Link: Pharm/Biotech Resources
Title: Method and tool for prognosticating HIV infection
in a subject by measuring soluble urokinase plasminogen activator
receptor, degradation products thereof, and urokinase plasminogen
activator receptor
United States Patent: 6,902,884
Issued: June 7, 2005
Inventors: Eugen-Olsen; Jesper (Hellerup, DK)
Assignee: Virogates ApS (Kbh K, DK)
Appl. No.: 148492
Filed: November 27, 2000
PCT Filed: November 27, 2000
PCT NO: PCT/DK00/00651
371 Date: August 22, 2002
102(e) Date: August 22, 2002
PCT PUB.NO.: WO01/38871
PCT PUB. Date: May 31, 2001
Abstract
Method of diagnosing and/or prognosticating HIV infection in a subject comprising the steps of: (a) performing in vitro a measurement of the level of a marker in the form of (i) urokinase plasminogen activator receptor (uPAR), (ii) soluble urokinase plasminogen activator receptor (suPAR), (iii) urokinase-type plasminogen activator (uPA), (iv) one or more degradation products of (i), (ii), or (iii), and/or (v) an mRNA for (i), (ii) or (iii), in a biological fluid sample from a subject, and (b) using the measurement value obtained to evaluate the state of the subject.
Description of the Invention
TECHNICAL FIELD OF THE INVENTION
The present invention concerns the diagnosis and/or prognosis of HIV infection. More particular it concerns the measurements of the concentration of soluble urokinase plasminogen activator receptor (suPAR) in human biological fluids (sputum, cystic fluid, ascites, serum, plasma, urine) as a tool of diagnosing HIV infection as well as the prognosis of disease progression.
BACKGROUND OF THE INVENTION
The cellular receptor for urokinase (uPAR, CD87) plays multiple functions
in cell migration, cell adhesion, pericellular proteolysis and tissue
remodeling [Blasi, 1997]. uPAR is expressed by most leukocytes including
monocytes, macrophages, neutrophils and platelets. uPAR is an activation
antigen in monocytes and T cells [Min, 1992][Nykjaer, 1994] and T-cells from
HIV-1 infected individuals express elevated levels of uPAR [Nykjaer, 1994].
HIV-1 infection of leukocytes in vitro causes up-regulation of uPAR cell
surface expression in a process which appear to be coordinated temporally
with the onset of viral replication [Frank, 1996], [Speth, 1998].
uPAR may be shed from the cell surface generating a soluble form of the
receptor (suPAR) lacking the GPI-anchor. The shedding mechanism is poorly
understood but may occur by GPI-specific phospholipase D catalyzed
hydrolytic cleavage of the GPI-anchor (Wilhelm, 1999). Soluble forms of uPAR
(suPAR) has been identified in cell culture supernatants and in diverse
biological fluids such as tumor ascites, cystic fluid, serum, plasma and
recently also in urine [Pedersen, 1993], [Rønne, 1995], [Stephens, 1997], [Sier,
1998], [Chavakis, 1998], [Stephens, 1999], [Wahlberg, 1998], [Sier, 1999].
Serum, plasma and urine levels of suPAR are elevated in patients suffering
from different types of cancer [Stephens, 1997], [Sier, 1998], [Stephens,
1999], [Sier, 1998], the paroxysmal nocturnal hemoglobinuria syndrome (PNH)
syndrome [Rønne, 1995], [Ninomiya, 1997], and in rheumatoid arthritis
patients [Slot, 1999]. The plasma level of suPAR is furthermore a prognostic
marker for overall survival in patients suffering from ovarian and
colorectal cancer [Sier, 1998], [Stephens, 1999] and for the response to
therapy in leukemia [Mustjoki, 1999].
The cellular origin of circulating suPAR is not known. Many, if not all,
cells which express uPAR also shed soluble forms of the receptor when
cultured in vitro. The source of excess serum suPAR in cancer patients has
been suggested to derive from the cancer cells and/or tumor-infiltrating
macrophages as these cells often express high levels of uPAR [Stephens,
1997] and experiments using xenografted mice carrying human tumors have
indeed demonstrated that the tumor tissue does release suPAR to the
circulation and urine [Holst-Hansen, 1999].
Persons infected with HIV-1 probably represents the single most well
medically monitored group of patients ever in history. Despite these very
intensive studies only few prognostic markers, providing long-term
predictions of clinical outcome, have proven strong enough to obtain broad
clinical acceptance. Currently used prognostic markers for disease
progression in untreated HIV-1 infected patients are essentially restricted
to the blood concentration of CD4 positive lymphocytes (the CD4 cell count),
the plasma level of HIV-1 RNA (the viral load), and the age of the patient.
CD4+ T cell counting is a general marker of immune deficiency, whereas HIV
viral load provides direct information about viral replication. Although
these two parameters are based on different approaches, they both provide
information about the expected clinical outcome and prognosis of the
patient. In specific cases one of the two assays is more useful than the
other, which is the reason that both markers are found to be independent
parameters in multivariate analysis. Hence, preferably both markers should
be used together. Other potential prognostic parameters like age of the
patients or β2 microglobulin level give additional information, but usually
their significance decrease if they are used in multivariate analyses
together with CD4+ or HIV viral load.
After the introduction of highly active anti-retroviral therapy (HAART)
therapy, viral load is dramatically reduced and CD4 T cell counts become
stable, which decreases the clinical use of these markers for
prognostic/diagnostic purposes. Hence, there is a strong need for novel
markers useful for monitoring patients in HAART therapy.
Measurements of CD4 cell counts and HIV-1 viral load are expensive and
require equipment (flow-cytometers and PCR-machines) which is unlikely to be
affordable for wide use in developing countries such as Uganda, Rwanda and
Namibia. In these countries, around 30% of individuals between the age of 20
and 49 years are infected (October 1999 data).
SUMMARY OF THE INVENTION
The technical problem addressed by the present invention is to provide a
novel marker for diagnosing and prognosticating HIV infection. A further
technical problem addressed by the present invention is to provide a marker
of the said type, which is simple and affordable to measure.
The present invention has provided a solution to the above technical
problems, the invention being directed to a method of diagnosing or
prognosticating HIV infection in a subject comprising the steps of
The invention is based on the discovery that soluble uPAR (suPAR) is present in elevated levels in serum, plasma and urine of HIV infected individuals, and that the level of suPAR is useful as a diagnostic marker. Also, the level of suPAR in HIV infected individuals is prognostic for the development of AIDS, the CD4 T cell decline, and death. The suPAR level is a novel and highly diagnostic and prognostic factor, even in the context of other known prognostic factors related to HIV disease. In fact, in various multivariate analyses including all presently known relevant survival-related parameters, suPAR was the second strongest parameter after CD4 T-cell count. In a comparative analysis against CD4, suPAR kept its highly significant prognostic value (Hazard Rate (HR) 2.5, P<0.001), whereas the other markers became less significantly associated with survival.
Moreover, the present invention is based on the discovery that, at least for some HIV patients, not only was the total suPAR level enhanced, but there was also a change in the ratio between various degradation product variants of suPAR. The pattern of these variants was clearly different in some subgroups of patients compared to healthy blood donors. Therefore, the measurement of one or more degradation product variants of suPAR may also be used as a marker for diagnosing and prognosticating HIV infection.
Furthermore, the present invention is based on the recognition that the amount of suPAR is correlated to the amount of uPAR as well as to the amount of uPA, and that therefore the amounts of uPAR and uPA are equally suitable as diagnostic and prognostic indicators of HIV infection.
Finally, when uPAR, suPAR and uPA are present at high levels, i.e. expressed at high levels, this means that mRNA for the said proteins are present at high levels, and therefore said mRNA are equally suitable as diagnostic and prognostic indicators of HIV infection.
A further advantage of the invention is that measurement of suPAR can be performed using e.g. a simple ELISA technique or even a stick and may therefore provide a very inexpensive, simple and quick supplement to the currently used prognostic tools for HIV-1 infected persons. Thus, in developing countries without the financial possibility to carry out the costly assays used in the western world, suPAR levels could be used 1) to determine HIV status (diagnosis), 2) to select patients for treatment (prognosis), and 3) to monitor the progress of treatment.
Furthermore, the present invention involves the advantage that while suPAR levels also can be measured in a urine sample, a blood sample is necessary for measurement of CD4 counts and HIV viral load, and of course a urine sample is much more easy to obtain.
The invention further relates to a method of evaluating the progression of the state of a subject suffering from HIV infection comprising the steps of
This method may be used to continuously monitor the state of the patient.
DETAILED DESCRIPTION OF THE INVENTION
In the following, the invention is explained in particular with reference to
suPAR for reasons of simplicity. This should not be understood as a
limitation of the scope of the present invention to suPAR. Furthermore,
suitable extrapolations to uPAR, uPA and the degradation products of all the
said three substances lie well within the skill of a person skilled in the
art.
The inventors have surprisingly found that the concentration of suPAR, a
molecule that is in general known to be involved in cell migration and
adhesion, is increased in serum and urine from HIV-infected persons compared
with healthy controls. Enhanced serum suPAR levels detected 91.6% of the
HIV-patients (n=191) with a specificity of 93.3%, cf. Example 1. Moreover,
subgroups of patients with low, intermediate and high levels of suPAR,
respectively, showed significant differences in survival, cf. Example 2.
Patients in the intermediate suPAR group had an enhanced hazard ratio (HR)
of 2.17, whereas high suPAR serum levels were even more hazardous (HR 3.29,
see Table 1 below). The prognostic value of suPAR stayed significant even
when it was tested in a multivariate analysis against the strongest known
parameters presently available. In fact, suPAR was found to be the second
strongest prognostic factor, directly after CD4+ C-cell counts, and even in
a direct comparison with CD4 (or any of the other parameters) suPAR kept its
independent prognostic value (P<0.001). These results suggest that the serum
levels of suPAR alone (preferably in combination with CD4+ and HIV-viral
load) could be a strong clinical tool for the diagnosis/prognosis of
HIV-patients. The same is true for urinary suPAR values as indicated in FIG.
3. Also, analysis of the fragmentation of suPAR in body fluids
provides additional information.
The biological fluid sample may be any fluid that can be obtained from
humans i.e. sputum, cystic fluid, ascites, blood, serum, plasma, and urine.
Urine is preferred due to the fact that it is easy to obtain. When urine is
used as the biological fluid sample, the measurement of the marker should be
correlated to the total concentration level of the sample, e.g. by
correlating the measurement of the marker to the content of creatinine in
the sample.
Preferably, the biological fluid sample is stored at a temperature of below
0° C., more preferably from -20° C. to -80° C., until measurement.
The measurement of the marker in the biological fluid sample may be carried
out using any available method/device therefore. Examples of such
measurement methods/devices are ELISA, RIA (radioimmunoassay), western
blotting, FACS analysis, sticks, etc. Also, measurement may be carried out
by determining mRNA expression using RT-PCR, northern blotting, RNase
protection assay or using micro-array techniques etc. Preferred measurement
methods/devices are ELISA and sticks.
An ELISA may be carried out in a number of different embodiments, many of
which are applicable in the present invention. One ELISA embodiment, which
is particularly suitable for use in the present invention, is the one
described by [Holst-Hansen, 1999] and [Stephens, 1999], which are included
herein by this reference.
The measurement of the marker may be carried out using any suitable stick.
Preferably, the stick is a stick comprising an antibody to the marker as a
capture agent.
Preferably, the measurement of uPAR or uPA in the biological fluid sample is
carried out by FACS analysis, western blotting or ELISA. mRNA levels may be
determined using RT-PCR, Northern blotting, Micro arrays or protection
assays.
Preferably, the measurement of uPAR/suPAR/uPA degradation products is
carried out using western blotting.
The measurement of uPAR is carried out in biological fluid samples
containing uPAR expressing cells, i.e. blood samples.
The measurement value of the level of marker obtained in step (a) may be
used to evaluate the state of the subject by comparing the measurement value
with the level of the marker in subjects not infected with HIV virus.
As mentioned above, one aspect of the present invention relates to a method
of evaluating the progression of the state of a subject suffering from HIV
infection. In particular, this method is suitable for monitoring subjects
undergoing treatment, e.g. highly active anti-retroviral therapy (HAART).
The measurement values of the level of marker obtained in step (a) may be
used to evaluate the progression of the state of the subject by comparing
the measurement values with the level of the marker in subjects not infected
with HIV and/or by comparing the temporal course of measurement values with
that of subjects not infected with HIV.
The invention further relates to an ELISA-kit for evaluating the physical
state of a subject suffering from HIV infection comprising a) an immobilised
capture agent capable of capturing a marker in the form of (i) urokinase
plasminogen activator receptor (uPAR), (ii) soluble urokinase plasminogen
activator receptor (suPAR), (iii) urokinase-type plasminogen activator (uPA),
(iv) one or more degradation products of (i), (ii) or (iii), and/or (v) an
mRNA for (i), (ii) or (iii), and b) a binding partner capable of binding to
the marker, the binding partner comprising c) a label system.
The capture agent may be an antibody to the marker.
The binding partner may be an antibody to the marker.
Preferably, the label system is conjugated to the binding partner. The label
system may be any conventionally used label system, such as antibody to the
binding agent conjugated to an enzyme, e.g. an immunoglobulin-alkaline
phosphatase conjugate.
Furthermore, the invention relates to a stick for evaluating the physical
state of a subject suffering from HIV infection comprising a) an immobilised
capture agent capable of capturing a marker in the form of (i) urokinase
plasminogen activator receptor (uPAR), (ii) soluble urokinase plasminogen
activator receptor (suPAR), (iii) urokinase-type plasminogen activator (uPA),
(iv) one or more degradation products of (i), (ii) or (iii), and/or (v) an
mRNA for (i), (ii) or (iii), and b) a binding partner capable of binding to
the said marker, the binding partner comprising c) a label system.
The capture agent may be an antibody to the marker.
The binding partner may be an antibody to the marker.
Preferably, the label system is conjugated to the binding partner. The label
system may be any conventionally used label system, such as antibody to the
binding agent conjugated to an enzyme, e.g. an immunoglobulin-alkaline
phosphatase conjugate.
Claim 1 of 13 Claims
1. A method of prognosticating HIV infection in a subject comprising the
steps of
(a) taking a serum, blood, or urine sample from an HIV infected subject,
(b) performing in vitro a measurement of the level in the serum, blood, or
urine sample of soluble urokinase plasminogen activator receptor (suPAR),
and
(c) using the measurement value obtained to evaluate and provide prognosis
for the subject.
____________________________________________
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