CA2541872A1 - H5 pseudotyped viruses and uses thereof - Google Patents

Abstract

The present invention relates to a new tool to detect human, animal and avian blood samples for the presence of Influenza antibodies. More particularly, the present invention relates to the use of H5 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 H5 pseudotyped viruses in methods for inhibitors that prevents the entry of the influenza virus in a cell.

Description

FIELD OF THE INVENTION

The present invention relates to a new tool to detect human, animal and avian blood samples for the presence of Influenza antibodies. More particularly, the present invention relates to the use of H5 pseudotyped viruses in methods for detecting the presence or absence of Influenza antibodies in a sample. The present 1 o invention also relates to the use of the H5 pseudotyped viruses in methods for inhibitors that prevents the entry of the influenza virus in a cell.

BACKGROUND OF THE INVENTION

The pandemic spreading of the avian flu in poultry farms and the endemic presence in Asia have great economic and social impacts on several countries.
Sporadic human cases have been confirmed in such countries and proven to be highly pathogenic and lethal in about 50 % of the cases. Concerns are expressed that the virus might soon acquire new characteristics that will facilitate human-to-2 o human transmission. Closely monitoring of the disease and the spreading of it, both in chickens and humans, is a prerequisite to contain infection herds and to be able to take the right preventive measurements. Until now the process of large scale screening of blood samples for neutralizing antibodies was tedious, requiring facilities for growth of the virus.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Production of lentiviral particles pseudotypes with a synthetic H5 envelope protein.

3 o A. 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.
B.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. C.
24 h later, the supernatant containing the secreted pseudotyped viruses is harvested, filtered and incubated with the target cells. D.The synH5 pp will interact with their receptors and the HA2 will fuse with the plasma membrane. E. After endocytosis, the nucleopcapsid is released into the cytoplasm. F. 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 1 o FACS analysis for the luciferase or GFP gene respectively.

Figure 2 a: The yield of SynH5 pp in the supernatant is increased in the presence of NA.
NA was added to the HEK293T producer cell line after transfection. 24 h later, the supernantant was harvested, filtered and incubated with Huh7 cells. The yield is expressed in relative luciferase untis (RLU).

Figure 2 B.: Different cell types are permissive for syn H5 pp and the entry is dependent on sialic acid.
Cells were pretreated with medium containing 0,025 U/ ml NA for 1 h at 37 C.
Then, the cells were incubated with concentrated syn H5pp in the presence or absence of NA. Infection was diminished in the presence of NA for all cell types.
Infection is expressed in relative luciferase untis (RLU).

Figure 2 C.: Syn H5 pp entry id pH-dependent.
Cells were pretreated with medium containing the indicated concentrations of for 1 h at 37 C. Then, the cells were incubated with concentrated syn H5pp in the presence or absence of NH4CI for 2h. SynH5 pp, but not AMLVpp infection was diminished in the presence of NH4CI in a concentration-dependent manner.
Infection 3 o is expressed in percent relative to the non-treated sample.

Figures 3a to 3e show preferred nucleotide sequences of an hemagglutinin used according to the present invention.

Figure 4 a: Infection of synH5pp can be neutralized by pre-incubation of synH5pp with sera from H5N1 infected patients.
Pseudotyped viral particles were incubated with indicated dilutions of the sera for 1 h at 37 C. Then, the virus/antibody complexes were added to Huh7 cells and the infection rate assessed by the luciferase expression. The yield is expressed in relative luciferase untis (RLU). The sera from the patient from Vietnam who recovered (TH001), has high neutralizing capacity on syn H5pp, but not on VSV-Gpp pseudotyped viral particles and the neutralization potency is dose-dependent.
The 1 o sera of patient p0322095 has much lower neutralizing capacity, but there is a dose-dependent decrease in infection.

Figure 4 b: Sera from H5N1 - infected patients, but not from a non-infected control group neutralize synH5pp infection.
Pseudotyped viral particles were incubated with indicated dilutions of the sera for 1 h at 37 C. Then the virus/antibody complexes were added to MDCK cells and the infection rate assessed by the luciferase expression. The yield is expressed in relative luciferase untis (RLU). The sera of a patient infected with H5N1 (KH1JF) clearly diminishes the infection of synH5pp, while non-infected control sera (136 1 B
2 0 1305 and 149 1 B1704) do not affect synH5pp infection. 147 1 B1702 gives a minor, but reproducible decrease in infection, which correlates with the results obtained in a microneutralization test on the H5N1 virus (communicated with IP Cambodia).
Figure 4 c: Sera from H5N1 infected ducks neutralize the synH5pp very efficiently.
Pseudotyped viral particles were incubated with indicated dilutions of the sera for 1 h at 37 C. Then the virus/antibody complexes were added to MDCK cells and the infection rate assessed by the luciferase expression. The yield is expressed in relative luciferase untis (RLU). The sera of a p41CTF1-D5 completely neutralize 3 o synH5pp up to a dilution og 2560 times, while the titers of a second duck (41 CTF1-D1 are considerably lower. As a comparison, the sera of 2 humans are included (KH1JF and 147 1 B1702). Again, these results correlate with the results obtained by the microneutralization test on the H5N1 virus (communicated with IP

Cambodia).

Figure 5. Screening of 8000 small molecules for inhibitory activity on H5pp.
Greater than 99% of the compounds do not inhibit H5pp infection. Horizontal lines indicate 2 and 3 SD respectively.

DESCRIPTION OF A PREFERRED EMBODIMENT
lo HApp displays key features of Influenza virus entry The inventors have developed a pseudotyped lentiviral system where the hemaglutinnin of isolate H5 p0408008, described by Buchy et al (Institut Pasteur Cambodia), hereafter named (H5) is expressed on the virion surface of defective HIV
particles. The basic technoloy of generating pseudotype viruses have been described previously, including reference Lozach et al, (JBC 2004, Vol. 279, No. 31, pp. 32035-32045) and is described in detail in Example 2. Specifically for the production of H5pp the hemagglutinin sequence has been optimised in order to eliminate splice site, cryptic splice sites, RNA instability motifs and in order to optimise the codon and signal peptide (Fig 3a). The same gene optimisation strategy has been pursued to generate H1, H3, H5 Indonesia and H7 (Figs 3c to 3e).

H5pp reproduce the key steps in Influenza virus entry into cells (Figs 1, 2, 4 and 5).
Entry of the viruses is then detected by means of the expression/functionality of the luciferase reporter gene. Infectious particles containing H5 and the p24 capsid protein are co-eluted/migrating on a 20 % to 60 % sucrose gradient showing that reporter gene expression is due to infection with pseudotyped virus. Several experiments demonstrate that the entry of these particles faithfully mimics the entry of the influenza virus. First, pre-treatment of the target cells with neuroaminidase aiming to remove the sialic acids on the cell surface, diminish the infection by H5pp.
Secondly, the entry is a pH dependent process that could be decreased by inhibiting the endosomal acidification by the presence of NH4CI. Third, siRNA experiments show that the entry of H5 pseudotyped viruses is a clathrin-independent, dynamin-dependent process. Fourth H5pp are neutralised by sera from H5N1 infected humans or animals.

5 The inventors used our H5 pseudotyped viruses as a tool to screen different human and avian blood samples for the presence of H5 neutralizing antibodies.
These results correlate with the traditional microneutralization test. The inventors identified 100 % of previously H5N1 confirmed cases (7 out of 7, 4 duck sera and 3 human) in their test with titres varying from 1/40 to > 1/1280. None of these sera 1 o were neutralizing for VSV-G pseudotyped viruses. Based on positive results with H5pp lentiviral particles pseudotyped with different subtypes of influenza including H3, H1, H7 and H9 and different isolates originating from 2 different clades (Indonesia and Qinghai origin) of H5 can be prepared. The initial tests show the feasibility of the present screening method, particularly on a large scale under BSL2 conditions.

Potential applications 1. Diagnosis of H5N1 infections.
This includes:
- serodiagnosis of H5N1 exposed or infected humans and animals in outbreak or epidemic situations - serodiagnosis in large scale seroprevalence in humans and animals to determine the degree of protective immunity against H5N1 in the general population. H5pp technology requires only BSL2 containment and therefore renders seroneutralisation tecnhiques for H5N1 accessible to countries and institutions which do not have BSL3 laboratories required for microneutralisation.
- Serodiagnosis of large number of sample in HTS format in BSL laboratories.

2. Detection of protective H5N1 antibody levels in H5N1 exposed or vaccinated 3 0 humans and animals 3. Identification of virus entry factors and inhibitors.

HApp specifically reproduces the entry step of H5NI and can therefore be used to identify cellular factors (receptors, etc) that play a role in H5N1 entry. In the same line of thought HApp can be used to identify H5N1 entry inhibitors, eg small molecule inhibitors.
EXAMPLES
Example 1:

1 o Materials and methods HEK293T cells are maintained in DMEM + 5 % FBS + 1% P/S (= complete medium) Production of pseudoparticles HEK293T cells are transfected with plasmids pNL env- luc and pCDNA-synH5 (ratio 3:1) using the calcium phosphate method (Clontech, PT3025, catalog # K2051-1) on the evening of day 1. The next day, the medium is replaced with fresh medium supplemented with neuraminidase (6,2 mU/mI) (Roche, HK). 24h later, the supernatant is harvested, filtered through a 0,22 uM filter and put immediately on the target cells or alternatively concentrated. For concentration, the supernatant is loaded on a 20 % sucrose cushion in Ultra-Clear sterile tubes (Beckmann) and centrifuged for 2,5 h at 28000 rpm at 4 C. The supernatant is then decanted and the pellet solved in 1/100 of the original volume of DMEM. The concentrated viral particles are subsequently aliquoted and stored at -80 C.

Infection of target cells MDCK, Huh7 or other cell types are plated 16 h before infection in a white 96 well plate in 100 ul of medium. The next day, viral particles are thawed at 37 C and subsequently kept on ice. The appropriate dilution is then added to the target cells.

72 h later, the luciferase assay is performed (Flash glow luciferase assay reagent, Promega).

Neutralization assay On day 0, MDCK cells are seeded in a 96 well white plate (4000 cell /well) in 100 ul DMEM+FBS (5% + 1% P/S) (="infection" plate). The day after, a clear 96 well plate ("incubation plate") is prepared as follows. Thirty uI of complete DMEM
is added to each well and an additional 27 ui of complete DMEM to column 1 of the i o well plate. Then, 3ul of the sera is mixed to column 1 in the appropriate row (A,B, C:
triplicate). The solution is mixed and 30 ul is transferred from column 1 to column 2.
This serial dilution is continued until the end of the row. From the last well, 30 ul is discarded.
The HApp are taken from the -80 C freezer and thawed at 37 C. The appropriate dilution of HApp is prepared to obtain RLU of 1 x 104 to 105 for the non-sera treated samples.
In each well, 30 pl of the HApp solution is added. After incubation for 60 minutes at 37 C, 40 ul of each well is transferred to the appropriate well on the infection plate. The luciferase assay is performed 72 h later.

Example 2: Title of procedure : Production of InfluenzaH5 Virus Pseudo Particles (HApp) Number of procedure : HKIP-03 Aim of the procedure :
This procedure describes the different steps allowing production of human retroviral particules that bear the hemagglutine envelope protein H5 (p0408008) that is expressed from an optimised gene (Gene-art) cloned into the pcDNA3.1 (+) plasmid (pHKIP 136).

3 0 Useful definition :
BSC = Biological Safety Cabinet DMEM = Dulbecco's Modified Eagle's Medium FBS = Foetal Bovine Serum HBS = HEPES-Buffered Saline Solution HA = Influenza virus HIV = Human Immunodeficiency Virus PBS = Phosphate-Buffered Solution pp = Pseudo-particle CF = cell factory Useful references :

l o Chimeric Avian Retrovirus Containing the Influenza VirusHemagglutinin Gene Has an Expanded Host Range. JIANYUN DONG, 1 MICHAEL G. ROTH,2 AND
ERIC HUNTER"* JOURNAL OF VIROLOGY, Dec. 1992, p. 7374-7382 I/ Materials needed 1) Facilities - 1 C02 incubator, 37 C
- 1 fridge, +4 C
- 1 freezer, -20 C or -80 C
- 1 Class II BSC
2 0 - 1 ultracentrifuge 2) Materials - 1 vortex - 1 pipet-aid - 1 tissue culture microscope - 1 vacuum pump - pipetman 3) Consumables 3 0 - 50-mL sterile plastic tubes - 5-, 10-, 25- & 50-m1 sterile pipettes - 1.8-mL sterile eppendorf tubes - Sterile cell factory i. NUNC #167695 Denmark Cell factory, 2-trays ii. Corning 2 stack cell factory: Culture Chamber; 2-Stack; Polystyrene;
Rectangular; Standard Tissue Culture Surface; Graduated Marking Interval:
50mL; Cell Growth Area: 1272cm2; Recommended Medium Volume 250-380mL; Sterile; With Vent Caps;

- Disposable filter discs i. HEPA-Vent. (pkt/10) WHATMAN "COPE" #FC445-10 England or ii. Corning Venting Filling cap, 33 mm threaded cap with 3/8 (9,5 mm) ID
tubing and 50 mm filter with 0,2 pm hydrophobic membrane 5 Pk/Cs - Ultra-clear SW28 tubes (Beckmann) - 20-, 200-, 1 000-pL sterile tips - 250 ml Filter System, 0,22 pm PES (polyethersulfone) sterilizing low protein binding membrane, polysterene (Corning, #431096) 4) Medium & Reagents - FBS (Invitrogen #10270-106) - DMEM (Invitrogen #42430-025) - GMEM with L-glutamin, without tryptose phosphate broth (Gibco, # 11-710-035) 2 0 - Tryptose phosphate broth (Sigma, T8159, 100 ml) - HEPES (Gibco, # 15630-080) - Penicillin (100 U/ml)/Streptomycin ( 100 pg/mI) - PBS (Invitrogen #10010-023) - Trypsine-EDTA 1 X (#25200-072) - 20% sucrose in PBS
- neuroaminidase (Sialidase) from V. cholerae (Roche, #11080725001), 1 U/ ml, 4 C

5) Glassware 6) Biological materials - 293T human cell line (kidney, ATCC CRL-1573) - pcDNA-HA08 #9 plasmid = pHKIP 136 containing an optimised H5 gene from patient 0408008 (IP, Cambodja) with a C-terminal FLAG tag - pNL4.3.Luc R-E-pro- Luc plasmid (HIV provirus ~env ~nef, Institut Pasteur Paris, France) (packaging-competent luciferase retroviral transfert vector, 5 Charneau P., Virologie moleculaire et Vectorologie, Institut Pasteur Paris, France) named pHKIP49 - note : 3 plasmid system with pcDNA-HA08 #9 plasmid = pHKIP 136, pTRIP-EGFP= pHKIP 29 and p8.71 plasmid = pHKIP27 (gag-pol packaging construct, Charneau P., Virologie moleculaire et Vectorologie, Institut 10 Pasteur Paris, France) works also but much less signal then VSV-G

7) Commercial kits - CalPhos Mammalian Transfection Kit, Clontech, PT3025, catalog #

II/ Protocol 1) Cell culture Plate the 293T cells 24 hr before the transfection experiment. The cells should be 50-80% confluent the day of transfection.
- in a class I I BSC, plate 45 x 106 cells/Cell factory with 150 mL DMEM, FBS 10%, Glutamax, antibiotics - incubate plates at 37 C for 24 hr in a C02 incubator 2) Transfection To produce HApp, plasmids are transfected in 293 cells:
- 1 h before transfection change medium to 150 ml fresh DMEM, FBS 10%, Glutamax, antibiotics a) 2 plasmids-transfection system - (1) the pcDNA-HA08 #9 plasmid = pHKIP 136 (Isabelle Nefkens, HKUIP), containing an optimised H5 gene from patient 0408008 (IP, Cambodja) with a C-terminal FLAG tag (2) the pNL4.3.Luc R-E-pro- packaging construct encoding the HIV-1 gag and pol genes and containing a luciferase transcription unit - for each transfection, prepare solution A and solution B in separate sterile tubes - solution A: add these components in the following order: plasmids, water then Calcium Phosphate (plasmid ratio and quantity are experimentally determined: 2 to 1(provirus/envelope plasmid) was found for HApp). The amount for one cell factory equals that of 16 x 100 mm petridishes - solution B: 7200 pl 2 M HBS
Note 1: yield of production is tightly related to the transfection efficacy.
Efficacy of transfection of 2 plasmids is higher than transfection of 3 plasmids.
Nevertheless, in the 3-plasmids system, the reporter gene is under the control of a CMV-promoter leading to higher protein expression Note 2 use the same batch of Maxi-prep for each plasmid DNA
Note 3 do not transfect more than 400 pg of plasmids DNA per cell factory Note 4 for calculation, please see transfection at a glanceHApp.xls file - in a 50-mi sterile tube, carefully and slowly vortex solution B while adding solution A dropwise 2 0 - incubate the transfection solution at room temperature for at least 15 min and 20-30 min maximum - in a class II BSC, take out the medium of the cell factory in a sterile (autoclaved) glass beaker - gently mix transfection (up & down with the tip) and then add transfection solution dropwise to the medium in the glass beaker - after incubation time, gently move glass beaker back and forth to distribute transfection solution evenly (Do not rotate as this will concentrate transfection solution precipitate in the center) - incubate CF at 37 C for 12 hr maximum in a C02 incubator 3 o Note : the cells should be maximum 80% confluent and a fine transfection precipitate should be observed under the microscope - in a class II BSC, replace culture medium on CF transfected with 115 ml complete GMEM (GMEM + 5 % FBS + 20 mM HEPES + 10 % tryptose phosphate broth+ 1% P/S) with 0,00625 U/mI neuroaminidase (Sialidase) from V. cholerae (Roche, #11080725001), (1/160 from the Roche stock solution (1 U/ ml)) - Note : the cells should be maximum 80% confluent and a fine transfection precipitate should be observed under the microscope - incubate plates at 37 C for 24 hr in a C02 incubator 3) Pseudo-particules harvest - 36-40 hr post transfection (24 h after end of transfection = change of medium), in a class II BSC, gently move CF back and forth the plates and harvest all the supernatants containing the pseudo-particules in a 250 ml Bottle Filter System - filter supernatants through 250 ml Filter System (0,22 pm PES
(polyethersulfone) sterilizing low protein binding membrane, polysterene (Corning, #431096)) by attaching the bottle filter to the vacuum aspirator and switching the aspirator on - keep the bottle on ice Note : if you wish to stop the procedure and continue later, store the filtered 2 o supernatant at 4 C for 24 hr maximum (HApp seems to be very stable at 4 C
for short periods of time) - replace culture medium on CF transfected with 115 ml complete GMEM
(GMEM + 5 % FBS + 10 mM HEPES + tryptose broth+ 1% P/S) with 0,00625 U/mI neuroaminidase (Sialidase) from V. cholerae (Roche, #11080725001) - incubate plates at 37 C for 24 hr in a C02 incubator - harvest and filter all the supernatants (cfr. beginning of point 3) 4) Purification Purified virus sample were obtained by ultracentrifugation of viral supernatants through a 20% sucrose cushion in a SW 28 Beckman rotor (28,000 rpm, 2.5 hr, 4 C).
- put 3 Ultra-Clear SW 28-type sterile tubes in each of the metal ultracentrifuge containers - at the bottom of 3 Ultra-Clear SW 28-type sterile tube gently add 2,5 mL of filtered 20% sucrose - in a class II BSC, add 37.5 mL of filtered viral supernatant -or until ultra-Clear tube is full- to each ultra-Clear tube. Balance tubes (ultracentrifuge containers plus ultra-clear tubes) one by one on the balance that is placed under the hood (use 500 mi Erlenmeyer to put the ultracentrifuge containers in). Do not forget to include the lid on the balance. Make sure that the tubes don't differ by more then 0.05 g in weight.
- Note : if you don't fill the tubes completely they will brake during centrifugation. If necessary add complete GMEM (GMEM + 5 % FBS + 10 mM HEPES + tryptose broth+ 1% P/S) until the tube is full and the weight is equal +/- 0.05 g to the other containers.
- centrifuge at 28,000 rpm for 2.5 hr at 4 C
- in a class II BSC, carefully decant the supernatant into bleach solution without disturbing the pellet - invert tubes on absorbant paper for 2 min and wipe top of the walls - add 1/100 of the initial volume of the viral supernatant (375 pl) of DMEM, FBS free (or TNE buffer) onto the pellet of each of the tubes - incubate the SW28-type tube sealed with parafilm on ice for 1 hr 2 0 - rinse the walls for 3-4 min (with a 100 _I tip to avoid bubbles) and combine the solution of the 3 different tubes in one tube - mix and aliquot into 20 _I aliquots (in total 75 tubes) in I ml eppendorfs and store the viral samples at -80 C. Also mark one specific "p24" tube with 10 ul for the use in the p24 test. Put date, number of cell factory production, aliquot size and number of aliquots on top of the lid of the box and put the date on minimum 2 sides on box itself so it can been seen when the box is closed.

Cell factory transfection Date of experiment:
Saiut&on A

Happ Component 14taxiprep Conc Quanritp of R {280126.0 Volume ( L) for Num6er of Tot31 ( frgtpL) plasm'sd (}rg( nm) 1 D#}-snm Plate Plates Volume (pL) FiaT~1S(1 1 8 1a pFl< I Z%': :' PI3=-;~ d 2: pHiti? ~ 13 #2F
H~0 2M Ca'.; u ,. 55 19 880 Phasp~xe Total 45C 1{s 7200 Solution B

C'oanponent Volume (pL) for Number of Total Vdlunie (PL) 900-mnz Plate Plates 2X Fii3S 45C t,9 7230 Procedure:

1) Prepare solution A & B
2) Mix solution A dropwise into solution S, while gently vortexing solution B
3) Incubate at room temperature for maximum 20 min (mininnum 15 min) 4) Mix solution gently (up & down) 5j Add transfection solution dropwise to 150 ml of medium in beaker that was taken from cell factory 6) Gently move back and forth to distribute solution evenly 7) remove medium from cell factory and incubate in incubator 8) Incubates plates at 37 C for 12 hr in C02 incubator 9) Change medium to fresh 150 rn1 complete GMEM with NA

Although preferred embodiments of the present invention have been described in detail herein and illustrated in the accompanying drawings, it is to be understood that the invention is not limited to these precise embodiments and that various changes and modifications may be effected therein without departing from the scope or spirit of the present invention.

Claims (6)

1. 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 for a time and under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex form in a).

2. The method according to claim 1, wherein the sample consists of a blood sample from a subject selected from the group consisting of a human, an animal and an avian.

3. The method according to claim 1, wherein the hemagglutinin consists of at least one hemagglutinin selected from the group consisting of H1, H3, H5 and H7.

4. The method of claim 3, wherein the hemagglutinin comprises a sequence as set forth in any one of SEQ ID NOS:1 to 5.

5. Use of an H5 pseudotyped virus for the detection of Influenza antibodies.

6. Use of an H5 pseudotyped virus for the detection of a compound that inhibits the entry of an Influenza virus in a cell.