg Tyr Asn Lys Ser Lys Ser Lys Ser Lys Ser Lys Asn 20 25 30Arg Trp
Glu Trp Arg Pro Asp Phe Glu Ser Glu Lys Cys 35 40
45245PRTArtificial sequenceSynthetic polypeptide 2Cys Asn Arg Trp
Glu Trp Arg Pro Asp Phe Glu Ser Glu Lys Ser Lys1 5 10 15Ser Lys Ser
Lys Ser Met Gln Glu Leu Gly Ser Asn Gln Asn Gln Phe 20 25 30Phe Ser
Lys Val Pro Pro Glu Leu Trp Lys Arg Tyr Asn 35 40
45346PRTArtificial sequenceSynthetic polypeptide 3Cys Trp Glu Trp
Arg Pro Asp Phe Glu Ser Glu Arg Glu Leu Gly Ser1 5 10 15Asn Gln Asn
Gln Phe Phe Ser Lys Ser Phe Phe Gln Asn Gln Asn Ser 20 25 30Gly Leu
Glu Leu Gly Ser Asn Gln Asn Gln Phe Phe Ser Lys 35 40
45442PRTArtificial sequenceSynthetic polypeptide 4Cys Asn Arg Trp
Asp Trp Arg Pro Asp Phe Glu Ser Lys Lys Ser Lys1 5 10 15Thr Ala Phe
Ala Met Gln Glu Leu Gly Ser Asn Gln Asn Gln Phe Phe 20 25 30Ser Lys
Ile Pro Leu Glu Leu Trp Thr Arg 35 40538PRTArtificial
sequenceSynthetic polypeptide 5Cys Asn Arg Trp Glu Trp Arg Pro Asp
Phe Glu Ser Glu Lys Met Gln1 5 10 15Glu Leu Gly Ser Asn Gln Asn Gln
Phe Phe Ser Lys Val Pro Pro Glu 20 25 30Leu Trp Lys Arg Tyr Asn
35618PRTArtificial sequenceSynthetic polypeptide 6Thr Ala Phe Ala
Met Gln Glu Leu Gly Ser Asn Gln Asn Gln Phe Phe1 5 10 15Ser
Lys718PRTArtificial sequenceSynthetic polypeptide 7Thr Ala Phe Ala
Met Gln Glu Leu Gly Cys Asn Gln Gln Gln Phe Phe1 5 10 15Cys
Ala819PRTArtificial sequenceSynthetic polypeptide 8Tyr Thr Ala Phe
Ala Met Gln Glu Ile Gly Cys Asn Gln Asn Gln Phe1 5 10 15Phe Cys
Ala912PRTArtificial sequenceSynthetic polypeptide 9Glu Leu Gly Cys
Asn Gln Asn Gln Phe Phe Cys Lys1 5 101012PRTArtificial
sequenceSynthetic polypeptide 10Cys Glu Gly Ser Asn Gln Asn Gln Phe
Phe Ser Lys1 5 101122PRTArtificial sequenceSynthetic polypeptide
11Glu Leu Gly Ser Asn Gln Asn Gln Phe Phe Ser Lys Val Pro Pro Glu1
5 10 15Leu Trp Lys Arg Tyr Asn 201222PRTArtificial
sequenceSynthetic polypeptide 12Met Gln Glu Leu Gly Ser Asn Gln Asn
Gln Phe Phe Ser Pro Pro Glu1 5 10 15Leu Trp Lys Arg Tyr Asn
201312PRTArtificial sequenceSynthetic polypeptide 13Glu Leu Gly Ser
Asn Gln Asn Gln Phe Phe Ser Lys1 5 101410PRTArtificial
sequenceSynthetic polypeptide 14Leu Gly Ser Asn Gln Asn Gln Phe Phe
Ser1 5 101526PRTArtificial sequenceSynthetic polypeptide 15Thr Ala
Phe Ala Met Gln Glu Leu Gly Ser Asn Gln Asn Gln Phe Phe1 5 10 15Ser
Lys Ile Pro Leu Glu Leu Trp Thr Arg 20 251614PRTArtificial
sequenceSynthetic polypeptide 16Asn Arg Trp Glu Trp Arg Pro Asp Phe
Glu Ser Glu Lys Cys1 5 101714PRTArtificial sequenceSynthetic
polypeptide 17Cys Asn Arg Trp Glu Trp Arg Pro Asp Phe Glu Ser Glu
Lys1 5 101812PRTArtificial sequenceSynthetic polypeptide 18Cys Trp
Glu Trp Arg Pro Asp Phe Glu Ser Glu Arg1 5 101914PRTArtificial
sequenceSynthetic polypeptide 19Cys Asn Arg Trp Asp Trp Arg Pro Asp
Phe Glu Ser Lys Lys1 5 102013PRTArtificial sequenceSynthetic
polypeptide 20Cys Glu Leu Gly Cys Asn Gln Asn Gln Phe Phe Cys Lys1
5 10
* * * * *