Note: Descriptions are shown in the official language in which they were submitted.
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H5 PSEUDOTYPED VIRUSES AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to the field of Influenza in the presence of
Influenza
antibodies and, more particularly, the present invention relates to the use of
hemagglutinin pseudotyped viruses in methods for detecting the presence or
absence of Influenza antibodies in a sample. The present invention also
relates to
the use of the hemagglutinin pseudotyped viruses in methods for the detection
of
modulators of influenza virus entry in a cell.
BACKGROUND OF THE INVENTION
The global spread of highly pathogenic avian influenza (HPAI) A H5N1 viruses
in poultry and its transmission to humans poses a pandemic threat. Since 2003
there
have been over 270 human cases with over 160 deaths. Binding of influenza
virus to
cellular receptors is determined by the viral hemagglutinin. The HAl subunit
of
hemagglutinin binds to terminal sialic acids of glycoproteins and glycolipids
at the cell
surface (Skehel and Wiley, 2000; Skehel and Wiley, 2002). Avian influenza
viruses
preferentially bind a2-3-linked sialic acids (SA) (Skehel et al., 1982; Skehel
et al.,
1983, Russell et al., 2006) while human influenza viruses preferentially
recognize a2-
6-linked SA. Subsequent virus entry and uncoating is dependent on low pH
(Skehel
and Wiley, 2000). During the final stage of the virus life cycle the HA binds
to the SA
receptor requiring the enzymatic activity of the neuraminidase for the release
of the
viruses from the cell surface (Dong et al. 1992).
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Twelve percent of household contacts of confirmed H5N1 patients during the
H5N1 outbreak in Hong Kong in 1997 had neutralizing antibodies against the
H5N1
(Katz et al., 1999). However, seroepidemiologic studies during the recent H5N1
outbreaks indicate a low transmission rate of H5N1 virus to humans in spite of
extensive exposure to infected poultry (Vong et al., 2006). At present,
microneutralization tests confirmed by western-blot assays are the gold-
standard for
detection of anti-H5 specific antibodies in humans (Rowe et al., 1999).
However
H5N1 microneutralization tests require BSL-3 containment which precludes such
studies in many affected countries. The conventional haemagglutination
inhibition
(HI) test is not suitable for serodiagnosis of avian H5N1 infections in humans
(Rowe
et al 1999). The modified HI test using horse erythrocytes (Stephenson et al.,
2003)
is currently under evaluation but is not a functional assay for inhibition of
entry and is
not amenable to high throughput testing.
There is thus a need to definite need for the development of new tools for the
serodiagnosis of Influenza antibodies, and for the screening of modulators
with
respect of Influenza entry into cells.
SUMMARY OF THE INVENTION
An aspect of the invention is to provide new serodiagnostic tools and new
inhibiting or promoting compounds in the field of Influenza.
Such an aspect is particularly achieved by providing a method for detecting
the
presence or absence of Influenza antibodies in a sample, comprising the steps
of:
a) contacting the sample with an hemagglutinin pseudotyped virus under
conditions sufficient to form an immune complex; and
b) detecting the presence or absence of the immune complex form in a).
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Another aspect of the invention concerns isolated and purified hemagglutinin
pseudotyped virus, wherein said hemagglutinin is encoded by :
- a polynucleotide encoding an H1 hemagglutinin having a nucleotide
sequence as set forth in SEQ ID NO 2;
- a polynucleotide encoding an H3 hemagglutinin having a nucleotide
sequence as set forth in SEQ ID NO 3;
- a polynucleotide encoding an H7 hemagglutinin having a nucleotide
sequence as set forth in SEQ ID NO 4 or
- a polynucleotide encoding an H5 hemagglutinin having a nucleotide
sequence as set forth in SEQ ID NO1 or 5.
Further aspects of the invention concern the use of an hemagglutinin
pseudotyped virus as defined above in methods for the detection of a compound
that
either inhibits or promotes the entry of an Influenza virus in a cell.
Yet, another aspect of the invention provides a kit for the detection of the
presence or absence of antibodies indicative of Influenza virus, comprising:
- an hemagglutinin pseudotyped virus as defined above;
- a reagent to detect an hemagglutinin-antibody immune complex;
- optionally a biological reference sample lacking antibodies that
immunologically bind with said hemagglutinin; and
- optionally a comparison sample comprising antibodies which can
specifically bind to said hemagglutinin;
wherein said hemagglutinin pseudotyped virus, reagent, biological reference
sample,
and comparison sample are present in an amount sufficient to perform said
detection.
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BRIEF DESCRIPTION OF THE FIGURES
Figure IA: Production of lentiviral particles pseudotypes with a synthetic H5
envelope protein.
1. HEK293 T cells ("producer cells") are transfected with provirus expressing
the
luciferase or GFP reporter gene driven by the LTR promoter and a plasmid
expressing the synthetic H5 gene. The cells will subsequently secrete
pseudotyped
viruses containing the proviral RNA and surrounded by the synthetic H5 gene.
2.
12 h after transfection NA was added to the medium to release the particles
that
were bound to the sialic acids that are present on the cell surface of the
producer
cells. 3. 24 h later, the supernatant containing the secreted pseudotyped
viruses is
harvested, filtered and incubated with the target cells. 4.The synH5 pp will
interact
with their receptors and the HA2 will fuse with the plasma membrane. 5. After
endocytosis, the nucleopcapsid is released into the cytoplasm. 6. The
nucleocapsid
will then brake down and the viral mRNA is reverse transcribed into DNA.
Finally, the
viral DNA is transported into the nucleus where it will be integrated into the
DNA of
the host cell. There it will express the reporter gene which can be detected
by a
lucifease assay or FACS analysis for the luciferase or GFP gene respectively.
Figure IB: Expression of H5 in the producer cells.
A western assay was performed on cell lysates from the HEK293T producer cells.
The blot was then stained with a human serum against H5N1. Lane 1: HEK293T
cells, lane 2: HEK293T cells transfected with pNL Luc E- R- and pCDNA-synH5.
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Figure 2A: The yield of H5 pp in the supernatant is increased in the presence
of
sNA.
sNA was added to the HEK293T producer cell line after transfection. 24 h
later, the
supernatant was harvested, filtered and incubated with Huh7 cells. The yield
is
5 expressed in relative luciferase untis (RLU).
Figure 2B and C:B. FACS analysis of MDCK cells infected with HSpp
expressing the eGFP reporter gene.
Supernatant of the producer cells were harvested and concentrated 100 times
over a
sucrose cushion. MDCK were incubated with Denvpp or different dilutions of
H5pp.
Incubation of the MDCK cells with a two fold, 16 fold or 256 fold dilutions of
the H5pp
resulted in 92.3 %, 55,16% or 7 % eGFP-positive cells respectively. (i) FACS
analysis (ii) fluorescence images of infected cells. C. EM image of 293T
producer
cells with budding H5pp. The bar represents 120 nm.
Figure 3: Infectious particle containing secreted H5 protein and p24 proteins
can be purified on a sucrose gradient.
Concentrated supernatant was loaded on a 20-60 % sucrose gradient. Fractions
were analyzed for (A) infectivity (luciferase activity) (B) HA expression and
(C) p24
expression. Infectivity, HA and P24 are detected in the same fractions 10 to
16.
Figure 4A: Different cell types are permissive for HSpp and the entry is
dependent on sialic acid.
Cells were pretreated with medium containing 0,025 U/ mi sNA for 1 h at 37 C.
Then, the cells were incubated with concentrated H5pp in the presence or
absence
of sNA. Infection is expressed in relative luciferase untis (RLU).
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Figure 4B: Infection with HSpp depends on expression of a2-3- linked sialic
acids.
Huh7 cells were pre-incubated for various time periods with sNA (0.025 U/mi)
prior to
infection with H5pp. At the time of infection, SNA and MAA staining was
performed to
determine a2-6- linked and a2-3- linked sialic acid (SA) expression
respectively. All
data are expressed in percentage compared to the no treatment control, which
is
considered 100 %.
Figure 5: H5 pp entry is pH-dependent.
Cells were treated with medium containing the indicated concentrations of
NH4CI as
described in materials and methods prior to infection with H5pp or AMLVpp.
Infectivity was measured by luciferase expression and are expressed as % of
control
(untreated cells).
Figure 6A: Infection of H5pp, but not VSV-G are neutralized by pre-incubation
of HSpp with sera from H5N1 infected patients.
Infectivity of pseudotyped viral particles incubated with indicated dilutions
of the sera
for 1 h at 37 C is visualized. The yield is expressed in relative luciferase
untis (RLU).
Figure 6B: Sera from H5N1 infected persons and vaccinated persons neutralize
the infection of H5pp.
Infectivity of pseudotyped viral particles incubated with indicated dilutions
of the sera
for 1 h at 37 C. The luciferase expression is presented as downregulation
compared
to no serum control. NIH #1-#5 are sera from H5N1 vaccinated persons.
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Figure 6C: Sera from H5N1 infected and vaccinated poultry neutralize the
infection of H5pp.
Infectivity of pseudotyped viral particles incubated with indicated dilutions
of the sera
for 1 h at 37 C. The luciferase expression is presented as downregulation
compared
to no serum control.
Figures 7A shows a preferred nucleotide sequence of an hemagglutinin used
according to the present invention and set forth as SEQ ID NO 1.
Optimised H5 P0408008 (A/Cambodia/2005) from a H5N1 infected patient from
Cambodia. Negative cis-acting motifs have been removed and codon as well as
signal peptide optimised.
Figures 7B shows a preferred nucleotide sequence of an hemagglutinin used
according to the present invention and set forth as SEQ ID NO 2.
Optimised sequence CAC86622 (A/New Caledonia/20/99(H1 N1). Negative cis-acting
motifs have been removed and codon as well as signal peptide optimised.
Figures 7C shows a preferred nucleotide sequence of an hemagglutinin used
according to the present invention and set forth as SEQ ID NO 3.
Optimised sequence of H3N2 5504. Negative cis-acting motifs have been removed
and codon as well as signal peptide optimised.
Figures 7D shows a preferred nucleotide sequence of an hemagglutinin used
according to the present invention and set forth as SEQ ID NO 4.
Optimised sequence of H7N7 Netherlands. Negative cis-acting motifs have been
removed and codon as well as signal peptide optimised.
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Figures 7E shows a preferred nucleotide sequence of an hemagglutinin used
according to the present invention and set forth as SEQ ID NO S.
Optimised sequence of H5 Indonesia. Negative cis-acting motifs have been
removed
and codon as well as signal peptide optimised.
DETAILED DESCRIPTION OF THE INVENTION
Pseudotyped lentiviral particles expressing heterologous viral glycoproteins
have been described for several viruses including Hepatitis C (Bartosch, et
al., 2003),
SARS (Simmons et al., 2004) and the avian influenza virus H7N7 (Duisit et al.,
2002). The principle of production is schematically presented in Figure 1A. H5
pseudotyped particles (H5pp) preferably contemplated by the present invention
are
capable only of a single-round infection and do not produce progeny virus, and
can
therefore be produced under lower biosafety requirements than the wild-type
virus.
The inventors have thus developed a system to produce lentiviral particles
pseudotyped with an hemagglutinin, preferably H5 (H5pp) isolated from a
patient in
Cambodia. H5pp show similar entry characteristics with respect to receptor
usage,
pH requirement and neutralization compared to the wild type H5N1. The system
described herein finds a particular use in serodiagnostic assays and analysis
of
cellular pathways of influenza entry, and more particularly for H5N1 subtype.
It is therefore an embodiment of the present invention to use an hemagglutinin
pseudotyped virus as described further below for the detection of Influenza
antibodies. In this connection, the present invention provides a method for
detecting
the presence or absence in a sample of Influenza antibodies.
The detecting method of the present invention comprises the following steps:
a) contacting the sample with an hemagglutinin pseudotyped virus under
conditions sufficient to form an immune complex; and
b) detecting the presence or absence of the immune complex form in a).
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It will be understood that the method of the invention may detect non-
neutralizing and/or neutralizing antibodies with respect to Influenza. In a
case where
the detection of Influenza neutralizing antibodies are preferred, the present
method
of the invention may consists of a neutralizing assay, such as the one
described in
the Examples Section. Alternatively, if anti-hemagglutinin antibodies in
general have
to be detected, the present method of the invention may take the form of an
ELISA
for instance. Of course, one skilled in the art will appreciate that the
present method
of the invention may also consist of any other suitable type of assay for the
detection
of anti-Influenza antibodies.
It will be further understood that by "immune complex" it is meant that the
antibodies in the sample bind, in a specific manner, to the hemagglutinin.
As used herein, the term "sample" refers to a variety of sample types obtained
from an individual and can be used in the method of the invention. The
definition
encompasses blood and any other liquid samples of biological origin which may
be
suspected of containing anti-Influenza antibodies. Preferably, the sample
consists of
a blood sample from a subject selected from the group consisting of a human,
an
avian and any animal subject of being infected by an influenza virus, and
preferably
of a H5N1 subtype.
As used therein, the expression "under condition sufficient to form an immune
complex" refers to the conditions in terms of time and temperature for
instance that
are used to allow antibodies in the sample to specifically bind to the
hemagglutinin of
the pseudotyped virus. For instance, conditions that are preferably
contemplated in a
neutralizing assay are shown in the Examples Section.
A preferred hemagglutinin pseudotyped virus contemplated by the present
invention consists of a viral expressing vector. For instance, such a
contemplated
hemagglutinin pseudotyped virus consists of an isolated and purified
hemagglutinin
pseudotyped virus, such as an hemagglutinin pseudotyped lentivirus. More
particularly, the isolated and purified hemagglutinin pseudotyped virus
comprises an
hemagglutinin which is encoded by :
CA 02586302 2007-04-26
- a polynucleotide encoding an H1 hemagglutinin having a nucleotide
sequence as set forth in SEQ ID NO 2;
- a polynucleotide encoding an H3 hemagglutinin having a nucieotide
sequence as set forth in SEQ ID NO 3;
5 - a polynucleotide encoding an H7 hemagglutinin having a nucleotide
sequence as set forth in SEQ ID NO 4, or
- a polynucleotide encoding an H5 hemagglutinin having a nucleotide
sequence as set forth in SEQ ID NO 1 or 5.
As it may be appreciated, the hemagglutinin sequences preferably
10 contemplated by the present invention have been advantageously optimised in
order
to eliminate splice site, cryptic splice sites, RNA instability motifs and in
order to
optimise the codon and signal peptide (Fig 7A-7E).
In accordance with a preferred embodiment, the hemagglutinin is H5 and has a
nucleotide sequence as set forth in SEQ ID NO 1 or 5.
As one skilled in the art may appreciate, the hemagglutinin pseudotyped virus
contemplated by the present invention advantageously comprises a reporter
protein,
such as luciferase. Any other reporter gene, such as GFP, that is suitable
according
to one skilled in the art is within the scope of the present invention.
It will thus be appreciated by one skilled in the art that the method of the
present invention finds particular advantageous applications in the
serodiagnosis of
H5N1 exposed or infected humans and animals in outbreak or epidemic
situations,
and in the serodiagnosis in large scale seroprevalence in humans and animals
to
determine the degree of protective immunity against H5N1 in the general
population.
The methods of the invention such as the H5pp technology requires only BSL2
containment and therefore renders seroneutralisation techniques for H5N1 for
instance accessible to countries and institutions which do not have BSL3
laboratories
required for microneutralisation. The method of the present invention further
finds a
particular advantageous application in the serodiagnosis of large number of
sample
in HTS format in BSL laboratories.
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Yet another advantageous application of the present method consists in the
detection of protective H5N1 antibody levels in H5N1 exposed or vaccinated
humans, animals and avians.
A further embodiment of the invention consists of the use of an hemagglutinin
pseudotyped virus as defined above for the detection of modulators of
Influenza
virus entry in a cell such as a compound that inhibits or promotes such entry.
Indeed,
as one skilled in the art will appreciate, the contemplated hemagglutinin
pseudotyped
virus can be advantageously used in order to identify Influenza entry factors
and
inhibitors. For instance and as shown in the Examples Section, since the
inventors
have surprisingly found that, the H5pp specifically reproduces the entry step
of H5N1
and it can therefore be used to identify cellular factors (receptors, etc.)
that play a
role in H5N1 entry. In the same line of thought, H5pp can be used to identify
H5N1
entry inhibitors, e.g., small molecule inhibitors.
Therefore, in closely related aspects, the present invention provides methods
for identifying a compound that inhibits or promotes the entry of an Influenza
virus
into a cell; the method comprises the steps of :
a) providing a mixture containing said cells and the compound to be
tested;
b) contacting said mixture with an hemagglutinin pseudotyped virus as
define above under conditions to allow entry of said virus into the cell;
and
c) evaluating the capacity of the compound to inhibit or to promote the
entry of the hemagglutinin pseudotyped virus into said cell.
It also provides methods for identifying a compound that promotes or inhibits
the entry of an Influenza virus into a cell; the method comprising the steps
of :
a) providing a mixture containing said an hemagglutinin pseudotyped
virus as defined above and a compound to be tested;
b) adding said mixtures into a cell culture under conditions to allow the
entry of said virus into the cells; and
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c) evaluating the capacity of the compound to promote or inhibit the entry
of the hemagglutinin pseudotyped virus into said cell.
As used herein, the expression "compound that inhibits" refers to a regulatory
compound that inhibits, interferes with the entry into a cell of an Influenza
virus, such
as one of the H5N 1 type. A "compound that promotes" as used herein is a
regulatory
compound that promotes, induces, or facilitates the entry into a cell of an
Influenza
virus, such as one of the H5N1 type.
The present invention further provides a kit for use within the detection
method
of the present invention. Such a kit typically comprises two or more
components
necessary for performing an antibody detection assay. Components may be
compounds, reagents, containers and/or equipment. For example, one container
within a kit may contain an hemagglutinin pseudotyped virus, such as H5pp,
that will
allow the binding onto it of a specific antibody such as an anti-H5 antibody.
One or
more additional containers may enclose elements, such as reagents or buffers,
to be
used in the assay. In this connection, a further embodiment of the present
invention
concerns a serodiagnostic kit for the detection of the presence or absence of
antibodies indicative of Influenza virus, comprising:
- an hemagglutinin pseudotyped virus as defined above;
- a reagent to detect an hemagglutinin-antibody immune complex;
- optionally a biological reference sample lacking antibodies that
immunologically bind with said hemagglutinin; and
- optionally a comparison sample comprising antibodies which can
specifically bind to said hemagglutinin;
wherein said hemagglutinin pseudotyped virus, reagent, biological reference
sample,
and comparison sample are present in an amount sufficient to perform said
detection.
With respect to the antibodies of the invention, the expression "specifically
binds to" refers to antibodies that bind to one or more epitopes of an
hemagglutinin
of interest, but which do not substantially recognize and bind other molecules
in a
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sample containing a mixed population of antigenic biological molecules.
EXAMPLES
Highly pathogenic avian influenza (HPAI) H5N1 has spread globally in birds and
infected over 270 humans with an apparently high mortality rate. Serologic
studies to
determine the extent of asymptomatic H5N1 infection in humans and other
mammals
and to investigate the immunogenicity of current H5N1 vaccine candidates have
been hampered by the biosafety requirements needed for H5N1 micro-
neutralization
tests. Objective: Development of a serodiagnostic tool for highly pathogenic
influenza that reproduces H5N1 biology but can be used with less biohazard.
Study
Design: We have generated and evaluated H5 hemagglutinin pseudotyped
lentiviral
particles encoding the luciferase reporter (H5pp). Results: H5pp entry into
target
cells depends on a2-3 cell surface sialic acids and requires low pH for
membrane
fusion. H5pp infectivity is specifically neutralized by sera from patients and
animals
infected with H5N1 and correlates well with conventional microneutralization
test.
Conclusions: H5pp reproduce H5N1 influenza virus entry into target cells and
potentially provides a high-throughput and safe method for sero-epidemiology.
Experimental procedures
Production and purification of H5pp
The pseudotyped particles production and sucrose gradient purification were
performed as described previously (Lozach et al., 2004). Briefly, HEK293T
cells were
transfected with pNL Luc E- R- and pCDNA-synH5 (for luciferase) or pcHMWS-
EGFP, pCDNA-synH5 and pCMV-dR8.91 (for eGFP) and grown in the presence of
soluble Vibrio cholerae neuraminidase (sNA) (6,2 mU/mi; Roche). Supernatant
was
harvested 24 hours posttransfection, filtered and concentrated and particles
were
titrated in an infectivity assay using luciferase and the HIV p24 as readout.
For
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sucrose purification, 200 ng of p24 equivalent of H5pp were centrifuged
overnight
over a 20-60 % discontinuous sucrose gradient. Fractions of 600 pi were taken
and
pelleted in the presence of 0.05% BSA. The pellet was dissolved in 75 pl DMEM;
1/10 was used to infect MDCK cells and 1/5 was used to perform a western-blot
as
described (Lozach et al., 2004). Immunostaining was performed with an anti-
H5N1
duck serum, a human anti-H5 serum (TH001) or an anti-p24 (Abcam, ab9044).
Neutralization assay
MDCK cells (4000 cells/well) were seeded in 96-well plates in 100 uL of DMEM.
104 RLU of H5pp were incubated with twofold serial dilutions of serum
(starting
dilution 1:20) for 1 hour at 37 C (C02 incubator) in 60 uL total DMEM.
Subsequently,
100 uL of fresh medium was added and 140 uL of the virus-antibody mixture was
transferred to the cells. The luciferase assay was performed 48h later by
direct
addition of Steady-Glow Luciferase substrate (Promega). Sera were scored
positive
when they inhibited the H5pp infectivity _80% at serum dilutions >1/20.
Lectin staining
Huh7 cells were treated with 0.025 U/mi sNA (Roche) for the indicated periods
of times. They were harvested by Accutase (Sigma) treatment, washed and
distributed over a 96-well plate. Cells were incubated with Fluorescein SNA
(Vector
laboratories, 10 ug/ml), Biotinylated MAA II (Vector laboratories, 20 Ng/mi)
or PBS
containing 1% BSA (1 h, 4 C) and washed three times. When needed, secondary
staining was performed with Streptavidin-FITC (BD, 25 pg/mI) prior to analysis
by
FACScan.
Sera
Human sera from confirmed H5N1 patients were provided by Institut Pasteur
Cambodia and the University of Hong Kong. Sera from H5NI vaccinated
volunteers,
collected at the 28 day post second dose of the vaccine (Treanor et al., 2006)
were
CA 02586302 2007-04-26
provided by Linda Lambert (National Institute of Allergy and Infectious
Diseases,
Rockville, MD). Avian sera were provided by Institut Pasteur Cambodia and by
Robert Webster (St. Jude Children's Research Hospital, Memphis, TN).
Serostatus
for mammalian sera was confirmed by microneutralization tests and for avian
sera by
5 haemagglutination inhibition tests (WHO 2002; OIE 2004).
Example 1: Production of infectious lentiviral H5 pseudotyped particies (H5pp)
The HA of A/Cambodia/408008/05 (H5N1) virus was used. Sequence analysis
confirmed that this is a clade 1 H5N1 virus with no known mutations relevant
for
10 receptor binding including E190 and G225, suggesting that the H5 protein
retained
the binding characteristics for a2,3-linked SA. The HA sequence was codon-
optimized (Geneart, Germany), cloned into a eukaryotic expression vector and
protein expression verified after transfection into HEK293T cells by Western
(Figure
1 B). Three main bands were seen which is consistent with the expected
molecular
15 weight for the uncleaved HAO and the cleaved subunits HAl and HA2.
H5pp were generated as described in Figure 1A. Figure 2A shows that H5
pseudotyped but not non-enveloped particles yielded a luciferase signal in
MDCK
cells. Addition of sNA during the production of H5pp increased the infectivity
by more
than 1 log (Figure 2A). FACS and microscopy analysis show that H5pp
infectivity
couid also be observed when using a different reporter gene (eGFP) (Figure
2B).
Electron microscopy confirmed that viral particles with an average size of 130
nm
were generated in transfected cells and could be visualized at the plasma
membrane
(Figure 2C).
To confirm that the luciferase signal was due to H5pp, concentrated
supernatants of producer cell lines were separated over a sucrose gradient and
fractions tested for infectivity in MDCK cells and for the presence of HA and
p24
antigens. The peak of infectivity, H5 and p24 protein detection was detected
in the
same fractions 10 to 16 (Figure 3A and 3B).
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In addition to MDCK cells Huh7, 293T, BHK, Vero, HeLa and J774 celis were
also susceptible to H5pp infection (Figure 4A) which is in accordance with
previous
reports (Matlin et al., 1981; Kaverin and Kienk, 1995; Kaverin and Webster,
1995,
Govorkova et al., 1996; Schultz-Cherry et al., 1998; Kuiken et al., 2003;
Rimmelzwaan et al., 2003).
Example 2: HSpp reproduce key steps of H5N1 entry mechanism
Removal of the sialic acids from the cell surface, by pre-incubation of cells
with
sNA treatment, decreased infection (Figure 4A). Sambucus Nigra lectin (SNA)
was
used to label a2-6-linked SA and Maackia amurensis lectin II (MAA) to detect
a2-3-
linked SA. sNA treatment decreased the expression of both a2-3-linked and a2-6-
linked SA, but with different kinetics. Treatment for 30 min diminished the
expression
of a2-3-linked SA (Figure 4B grey bars), while the a2-6-Iinked SA were not
affected
until more prolonged incubation times (Figure 4B black bars). The reduction of
the
infection upon sNA treatment closely correlates with the expression level of
a2-3-
linked SA. A 30 min pre-incubation period with sNA resulted in a decrease in
infection (Figure 4B white bars) while longer incubation times did not further
decrease the levels of infection.
The present results show that infectivity by H5pp, but not AMLVpp was greatly
reduced by NH4CI treatment which inhibits acidification of the endosomes in a
dose-
dependent manner (Figure 5). Similar results were obtained with Bafilomycin A,
another inhibitor of endosomal acidification. Altogether the present results
show that
H5pp uses a2-3-linked SA as receptor and infect cells in a pH-dependent
mechanism.
Example 3: HSpp are neutralized by patient sera
Initial neutralization experiments showed that a convalescent serum from a
H5N1 infected patient (TH001) reduced H5pp infectivity to background levels
while
infectivity of VSV-Gpp was not affected (Figure 6A). A serum taken from a
another
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H5N1 patient (p0302295) at day 10 after the onset of disease was less potent
in
neutralizing H5pp but still inhibited infectivity of H5pp. These findings were
extended
by analyzing the neutralizing capacity of additional human and avian sera in
the
H5pp neutralizing assay (Figure 6 and Table 2).
15/15 sera from confirmed H5N1 patients, 5/5 post-H5N1 human vaccinee sera
and 2/227 human sera from healthy controls inhibited H5pp infectivity
(sensitivity
100%; specificity 99%) (Table 1A). The two false positive human sera were from
persons >65 years of age. Two other human sera (#260, elderly and #195 child)
had
indeterminate neutralizing activity.
12/12 confirmed H5 seropositive avian sera and 0/41 seronegative avian sera
neutralized H5pp. 8/10 sera from H5 vaccinated chicken and ducks tested
positive.
The two negative sera were also negative in conventional microneutralization
tests.
General comments
The inventors have developed lentiviral vectors expressing H5 hemagglutinin
(H5pp). H5pp entry requires a2,3-linked SA and low endosomal pH and can be
abrogated by sera containing H5N1-specific antibodies. Selective removal of
cell
surface a2,3-linked SA (Ada, et al., 1961; Tomlinson et al., 1992)
downregulates
H5pp infection highlighting that H5pp are an ideal tool to study influenza
cell tropism
such as the role of critical HA residues in receptor usage.H5pp encode a
reporter
gene which allows for high throughput analyses under lower biosafety
requirements
than wild type H5N1.
Using a novel H5pp neutralization assay, the inventors detected neutralizing
antibodies in sera from previously confirmed avian and human H5N1 cases. The
inventors have observed a tight correlation of the H5pp neutralization assay
with
characterized positive and negative human and avian (Table 1AB). The two false
positive sera were detected in the H5pp assay came from elderly persons and it
was
previously reported that false positive results in the H5 microneutraliztion
test are
also more common in the elderly (Rowe et al., 1999). While a more
comprehensive
CA 02586302 2007-04-26
18
clinical and epidemiological evaluation is needed, the results presented
herein
indicate the H5pp neutralization assay is a novel approach that can be used
for
large-scale H5 seroprevalence studies on human and animal sera. The advantages
over existing techniques for detection of H5N1 neutralizing antibodies include
easy
and flexible read-out, handling under BSL2 condition and the requirement of
small
quantities of serum. The system is suited for automated high throughput
screening in
a 96-well plate or 384-well plate format. The H5pp particles can be produced
from
synthetic codon optimized DNA without access to the live H5N1 virus or cloned
viral
genes. The flexibility of the system encourages the development of a multiplex
assay
where different subtypes or clades of influenza viruses can be screened
simultaneously. In conclusion, the method of detecting anti-Influenza
antibodies of
the present invention is clearly a valuable tool in global efforts to increase
the
surveillance of influenza viruses in avians, animals and humans.
Table IA: Overview of the human sera tested in the HSpp neutralization test.
# Positive / # Tested
H5N1 Status
H5N1 confirmed patients 15/15
H5N1 vaccinated volunteers 5/5
Healthy controls -<_ 18 years 0/106
Healthy controls - Young adults 0/20
Healthy controls -> 65 years 2/121
The criterion for H5pp positive is > 80% reduction of infectivity at
a serum dilution of 21/20
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19
Table 113: Overview of the avian sera tested in the HSpp neutralization test.
# positive I # Tested
H5N1 status
H5N1 positive sera* 12/12
H5N1 negative sera* 0/41
H5N1 vaccinated chicken or ducks 8/10**
The criterion for H5pp positive is > 80% reduction of infectivity at a serum
dilution of _ 1/20
* Based on hemagglutination inhibition and microneutralization test
** The two negative sera were also negative on microneutralization tests.
Abbreviations:
HI: hemagglutination inhibition
HA: hemagglutinin
H5: hemagglutinin of H5N1
H5pp: H5 pseudotyped viral particles
AMLV: amphotrophic envelope of the murine leukemia virus
sNA: soluble recombinant neuraminidase from Vibrio cholerae
SNA: Sambucus Nigra (elderberry) Bark lectin
MAA: Maackia amurensis lectin II
HPAI: highly pathogenic avian influenza
SA: sialic acids
CA 02586302 2007-04-26
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