The subject of the present invention is a method for the early
5 prediction of severe dengue or hemorrhagic dengue using protein
markers.
Over the past 30 years, dengue, a viral disease transmitted by
urban hematophagous mosquitoes of the Aedes genus has worryingly
10 spread throughout the world. It is currently a real public health
problem for more than one hundred countries located in the subtropical
zone, particularly in the Pacific West, South America and South-East
Asia zones. The emergence of the disease is largely due to the
population explosion and to chaotic urbanization. Climatic
15 abnormalities also play a not insignificant role.
In this respect, dengue could emerge in the western regions of
the world which until now have been spared the virus. Thus, Aedes
albopictus, one of the vectors of the disease, has recently been found
in the North of Italy and in the South of France. Most recently,
20 autochthonous cases of dengue have been recorded in the South of
France. It is estimated that close to three billion people are exposed
to the risks of dengue. Close to one million hospitalizations are
registered yearly and there have been thousands of deaths. Children
are the main victims of the disease.
25 The dengue virus is' a single-stranded, positive-polarity
enveloped RNA virus of the family Flaviviridae. The genome of the
virus (11 000 nucleotides) encodes a polyprotein of approximately
3400 amino acids which undergoes co- and post-translational cleavage
which results in structural proteins (C, prM, E) and non-structural
30 proteins (NS1, NSZA, NSZB, NS3, NS4A, NS4B, NS5). There are 4 viral
serotypes (DV1 to DV4), which can coexist in endemic zones. There is
approximately 70% sequence homology between the various serotypes.
Infection by a given serotype confers long-term .immunity for this
serotype. Cross-protection lasts only a few months: reinfection is
35 therefore possible with a different serotype.
Infection begins with a bite from a mosquito infected with one of
the dengue viruses. Incubation, the period during which the virus
replicates in the blood without however giving rise to any symptoms,
generally lasts from 4 to 10 days. The first signs occur after the
incubation period.
In its conventional form ("conventional" dengue fever: Dr),
dengue is characterized by sudden-onset hyperthermia accompanied by
5 one or more of the following symptoms: shivering, headaches, joint
and/or muscle pain, nausea and vomiting. A rash may also appear,
generally on around the 5th day of symptoms. This acute febrile stage,
which corresponds to the viremic phase, generally lasts from 3 to 5
days (extremes: 2 to 7 days). More than 95% of cases will have no
10 signs of severe illness and will recover with no complications in
under 7 days.
In 2 to 4% of cases, the patient may develop a critical phase
characterized by a more or less severe plasma leakage syndrome and an
increased hematocrit level, leading to dengue hemorrhagic fever: DHF.
15 This phase typically (but not necessarily) appears at the time of
defervescence, around the 4th or 5th day. It is generally brief (24 to
48 h) but may develop into a severe form characterized by major
hemorrhagic manifestations, a state of shock and/or the failure of one
or more organs. Development into a severe form is most often signalled
20 by one (or more) warning sign (s) , such as:
- fever (temperature of greater than 39°C) after the 5th day;
- intense abdominal pain, persistent diarrhea, uncontrollable
vomiting and complete refusal of food;
- edemas and/or minor effusion;
f
25 - bleeding of mucous membranes which does not stop
automatically;
- pronounced lethargy or restlessness;
- thrombocytopenia;
- signs of hemoconcentration.
30 In the most severe cases, the leaking of plasma can lead to
deadly hypovolemic shock (Dengue Shock Syndrome: DSS) if the patient
is not rapidly treated. Rare but deadly hepatic and neurological
involvement is also associated with the severity of the disease. The
mortality rate, which is variable according to epidemics, can reach 5%
35 of established DHF cases. This rate can increase up to 20% without
hospital care or appropriate treatments.
To simplify, these severe cases will be referred to as severe
dengue, SevD in the remainder of the description, as opposed to
conventional dengue, DF.
I 90% of cases of severe dengue occur during secondary infection
5 with a heterologous serotype, and 10% during primary infection,
generally in infants aged from 6 months to 1 year. There are several
factors which influence the severity of the infection, such as the
factors of the host, serotype and genotype of the virus, the order and
time between successive infecting viruses, the quality and quantity of
10 cross-reactive antibodies and the CD4/CD8 response. Studies have shown
a correlation between viral load and severity of the disease. The
exact causes of the occurrence of severe dengue are, however, still
not known. Up until now, no specific determining factor for virulence
has been demonstrated. Furthermore, since there is no vaccine against
I
!
15 the dengue virus, the only treatments available are symptomatic
I
I
I treatments. Consequently, it is important to be able to monitor
!
epidemics and to predict severe cases for appropriate hospital care.
I The methods currently used to diagnose dengue do not make it
possible to predict the development of severe dengue. At the very most,
20 serological methods make it possible to distinguish between primary
and secondary infections and molecular methods make it posslble to
detect the virus and to carry out serotyping [I, 2, 3, 41.
The present invention provides a solution to the problems
presented above by means of a method which allows both early and
25 specific detection of protein& in a blood sample making iL possible to
predict patients dev~loping severe dengue. Indeed, the inventors found,
surprisingly, that proteins from the host were expressed more or less
abundantly (overexpressed/underexpressed) in cases of parients
developing severe dengue, compared to the amount or expression thereof
30 in cases of patients remaining with conventional dengue (that is to
say not developing severe dengue) in blood samples consisting, for
' example, of plasma. Most particularly, they have demonstrated for the
first time and completely unexpectedly that platelet factor 4 (PF4) is
underexpressed in the case of patients developing severe dengue and
35 thus constitutes a marker for predicting severe dengue.
Thus, a subject of the present invention is a method for
predicting, in vitro, the probability of a patient developing severe
dengue, based on a blood sample, wherein:
a) the quantity in said blood sample of at least one marker, which.
is platelet factor 4, is determined,
b) the quantity of platelet factor 4 determined in step a) is
compared with a reference quantity of said marker obtained from a
5 group of individuals who have been diagnosed with non-severe dengue,
wherein, if the quantity of platelet factor 4 determined in step a)
is less than the reference quantity established in step b), it is
predicted that the patient will develop severe dengue.
According to the method of the invention, it is also possible to
10 determine, in step a), the quantity in the blood sample of at least
one other marker chosen from olfactomedin 4 (OLFM4) and a2-
macroglobulin (A2M) or the respective quantities of the two markers
and, in step b), t.he quantity of the marker or of the two markers of
step a) is compared with a reference quantity obtained from a group of
15 individuals who have been diagnosed with non-severe dengue and, if the
quantity of olfactomedin 4 in step a) is greater than the reference
quantity established in step b) and/or the quantity of 012-
macroglobulin is less than the reference quantity established in step
b), it is determined that the patient will develop severe dengue.
20 The invention also relates to a kit for the in vitro prediction of
severe dengue, comprising:
- a binding partner for platelet factor 4,
- a binding partner for the dengue virus NS1 protein.
The kit may also comprise a binding partner for OLFM4 and/or a
25 binding partner for A2. I
Definitions
The term "blood sample" is intended to mean whole blood, serum
and plasma.
30 The term "group of individuals who have been diagnosed with nonsevere
dengue", used to determine the reference quantity of the marker
of interest, is intended of course to mean that the group of
individuals has not developed severe dengue. Thus, in step b) the
quantity of platelet factor 4 determined in step a) is compared with a
35 reference quantity of said marker obtained from a group of individuals
who have been diagnosed with dengue without having developed severe
dengue.
The term "binding partner" is intended to mean, for example,
receptors, antibodies, antibody fragments, antibody analogs and any
other ligand capable of binding to a protein.
The binding-partner antibodies are, for example, either polyclonal
antibodies or monoclonal antibodies.
The polyclonal antibodies may be obtained by immunization of an animal.
with the appropriate immunogen, followed by recovery of the desired
antibodies in purified form, by taking the serum of said animal, and
separation of said antibodies from the other serum constituents,
especially by affinity chromatography on a column to which is bound an
antigen specifically recognized by the antibodies.
The monoclonal antibodies may be obtained by the hybridoma
technique, the general principle of which is summarized below.
Firstly, an animal, generally a mouse, is immunized with the
appropriate immunogen, and the B lymphocytes of said animal are then
capable of producing antibodies against this antigen. These antibodyproducing
lymphocytes are then fused with "immortal" myeloma cells
(murine in the example) so as to give rise to hybridomas. Using the
heterogeneous mixture of cells thus obtained, a seLection of the cells
capable of producing a particular antibody and of multiplying
indefinitely is then carried out. Each hybridoma is multiplied in the
form of a clone, each resulting in the production of a monoclonal
antibody of which the recognition properties with respect to the
protein may be tested, for example, by ELISA, by one-dimensional or
two-dimensional Western blotting, by immunofluorescence, or by means
of a biosensor. The monoclonal antibodies thus selected are
subsequently purified, especially according to the affinity
chromatography technique described above.
The monoclonal antibodies may also be recombinant antibodies
obtained by genetic engineering, using techniques well known to those
skilled in the art.
The term "antibody analogs" is intended to mean biological and/or
chemical compounds which have the same binding capacities as the
antibodies or antibody fragments or similar binding capacities. In
particular, the antibody analogs include small proteins which, like
antibodies, are capable of binding to a biological target thus making
it possible to detect it, to capture it or quite simply to target it
within an organism or within a biological sample. The fields of
application of these antibody analogs are virtually as vast as those
of antibodies. By way of example, mention may be made of the
ano of it ins'^, which are small proteins sold by the company Affilogic.
The binding partners specific for the desired protein can be used
5 as a capture reagent, as a detection reagent or as capture and
detection reagents.
The visualization of the immunological reactions, i.e. the
proteidbinding partner binding, can be carried out by any means of
detection, via labeling, of the binding partner.
The term "labeling" is intended to mean the binding of a label
reagent capable of generating a detectable signal, i.e. a compound, a
substance or a particle which can be detected by visual, fluorescent
or instrumental means.
A nonlimiting list of these label reagents consists of:
- metal or alloy particles, such as colloidal gold particles,
- polymer particles, such as colored latex particles,
- magnetic particles,
- fluorescent molecules,
. chemoluminescent molecules.
By way of example of immunological tests as defined above, mention may
be made of "sandwich" and "competition" methods.
Figures :
The majority of the figures illustrate the validation of the results
I
by a quantitative F,I,ISA assay carried out on individual samples taken
from patients during the acute febrile phase of the disease, before
defervescence. The patients having remained with conventional dengue
are denoted DF and the patients having then developed severe dengue
are denoted SevD. In all cases, the reading is carried out at an
optical density (OD) of 450 nm. The results were obtained on samples
with different geographical origins: Columbia and Cambodia. On the
graphs obtained (Graphpad Prism software, V4.03), the median
calculated is represented by a horizontal line. The box illustrates
the values encompassing 50% of the individuals. The maximum and
minimum values are also illustrated. The values taken into account
correspond to the mean of two independent tests carried out in
duplicate.
Figure 1 illustrates the presence of virus in the fractions purified
from plasma from patients but not in the control (C), demonstrated
with Western blotting using a monoclonal. antibody directed against the
viral protein E.
5 Figure 2 illustrates the results obtained for the quantitative
assaying by means of an ELISA assay of the PF4 marker on plasma
samples from Colombian patients.
Figure 3 illustrates the results obtained for the quantitative
assaying by means of an ELISA assay of the PF4 marker on plasma
10 samples from Cambodian patients.
Figure 4 illustrates the results obtained for the quantitative
assaying by means of an ELISA assay of the OLFM4 marker on plasma
samples from Colombian patients.
Figure 5 illustrates the results obtained for the quantitative
15 assaying by means of an ELISA assay of the OLFM4 marker on plasma
samples from Cambodian patients.
Figure 6 illustrates the results obtained for the quantitative
assaying by means of an ELISA assay of the A2M marker on plasma
samples from Cambodian patients.
20
Example 1: Characterization of the samples
15 Colombian plasma samples positive for dengue were selected, from
which 8 originate from patients remaining with conventional dengue
without developing severe dengue (patients/samples referred to
25 hereinafter as DF or convehtional dengue) and 7 originate from
patients having then developed severe dengue (patients/samples
referred to hereinafter as SevD or severe dengue). The various plasmas
were grouped together, composing respectively a pool of conventional
dengue DF plasma and a pool of severe dengue .SevD plasma for those
30 having developed severe dengue. All the plasmas were taken during the
acute febrile phase of the disease, before the critical phase, from
patients having a secondary infection. The serotypes concerned were
serotypes 1, 2 and 3. All the patients having developed severe dengue
were hospitalized and had signs of hemorrhaging. No comorbidity was
35 reported 151. All the plasmas were verified as being NS1-positive
(Platelia dengue kit, Bio-rad) and the viral load was also verified by
Q-RT-PCR with a commercially available kit (PrimerDesign) following
supplier instructions: the mean number of viral RNA copies was
estimated at 4 x lo6 and 4.1 x 10' for the conventional dengue (DF)
pools and severe dengue (SevD) pools, respectively. The pools composed
correspond to a volume of approximately 2 ml of plasma. Before
purification, the plasma mixtures were centrifuged for 5 mins at 1000
5 x g and at 4 " s~o as to remove the impurities present in the sample
and to obtain clarified samples.
The plasma selection criteria are described in table 1 below. The
samples were taken after appearance of symptoms.
T a b l e 1
Colombian plasma DF POOL SevD POOL
General Number 8 7
Age (mean) 26.8 33.7
M/F ratio 6/2 3/4
Day 3.3 2.7
Secondary infection Yes Yes
NS1 Positive Positive
Serotvwe DV1 1 0
These plasma sample pools were then purified to obtain virus-enriched
fractions as described below.
Example 2: Purification of the samples
15 All the steps are carried hut at 4'C. The cl-arified samples are
supplemented with 8 ml of cold pH8 PBS (PBSR), then centrifuged for 2h
at 41 000 x g in an Optima L90 ultracentrifuge (Beckman). The rotor
used is the SW41 rotor (Beckman). After centrifugation, the
supernatant is removed and the viral pellet obtained is resuspended in
20 200 microliters of PBS8 then loaded onto a discontinuous gradient
composed of 5 ml of 60% (w/w) sucrose in PBS8 and 5 ml of 20% (w/w)
sucrose in PBS8. After renewed centrifugation for 2 h at 41 000 x g, a
virion-enriched ring located at the interface between the two sucrose
solutions is taken off with a pipette, diluted 10 times with PBS8 and
25 finally centrifuged one last time for 2 h at 41 000 x g. The pellet
obtained is resuspended in 200 microliters of PBS8.
This resuspension is then purified using an insoluble polyelectrolyte,
Viraffinity (BioSupportGroup, USA). For this purpose, 200 microliters
of an MN buffer (60 mM MES pH 6.5, 150 mM NaCl) are added to the viral
suspension along with 100 microliters of Viraffinity. The mixture is
incubated for 5 min at room temperature then centrifuged for 10 min at
1000 x g, following supplier instructions. The supernatant is removed
5 and the polymer pellet is rinsed 3 times with 200 microliters of MN
buffer. The viral proteins are recovered by heating the polymer for 5
min / 70°C in the presence of 50 microliters of a buffer containing
SDS (Novex InVitrogen) then centrifugation for 5 min at 1000 x g.
The presence of, the virus in the final samples was verified by
10 immunoblotting with a monoclonal antibody directed against the
envelope protein of the dengue virus (E protein). As illustrated in
figure 1, strong signals at 60 KDa and 120 KDa, corresponding
respectively to the monomeric and dimeric forms of the envelope
protein, are specifically detected by the monoclonal antibody in the
15 plasma pools. On the contrary, the envelope protein was not detected
on a control corresponding to a pool of healthy (non-dengue) plasma
purified in the same way as has been described above.
Example 3: Identification of the specific proteins for each plasma
20 pool, conventional dengue DF and severe dengue SevD, by mass
spectrometry (MS)
Method:
The viral preparations and the control sample obtained according to
example 2 are deposited on non-denaturing polyacrylamide gel and
25 migrated until the proteins penetrate into the gel, in order to
desalify the sample. The band containing the proteins is excised
manually then washed three times in a buffer containing 50%
acetonitrile then finally dried in 100% acetonitrile. The gel is then
rehydrated in a 7% H202 solution before being washed again. A solution
30 of trypsin diluted in 25 mM NH,HCO, is then added for hydrolysis at
37°C overnight. The peptides thus obtained are extracted by 15 minute
sequential extractions with 30 microliters of 50% acetonitrile, 30
microliters of 5% formic acid and 30 microliters of 100% acetonitrile.
These sequential extractions are mixed, dried under vacuum and
35 resuspended in a solution containing 5% acetonitrile and 0.1%
trifluoroacetic acid. After quantification of the samples, a defined
quantity of peptides is analyzed by nano liquid chromatography coupled
together with mass spectrometry (Ultimate 3000, Dionex and LTQOrbitrap
VelosPro, Thermo Fisher Scientific). The results are acquired
by virtue of the Xcalibur software (Thermo Fisher) and automatically
converted by the Mascot Daemon V2.2 software (Matrix Science).
5 Searching is then carried out on the Swissprot and Trembl databases
via Mascot 2.2. Each experiment was carried out twice, independently.
The proteins were identified by the EDyP Service laboratory (CEA
Grenoble, France).
Results :
The viral envelope protein E was repeatedly identified in the samples
containing virus. The predominantly identified peptide sequence is
15 GWGNGCGLLFKG. This result confirms the presence of the virus in the
purified fraction.
For the proteins of cellular origin, identified by proteomics on
purified plasma pools, the results obtained are summarized in tables
2a and 2b. In these tables, only those proteins having a variance of
20 less than 25% for the number of peptides found from one experiment to
the other have been considered. Similarly, for the severe dengne
sample, a number of peptides of greater than 2 was required. According
to these criteria, 189 proteins were finally selected. These proteins
are described in tables 2a and 2b below. A ratio of "number of
25 peptides in severe dengue (dev~) sample" / "number of peptides in
conventional dengue (DF) sample" (SevD/DF) could be calculated for the
majority of these proteins (cf. table 2a). Some proteins were only
identified in the SevD sample (cf. table 2b); in this case, the
SevD/DF ratio could not be calculated.
30
Table 2a
SevD/DF
peptides
ratio
Accession
number
PO9871
PO7225
PO1008
Protein name
Complement Cls subcomponent (C1
esterase)
Vitamin K-dependent protein S
Antithrombin-111 (ATIII) (Serpin C1)
Mean
number
of
peptides
DF
3.5
1
3
Mean
number
of
peptides
SevD
14.5
4
9.5
PO8567 (~47) 1.5 2 1.33
PO7996 Thrombospondin-1 19.5 26 1.33
Tubulin beta chain, (Tubulin beta-5
PO7437 chain) 4.5 6 1.33
A2MYD4 V2-7 protein (Fragment) 3 4 1.33
Q16610
P2791.8
B4ElB2
Q53H26
B4DPQ0
PO1019
B4DDU2
1
1.5
11
11
Extracel1.ul.ar matrix protein I
(Secretory component p85)
Properdin (Complement factor Pi
Serotransferrin
Transferrin variant (Fragment)
Complement Clr subcomponent
Angiotensinogen (Serpin A8)
Tubulin alpha-ubiquitous chain
3
4
2 9
2 9
-
3.00 ~-
2.67
2.64
2.64
5
1
2.5
13
2.5
6
2.60
2.50
2.40
PO0488
Q71U36
P68363
Q9BQE3
Q9H4B7
I Tubulin alwha-1A chain (Aloha- I 1 1
Coagulation factor XI11 A chain (EC
2.3.2.13)
Q96K68
PO8107
P34931
P11142
Q6N089
. L
Lubulin 3)
Tubulin alpha-1B chain (Alphatubulin
ubiquitous)
Tubulin alpha-1C chain (Alphatubulin
6)
Tubulin beta-1 chain
cDNA FLJ14473 fis. clone
PO2675
PO0739
08 6UX7
3.5
MAMMA1001080
Heat shock 70 kDa protein 1A/lB
Heat shock 70 kDa protein 1-like
Heat shock cognate 71 kDa protein
Putative uncharacterized protein
DKFZp686P15220
-
PO2751
B7ZLE5
Q6MZM7
Q68CX6
P68032
rypsln- E .
pro eln omp
3.5
3.5
3.5
3.5
Fibrinogen beta chain
Haptoglobin-related protein
Fermitin family homolog 3 (Kindlin-
3 1
Q562R1
POCOL4
POCOL5
4.5
12
2
2
2
15
,
Fibronectin (FN)
EN1 protein
Putative uncharacterized protein
DKFZp686012165
Putative uncharacterized protein
DKFZp686013149
Actin, alpha cardiac muscle 1
(Alpha-cardiac actin)
Beta-actin-like wrotein 2 IKaowa-
1.29
4.5
4.5
4.5
4.5
11
12
5
L &
actin)
Complement C4-A (Acidic complement
c4
Complement C4-B (Basic complement
c4
Complement component 4B (Childo
Q7Z351
PO2649
PO1009
Q08380
1.79
1.29
1.29
1.29
15
2.5
2.5
2.5
18.5
1.25
1.25
1.25
1.25
1.23
13.5
14.5
6
1.18
1.18
1.18
1.18
1.18
61.5
61.5
61.5
61.5
8.5
8.5
5 2
5 2
DKFZp686N02209
Apolipoprotein E (Apo-E)
Alpha-1-antitrypsin (Alpha-1
protease inhibitor) (Seroin Al)
Galectin-3-binding protein (Basement
membrane autoantigen p105)
1.23
1.21
1.20
72.5
72.5
72.5
72.5
10
10
6 1
6 1
15
4
8.5
12
1.18
1.17
1.17
17
4.5
9.5
13
1.13
1.13
1.12
1.08
P30447
F61QP2
F6IR35
A2NKM7
P26022
PO2760
HLA class I histocompatibility
antigen, A-23 alpha chain
MHC class I antigen (Fragment)
MHC class I antigen (Fragment)
NANUC-2 heavy chain (Fragment)
Pentraxin-related protein PTX3
(Pentaxin-related protein PTX3)
Protein AMBP
Rheumatoid factor RF-IP12
3
3
3
3
2
2.5
3
3
1.00
1.00
3
3
2
2.5
1.00
1.00
1.00
1.00
DKFZp781M0386
Inter-alpha-trypsin inhibitor heavy
chain H2 (ITI-HCZ)
Keratin (Fragment)
HLA class I , B-15 alpha chain (MHC
class I antigen B*15)
HLA class I , B-27 alpha chain (MHC
class I antigen B*27)
Myosin-reactive immunoglobulin light
chain variable region
Myosin-9 (Myosin heavy chain 9)
Putative uncharacterized protein
Clusterin (Apolipop~oteinJ ) (Apo-J)
Anti-RhD monoclonal'~125 kappa light
chain
Full-length cDNA clone CSODD006YL02
of Neuroblastoma
- Apolipoprotein Ll (ApoL-I)
Cold agglutinin FS-1 L-chain
(Fragment)
Cold agglutinin FS-2 H-chain
(Fragment)
Cryocrystalglobulin CC1 kappa light
chain variable region
Full-length cDNA clone CSODL004YM19
of B cells
Rearranged VH4-34 V gene segment
(Fragment)
Rheumat0j.d factor D5 light chain
(Fragment)
6.5
9.5
9.5
3
3
3
8.5
8.5
Uncharacterized protein
5.5
16
28
2.5
2.5
2.5
2.5
2.5
2.5
2.5
chain H4 (ITI-RC4)
5.5
8
8
2.5
2.5
2.5
7
7
Inter-alpha-trypsin inhibitor heavy / 1 I
2.5
16 / 12.5 1 0.78
0.85
0.84
0.84
0.83
0.83
0.83
0.82
0.82
4.5
13
22.5
2
2
2
2
2
2
2
2 1 0.80
0.82
0.81
0.80
0.80
0.80
0.80
0.80
0.80
0.80
0.80
14-3-3 rotei in beta/alpha (KCIP-I) / 4.5 3.5 0.78
P62258
004917
P61981
14-3-3 protein epsilon (14-3-32)
14-3-3 protein e t a (Protein ASI)
14-3-3 protein gamma (KCIP-1)
4.5
4.5
4.5
3.5
3.5
3.5
0.78
0.78
0.78
Spectrin alpha chain, erythrocyte
PO2549 (Erythroid alpha-spectrin) 3 3 1.5 0.05 --
Table 2b
Accession
Example 4: Confirmation ELISA
5 Method:
So as to confirm the mass spectrometry results, specific quantitative
ELISAs were carried out in duplicate on individual plasmas. The
proteins selected and tested, from those identified in tables 2a/2b,
are those with a severe dengue (SevD) / conventional dengue (DF) ratio
10 of greater than or equal to 1.33 and less than or equal to 0.75 with a
mean number of peptides of greater than 1 for each sample and a
potential link to dengue pathogenesis. This first screening made it
possible to only assay those proteins most of interest. According to
these criteria, the following proteins were selected:
15 - ceruloplasmin,
. protein S ,
properdin complement factor,
secretory component p85,
20 complement Clr protein,
complement Cls protein,
angiotensin,
factor 11,
CFB,
anti-factor VIII,
serum amyloid P-component,
5 olfactomedin 4 (OLFM4),
throrqbospondin,
platelet factor 4 ( P F ~ ) ,
complement Clq protein,
moesine, and
10 complement C8 protein.
Multimerin-1, apolipoprotein B-100 and von Willebrand factor were also
assayed.
It should be noted that these proteins are predominantly elements of
the coagulation pathway or the complement cascade.
15 These ELISAs were carried out by virtue of commercially available kits
(USCN, China), following supplier instructions. Statistical analyses
(Mann-Whitney test and ROC/AUC curve) were carried out by means of
GraphPad Prism V4.03 software. I
Each candidate marker was assayed on individual plasma samples. These
20 samp1.e~ are plasma sample:: taken during the acute febrile phase of the
disease (viremic phase), these samples either originating from
patients having remained with conventional dengue DF, without
developing severe dengue, or from patients having developed severe
dengue SevD. All 'the patients had secondary dengue. Only serotypes 1,
25 2 and 3 were represented (no serotype 4). These samples originated
from I'Universidad Industrial de Santander (Bucaramanga, Colombia) [51
or from the Institut Pasteur in Cambodia (Phnom-Penh). The latter were
part of a prospective study carried out in agreement with the local
ethics committee. The characteristics of the two sampling sources are
given in tables 3 and 4 below. The samples were collected after
appearance of symptoms.
Table 3
5
Table 4
test 19/23 (82%) 1 18/26 (82.6%) 1 ns
HGB
Hematocrit
Hepatomegaly
(ultrasound)
Serotype
Secondarv denwe
1 omo orb id it^
11.63+-0.15
38.23t-0.57
8/12 (66%)
DV1
Yes
No
SD: standard deviation
ns: non-significant p value
13.3+-0.29
41.62t-0.8
16/26 (61.5%)
DV 1
Yes
0.1) .
On the other hand, for two markers, the results make it possible to
clearly distinguish those patients who then developed severe dengue
SevD from those who remained solely with conventional dengue without
developing severe dengue. The first marker is PF4 (platelet factor 4).
10 For the Colombian samples, a difference in plasma concentration in
favor of the DF samples is observed (p<0.001) (figure 2). The AUC is
0.88 (95% CI: 0.7305-1).
This is confirmed for the Cambodian samples for which there is a
significant difference in plasma concentration in favor of the DF
15 samples (p< 0.0001) (figure 3) . The AUC is 0.94 (95% CI = 0.87-1). The
ROC curve made it possible to determine the best specificity for a
sensitivity close to 100%. The results are summarized in table 5 below:
for this marker and for a sensitivity of 95%, a specificity of close
to 78% is reached.
20 Table 5
The second marker is OLFM4 (olfactomedin 4)
l~ora sensitivity of: 1
For the Colombian samples, the plasma concentration of the marker is
higher in the SevD samples compared to the DF samples (p=0.07; cf.
25 figure 4).
The best specificity is:
This is confirmed on the Cambodian samples with an extremely
significant difference in concentration (p<0.0003) and a median that
is more than twice as high for the SevD samples compared to the DF
samples (figure 5). The AUC is 0.858 (95% CI = 0.7307-0.985). The ROC
30 curve made it possible to determine the best specificity for a
PF4 100 6 1
sensitivity close to 100%. The results are summarized in table 6: for
this marker and for a sensitivity close to 95%, a specificity of
greater than 72% is reached.
Table 6
In parallel, another marker, a-2 macroglobulin (A2M) was identified
from unpurified Cambodian plasma samples by a SILAC-type differential
proteomic method (Stable Isotope Labelling by Aminoacids in cell
Culture) [61. The identification of this third marker is described in
10 the following examples.
OLE'MQ
Example 5: Characterization of the samples
The composition of each plasma pool or group used in this experiment
is summarized in table 7. All the Cambodian plasmas selected to
15 compose the pools were taken during the acute febrile phase of the
disease, before the critical phase, from patients having a secondary
infection. The serotype concerned was serotype 1. All the SevD
patients were hospitalized and had signs of hemorrhaging. No
comorbidity was reported. All the plasmas were verified as being NS1-
20 positive (Platelia dengue kit/, Bio-rad) and the viral load was also
verified by Q-RT-PCR with a commercially available kit following
supplier instructions. The pools composed correspond to a final volume
of approximately 2 ml. of plasma. The plasma groups are inactivated
beforehand with heat (56"C/20 minutes) then preclarified by
25 centrifugation for 5 mins at 1000 x g and at 4"C, so as to remove
impurities present in the sample.
5
For a sensitivity of:
100
94.4
Table 7
The best specificity is:
6 1
72.2
Serotype
Secondary dengue
DF (n=6)
DV1
YES
SevD (n=6)
DVl
YES
Age in years (mean)
I I
,
Severity grade 0-1 3-4 7
6-12 (8.6)
I I
6-8 (7)
NS1 positive/virus positive YES/YES
Example 6: Differential proteome analysis
The method used is a semi-quantitative proteomic method of SILAC type
5 (Stable Isotope Labelling by Aminoacids in cell Culture) [6] developed
by Pronota (Ghent, Belgium) using the M~s~termindpl~a~tf orm and
carried out on conventional dengue DF or severe dengue SevD plasma
groups. Each group is composed of a mixture of 6 samples, as detailed
in example 5.
10 These plasma mixtures have been depleted beforehand in the 14 most
abundant plasma proteins by affinity chromatography. The quantity of
proteins recovered in the end was obtained by a colorimetric assay
based on bicinchoninic acid (BCA assay Thermo Fisher Scientific Inc.,
USA) .
15 The M~SSterrnind~s~t udy compared each sample to a reference sample
which groups together all the samples. This method provides
information on the relative' levels, and presence or absence, of
peptides/proteins in the severe dengue SevD samples compared to the
conventional dengue DF samples. The differential analysis is carried
20 out by mixing the samples labelled with different isotopes and by
analyzing, by mass spectrometry, each matched peak.' The isotope label
is introduced by tryptic hydrolysis which incorporates 2 ''0 atoms
("heavy" labeling) onto the C-terminal arginine of a peptide, which
' leads to a mass difference of 4 daltons to the same peptide labeled
25 with 160 ("light" labeling). The reference sample is labeled with 160,
whereas the individual samples are labeled with ''0. The MS/MS data are
then submitted to the MASCOT software for identification of the
peptides and proteins in each sample.
Following MS/MS analysis, more than 250 quantifiable proteins were
30 identifiable, 10 proteins of which had at least 1 peptide found to be
YES/YES
Mean day of sampling 3.85
I I
4
Comorbidity NO NO
differential. For each protein identified, the SevD/DF ratio is
calculated as the weighted mean of the coefficients of all the
peptides identified for the given protein. Overall, the results showed
a high degree of similarity between the two proteomes and only a few
5 proteins were found to be expressed differentially. For three of these
ten proteins, the peptides identified are systematically expressed
differentially and have a mean SevD/DF ratio which deviates from 1 (cf.
table 8). The three proteins identified are: a-2 macroglobulin (A2M),
complement C3f and heparin cofactor 2 . These proteins are
10 predominantly elements of the coagulation pathway or the complement
cascade.
Proteins
a-2 macroglobulin
Complement C3f
Heparin cofactor 2
Table 8
Accession
number
I
15
Example 7: Confirmation ELISA
SevD/DF ratio
C03- H UMAN
I I
Method:
1
95% CI on
mean of
HEP2- H UMAN
So as to confirm the mass spectrometry results, specific quantitative
0.89
ELISAs were carried out in duplicate on individual plasmas. The
0.68-1.17
2.25
20 proteins assayed are those identified in example 6: a-2 ~nacroglobulin
1.71-2.95
(AZM), complement C3 protein, and heparin cofactor 2
These ELISAs were carried out by virtue of commercially available kits
(USCN, China), following supplier instructions. Statistical analyses
(Mann-Whitney test and ROC/AUC curve) were carried out by means of
25 GraphPad Prism V4.03 software.
Each candidate marker was assayed on individual samples. These samples
were plasma samples taken from patients during the acute febrile phase
of the disease (viremic phase). Clinical follow-up of the patients
showed that some finally remained with conventional dengue DF without
developing severe dengue, whereas others had developed severe dengue
SevD. All the patients had secondary dengue. Only serotype 1 was
represented. These samples originated from the Institut Pasteur in
Cambodia (Phnom-Penh) and were part of a prospective study carried out
5 in agreement with the local ethics committee. The characteristics of
the sampling source are given in table 9.
T a b l e 9
DF (n=23) I SevD (n=26)
Mean weight t- SD (kg) 120.48i-0.93 1 18.86+-0.98 / ns
Total cholesterol (mmol/l) 13.32t-0.14 1 2.37t-0.12 / <0.0001
P value
% male
Mean age i- SD (years)
56%
HDL (mrnol/l)
Triglycerides (g/l)
AST (IU/L)
ALT (IU/L)
Viral load (copies/ml)
8.6i-0.38 1 7.3+-0.45 1 ns
Sampling (day)
Positive tourniquet test
HGB
Hematocrit
SD: standard deviation
35.6%
0.82+-0.07
1.37t-0.17
133.3t-
23.18
66.9+-13.4
7.1 lo8+-6
Hepatomegaly (ultrasound) f
Serotype
Secondary dengue
Comorbidity
ns: non-significant p value
ns
2.6
19/23 (62%)
11.63+-0.15
38.23t-0.57
Results:
0.31+-0.02
2.8t-0.2
302.51-50.7
106.3+-21.2
2.06 108t-2
18/26
3G: plasma hemoglobin
8/12 (66%)
DV1
Yes
No
15 For the majority of the ELISA-assayed markers, (complement C3f and
heparin cofactor 2) no difference in plasma concentration was observed
between the DF and SevD plasmas (p>O. 1) .
3.2