Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CnDgTNE nRTARTTTS-ENCEPIiALITIS VIRUS
PROVIDES IIrMUNOPROTECTION AGAINST HIV-1 INFECTION
This work was supported, in part, by a grant
awarded by the National Institutes of Health. The United
States Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
FTET,D OF THE INVENTION
This invention relates generally to the fields
of immunology and medicine and more specifically to
methods for providing immunoprotection against HIV-1
infection.
BACKGROTJND INFORMATTON
The incidence of acquired immunodeficiency
syndrome (AIDS) has reached an epidemic level,
particularly in third world countries in Africa, Asia and
the Caribbean. Despite the expenditure of billions of
dollars for research to discover drugs to treat or cure
the disease, however, only modest progress has been made
in identifying drugs that can delay the progress of the
disease.
The causative agent of AIDS is the human
immunodeficiency virus (HIV-1). HIV-1 infects a
particular cell type of the immune system, the T cell.
Upon entering a T cell, the HIV-1 genomic DNA is
incorporated into the T cell genome, where it directs
synthesis of viral proteins. New copies of the HIV-1
virus then are released from the infected T cell and
further infect additional T cells. Ultimately, the
infected T cells die and the HIV-1 infected individual's
immune system becomes depleted and cannot ward off
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subsequent infections. As a result, victims of AIDS
typically die from infections that normally would cause,
at worst, a mild illness in a healthy individual.
Initially, treatments for AIDS were limited to
methods of treating the infections that resulted from the
depleted immune response. Later, drugs such as AZT and
ddI, which are known as nucleoside analogs, were
identified that could interfere with replication of the
HIV-1 DNA. These drugs, particularly in combination, can
prolong the life of AIDS patients. More recently, a
class of drugs called protease inhibitors have been
reported to be even more effective in inhibiting HIV-1
replication. It is hoped that the protease inhibitors,
perhaps in combination with the nucleoside analogs, will
further prolong the life of AIDS patients and even result
in cures.
While the use of drugs as described above
provides a means to kill the virus in HIV-1 infected
individuals, such drugs are useful only after a person
has become infected; they have no enhancing effect on the
immune system. Clearly, a more preferable approach to
stifling the AIDS epidemic would be to prevent HIV-1
infection in the first place. Vaccines, which stimulate
a person's immune response against the vaccinating agent,
are the logical choice for preventing HIV-1 infection.
For example, vaccines have been used to prevent or reduce
the severity of various viral diseases, including polio,
measles, smallpox and influenza. In addition, a vaccine
can stimulate the immune system in individuals already
infected with a virus.
Numerous approaches have been made to develop a
vaccine that would increase a person's resistance to
infection with the AIDS virus. However, while various
types of HIV-1 vaccines have been designed and have been
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tested in clinical trials, each suffers from a serious
limitation. For example, subunit vaccines, composed of
portions of an HIV-1 protein, provide only transient or
ineffective protection. One of the obstacles to vaccine
development is that HIV-1 undergoes rapid mutation as it
reproduces. As a result, an immune response that is
stimulated against a particular HIV-1 protein or strain
of HIV-1 is ineffective against a changed form of the
virus. Furthermore, antibodies produced to portions of
the HIV-1 surface, which would be most effective in a
vaccine, in fact stimulate HIV-1 infection.
Attenuated vaccines, which consist of live but
reproductively defective viruses, also have been
proposed. However, there is justified concern for
injecting such an HIV-1 virus into an individual,
particularly an otherwise healthy person. Thus, a need
exists for a vaccine that provides a broad based immune
response against HIV-1 but does not carry the attendant
risks and limitations associated with the use of HIV-1 as
the vaccinating agent. The present invention satisfies
this need and provides additional advantages.
Caprine arthritis-encephalitis virus (CAEV) is
a retrovirus that is closely related to the human
immunodeficiency virus (HIV-1), which causes AIDS in
humans. CAEV is known to infect goats, where it causes
various pathologic conditions, including arthritis and
encephalitis. CAEV infection occurs world wide and can
result in costly losses to the goat farming industry.
In many parts of the world, goat milk commonly
is consumed and, particularly, in less developed
countries, is an important source of nutrition. However,
goat milk seldom is pasteurized in such countries prior
to human consumption. Also, the consumption of raw goat
milk is increasing in the United States due to its
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availability in health food markets. In accordance with
the present invention, it now has been recognized that
CAEV-like viral forms can infect humans (h-CAEV),
particularly those individuals who are occupationally
exposed to goats or consume raw goat milk. While it has
not yet been determined whether CAEV can cause a
pathologic condition in humans, the close relationship of
CAEV and HIV-1 suggests that it would be best to minimize
the occurrence of CAEV infection in humans.
Although efforts have been made in the dairy
industry to prevent spread of CAEV in goats, the
infection of humans with human adapted forms was
heretofore unknown. Therefore, a diagnostic method which
applies to humans is necessary. Such a method of
diagnosing CAEV infected humans would be useful for
identifying CAEV infected blood supplies. In particular,
it is important to have a method of detecting CAEV
infection in the blood supply because encephalitis
associated with CAEV infection may occur only in a very
young child receiving infected blood through a
transfusion. Unfortunately, no convenient method is
available for identifying the presence of a CAEV
infection in humans. Thus, a need exists for a simple
and inexpensive method for diagnosing the presence of
CAEV infection. The present invention satisfies this
need and provides additional advantages.
SUMMARY OF THE INVENTION
The present invention provides a vaccine
comprising a caprine arthritis-encephalitis virus (CAEV)
immunogen, preferably a human-CEAV, and a
pharmaceutically acceptable carrier. A vaccine of the
invention is useful, for example, to stimulate an
immunoprotective response against CAEV or a cross-
protective immune response against human immunodeficiency
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virus-1 (HIV-1) in a human individual and to vaccinate a
human, or other non-goat mammal against CAEV infection.
The invention also provides a method of
stimulating an immune response in an individual against
5 CAEV or HIV-1 by administering a CAEV immunogen to the
individual. Such a method is useful, for example, to
increase the resistance to HIV-1 infection or to CAEV
infection of an individual not previously exposed to
HIV-1 or to CAEV, respectively, or to reduce the severity
of a pathology caused by HIV-1 in an HIV-1 infected
individual or of CAEV in a CAEV infected individual. In
addition, the invention provides a method of stimulating
an immune response in vitro by contacting a lymphocyte
with a CAEV immunogen.
The present invention also provides methods of
diagnosing caprine arthritis-encephalitis virus (CAEV)
infection in a subject suspected of being infected with
CAEV. For example, the invention provides an enzyme
linked immunoadsorption assay (ELISA), wherein CAEV gp135
is bound to a solid support and wherein, upon contacting
the CAEV gp135 with a sample such as a serum sample
obtained from an individual suspected of being infected
with CAEV, the detection of an antibody in the serum that
binds to CAEV gp135 is diagnostic of CAEV infection in
the individual. Other methods include PCR-based assays.
The invention also provides a kit for performing such an
ELISA or nucleic acid-base diagnostic.
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5a
Various embodiments of this invention provide
a vaccine, comprising live CAEV and a pharmaceutically
acceptable carrier.
Various other embodiments of this invention
provide a vaccine, comprising a CAEV immunogen and a
pharmaceutically acceptable carrier, wherein said CAEV
immunogen is an amino acid sequence selected from the
group consisting of CAEV immunogens shown as SEQ ID NOS:
1 to 13.
Various other embodiments of this invention provide
use of a CAEV immunogen for the preparation of a medicament
for use in the prophylaxis or treatment of infection by HIV.
Various other embodiments of this invention
provide use of a CAEV immunogen for use in the prophylaxis
or treatment of infection by HIV.
Various other embodiments of this invention
provide use of a lymphocyte contacted in vitro with a
therapeutically effective amount of a CAEV immunogen for
the preparation of a medicament for use in the prophylaxis
or treatment of infection by HIV.
Various other embodiments of this invention
provide use of a lymphocyte contacted in vitro with a
therapeutically effective amount of a CAEV immunogen for
use in the prophylaxis or treatment of infection by HIV.
Various other embodiments of this invention
provide a composition comprising a CAEV immunogen and a
pharmaceutically acceptable carrier for use in the
prophylaxis or treatment of infection by HIV.
Various other embodiments of this invention
provide a composition comprising a pharmaceutically
acceptable carrier and a lymphocyte contacted in vitro with
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5b
a therapeutically effective amount of a CAEV immunogen for
prophylaxis or treatment of infection by HIV.
various other embodiments of this invention provide
use of the vaccine disclosed herein for prophylaxis or
treatment of infection by HIV.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a phylogenetic tree analyses comparing CAEV
gag sequences to other lentiviral gag sequences. PCR and
RT-PCR, and cloning and sequencing of PCR products were
performed as described in Example I.D. The various
sequences analyzed, and their accession numbers,
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are as described in Example I.D. All CAEV-related
sequences are enclosed in a box in Figure 1. All other
sequences are lentiviral except for JSR, the Jaagsiekte
sheep retrovirus, which serves as the designated
ancestral sequence in Figure 1. The PAUP 3.1.1 parsimony
algorithm was used to analyze 173 aligned gag sites, of
which 157 wee varied in Figure 1 and 65 were varied in
Figure 2. Randomization of the sequence input and
MULPARS were two of the options employed. Inversely
weighted parsimony was conducted in Figure 1, as
described in Example I.D., yielding branch lengths that
are not proportional to single nucleotide changes but
which provide superior relative distances for the highly
divergent sequences that are involved. One most-
parsimonious tree was found in Figure 1, and its
branching order was further supported by neighbor-joining
analysis and bootstrapping, and by tree analyses
utilizing pol gene fragments.
Figure 2 shows a phylogenetic tree analyses comparing
CAEV gag sequences to one another as described in Example
I.D. Ordinary parsimony is shown in Figure 2 (since
these sequences are all closely related) and accordingly
the branch lengths are given in units of minimal
nucleotide changes. The maedi-visna sequence is the
designated ancestral sequence. Two equally parsimonious
trees were obtained in Figure 2 that differed in no
substantive way.
Figure 3 compares the nucleotide sequence of PCR
apmplifed Hmc4-derived human-CAEV gag clones with the
corresponding region of a goat infecting CAEV as
described in Example II. The clones analyzed were Hmc4
genomic clones g-30, g-39, g-40, g-46 and g-50; Hmc4
plasma clones p-254 and p-256; clones from the infection
experiment i-238 and i-239. The "i" clones were derived
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from the cytoplasm of infected cultures. The "p" clones
were derived from the plasma of infected individuals.
Figure 4 compares the nucleotide sequence of PCR
apmplifed Hmc4-derived human-CAEV pol clones with the
corresponding region of two goat infecting CAEVs as
described in Example II.
Figure 5 shows a direct sequence comparison of the goat-
derived CAEV strains CAEV-CO and gWa19 (i.e., CAEV-79-63)
and the human-derived h-CAEV strains Hmcl and Hmc4.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a vaccine
comprising a caprine arthritis-encephalitis virus (CAEV)
immunogen, preferably a human adapted strain of CAEV, and
a pharmaceutically acceptable carrier. As disclosed
herein, a vaccine of the invention is useful to stimulate
an immunoprotective response against HIV-1 in a human
individual. In addition, a vaccine of the invention is
useful to vaccinate a human, or other non-goat mammal
against CAEV infection.
As used herein, the term "CAEV immunogen" means
a CAEV viral particle, which can be a live, attenuated or
killed CAEV, or an immunogenic portion of a CAEV viral
particle, which can be a portion of a CAEV viral particle
or a peptide fragment of a CAEV protein such as a peptide
fragment of the gp135 envelope glycoprotein (see Table 1;
env). A CAEV immunogen is characterized in that it can
stimulate an immune response, either in vivo or ex vivo.
In particular, a CAEV immunogen can stimulate an immune
response against CAEV when administered to a human and
can stimulate an immune response that is cross-reactive
against HIV-1. The cross-reactive immune response
against HIV-1 that is generated by CAEV infection is
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reminiscent of the cross-reactive immune response against
smallpox virus that is generated by cowpox infection.
TABLE 1
CAEV IMMUNOGENS
env
26 ERKREGFTAG (SEQ ID NO: 1)
48 SHHGNDSRRR (SEQ ID NO: 2)
54 SRRRRRKS (SEQ ID NO: 3)
514 RKETGTLGG (SEQ ID NO: 4)
620 RKKRELSHKRKKR (SEQ ID NO: 5)
pol
13 RMQRKERHK (SEQ ID NO: 6)
37 VRSSYGITSA (SEQ ID NO: 7)
83 GRIKLQGIGG (SEQ ID NO: 8)
307 QEILEDWIQQ (SEQ ID NO: 9)
1033 KRINNKYNKNS (SEQ ID NO: 10)
gag
123 DGLLEQEEKK (SEQ ID NO: 11)
141 SVFPIVVQAA (SEQ ID NO: 12)
306 AIDAEPTV (SEQ ID NO: 13)
- env, pol and gag refer to the envelope, polymerase
and nucleocapsid core protein coding regions,
respectively, of CAEV. Numbers preceding each sequence
indicates the amino acid position in each CAEV protein
that corresponds to the first amino acid shown for each
peptide.
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A peptide fragment of the 70K protein, which is
present in the U1 ribonucleoprotein complex, that is
homologous to an amino acid sequence present in HIV-1 can
act as a surrogate immunogen, which stimulates an immune
response that cross-reacts with HIV-1 (Douvas and
Takehana, AIDS Res. Hum. Retrovir. 10:253-262 (1994)).
As disclosed
herein, various peptide fragments of CAEV proteins are
homologous to 70K and HIV-1 sequences and, therefore, can
be useful as CAEV immunogens for stimulating an immune
response in an individual that cross-reacts with HIV-1.
Examples of peptide fragments of CAEV that are
homologous to amino acid sequences present in 70K and
HIV-1 are provided in the Table 1. Other exemplary CAEV
peptides fragments contemplated herein with corresponding
structural features in HIV-1(HIV-1-CAEV), include, for
example: 220-FAILKC-200-FAILKC (hinge region); 99-
DMVEQM-415-DMVEHM (a-helix 1); 129-LKCTDL-203-LKCTKW
(conserved turn); 300-NNNTRT-480-NNNT.IT (conserved turn),
and the like. Still other such CAEV peptides useful as a
CAEV immunogen can be identified by searching the CAEV
DNA or amino acid sequence to identify CAEV peptides that
are homologous to the immunologically homologous
sequences of 70K and HIV-1 (see Douvas and Takehana,
supra, 1994). Furthermore, methods for determining which
CAEV peptides that are homologous to 70K and to HIV-1
peptides also are useful as CAEV immunogens are disclosed
herein, including methods such as ELISA or western blot
or viral neutralization assays (Example I), or otherwise
known in the art (see, for example, Harlow and Lane,
Antibodies: A laboratory manual (Cold Spring Harbor
Laboratory Press 1988)).
As used herein, the term "vaccine" means a
composition containing an immunogen which, upon
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administration to an individual, which generally is a
non-goat mammal such as a human, and the like, stimulates
an immune response in the individual. In particular, a
vaccine of the invention contains a CAEV immunogen, which
5 can be administered, for example, to a human, wherein an
immune response against CAEV that is cross-reactive
against HIV-1 is stimulated. Thus, a CAEV immunogen is
useful as a surrogate immunogen for stimulating an immune
response against HIV-1. A particularly preferred vaccine
10 is an attenuated human-CAEV, such as those set forth in
Figures 3 and 4.
Although any CAEV infectious agent is
contemplated for use herein, particularly preferred CAEVs
for use herein are the novel human adapted strains of
CAEV, referred to herein as "human-CAEV", such as the
human strains obtained from patients Hmcl and Hmc4
described in Example II, whose partial genomic sequence
is set forth in Figures 3 and 4. It has been found that
the human-CAEVs are distinct from the CAEVs able to
infect goats. For example, within the genomic region
corresponding to the 173 nucleotide fragment amplified by
primers set forth in SEQ ID NOs: 16 and 17, it has been
found that the this particular region of a human-CAEV
virus has at least two mutations that are uniquely human
in origin. These mutations correspond to a guanine at
position 56 and a cytosine at position 77 within the 173
gag-region amplification product.
Thus, in accordance with another embodiment of
the present invention, novel isolated human-CAEVs are
provided. Invention human-CAEVs can be isolated by the
methods described in Example III or employing methods
well-known in the art.
It is recognized that a vaccine of the
invention can be administered to an individual that is
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not infected with HIV-l, in which case the vaccine
stimulates an immune response that can protect the
individual from infection due to subsequent exposure to
HIV-1, or can be administered to an HIV-1 infected
individual, in which case the vaccine can stimulate an
immune response so as to counter the immunopathology of
HIV-1 (see, for example, Cease and Berzofsky, Ann. Rev.
Immunol. 12:923-989 (1994)). Thus, a vaccine of the
invention can be useful to increase an individual's
resistance to HIV-1 infection or to reduce the severity
of a pathology due to HIV-1 in an HIV-1 infected
individual.
Prior to the present disclosure, it was not
known that CAEV could infect humans. As disclosed
herein, however, CAEV infection of humans is prevalent,
particularly in populations where consumption of raw goat
milk is common or where individuals in the population are
otherwise exposed to goats (see Example I.C.). While it
has not yet been determined whether CAEV infection causes
a pathology such as arthritis or encephalitis in humans
as it does in goats, if such a CAEV induced pathology
exists, then it will be important to vaccinate persons
involved in the goat farming industry against CAEV
infection. A vaccine of the invention can be useful for
this purpose. In addition, the diagnostic ELISA assay
disclosed herein can be useful to identify CAEV infected
individuals and can be particularly useful, for example,
to screen blood supplies to identify CAEV infected blood,
thereby preventing the spread of CAEV infection through
administration of CAEV contaminated blood.
Since it has now been determined that CAEV
infects humans, it is recognized that there is a
likelihood that CAEV also can infect other "non-goat"
mammals, including, for example, non-human primates
(e.g., monkeys, and the like), cows, horses, sheep, dogs,
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cats or other mammals. Thus, an ELISA of the invention
can be useful for diagnosing CAEV infection in any such
non-goat mammal. Furthermore, if it is determined that
CAEV infects other such mammals, a vaccine of the
invention can be useful for preventing or limiting the
spread of CAEV infection in these non-goat mammals.
As disclosed herein, exposure of a human
individual to CAEV also can stimulate an immune response
to CAEV that is cross-reactive with HIV-1 (Example I.E.).
CAEV is a retrovirus, subtype lentivirus, that is related
to human immunodeficiency virus-1 (HIV-1). CAEV infects
goats and causes arthritis and abnormalities of the
immune system of infected animals (see, for example,
Banks et al., Arthrit. Rheum. 30:1046-1053 (1987);
Crawford et al., Science 207:997-999 (1980)).
Visna maedi virus (VMV) is another lentivirus
that is closely related to CAEV and HIV-1 and causes a
disease in sheep similar to that caused by CAEV in goats.
Thus, it is contemplated herein that VMV or an
immunogenic fragment of VMV can be substituted for a CAEV
immunogen or, if desired, can be used in combination with
a CAEV immunogen in a vaccine of the invention. A
vaccine containing a VMV immunogen is useful for
stimulating an immune response in a human that cross-
reacts against HIV-1.
CAEV causes a persistent infection in goats and
is associated with three disease syndromes, including
arthritis, which occurs in 20-30% of infected animals;
leukoencephalitis, which occurs in young animals; and
sporadic neurologic disease, which occurs in adult goats.
CAEV infection is found world wide and was identified as
the cause of arthritis and encephalitis in goats in 1980.
In 1985 and 1986, the relationship between the nucleotide
and amino acid sequences of CAEV and HIV-1 (then called
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HTLV-III) was described. CAEV and HIV-1 are closely
related phylogenetically and share a high degree of
homology, including, for example, between their RNA
dependent DNA polymerases (pol) and between gp120/41 in
HIV-1 and gp135/38 in CAEV (see, for example, Gonda et
al., Proc. Natl. Acad. Sci.. USA 83:4007-4111 (1986);
Gonda et a1., Retroviridae 3:83-109 (1994); Garry et al.,
Retroviridae 4:491-603 (1995)).
CAEV is transmitted among goats through
infected milk, particularly colostrum, and infection is
spread by the agricultural practice of pooling colostrum
to feed young animals. CAEV multiplies in cells of the
monocyte/macrophage lineage and in fibroblast cell lines,
but does not infect T cells. Macrophages expressing CAEV
are distributed in,the synovia, lungs-, central nervous
system, lymph nodes, spleen, gastro-intestinal tract and
mammary glands of infected goats.
Prior to the present disclosure, CAEV was not
known to infect humans. However, the detection of
seroconversion by ELISA and western blot analyses using
human blood samples (Example I.C.) and the detection of
proviral DNA, corresponding to CAEV gag and pol genes, by
polymerase chain reaction (PCR) analysis in genomic DNA
obtained from an MCTD patient (Example I.D.) provides
demonstrative evidence that CAEV infects humans. The
incorporation of viral DNA sequences into host cell DNA
is a hallmark of infection by the lentiviruses, including
CAEV and HIV-1.
Based on the results disclosed herein, CAEV may
infect as many as half the population of Mexico and
Central America, particularly those who consume raw goat
milk, which is common in these areas, or are otherwise
exposed to goats-(see Example I.C.). Although there is
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no evidence that CAEV causes a pathologic condition in
humans, it is important, nevertheless, to determine the
scope of CAEV infection in the human population in order
to identify whether, for example, a pathology of unknown
etiology correlates with CAEV infection. The ELISA assay
provided herein allows for routine screening for CAEV
infection in humans and provides the additional advantage
of being useful for screening other non-goat mammals to
identify the presence of CAEV infection.
PCR analysis also was performed on genomic DNA
obtained from three groups of goats from different
geographical regions and from a CAEV infected human (see
Example I.D.). The PCR analysis of human and goat
genomic DNA identified at least two potential strains of
CAEV, which differ from the DNA sequence of a reference
CAEN(-strain (reference CAEV DNA sequence is available as
Ge.n$ankT" :Accession No. 1433677).
Remarkably, the immune responses
generated in two of the three groups of CAEV infected
goats and in several CAEV infected human individuals were
cross-reactive with HIV-1, indicating that either of
these strains can be used as a source of a CAEV immunogen
for a vaccine of the invention.
CAEV infected goats have been used as an animal
model for human rheumatoid-arthritis, which is the most
well known and prevalent syndrome in a cluster of
arthritic/autoimmune disorders, the systemic rheumatic
disorders. The systemic rheumatic disorders are about
three times more common in women than men.
Mixed connective tissue disease (MCTD) is
another systemic rheumatic disorder. MCTD is
characterized by autoantibodies to the Ui snRNP splicing
complex. MCTD is considered to be an overlap syndrome,
in that it embraces clinical and serologic features
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common to three other systemic rheumatic disorders -
systemic lupus erythematosus (SLE), scleroderma and
polymyositis. The clinical profile of MCTD includes
arthritis, lymphadenopathy, vasculitis, myositis,
5 Sjogren's syndrome (lymphocytic infiltration of salivary
and lacrimal glands) and immune dysregulation.
The clinical profile manifest in MCTD
individuals also occurs in HIV-1 infected individuals.
However, these clinical manifestations have a more severe
10 course and are more refractory to therapy in HIV-1
infected persons. For example, Sjogren's syndrome in
MCTD individuals is characterized by localized
lymphocytic infiltration, whereas widespread glandular
infiltration, referred to as diffuse infiltrative
15 lymphocytosis syndrome, occurs in the corresponding HIV-1
associated syndrome.
Severe necrotizing vasculitis accompanied by
neuropathy, inflammatory polymyopathy unrelated to drug
therapy and other forms of vascular and neuromuscular
disease associated with rheumatic disorders also occur in
HIV-1 infected individuals. Necrotizing lymphadenopathy,
which results in massive destruction of lymph node
structure, occurs in MCTD, SLE and HIV-1 infection. Two
classical rheumatic disorders, psoriatic arthritis and
Reiter's syndrome, are the earliest manifestations of
HIV-1 infection and are more refractory to
chemotherapeutic agents when associated with HIV-1
infection.
The clinical similarities of HIV-1 infection
and the systemic rheumatic disorders such as MCTD suggest
that HIV-1 can be a precipitant of rheumatic pathology or
that a phylogenetically related, but more clinically
benign lentivirus is an etiologic factor in rheumatic
disorders. Serologically, MCTD is characterized by the
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presence of anti-RNP (ribonucleoprotein) antibodies
specific for the U1 snRNP particle, which is positioned
at 5'-end of introns during nuclear RNA splicing.
Anti-RNP antibodies also are present in some SLE and
scleroderma patients. The U1 snRNP particle is composed
of a core of U1 RNA and a cluster of RNA binding
proteins, including the 70K, A and C proteins. Anti-RNP
antibodies inhibit RNA splicing by binding Ul snRNP.
Although the humoral anti-RNP autoantibody
response is pathognomonic in MCTD, the 70K protein, for
example, contains both T cell and B cell epitopes and the
humoral and cellular autoimmune manifestations are
interdependent. T cell clones responsive to 70K epitopes
have been isolated and include CDB' T cells having
cytotoxic T lymphocyte activity and CD4' T cells having
helper T cell activity. Some of the 70K T cell epitopes
are homologous to T cell epitopes of HIV-1 (see Douvas
and Takehana, supra, 1994). As disclosed herein, CAEV
proteins, including CAEV gp135, also contain epitopes
that are homologous to 70K and HIV-i T cell epitopes (see
Table 1, supra). Thus, a CAEV immunogen comprising such
an epitope can induce a broad based cross-reactive immune
response against HIV-1.
In view of the clinical similarities of HIV-1
infection and MCTD and the identification of CAEV
infection in MCTD patients and in otherwise healthy
individuals, it was important to determine whether
individuals infected with CAEV develop an immune response
that cross-reacts with HIV-1. The level of immunity to
the HIV-1 protein, gp120, in blood obtained from CAEV
infected individuals was examined by ELISA and by western
blot analysis (see Example I.E.). In addition to
exploring the relationship between CAEV infection and the
development of an immune response to HIV-1, the
relationship between CAEV infection and MCTD was
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investigated. Since some MCTD patients from Mexico or
Central America have antibodies that react to HIV-1 gp120
and inhibit HIV-1 infectivity in vitro, it remained to be
established whether CAEV infected persons that do not
have MCTD also develop immunity to HIV-1.
As disclosed herein, CAEV infection in humans,
whether suffering from MCTD or otherwise healthy,
stimulated a humoral immune response that cross-reacted
with the HIV-1 gp120 or p24 viral antigen (see
Example I.E.). Significantly, none of the humans
examined was infected by HIV-1 as determined using
clinically certified assays. These results indicate that
immunization of an individual with a CAEV immunogen can
generate cross-reactive immunity to HIV-1, thereby
increasing the resistance to HIV-1 infection in an
uninfected individual or reducing the severity of a
pathology due to HIV-1 in an HIV-1 infected individual.
Furthermore, the presence of a systemic rheumatic
disorder such as MCTD in a CAEV infected patient
substantially increased the level of HIV-1 cross-
reactivity (Example I.E.). Thus, administration of a
CAEV immunogen, preferably in combination with an
autoantigen associated with MCTD, such as a 70K
immunogen, which generates a cross-reactive immune
response against HIV-1 (Douvas and Takehana, supra,
1994), can be particularly effective in stimulating an
immune response that cross-reacts with HIV-1 in a human
individual.
A CAEV immunogen can be immunogenic by itself
or can be attached to a carrier molecule such as bovine
serum albumen or an inert carrier such that the CAEV
immunogen-carrier complex can stimulate an immune
response (see, for example, Harlow and Lane, supra,
1988). For example, where a vaccine contains the entire
CAEV virus, the virus is immunogenic and can stimulate an
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18
immune response in a vaccinated individual. CAEV is
readily available from the American Type Culture
Collection (ATCC VR-905).
A vaccine of the invention also can contain a
peptide portion of a CAEV protein such as gp135 (see
Table 1, supra). It is recognized that each of the
peptides exemplified in the Table 1 or otherwise useful
in the invention may not be immunogenic by itself.
However, it is well known that such haptens can be
attached to a carrier molecule so as to be rendered
immunogenic (see, for example, Harlow and Lane, supra,
1988). An immune response can be stimulated in vivo or
ex vivo. For example, immune cells, including T cells
and B cells, can be obtained from a subject and placed in
a tissue culture medium. The cells then can be contacted
with a CAEV immunogen, which can stimulate the immune
cells and induce a T helper cell and cytotoxic T cell
immune response.
A vaccine of the invention contains, in
addition to a CAEV immunogen, a pharmaceutically
acceptable carrier. Pharmaceutically acceptable carriers
are well known in the art and include aqueous solutions
such as physiologically buffered saline or other solvents
or vehicles such as glycols, glycerol, oils such as olive
oil or injectable organic esters. If desired, a vaccine
of the invention can contain a pharmaceutically
acceptable carrier that is an adjuvant. Adjuvants such
as alum or Freund's complete or incomplete adjuvant, or
other proprietary adjuvants are known in the art and
commercially available (Ribi Immunochem Research, Inc.;
Hamilton MT). The addition of an adjuvant generally
decreases the amount of a CAEV immunogen required to
stimulate an immune response.
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A pharmaceutically acceptable carrier also can
contain a physiologically acceptable compound that acts,
for example, to stabilize the CAEV immunogen or increase
its absorption. Physiologically acceptable compounds
include, for example, carbohydrates, such as glucose,
sucrose or dextrans; antioxidants, such as ascorbic acid
or glutathione; chelating agents; low molecular weight
proteins; or other stabilizers or excipients. One
skilled in the art would know that the choice of a
pharmaceutically acceptable carrier, including a
physiologically acceptable compound, depends, for
example, on the route of administration of the vaccine
and on the particular physico-chemical characteristics of
the CAEV immunogen. Various routes for administering a
vaccine to stimulate an immune response are known in the
art and include, for example, intravenous, intradermal
and subcutaneous injection, oral administration and
transdermal administration.
If desired, a vaccine of the invention also can
contain a 70K immunogen, or peptide fragment thereof,
which is a surrogate immunogen that stimulates an immune
response that cross-reacts against HIV-1 (Douvas and
Takehana, supra, 1994). The addition of a 70K immunogen
to the vaccine is advantageous because it is an
autoimmune protein that represents an enrichment of
sequences that can stimulate early shared clones, which
are anti-70K latent T cells and B cells that can be
rapidly activated to react with HIV-1 or can produce
cross-reactive anti-gp120/41 antibodies that can
neutralize HIV-1. The addition of a 70K immunogen also
can be advantageous in that it can provide co-
amplification or synergistic amplification of shared
clones with gp120/41. In addition, immunogenic fragments
of 70K that do not contain amino acid sequences that
stimulate antibodies that mediate the deleterious effects
CA 02249736 2004-01-21
associated with HIV-1 infection can be selected (Douvas
and Takehana, supra, 1994).
A CAEV immunogen can be produced by various
methods, including, for example, by recombinant DNA
5 methods or by methods of chemical synthesis. For
example, if a CAEV protein or a peptide fragment thereof
is desired, the protein or peptide fragment can be
produced by cloning the appropriate CAEV coding sequence
into an expression vector such as a baculovirus vector
10 and the protein or peptide can be expressed in and
isolated from the appropriate host cell. The nucleic
acid sequence of CAEV is available as GenBank Accession
No. M33677 (see, also, Saltarelli et al., Virology
179:347-364 (1990); Knowles et al., J. Virol. 65:5744-
15 5750 (1991)).
In addition, it can be desirable to produce the
recombinant protein or peptide in a mammalian cell, which.
can perform a desired post-translational modification.
Appropriate expression vectors and host cells are well
20 known in the art and commercially available, and the
skilled artisan would know how to select an appropriate
vector/host cell system based on a particular need.
Where the CAEV immunogen selected is a peptide
immunogen such as, for example, the CAEV immunogens
exemplified in the Table 1,' the peptide can be
synthesized using well known method of chemical
synthesis, including, for example, automated solid phase
methods. Chemical synthesis of a CAEV immunogen can be
particularly desirable because the method allows for the
introduction of amino acid analogs into the peptide. For
example, it can be desirable to synthesize a CAEV
immunogen containing one or a few (D) -amino acid
substitutions for corresponding naturally occurring (L) -
amino acids. The incorporation of a(D) -amino acid can
confer desirable properties on the CAEV immunogen,
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21
including, for example, increasing the stability of the
peptide, which can be particularly useful for preparing a
vaccine that is to be distributed to regions of the world
where refrigeration is not always available or
dependable. In addition, a CAEV peptide can be
synthesized and, if desired, can include an additional
cysteine residue, which can facilitate specific
attachment of the peptide to a matrix or to a carrier
molecule such as keyhole limpet hemocyanin using
appropriate oxidizing conditions.
The present invention also provides a method of
stimulating an individual's immune response against CAEV
or stimulating an immune response that is cross-reactive
against HIV-1 by administering a therapeutically
effective amount of a CAEV immunogen to the subject. If
desired, VMV or an immunogenic fragment of VMV can be
substituted for the CAEV immunogen. In addition, it can
be advantageous to administer a 70K immunogen to the
individual, either in combination with the CAEV or VMV
immunogen or as a separate treatment.
Although reference is made herein to the
administration of a CAEV immunogen to an individual, or
to vaccination of an individual, it is recognized that an
immune response against HIV-1 can be stimulated in vivo
or ex vivo. Each of these methods is encompassed within
the present invention. For example, it can be desirable
to stimulate an immune response against HIV-1 ex vivo
where the individual to be treated has AIDS. In this
case, lymphocytes can be removed from the individual and
immunized in culture. At the same time, the lymphocyte
population can be expanded. The stimulated, expanded
immune cells then can be reinfused into the individual,
thereby providing a therapeutic advantage to the
individual. Furthermore, even if a method of the
invention such as the ex vivo immunization and reinfusion
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22
method is not curative, such a treatment can be
palliative and, therefore, can increase the quality of
life of an individual suffering from AIDS.
As disclosed herein, vaccination of an
individual with a CAEV immunogen stimulates an immune
response that is cross-reactive against HIV-1.
Vaccination against HIV-1 using a CAEV immunogen, either
alone or in combination with a 70K immunogen, provides
significant advantages over the use, for example, of a
killed or attenuated form of HIV-1 or an HIV-1 protein
antigen as a vaccine in that CAEV is not known to be a
human pathogen and does not infect T cells. In addition,
CAEV infection, particularly in individuals suffering
from MCTD, provides a broad based immunologic resistance
to HIV-1, including a B cell response and a T cell
response against various strains of HIV-1. For example,
in the population of Hispanic individuals examined in the
study disclosed in Example I, greater than 60% of the
individuals were infected with CAEV (see Example I.D.).
In addition, 22% of the CAEV infected, but otherwise
healthy, individuals demonstrated cross-reactivity to
HIV-1 and 55% of the CAEV infected, MCTD patients
demonstrated cross-reactivity to HIV-1 (Example I.E.).
Controlled methods of immunization with a CAEV immunogen,
alone or in combination with a 70K immunogen, can
increase the frequency of individuals demonstrating
cross-reactive immunity to HIV-1 (see Example II).
In order to stimulate an immune response
against HIV-1, a therapeutically effective amount of a
CAEV immunogen is administered to an individual. As used
herein, the term "therapeutically effective amount" means
an amount of an immunogen required to stimulate an immune
response. The amount of an immunogen such as a CAEV
immunogen that constitutes a therapeutically effective
amount will vary, depending, for example, on whether
CA 02249736 2004-01-21
23
stimulation of the immune response is in vivo or ex vivo;
on whether the administration is a first administration
or a booster administration; whether an adjuvant is
administered with the immunogen; and, when administered
in vivo, on the route of administration. In general, a
therapeutically effective amount of a CAEV immunogen is
about 50 g to about 50 mg, preferably about 500 g to
about 5 mg. For example, where a CAEV virus is
administered, a therapeutically effective amount of the
particulate CAEV immunogen can be about 100 g to about
2 mg, whereas, where a soluble CAEV immunogen is
administered, a therapeutically effective'amount can be
about 1 mg to about 5 mg. Methods for determining a
therapeutically effective amount of an immunogen are
routine and well known in art (see Example II; see, also,
Powell and Newman, Vaccine Design: The subunit and
adjuvant approach (Plenum Pubi. Corp.; 1994)).
Methods for vaccinating an individual so as to
stimulate an immune response also are well known (Harlow
and Lane, supra, 1988). For example, the immunogen can
be administered intradermally, intramuscularly or
intravenously. In addition, it can be advantageous to
administer one or more booster immunizations. The need
to administer a booster immunization and the timing of
such booster immunizations can be determined
experimentally by measuring, for example, the presence of
anti-HIV-1 antibodies in a vaccinated individual's serum,
using the methods disclosed herein.
A method of the invention can be useful for
increasing the resistance to HIV-1 infection of an
individual not previously exposed to HIV-1. Similarly, a
method of the invention can be useful for increasing the
resistance to CAEV infection of a human or other
susceptible non-goat mammal not previously exposed to
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24
CAEV. As used herein, the term "increasing the
resistance," when used in reference to HIV-1 or CAEV
infection, means that the likelihood of infection by the
virus is reduced due to the stimulation of a immune
response against the virus and consequent generation of
memory immune cells, which can be rapidly mobilized
against and sequester subsequent viral infection. In the
case of CAEV, resistance is increased due to vaccination
with a CAEV or a VMV immunogen, respectively, whereas, in
the case of HIV-1, resistance is increased to vaccination
with a CAEV or a VMV immunogen, which stimulates an
immune response that cross-reacts with HIV-1.
In addition, a method of the invention can be
useful in an individual, including a human or a non-goat
mammal, that currently is infected with a virus and is
suffering a pathology due to the infection. In
particular, a method of administering a CAEV immunogen to
a human in order to stimulate an immune response in an
HIV-1 infected individual that is cross-reactive against
HIV-1 can be useful for decreasing the immunopathology of
HIV-1. Specifically, the stimulation of such a response
in an HIV-1 infected individual can prevent the further
spread of HIV-1 infection in the individual, thereby
reducing the depletion of T cells in the individual.
Since many of the pathologies observed in an HIV-1
infected individual are due to the depleted immune
system, a treatment that reduces depletion of the immune
system can provide a therapeutic advantage.
Based on the studies disclosed herein, a
significant population of persons, particularly in Mexico
and Central America, may be infected with CAEV. Thus, it
is important that a simple and inexpensive assay for
diagnosing an individual infected with CAEV be available.
CA 02249736 2004-01-21
Accordingly, the invention further provides
methods of diagnosing a CAEV infection in a non-goat
mammalian subject, preferably.human, suspected of being
infected with CAEV. Exemplary methods for diagnosing
5 CAEV infection include ELISA assays, PCR amplification
assays, nucleic acid probe*assays, and the like.
In one embodiment, for example, the invention
provides an enzyme linked immunoadsorption assay (ELISA)
useful for diagnosing the presence of a CAEV infection in
10 an individual suspected of having a CAEV infection, by
obtaining a sample such as a blood sample from the
individual, contacting the sample with a CAEV antigen and
detecting binding of an anti-CAEV antibody present in the
sample to the CAEV antigen, wherein such a detection is
15 diagnostic of CAEV infection. Thus, methods and reagents
for performing a diagnostic assay of the invention are
disclosed herein or otherwise known in the art (see, for
example, Litt, U.S. Patent No. 4,092,408; issued May 30,
1978).
20 A method of the invention is exemplified by the
ELISA (Example I.C.), in which CAEV gp135 was used as the
CAEV antigen. However, a CAEV antigen useful in a method
of the invention can be selected from essentially any
antigenic fragment of CAEV or can be the entire CAEV
25 viral particle. For purposes of the describing a method
of the invention, the term "CAEV antigen" is intended to
be synonymous with the term "CAEV immunogen" as defined
above.
A competition ELISA can be particularly useful
for diagnosing the presence of a CAEV infection in a
sample obtained from a subject. A competition ELISA is
performed similarly to a standard ELISA, except that one
or more monoclonal antibodies, each specific for a CAEV
epitope such as the epitopes defined by the CAEV
CA 02249736 2004-01-21
26
immunogens shown in Table 1, is added to the reaction to
compete with an anti-CAEV antibody present in the sample.
The use of a competition ELISA can produce enhanced
sensitivity for detecting the presence of an anti-CAEV
antibody in a sampl-- Methods for performing a
competition ELISA such as the threshold ligand receptor
assay of Buechler et al. (U.S. Patent No. 5,089,391, issued
February 18, 1992) are well known in the art.
Monoclonal antibodies specific for particular
CAEV epitopes can be prepared using well known methods
(see, for example, Harlow and Lane, supra, 1988). For
example, a mouse can be immunized with a CAEV immunogen
(see, for example, Table 1), then spleen cells are
collected from a mouse having a high titer of antibody
for the particular CAEV immunogen. Methods for
identifying an anti-CAEV immunogen antibody.having an
appropriate specificity and affinity and, therefore,
useful in the invention are disclosed herein or otherwise
known in the art and include, for example, enzyme-linked
immunoadsorption assays, radioimmunoassays and precipitin
assays (see Example I; see, also, Harlow and Lane, supra,
1988, chap. 14). The mouse spleen cells can be fused to
an appropriate myeloma cell line such as SP/02 or
P3x653.Ag8 myeloma cells to produce hybridoma cells.
Cloned hybridoma cell lines can be screened using a
labelled CAEV immunogen to identify clones that secrete
monoclonal antibodies specific for the CAEV immunogen.
Hybridomas that express antibodies having a desirable
specificity and affinity can be isolated and utilized as
a continuous source of monoclonal antibodies useful in
the competition ELISA.
As used herein, the term "sample," when used in
reference to a diagnostic method of the invention, means
a tissue specimen or a fluid specimen such as a blood,
CA 02249736 2004-01-21
27
which can be whole blood, plasma or serum, or a urine
specimen, which is obtained from an individual such as a
human, or other non-goat mammal to be tested for CAEV
infection. Methods of obtaining such a sample, including
an appropriate tissue sample, are well known and routine
in the art.
A method of the invention can diagnose CAEV
infection in an individual due, in part, to the presence
of anti-CAEV antibodies in a sample obtained from the
individual. A sample obtained from an individual
suspected of being infected with CAEV is contacted with a
CAEV antigen under suitable conditions, which allow the
CAEV antigen to bind with an anti-CAEV antibody, if
present, in the sample. Thus, a method of the invention
can be an ELISA, which is exemplified herein, or can be a
radioimmunoassay, western blot or other such assay based
on the detection of a specific antibody-antigen
interaction (see, for example, Harlow and Lane, supra,
1988; see, also, Gribnau et al., U.S. Patent No.
-20 4,373,932, issued February 15, 1983).
Methods for detecting an antigen-antibody
interaction such as the interaction of a CAEV antigen
with an anti-CAEV antibody present in a sample are well
known in the art and include, for example, the use of a
detectably labelled antigen or the use of a detectably
labelled second antibody, which is an antibody that
specifically binds a particular class of antibody such as
IgG, IgA, IgM, IgA, IgD or IgE from a particular
mammalian species (see, for example, Greene and David,
U.S. Patent No. 4,376,110, issued August 1983).
For example, if a
sample is a blood serum or blood plasma sample from a
human individual, a second antibody can be a goat, anti-
human IgG (see Example I.C.). Such second antibodies can
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28
be prepared using well known methods or can be purchased
from a commercial source. Methods for detectably
labeling a CAEV antigen or an antibody also are well
known and routine in the art.
CAEV gp135~was selected as the antigen in the
exemplified ELISA because the presence of anti-gp135
antibodies in antiserum collected from a human
individual, besides being indicative of infection of the
individual with CAEV, also indicates that the individual
can have antibodies that are cross-reactive with HIV-1.
In fact, 22% of the human individuals identified as CAEV
infected by the CAEV ELISA also had antibodies cross-
reactive to HIV-1 as determined HIV-1 ELISA and western
blot assays (see Example I.E.): In addition, 55% of the
MCTD individuals that were infected with CAEV as
determined using the CAEV ELISA also had antibodies
cross-reactive to HIV-1. An ELISA of the invention also
can be used to screen non-goat mammals other than humans,
including, for example, other mammals susceptible to CAEV
infection, to identify CAEV infected individuals.
A method of the invention such as an ELISA is
particularly convenient if it is available in the form of
a kit. Such an ELISA kit, for example, can contain a
CAEV antigen such as gp135 attached to a solid support
such as a plastic support. For example, the kit can
contain a microtiter plate such as a 96 well plate
containing a CAEV antigen attached to some or all of the
wells. Other solid supports useful in an ELISA assay are
known in the art. Use, for example, of a 96 well
microtiter plate in an ELISA provides the additional
advantage that, upon selection of an appropriate
detectable label such as a radioactive, fluorescent or
luminescent label, the assay can be automated, thereby
allowing rapid through put of a large number of samples,
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including, if desired, appropriate standard or control
samples.
A kit such as an ELISA kit also can contain, if
desired, a standard reagent such as a predetermined
amount of an anti-gp135 antibody. Such reagents can
provide a means to readily determine whether a sample
such as a blood sample or a blood serum sample obtained
from an individual contains an amount of circulating
anti-CAEV antibody. Accordingly, a kit of the invention
can contain, for example, an appropriate buffer, which
provides suitable conditions for binding of an anti-CAEV
antibody present in the sample to a CAEV antigen attached
to the solid support. By providing such reagents in the
form of a kit, the ELISA assay can be standardized such
that results of different tests performed in different
places at different times can be compared with each
other. It is recognized that, for a selected CAEV
antigen, a population of serum samples obtained from
uninfected individuals (controls) should be analyzed in
order to determine a base line level of reactivity to the
particular CAEV antigen.
A method of the invention such as the
exemplified CAEV ELISA can be useful to detect the
presence of circulating anti-CAEV antibodies or to follow
the development of an immune response. For example, an
individual can be vaccinated with a vaccine of the
invention and the development of the immune response can
be followed using the ELISA of the invention. Such a
method can be useful for determining whether a booster
immunization is required and, if necessary, the optimal
time for administering the booster. In addition, a CAEV
assay such as the exemplified ELISA can be useful for
screening a sample of blood provided by a blood donor in
order to identify and remove CAEV contaminated blood from
a blood bank.
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In accordance with another embodiment of the
present invention, there are provided nucleic acid-based
methods for diagnosing CAEV infection in a non-goat
mammalian subject, preferably a human subject. One such
5 method comprises:
detecting, in said subject, a genomic or
transcribed mRNA sequence corresponding to CAEV
nucleic acid.
In one embodiment, the CAEV nucleic acid
10 sequence detected includes sequences that are unique to
human isolates of CAEV (i.e., human-CAEVs). For example,
within the genomic region corresponding to the 173
nucleotide fragment amplified by primers set forth in SEQ
ID NOs: 16 and 17, it has been found that the human-CAEV
15 virus has at least two mutations that are uniquely human
in origin. These mutations correspond to a guanine at
position 56 and a cytosine at position 77 within the 173
gag-region amplification product. In addition to the
above-described uniquely human mutations, once additioal
20 human-CAEV sequence is elucidated, those of skill in the
art can readily obtain other unique nucleic acid regions
that are present in the human-CAEV isolates. Thus, a
variety of uniquely human nucleic acid regions within the
human-CAEV genome are contemplated for use herein.
25 In yet another embodiment of the present
invention, there is provided a method for diagnosing CAEV
infection in a subject, preferably human, said method
comprising:
a) contacting nucleic acid obtained from
30 a subject suspected of having CAEV infection with primers
that amplify a CAEV genomic nucleic acid fragment, under
conditions suitable to form a detectable amplification
product; and
b) detecting an amplification product
containing CAEV genomic nucleic acid, whereby said
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detection indicates that said subject has been infected
by CAEV.
Suitable primers for use heren in PCR
diagnostic assays include at least one pair of primers
that is capable of amplifying CAEV nucleic acid.
Preferably, the primers selected amplify nucleic acid
specific to CAEV and do not amplify HIV-1 sequences.
See, for example the nested set of PCR primers including
SEQ ID NOs: 16 and 17, which amplify a 173 nucleotide
region of the gag gene from human-CAEV; or the nested set
of primers including SEQ ID NOs: 20 and 21, which amplify
a 146 nucleotide region of the human-CAEV pol gene.
Although the use of single primer pair is
contemplated for use herein, in a particular embodiment
of the invention PCR-based diagnostic assay, a panel of
multiple sets of primers (e.g., primer pairs or nested
sets of primers) that amplify a variety of distinct
nucleic regions of human-CAEV nucleic acid are employed
for each subject being analyzed. Since retroviruses are
known to have a high rate of mutation, a panel of
multiple sets of primers are preferably used in the
invention diagnostic methods to increase the likelihood
that CAEV infection in an infected individual will in
fact be detected. The number of primer pairs can readily
be selected by those skill in the art to ensure that up
to at least 99% of CAEV-infectd subjects are detected.
For example, 1 up to about 100 or more primer pairs are
contemplated for use herein. In one embodiment, a panel
of at least about 10 up to 70 primer pairs, or nested
sets of primers, are employed herein. In another
embodiemnt at least about 20 up to 50 primer pairs are
employed.
In accordance with another embodiment of the
present invention, there are provided diagnostic systems,
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preferably in kit form, comprising at least one
diagnostic nucleic acid in a suitable packaging material.
The diagnostic nucleic acids are preferably derived from
human CAEV nucleic acid.
Invention diagnostic systems are useful for
assaying for the presence or absence of CAEV infection.
A suitable diagnostic system includes at least one
invention nucleic acid, preferably two or more invention
nucleic acids, as a separately packaged chemical
reagent(s) in an amount sufficient for at least one
assay. Instructions for use of the packaged reagent are
also typically included. Those of skill in the art can
readily incorporate invention nucleic probes and/or
primers into kit form in combination with appropriate
buffers and solutions for the practice of the invention
methods as described herein.
As employed herein, the phrase "packaging
material" refers to one or more physical structures used
to house the contents of the kit, such as invention
nucleic acid probes or primers, and the like. The
packaging material is constructed by well known methods,
preferably to provide a sterile, contaminant-free
environment. The packaging material has a label which
indicates that the invention nucleic acids can be used
for detecting CAEV genomic or transcribed mRNA nucleic
acid, thereby diagnosing the presence of CAEV infection.
In addition, the packaging material contains instructions
indicating how the materials within the kit are employed
both to detect a particular sequence and diagnose the
presence of CAEV infection.
The packaging materials employed herein in
relation to diagnostic systems are those customarily
utilized in nucleic acid-based diagnostic systems. As
used herein, the term "package" refers to a solid matrix
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or material such as glass, plastic, paper, foil, and the
like, capable of holding within fixed limits an isolated
nucleic acid, oligonucleotide, or primer of the present
invention. Thus, for example, a package can be a glass
vial used to contain milligram quantities of a
contemplated nucleic acid, oligonucleotide or primer, or
it can be a microtiter plate well to which microgram
quantities of a contemplated nucleic acid probe have been
operatively affixed.
"Instructions for use" typically include a
tangible expression describing the reagent concentration
or at least one assay method parameter, such as the
relative amounts of reagent and sample to be admixed,
maintenance time periods for reagent/sample admixtures,
temperature, buffer conditions, and the like.
The following examples are intended to
illustrate but not limit the present invention.
EXAMPLE I
CHARACTERIZATION OF HIV-1 CROSS-REACTIVITY
IN CAEV INFECTED INDIVIDUALS
This example describes the methods used to
demonstrate that CAEV infected individuals generate an
immune response that is cross-reactive with HIV-1 gp120.
A. Human subjects:
More than 50 human subjects were involved in
the studies disclosed herein. The subjects were
categorized based on their disease status (MCTD or
normal) and their place of origin (Mexico/Central America
or the United States) and were grouped as follows:
CA 02249736 2004-01-21
34
Group A. MCTD - Hispanic born females, clinically
diagnosed with MCTD.
Group B. MCTD - U.S. born black or caucasian females,
clinically diagnosed with MCTD.
Group C. MCTD - Guadalajara female residents, clinically
diagnosed with MCTD.
Group D. Normal (non-MCTD) - Hispanic born females.
Group E. Normal - non-Hispanic males and females,
including two workers in a veterinary service that treats
infected goats.
Group F. Normal - 4 female and 4 male residents of a
village located 30 km from Guadalajara, including the
owner of a herd of 150 goats and 5 members of his family.
Group G. Sclerode,rma patients, all having
anti-Scl-70/type 1 antibodies.
B. Blood Samples and Reagents:
Human and goat (see Section I.F., below) blood
samples were collected and the serum component was used
for immunologic testing. The antigens used in the ELISA
and western blot assays to measure levels of serum
antibodies included crude CAEV and affinity purified
gp135 and recombinant HIV-1 gp120 and HIV-1 p24.
Recombinant HIV-1 gp120 and p24 were purchased from
IntracelT'' (Shepreth UK).
Crude CAEV is prepared as described by Klevjer-
Anderson et al., (Virology 110:113-119 (1981)).
Essentially, CAEV is
grown in primary fetal goat synovial membrane (SM) cells
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grown in DMEM with a bicarbonate-HEPES buffer
supplemented with 2 mM glutamine, 100 mg/mi streptomycin
and 100 units/ml penicillin and containing 10o FBS.
Medium of CAEV infected SM cultures is collected and
5 clarified by centrifugation at 600 x g.
Aliquots of the crude CAEV preparation can be
stored frozen at -70 C. The crude CAEV preparation can be
used to isolate purified CAEV or can be used to prepare
purified CAEV proteins. Purified CAEV proteins such as
10 purified CAEV gp135 also can be prepared by cloning a
nucl'eic acid molecule encoding gp135 (GenBank Accession
NO. M33677; see, also, Saltarelli et al., supra, 1990;
Knowles et al., supra, 1991) into an expression vector
using routine methods of recombinant DNA technology and
15 purifying the gp135 as described below.
CAEV gp135 is affinity purified from the crude
CAEV preparation using an anti-gp135 antibody conjugated
to a chromatography matrix. Essentially, the crude CAEV
preparation is adjusted to 0.1 s SDS to lyse the CAEV
20 viral particles, then applied to an anti-gp135 antibody
affinity chromatography column or recombinantly produced
gp135 is applied to the affinity column.
Anti-gp135 antibody is isolated from CAEV
infected goat serum or from serum obtained from goats
25 immunized with a peptide fragment of CAEV gp135. If a
CAEV infected goat is used as the source of serum, a goat
having a high titer of anti-gp135 antibodies is used as
the source of serum. Such serum can be identified by
using western blot analysis to screen serum samples
30 obtained from several different goats. Anti-gp135
antibodies are isolated from the serum by ammonium
sulfate precipitation, followed by column chromatography
to obtain the IgG fraction. The anti-gp135 antibodies
are attached to a chromatography matrix such as cyanogen
CA 02249736 1998-09-14
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36
bromide activated SepharoseT''. Methods for isolating IgG
antibodies and attaching such antibodies to a matrix are
well known and routine in the art (see, for example,
Harlow and Lane, supra, 1988).
The crude CAEV preparation or recombinantly
produced gp135 in PBS is loaded onto the anti-gp135
antibody affinity column and gp135 is allowed to bind to
the antibodies overnight at 4 C. Following binding, the
column is washed with PBS, then bound gp135 is eluted
with 10 mM HC1, 0.15 M NaCl. Purity of the gp135 antigen
is determined by SDS-PAGE and silver staining.
C. CAEV ELISA and Western Blot Assays:
For ELISA assays, crude CAEV or affinity
purified gp135 antigen was diluted 1:20 with phosphate
buffered saline (PBS; final concentration gp135 approx.
1 g/ml) and 50 l was added to each well of a 96 well
plate (Corning). The plate was covered and incubated
overnight at 4 C, then antigen was removed and the wells
were washed 3x for 1 min each with 90 l washing solution
(1 mg BSA/ml PBS). 100 l blocking solution (10 mg
BSA/ml PBS) was added to each well and plates were
incubated 60 min at room temperature (RT), then the
blocking solution was removed and the wells were washed
3x for 1 min each with washing solution.
Fifty l of diluted serum sample (1:100 in
washing solution) was added to each well and plates were
incubated 60 min at RT. Blank (control) wells contained
washing solution, alone. Following incubation, wells
were washed as above, then 50 l diluted second antibody
was added and incubated as above. Where the serum sample
to be analyzed was from humans, the second antibody was
goat anti-human IgG conjugated to horse radish peroxidase
(HRP; Zymed Laboratories, Inc.; South San Francisco CA;
CA 02249736 2004-01-21
37
cat. # 62-8420) diluted 1:1000 in PBS. Where the serum
sample to be analyzed was from goats, the second antibody
was rabbit anti-goat IgG-HRP (Jackson ImmunoResearch
Labs, Inc.; West Grove PA; cat. # 305035003, lot # 28671)
diluted 1:3000 in PBS. Following incubation, wells were
washed 2x with washing solution, then 2x with PBS.
Seventy-five l color development solution
(10 mg O-phenyldiamine (Sigma) in 25 ml ELISA buffer;
50 l 30% H202, prepared fresh; ELISA buffer is 500 ml
0.1 M citric acid, 500 ml 0.2 M NaZHPO4, adjusted to
pH 5.0) was added to each well and incubated 10 min at RT
in the dark. Color development was terminated by adding
25 l 6N H2SO91 then absorption was measured at OD490 =
For CAEV western blot analysis, 0.1-0.5 g
aliquots of gp135 in SDS sample buffer (Laemmli, Nature
227:680-685 (1970)),
were separated by 10% SDS-PAGE, then
transferred electrophoretically onto a nitrocellulose
membrane. The membrane was incubated for 1 hr in 50 ml
blocking buffer (1% BSA in WBS; WBS is 9 g NaCl, 1.21 g
Tris-base, 0.25 ml NP-40 made up to 1 liter, pH 7.4),
then washed 3x with washing buffer (0.1% BSA in WBS).
The membrane was cut into strips, representing the lanes
on the gel.
Serum samples were diluted 1:100 (20 l
serum/2 ml washing buffer; 40 l/2 ml if plasma used),
then added to an incubation tray containing a
nitrocellulose strip and shaken for 60 min at RT.
Following incubation, the strips were washed 3x with
washing buffer, then 2 ml second antibody (as above, but
diluted with washing buffer) was added and incubation
continued for 60 min at RT. Strips were washed 3x with
washing buffer and the enzyme reaction was initiated by
adding 2 ml color development solution (10 mg 3,3-
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38
diaminobenzidine tetrahydrochloride (Sigma Chemical Co.;
St. Louis MO; cat. # D 5905), 1 ml 0.05 M Tris, pH 7.5,
39 ml WBS, 80 l H202, prepared fresh) . Incubation was
continued from 10 min at RT with gentle shaking, then
color development was terminated by transferring the
membrane to distilled water.
High reactivity to extracts of CAEV and to
purified gp135 was observed in 62% of the serum samples
obtained from Hispanic born MCTD patients (Table 2),
including those from LAC/USC (group A) and those from
Guadalajara, Mexico (group C). Serum samples from the
disease control (group G) and from non-Hispanic healthy
individuals, excluding the two veterinarians, were weakly
reactive or non-reactive. The human serum samples that
reacted to CAEV on western blots contained antibodies to
the same polypeptides as those reacting with serum
samples obtained from CAEV infected goats.
The reactivity to CAEV of serum samples from
the normal individuals correlated with a history of
drinking raw goat milk or other exposure to goats. The
highest reactivities were present in the owner of the
Guadalajara goat herd and in one of the veterinarians,
who, in addition to treating CAEV infected goats, also
reported regularly consuming raw goat milk. The second
veterinarian, who also treated CAEV infected goats, also
had positive reactivity.
In summary, 22 of 39 MCTD patients and 11 of 23
non-MCTD individuals had positive reactivity for CAEV
infection, including 20 of 33 Hispanic MCTD patients
(61%) and 9 of 14 Hispanic non-MCTD individuals (64%; see
Table 2). These results indicate that over 60% of the
Hispanic individuals examined in this study were infected
with CAEV. In the CAEV reactive individuals, the
reactivity of serum obtained from Hispanic MCTD patients
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was higher than that of the non-MCTD Hispanic
individuals.
Table 2
The relationship between CAEV gp135 and HlV-1 gp120 reactivity in MCTD
patients and normals
QQsitive an13 negative 00135
1. MCTD 3.MCTD
n=22; hispanic=20 n= 17; hispanic= 13
.total gp120(+) 12.= 55% total gp120(+) 0 = 0%
22 17
hispanic gp,12D(+) u= 55% his anic 120 + 0%
P 9P ().9= 0%
20 13
2.NL 4.NL
n=1 1; hispanic =9 n=9; hispanic=5
total gp120(+) --4-= 36% total gp120(+) Q= 0%
11 g
hispanic gp120(+) ,_2 = 22% hispanic gp120(+) 0%
9 5
Summary 1: CAEV(+)
Total Hispanic 29/47 = 62%
MCTD - hispanic 20/33 = 61 %
NL - hispanic 9/14 = 64%
Total Nonhispanic 2/15 = 13%
MCTD - nonhispanic 2I6 = 33%
NL - nonhispanic 2/6 = 33%
Summary 2: CAEV(+)/gp120(+)
hispanic MCTD 12/22 = 55%
hispanic NL 219 = 22%
CAEV(-)!gp 120(+)
hispanic MCTD and NL 0/20 = 0%
non-hispanic MCTD and NL 0111 = 0%
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D. PCR analysis:
PCR analysis for identification of CAEV gag and
pol genes was performed using genomic DNA isolated from
peripheral blood mononuclear cells (PBMC). PBMC were
5 obtained by Ficoll density gradient centrifugation of
whole citrated or heparinized blood collected from
selected human individuals and from WA, UC Davis and Gua
goats (see below).
CAEV-specific genomic DNA sequences encoding
10 regions of the gag protein were amplified in two stages.
The first stage produced amplification products
representing nucleotides 1057 to 1553 of CAEV. Primers
were 5'-GCAGTTGGCATATTATGCTACTAC-3' (SEQ ID NO: 14; CAEV
nucleotides 1057-1080) and 51-CTTGTTGTACTCTTTGTCCTAGTG-3'
15 (SEQ ID NO: 15; nucleotides 1530-1553). The second stage
produced amplification products within the product of the
first amplification product, from nucleotides 1329 to
1501. The primers for the second stage were
5'-GAGCAGTAAGACATATGGCGCAC-3' (SEQ ID NO: 16; nucleotides
20 1329-1351) and 5'-TGATGCATTTGTATAAGATAGTGTTAGCTTT-3' (SEQ
ID NO: 17; nucleotides 1471-1501).
DNA encoding the CAEV pol also was examined by
PCR analysis. Primers for the first stage of
amplification were 5'-GGATTTGAACTACACCCGCAG-3' (SEQ ID
25 NO: 18; CAEV nucleotides 2845-2865) and
5'-CCTGTTGATACTATGAACCCTAGAC-3' (SEQ ID NO: 19;
nucleotides 3494-3518); primers for the second stage were
5'-AAGAACCTAAGCATCCCGCAAC-3' (SEQ ID NO: 20; nucleotides
3223-3244) and 5'-GTGATGTTCCCTAATTGCAATTCTAGTC-3' (SEQ ID
30 NO: 21; nucleotides 3341-3368).
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Amplifications were performed using an
annealing temperature of 52 C and an elongation
temperature of 72 C and were allowed to proceed for
38 cycles. Taq polymerase or pfu Taq polymerase (Promega
Corp.; Madison WI) was used as recommended by
manufacturer. One l of the reaction mixture from the
first stage of amplification was used for the second
stage of amplification, which was performed under
identical conditions. The products following the second
stage of amplification were separated by agarose
electrophoresis and collected by eluting the appropriate
band. The amplified DNA samples then were cloned into a
TA3 pCRT"'vector (Invitrogen; La Jolla CA) and sequenced
using a SequenaseT"' Version 2.0 DNA sequencing kit (U.S.
Biochemical Corp.; Cleveland OH) as recommended by the
manufacturer. The cloned DNA sequences were compared to
a reference CAEV DNA sequence (GenBank Accession No.
M33677).
PCR analysis revealed that CAEV proviral DNA
was present in genomic DNA of individuals that were CAEV
positive as determined by ELISA or western blot analysis.
Specifically, DNA encoding CAEV gag and pol sequences was
amplified from genomic DNA of an MCTD patient (group A)
and from the CAEV infected UC Davis goat. Comparison
with the reference CAEV gag sequence demonstrated that
the gag sequence in the MCTD patient diverged from the
reference sequence by 8.2% and that the gag sequence in
the UC Davis goat diverged by 8.8%. In addition,
comparison with the reference CAEV pol sequence
demonstrated that the CAEV pol sequence in the MCTD
patient diverged by 7.6% and the CAEV pol sequence in the
UC Davis goat diverged by 7.6%.
The relatively high degree of similarity
between the CAEV sequences present in the human genomic
DNA samples and the reference goat CAEV sequence provides
CA 02249736 1998-09-14
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definitive evidence of CAEV infection in humans. The
average divergence of about 8% between the sequences
indicates that the human CAEV infection can be due to a
distinctive, human-adapted CAEV strain. Additional DNA
sequencing can clarify whether a human-adapted CAEV
strain is responsible for human CAEV infection.
To obtain further direct evidence for the
presence of CAEV in human PBMC, viral gag and pol gene
sequences were amplified by nested PCR from the DNA of 8
gp 135(+) subjects. CAEV gag was also amplified from
cell-free plasma of the individual human subject Hmc4 by
RT-PCR. Efforts to amplify CAEV gag and pol sequences
from PBMC DNA of 11 gp 135(-) individuals were negative.
All DNA samples used for CAEV gag and po1 PCR were also
subjected to PCR using HIV-1 gag and pol primers, and all
were HIV-1 negative, except for the HIV(+) control group.
CAEV gag sequences were also amplified from PBMC DNA of 4
gp 135(+) goats. Two CAEV gpl35(+) goats (gDl and gWA19)
were from U.S. veterinary herds; gT38 and gT42 were
Mexican gp135(+) goats. Twenty-two human PBMC gag
clones, 3 HMC4 plasma gag clones and 11 goat PBMC gag
clones were sequenced and compared to the published
sequence of CAEV-CO gag (M. Saltarelli, et al., Viroloav,
179:347 (1990).
A phylogenetic analysis of 8 human gag
sequences amplified from 7 individuals is shown in Fig.
1. Also included are 4 goat gag sequences. Clones and
the corresponding Genbank accession numbers for CAEV,
Visna and OMVVSA gag sequences analyzed in Figures 1 and
2 are: Hmc4-c30(U69922), -c39(U69923), -c40(U69924),
-c46(U69925), -c49(U69932), -c50(U69926),
-fp-c254(U69933), -fp-c256(U69934), -fp-c261(U69935);
gD1c12(U69927), -c13(1369928), -c52(U69929), -c53(U69930);
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43
Tn8-c144 (U69931), -c147(U69909), -c151(U69910),
-c155(U69911); gWa19-c74(U69918), -c75(U69919),
-c77(U69920), -c80(U69921); gT42-c84(U69916),
-c90(1369917); gT38-c93(U69915); Enl-c108(U69900),
-cllO(69901); Ocl-c120(U69905), -c136(U69906),
-c139(U69907); Oc2-c167(U69908); Umc5-c184(U69912),
-c185(U69913), -c190(U69914); Hmcl-c209(U69902),
-c212(U69903), -c213(U69904); CAEV-CO (M33677); Visna
(M18039); OMVVSA (M31646). All other sequences in Figure
1 are lentiviral sequences with exception of JSR, which
is from the Jaagsiekte sheep retrovirus. In addition,
CAEV pol clones and accession numbers corresponding to
pol sequences discussed in the text are: Hmc4-
c10(U69948),-c12(U69949), -c19(U69950), -c20(U69951),
-c28(U69952); gDl-cl(U69944), -c2(U69945), -c3(U69946),
-c30(U69947); gWa19-c51(1369953), -c52(U69954),
-c53(U69955), -c60(U69956); Enl-c43(U69940),
-c46(U69941), -c47(U69942), -c48(U69943);
Gmc15-c37(U69936), -c38(U69937), -c41(U69938),
-c42 (U69939) .
The relative distances of human CAEV gag
sequences from CAEV-CO gag and several other lentiviral
gag sequences are made apparent using inversely weighted
parsimony (D.M. Hillis et al., Science, 264:671 (1994).
B.T.M. Korber et al., J. Virol., 68:6730 (1994)).
Unweighted (ordinary) parsimony was performed using PAUP
3.1.1 (D.L. Swofford, Illinois Natural History Survey,
Urbana). The twelve distinct substitution frequencies
from the initial PAUP analysis were then ascertained
using Macclade (W.P. Maddison and D.R. Maddison,
"Macclade: Analysis of phylogeny and character
evolution", Sinauer Associates, Inc., Sunderland,
(1992)). An inverse weighting scheme was derived from
this matrix of substitution frequencies which was then
introduced into a subsequent parsimony analysis. The
resulting branch length estimates are compound quantities
CA 02249736 1998-09-14
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44
generated from the matrix of inversely weighted
substitutions. All viral gag sequences derived from
experimental subjects and the goat gag sequences
clustered with CAEV-CO gag.
Simple nucleotide distance measurements, i.e.
Hamming distances, were determined using the SIMILARITY
function of MASE (D.V. Faulkner and J. Jurka, Trends
Biochem. Sci., 13:321, (1988)). Only single nucleotide
differences were scored, gaps were not encountered in
these data sets. By Hamming distance measurement, the
range of sequence divergence of the 8 human gag clones
from CAEV-CO gag was 5.3 - 9.4%, compared to 23.4 - 28%
divergence from the published sequence of ovine maedi-
visna gag (P. Sonigo et al., Cell, 42:369 (1985)).
To further assess inter- and intra-individual
variation in human and goat gag sequences, an evaluation
of 33 clones was performed by ordinary parsimony (Fig.
2). As in Fig. 1, all human sequences clustered with
CAEV-CO. The mean sequence divergence of all 22 human
gag clones from CAEV-CO was 8.4 1.5%.
Viral sequences from 3 human subjects (Hmc4,
Gmc15 and Enl) and 2 goats (gDl and gWAl9) were also
amplified from PBMC DNA using CAEV nested pol primers.
The poI sequences were 4.8 - 8.2% divergent from the
published CAEV-CO pol sequence (M. Saltarelli, et al.,
Virology, 179:347 (1990)) and 22.6 - 26.0% divergent from
ovine maedi-visna pol (P. Sonigo et al., Cell, 42:369
(1985)). Parsimony analysis of these pol sequences
yielded a topology consistent with that of gag (Fig. 1).
Approximately 40% of gag and 17% of poI nucleotide
changes resulted in amino acid changes. All of the pol
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changes and >80% of the gag changes were conservative
(principally I4---V, K+--R and D+-E) . Thus, evaluation of
gag and pol sequences indicates that 8 CAEV gp 135(+)
human subjects examined in this study harbor a ruminant
5 lentivirus closely related to CAEV. Evidence for in vivo
replication of this agent as described herein in Example
II was obtained by RT-PCR amplification of CAEV gag
sequences from cell-free plasma of subject Hmc4 (Fig. 2).
R. HIV-1 ELISA and Western Blot Assays:
10 Certified HIV-1 ELISA and western blot
diagnostic kits were purchased from Organon Teknika
(Cambridge UK) and the assays were performed as
recommended by the supplier. Based on the clinical
criteria using the certified HIV-1 ELISA and western blot
15 assays, all of the human serum samples were negative for
HIV-1, except for known HIV-1 positive sera and
antibodies used as positive controls.
ELISA and western blot assays were performed
using the individual recombinant HIV-1 antigens, gp120
20 and p24, and HRP-conjugated rabbit anti-goat and goat
anti-human second antibodies (Zymed Laboratories Inc.) as
previously described (Douvas and Takehana, supra, 1994;
Crow et al., Cell. Immunol 121:99-112 (1989), which is
incorporated herein by reference). Subsets of sera that
25 reacted with CAEV gp135 also reacted with HIV-1 gp120,
including a total of 12 MCTD sera reacted with HIV-1
gp120 (see Table 2, supra).
As discussed in Section I.C., above, 22 of 39
MCTD patients and 11 of 23 non-MCTD individuals were
30 positive for CAEV infection, including 20 of 33 Hispanic
MCTD patients (61%) and 9 of 14 Hispanic non-MCTD
individuals (64%; Table 2). The results of the HIV-1
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46
assays further indicated that 12 of the 22 CAEV positive
Hispanic MCTD patients (55 s) and 2 of the 9 CAEV positive
Hispanic non-MCTD individuals (22%) also showed positive
reactivity to HIV-1 gp120 (see Table 2). In contrast,
none of the CAEV negative Hispanic MCTD or non-MCTD
individuals was positive for HIV-1 gp120.
These results indicate that CAEV infection of
otherwise healthy individuals occurs without the
concomitant development of clinical disease, including
without developing symptoms of MCTD. However, exposure
to CAEV results in the development of an immune response
to CAEV that generalizes to HIV-1 in a subset of infected
individuals. In addition, in CAEV positive MCTD
patients, the reactivity to CAEV is about 1.6 times
stronger than in CAEV positive non-MCTD individuals and
the frequency of cross-reactivity to HIV-1 gp120 is more
frequent. These results demonstrate that cross-
reactivity to HIV-1 develops as a result of infection
with CAEV and that the cross-reactive immune response is
increased in an individual with an autoimmune disease.
F. CAEV Infected Goats:
Three groups of goats were studied as follows:
Group I. WA - uninfected goats and experimentally
infected (CAEV) goats maintained at Washington State
University, Pullman WA.
Group II. UC Davis - a naturally infected (CAEV) goat
maintained at the University of California, Davis CA.
Group III. Gua - 20 naturally infected (CAEV) goats
belonging to a goat herder in Guadalajara Mexico (see
Group F, above).
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Serum samples collected from the various CAEV
infected or uninfected goats were examined. The Gua
goats (Group III) lacked reactivity to CAEV gp135 but
about 50% were reactive against CAEV gp38 and gag. In
addition, the Gua goats, but not the WA goats showed
strong reactivity to HIV-1 gp120 and p24. The UC Davis
goat was reactive against both gp135, gp120 and p24.
These results demonstrate that differences
exist between the immunologic reactivities of the WA and
Gua goats to CAEV and to HIV-1 antigens. These
differences may derive from different viral strains or
may be due to the different routes of infection. For
example, the WA goats were infected intravenously,
whereas the Gua goats and the UC Davis goat were
naturally infected via ingestion of infected milk.
G. HIV-1 Viral Neutralization Assay:
Virus neutralization assays are performed in
48 well tissue culture plates using PHA-activated normal
donor PBMC maintained in growth medium at a concentration
of 1 x 106 cells/mi as targets for infection. Various
dilutions of HIVIGT'', heat-inactivated (56 C, 30 min) MCTD
sera or purified IgG are incubated with 10 or 50 TCIDso
(tissue culture infecting dose-50) of virus for 30 min
at 37 C.
Negative controls for the MCTD sera and for
HIVIGT' include heat inactivated serum from individuals
not infected by HIV-1 and not affected by a systemic
rheumatic disorder, and IVIGT''', respectively. HIVIGT"',
which is a neutralizing IgG preparation from pooled serum
from 9 seropositive donors in early stages of HIV-1
infection, and IVIGT"', which is IgG from normal donors,
are obtained from North American Biologicals Inc. (Miami
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FL}. TCID50 is determined using routine methods of viral
titering.
Following incubation of the serum or IgG and
the virus, serum/IgG-virus mixtures are added to the
target cells in triplicate. After an overnight
incubation at 37 C, the plates are centrifuged at 1000
rpm for 5 min and the medium is completely replaced.
This washing step is repeated 3x to ensure complete
removal of the serum/IgG-virus inoculum. On day 4 of
culture, the medium is changed and on day 7 the
supernatant is harvested for quantitation of p24 core
antigen. Percent inhibition is determined by comparing
the mean p24 core antigen concentration in each serum/IgG
treated tissue culture well with that of the specific
negative control.
In parallel, a control is prepared to measure
any non-specific reduction in the amount of p24 core
antigen measured due to a serum or HIVIGT"' not being
completely removed by the washing steps. At the time
that the serum/IgG-virus mixtures are added to the target
cells, each serum, IVIGT"' or HIVIGT'"' also is added to
target cells without preincubation with virus. These
wells are treated in parallel with the experimental
cultures. After 7 days of culture, the supernatant from
each well is mixed 1:1 with that of the negative control
and the p24 antigen concentration is quantitated. A
measurement that is less than 50 s the value of the
negative control is considered non-specific inhibition of
p24 antigen, which can be due to the presence of residual
p24 antibodies.
The results of such an experiment are shown in
Table 3. Table 3 indicates that anti-RNP sera from MCTD
patients was able to neutralize infection by HIV-1
strains MN, JR-FL, and CO. Sample # 1 in Table 3
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corresponds to the CAEV(+)(i.e., CAEV-infected) patient
Hmcl. Sample # 6 in Table 3 corresponds to the CAEV(+)
patient Hmc4. The patient corresponding to sample # 4 in
Table 3 was also determined to be CAEV(+). The patients
corresponding to samples 2, 3, 5 and 7 could not be
assessed for CAEV-infectivity. Thus, since samples 1, 4
and 6 correspond to anti-CAEV sera from humans, it is
apparent that human anti-CAEV sera neutralizes HIV-1
infection.
TABLE 3
NEUTRALIZATION OF HIV-l STRAINS BY ANTI-RNP SERA
Strains
Antibody UK JR-FL
a. anti-RNP ~ neutralized
1 99 77 86
2 71 71 -
3 52 80 -
4 72 55 37
5 9 79 -
6 28 60 31
7 18 - -
8 0 - -
9 0 - -
b. HIV-1(+)
Pt. M 99 30 -
HIVIGtm 93 94 54
c. Normal
NL-1 11 - -
NL-2 8 - -
NL-3 1 - -
IVIGtm 19 20 -
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EXAMPLE II
In vitro infection of human PBMC with PCR(+) plasma from
a human sub.-j ect
This example demonstrates the ability of h-CAEV
5 to infect human PBMC cells. A comparison of rescued CAEV
gag sequences will be presented.
Cultures of 0.8 million human PBMC from a donor
who was negative for CAEV infection, as determined by PCR
(i.e., PCR-), were inoculated with 100 l of plasma from
10 patient Hmc4, who was shown to be positive for CAEV
infection (PCR+), in a total volume of 1 ml. After 4
days, the genomic DNA, cellular RNA and culture
supernatant RNA were extracted and analyzed by PCR and
RT-PCR using CAEV gag primers, as was 100 l of the
15 original plasma, diluted to 1 ml. Agarose gels of the
PCR reactions show CAEV gag signals in the genomic DNA,
RNA and culture supernatants, while levels of CAEV in the
inoculating plasma of the patient Hmc4 were below the
level of detection. This indicates that CAEV
20 productively infected human PBMC cells. A similar
infection of human PBMC with PCR(+) plasma from a human
subject designated Hmcl was performed with similar
results.
CAEV virus was rescued from infected cell
25 cultures in the above-described experiment. The
sequences rescued from the Hmc4 experiment are compared
to those of the genomic DNA and plasma RNA from the same
subject. CAEV gag sequences cloned from the infected
PBMC match those derived from the concentrated plasma of
30 patient Hmc4 as shown in Figure 3. Figure 3 compares the
following Hmc4-related CAEV gag clones, derived by PCR or
RT-PCR: Hmc4 genomic clones g-30, g-39, g-40, g-46 and
g-50; Hmc4 plasma clones p254 and p-256; clones from the
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51
infection experiment i-238 and i-239. The "i" clones
were derived from the cytoplasm of infected cultures.
The boxes indicate that there are certain similarities
between all of the "p" and "i" clones, and genomic clone
g-30 from Hmc4, suggesting that there is a subset of
particularly viable forms in this patient.
From the data presented in Figure 3, it is
readily apparent that CAEV subtype that infects humans is
distinct from the reference goat-infecting CAEV M33677
(also referred to herein as "CAEV-CO"). For example,
within the relatively small genomic region corresponding
to the 173 nucleotide fragment amplified by primers set
forth in SEQ ID NOs: 16 and 17, it has been found that
the human-CAEV virus has at least two mutations that are
uniquely human in origin. These mutations correspond to
a guanine at position 56 and a cytosine at position 77
within the 173 gag-region amplification product in the
Hmc4 clone.
Figure 4 shows a sequence comparision of a PCR
amplified 146 nucleotide POL gene region corresponding to
nucleotides 3223-3358 of the goat virus CAEV, strain 79-
63. The reference strain CAEV-CO comes from the brain of
a goat. The brain is an immunologically privileged site.
Therefore, the evolution of the virus was different from
virus isolated from joint fluid of goats with arthritis.
gWa19 was infected with virus from joint fluid, a strain
known as CAEV 79-63. The evolution of the human forms
should be most similar to CAEV-63. Figure 4 compares a
reference goat CAEV-CO, the human-CAEV isolated from Hmc4
and a goat gWal9 CAEV-63 virus. The differences between
the goat infecting viral sequences versus the human-
infecting CAEV virus in Figure 4 are readily apparent.
Thus, novel human-CAEV lentivirae capable of infecting
humans are contemplated herein.
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Figure 5 shows a direct sequence comparison of
the goat-derived CAEV strains CAEV-CO and gWa19 (i.e.,
CAEV-79-63) and the human-derived h-CAEV strains Hmcl and
Hmc4 for the same 173 nucleotide region set forth in
Figure 3. The percentage divergence between CAEV virus
isolated from human (human-CAEV) and goats is summarized
in Table 4. Hmci and Hmc4 correspond to human-CAEV,
whereas gWa19, CO and g147 correspond to goat-CAEV.
TABLE 4
% DIVERGENCE
gag (173 nucleotides)
% max % min # of clones
Hmc4/gWal9 4.0 1.7 7/1
Hmcl/gWa19 8.7 1.2 10/1
Tn8(g147)/gWal9 1.7 1.7 1/1
pol (146 nucleotides)
Hmc4/gWal9 8.2 2.1 5/4
gWa19/Co 4.0 4.0 1/1
(Uniquely human gag mutations include nucleotides at
positions 56 and 77).
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53
E7CAMPLE III
USE OF A CAEV VACCINE TO VACCINATE AN INDIVIDUAL
This example describes a method of
administering a CAEV vaccine to an individual in order to
stimulate an immune response against CAEV.
Human-CAEV virus is prepared by inoculation of
PBMC cells (see Example II) or monocyte cells. Cell
supernatants are harvested and CAEV virions are pelleted
by centrifugation at 150,000 x g at 4 C to obtain a
sterile suspension of approximately 2 x 108 pfu/ml,
containing 50% glycerol as a diluent (see Graham et al.,
J. Infect. Dis. 166:244-252 (1992)).
Recipients of live virus are
vaccinated intradermally, using a sterile bifurcated
needle, with 50 l CAEV suspension to a single skin site.
Vaccinations also can be performed using a killed or
attenuated CAEV preparation or other CAEV immunogen such
as CAEV gp135 (see, also, Table 1, supra). A sterile
transparent dressing is applied to the vaccination site,
then removed after a crust is formed.
Blood samples are drawn on or about days 14, 28
and 54 after administration of the CAEV immunogen and
evaluated to determine antibody titers (humoral response)
or T helper or cytotoxic T cell immune response (cellular
response). Methods for examining the humoral and
cellular responses are disclosed in Example I or
otherwise well known in the art (see, for example, Egan
et al., J. Infect. Dis. 171:1623-1627 (1995);
see, also, Harlow and
Lane, supra, 1988). If desired, a secondary (booster)
immunization is administered on or about day 56 after the
initial vaccination. Additional evaluations of humoral
and cellular responses are made on or about days 70, 90,
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54
160, 180, 270 and 355 after the initial vaccination. If
desired, a tertiary (booster) immunization is
administered on or about day 365.
For administration of a CAEV immunogen that is
a haptenic peptide, approximately 1 mg of peptide is
conjugated to keyhole limpet hemocyanin and administered
with an adjuvant, intradermally, as above, in a volume of
about 0.1 ml. Evaluations of the humoral and cellular
response are made as above and, if desired, secondary or
tertiary immunizations are administered.
Although the invention has been described with
reference to the examples above, it should be understood
that various modifications can be made without departing
from the spirit of the invention. Accordingly, the
invention is limited only by the following claims.
CA 02249736 1999-02-02
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: University of Southern California
(ii) TITLE OF INVENTION: Caprine Arthritis-Encephalitis Virus
Provides Immunoprotection Against HIV-1 Infection
(iii) NUMBER OF SEQUENCES: 21
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Smart & Biggar
(B) STREET: Box 11560, Vancouver Centre,
2200 - 650 West Georgia Street
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(D) SOFTWARE: Notepad
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,249,736
(B) FILING DATE: 14-MAR-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/616,855
(B) FILING DATE: 15-MAR-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/616,854
(B) FILING DATE: 15-MAR-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Smart & Biggar
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(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (604) 682-7295
(B) TELEFAX: (604) 682-0274
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02249736 1999-02-02
56
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Glu Arg Lys Arg Glu Gly Phe Thr Ala Gly
1 5 10
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Ser His His Gly Asn Asp Ser Arg Arg Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ser Arg Arg Arg Arg Arg Lys Ser
1 5
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Arg Lys Glu Thr Gly Thr Leu Gly Gly
1 5
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(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Arg Lys Lys Arg Glu Leu Ser His Lys Arg Lys Lys Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Arg Met Gln Arg Lys Glu Arg His Lys
1 5
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Val Arg Ser Ser Tyr Gly Ile Thr Ser Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02249736 1999-02-02
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(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Gly Arg Ile Lys Leu Gln Gly Ile Gly Gly
1 5 10
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Gln Glu Ile Leu Glu Asp Trp Ile Gln Gln
1 5 10
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Lys Arg Ile Asn Asn Lys Tyr Asn Lys Asn Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Asp Gly Leu Leu Glu Gln Glu Glu Lys Lys
1 5 10
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(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ser Val Phe Pro Ile Val Val Gln Ala Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Ala Ile Asp Ala Glu Pro Thr Val
1 5
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GCAGTTGGCA TATTATGCTA CTAC 24
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CA 02249736 1999-02-02
CTTGTTGTAC TCTTTGTCCT AGTG 24
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GAGCAGTAAG ACATATGGCG CAC 23
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
TGATGCATTT GTATAAGATA GTGTTAGCTT T 31
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GGATTTGAAC TACACCCGCA G 21
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
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CCTGTTGATA CTATGAACCC TAGAC 25
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
AAGAACCTAA GCATCCCGCA AC 22
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GTGATGTTCC CTAATTGCAA TTCTAGTC 28
