Note: Descriptions are shown in the official language in which they were submitted.
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1
H5 PSEUDOTYPED VIRUSES AND USES THEREOF
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
BSL3
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
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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
NH4CI
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.
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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
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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
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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.
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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.
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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
96
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
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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
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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)
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- 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 #
K2051-1
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
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(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
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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
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- 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.
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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.
