U.S. patent application number 11/746415 was filed with the patent office on 2007-11-15 for sphingoid polyalklamine conjugates, isomers and uses thereof.
This patent application is currently assigned to Yissum Research Development Company of the Hebrew University of Jerusalem. Invention is credited to Yechezkel BARENHOLZ, Eliezer Kedar, Sarit Samira.
Application Number | 20070264273 11/746415 |
Document ID | / |
Family ID | 33556649 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070264273 |
Kind Code |
A1 |
BARENHOLZ; Yechezkel ; et
al. |
November 15, 2007 |
SPHINGOID POLYALKLAMINE CONJUGATES, ISOMERS AND USES THEREOF
Abstract
The present application discloses methods for stimulating or
enhancing an immune response of a subject to protect against an
infection caused by an agent selected from Hepatitis B Virus (HBV),
avian influenza virus, the bacterium Bacillus anthracis or the
bacterium Streptococcus pneumoniae, the method comprising
administering to said subject a combination of
sphingoid-polyalkylamine conjugate and a biologically active
molecule, the combination being effective to provide said
stimulation or enhancement of the immune response. Also disclosed
are vaccines comprising a combination of the
sphingoid-polyalkylamine conjugate and a biologically active
molecule for stimulating or enhancing an immune response of a
subject to protect against an infection caused by such an agent.
Preferred conjugates are N-palmitoyl D-erythro
sphingosyl-1-carbamoyl spermine (C-1 CCS), N-palmitoyl D-erythro
sphingosyl-3-carbamoyl spermine (C-3 CCS) and mixtures thereof.
Also disclosed are uses of C-3 CCS for various applications.
Inventors: |
BARENHOLZ; Yechezkel;
(Jerusalem, IL) ; Kedar; Eliezer; (Jerusalem,
IL) ; Samira; Sarit; (Raanana, IL) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
Yissum Research Development Company
of the Hebrew University of Jerusalem
Jerusalem
IL
BioLab Ltd.
Jerusalem
IL
NasVax Ltd.
Ness Ziona
IL
|
Family ID: |
33556649 |
Appl. No.: |
11/746415 |
Filed: |
May 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11345624 |
Feb 2, 2006 |
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11746415 |
May 9, 2007 |
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10560928 |
May 5, 2006 |
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PCT/IL04/00534 |
Jun 17, 2004 |
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11345624 |
Feb 2, 2006 |
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60537553 |
Jan 21, 2004 |
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60545505 |
Feb 19, 2004 |
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60479185 |
Jun 18, 2003 |
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60505638 |
Sep 25, 2003 |
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Current U.S.
Class: |
424/184.1 |
Current CPC
Class: |
A61K 31/132 20130101;
A61K 48/0041 20130101; A61K 31/132 20130101; A61K 2039/543
20130101; A61K 2039/55561 20130101; C12N 15/88 20130101; A61K 38/47
20130101; A61P 43/00 20180101; A61P 37/02 20180101; C07C 271/20
20130101; A61K 38/47 20130101; A61K 2300/00 20130101; A61K 39/39
20130101; A61K 9/1272 20130101; A61K 2300/00 20130101; A61K
2039/55555 20130101 |
Class at
Publication: |
424/184.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Claims
1. A method for stimulating or enhancing an immune response of a
subject to provide protection against an infection, the method
comprising administering to said subject a combination of
sphingoid-polyalkylamine conjugate and a biologically active
molecule, the combination being effective to provide said
stimulation or enhancement of the immune response, wherein said
infection is caused by an agent selected from the group consisting
of hepatitis B virus (HBV), avian influenza virus (AIV), the
bacterium Bacillus anthracis and the bacterium Streptococcus
pneumoniae.
2. The method of claim 1, wherein said sphingoid-polyalkylamine
conjugate comprises a sphingoid backbone carrying, via a carbamoyl
linkage, at least one polyalkylamine chain.
3. The method of claim 1, wherein said biologically active molecule
is associated with said sphingoid-polyalkylamine conjugate.
4. The method claim 1, wherein said biologically active molecule is
selected from an antigenic protein, antigenic peptide, antigenic
polypeptide, carbohydrate, or antigenic glyco-protein.
5. The method claim 1, wherein: (i) when said infection is caused
by HBV, said biologically active molecule is an Hepatitis B
antigen; (ii) when said infection is caused by avian influenza
virus, said biologically active molecule is an AIV antigen; (iii)
when said infection is caused by the bacterium Bacillus anthracis,
said biologically active molecule is an antigen of said bacterium;
or (iv) when said infection is caused by the bacterium
Streptococcus pneumoniae said biologically active molecule is an
antigen of said bacterium.
6. The method of claim 5, wherein: (i) said HBV antigen is an HBsAg
particle; (ii) said AIV antigen is an inactivated purified whole
avian influenza virus; (iii) said bacterium antigen is the
protective antigen (PA) moiety of anthrax toxin; or (iv) said
Streptococcus pneumoniae antigen comprises a polysaccharide or
protein conjugate of an antigen selected from pneumococcal surface
protein A (PspA), pneumococcal adherence and virulence factor A
(PavA), pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin
binding protein A (CbpA), pneumococcal surface adhesion A
(PsaA).
7. The method of claim 1, further comprising administering to said
subject an immunostimulating agent.
8. The method of claim 1, wherein said sphingoid-polyalkylamine
conjugate forms a lipid assembly.
9. The method of claim 1, wherein said sphingoid is ceramide.
10. The method of claim 1, wherein said polyalkylamine is selected
from spermine, spermidine, a polyamine analog or a combination of
same thereof.
11. The method of claim 1, wherein said sphingoid-polyalkylamine
conjugate is N-palmitoyl-D-erythro-sphingosyl carbamoyl-spermine
(CCS).
12. The method of claim 1, wherein said sphingoid-polyalkylamine
conjugate has the following formula (I): ##STR5## wherein R.sub.1
represents a hydrogen, a branched or linear alkyl, saturated or
unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group
--C(O)R.sub.5; R.sub.2 and R.sub.5 represent, independently, a
branched or linear C.sub.10-C.sub.24 alkyl, hydroxyalkyl, alkenyl,
saturated or unsaturated cycloalkyl, aryl or polyenyl groups;
R.sub.3 and R.sub.4 are independently a hydrogen or group
--C(O)--NR.sub.6R.sub.7, R.sub.6 and R.sub.7 being the same or
different for R.sub.3 and R.sub.4 and represent, independently, a
hydrogen, or a saturated or unsaturated branched or linear
polyalkylamine, wherein one or more amine units in said
polyalkylamine may be a quaternary ammonium; provided that R.sub.3
and R.sub.4 are not simultaneously a hydrogen; or R.sub.3 and
R.sub.4 form together with the oxygen atoms to which they are bound
a heterocyclic ring comprising
--C(O)--NR.sub.9--[R.sub.8--NR.sub.9].sub.m--C(O)--, R.sub.8
represents a saturated or unsaturated C.sub.1-C.sub.4 alkyl and
R.sub.9 represents a hydrogen or a polyalkylamine of the formula
--[R.sub.8--NR.sub.9].sub.n--, wherein said R.sub.9 or each
alkylamine unit R.sub.8NR.sub.9 may be the same or different in
said polyalkylamine; and n and m, represent independently an
integer from 1 to 10; W represents a group selected from
--CH.dbd.CH--, --CH.sub.2--CH(OH)-- or --CH.sub.2--CH.sub.2--.
13. The method of claim 12, wherein R.sub.1 represents a
--C(O)R.sub.5 group; R.sub.5 represents a C.sub.12-C.sub.18 linear
or branched alkyl or alkenyl; W represents --CH.dbd.CH--; R.sub.2
represents a C.sub.12-C.sub.18 linear or branched alkyl or alkenyl;
R.sub.3 and R.sub.4 represent, independently, a hydrogen or a group
C(O)--NR.sub.6R.sub.7, wherein said R.sub.3 and R.sub.4 are not
simultaneously a hydrogen, and R.sub.6 and R.sub.7 represent,
independently, a hydrogen or a polyalkylamine having the general
formula (II): ##STR6## wherein R.sub.8 represent a C.sub.1-C.sub.4
alkyl; R.sub.9 represents a hydrogen or a polyalkylamine branch of
formula (II), said R.sub.8 and R.sub.9 may be the same or different
for each alkylamine unit, --R.sub.8NR.sub.9--, in the
polyalkylamine of formula (II); and n represents an integer from 3
to 6.
14. The method of claim 13, wherein R.sub.3 or R.sub.4 is a
hydrogen atom.
15. The method of claim 13, wherein both R.sub.3 and R.sub.4
represent the same or a different polyalkylamine.
16. The method of claim 13, wherein R.sub.1 represents a
C(O)R.sub.5 group; R.sub.5 represents a C.sub.12-C.sub.18 linear or
branched alkyl or alkenyl; W represents --CH.dbd.CH--; R.sub.2
represents a C.sub.12-C.sub.18 linear or branched alkyl or alkenyl;
R.sub.3 and R.sub.4 form together with the oxygen atoms to which
they are bonded a heterocyclic ring comprising
--C(O)--[NH--R.sub.8].sub.n--NH--C(O)--, wherein R.sub.8 represents
a C.sub.1-C.sub.4 alkyl, wherein for each alkylamine unit having
the formula --NH--R.sub.8--, said R.sub.8 may be the same or
different; and n represents an integer from 3 to 6.
17. The method of claim 13, wherein said R.sub.8 is a
C.sub.3-C.sub.4 alkyl.
18. The method of claim 1, comprising intranasal or parenteral
administration of said combination of sphingoid-polyalkylamine
conjugate and said biologically active molecule.
19. The method of claim 1, comprising intranasal or intramuscular
administration of a combination of said sphingoid-polyalkylamine
conjugate with said biologically active molecule, wherein: (i) when
said infection is caused by HBV, said biologically active molecule
is an HBV antigen; (ii) when said infection is caused by avian
influenza virus, said biologically active molecule is an AIV
antigen; (iii) when said infection is caused by the bacterium
Bacillus anthracis, said biologically active molecule is an antigen
of said bacterium; or (iv) when said infection is caused by the
bacterium Streptococcus pneumoniae said biologically active
molecule is an antigen of said bacterium.
20. The method of claim 19, wherein: (i) said HBV antigen is an HBV
surface particle (HBsAg); (ii) said AIV antigen is an inactivated
purified whole avian influenza virus; (iii) said bacterium antigen
is the protective antigen (PA) moiety of anthrax toxin; or (iv)
said Streptococcus pneumoniae antigen is a polysaccharide or
protein conjugate of an antigen selected from pneumococcal surface
protein A (PspA), pneumococcal adherence and virulence factor A
(PavA), pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin
binding protein A (CbpA), pneumococcal surface adhesion A
(PsaA).
21. The method of claim 13, comprising intranasal or intramuscular
administration of said N-palmitoyl D-erythro sphingosyl
carbamoyl-spermine together with said biologically active
molecule.
22. A vaccine comprising a combination of a
sphingoid-polyalkylamine conjugate and an amount of a biologically
active molecule, the amount of said biologically active molecule,
when combined with said sphingoid-polyalkylamine conjugate, being
effective to stimulate or enhance an immune response of a subject
to provide protection against an infection caused by an agent
selected from the group consisting of HBV, AIV, the bacterium
Bacillus anthracis and the bacterium Streptococcus pneumoniae.
23. The vaccine of claim 22, further comprising an
immunostimulating agent.
24. The vaccine of claim 22, wherein said sphingoid-polyalkylamine
conjugate comprises a sphingoid backbone carrying, via a carbamoyl
linkage at lest one polyalkylamine chain.
25. The vaccine of claim 22, wherein said sphingoid backbone is
selected from ceramide, dihydroceramide, phytoceramide,
dihydrophytoceramide, ceramine, dihydroceramine, phytoceramine,
dihydrophytoceramine.
26. The vaccine of claim 22, wherein said sphingoid is ceramide and
said polyalkylamine chain is selected from spermine, spermidine or
a polyalkylamine analog of spermine or spermidine.
27. The vaccine of claim 22, wherein said sphingoid-polyalkylamine
conjugate comprises N-palmitoyl-D-erythro-sphingosyl
carbamoyl-spermine (CCS).
28. The vaccine of claim 22, wherein: (i) when said infection is
caused by HBV, said biologically active molecule is an HBsAg
particle; (ii) when said infection is caused by avian influenza
virus, said biologically active molecule is an inactivated purified
whole avian influenza virus; (iii) when said infection is caused by
the bacterium Bacillus anthracis, said biologically active molecule
is a protective antigen (PA) moiety of anthrax toxin or (iv) said
Streptococcus pneumoniae antigen is a polysaccharide or protein
conjugate of an antigen selected from pneumococcal surface protein
A (PspA), pneumococcal adherence and virulence factor A (PavA),
pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin binding
protein A (CbpA), pneumococcal surface adhesion A (PsaA).
29. A vaccine comprising a combination of N-palmitoyl D-erythro
sphingosyl carbamoyl-spermine (CCS), being a single isomer of CCS
or mixture of CCS isomers, with a biologically active molecule
selected from the group consisting of HBsAg particle; an
inactivated purified whole avian influenza virus comprising
haemagglutinin (H5) antigen; the protective antigen (PA) moiety of
anthrax toxin and pneumococcal surface protein A (PspA), or
pneumococcal surface adhesion A (PsaA).
30. The vaccine of claim 22, wherein said sphingoid-polyalkylamine
conjugate has the following formula (I): ##STR7## wherein R.sub.1
represents a hydrogen, a branched or linear alkyl, saturated or
unsaturated cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group
--C(O)R.sub.5; R.sub.2 and R.sub.5 represent, independently, a
branched or linear C.sub.10-C.sub.24 alkyl, hydroxyalkyl, alkenyl,
saturated or unsaturated cycloalkyl, aryl or polyenyl groups;
R.sub.3 and R.sub.4 are independently a hydrogen or a group
--C(O)--NR.sub.6R.sub.7, R.sub.6 and R.sub.7 being the same or
different for R.sub.3 and R.sub.4 and represent, independently, a
hydrogen, or a saturated or unsaturated branched or linear
polyalkylamine, wherein one or more amine units in said
polyalkylamine may be a quaternary ammonium; wherein said R.sub.4
and R.sub.3 are not simultaneously a hydrogen; or R.sub.3 and
R.sub.4 form together with the oxygen atoms to which they are bound
a heterocyclic ring comprising
--C(O)--NR.sub.9--[R.sub.8--NR.sub.9].sub.m--C(O)--, R.sub.8
represents a saturated or unsaturated C.sub.1-C.sub.4 alkyl and
R.sub.9 represents a hydrogen or a polyalkylamine of the formula
--[R.sub.8--NR.sub.9],-, wherein said R.sub.9 or each alkylamine
unit R.sub.8NR.sub.9 may be the same or different in said
polyalkylamine; and n and m, represent independently an integer
from 1 to 10; W represents a group selected from --CH.dbd.CH--,
--CH.sub.2--CH(OH)-- or --CH.sub.2--CH.sub.2--.
31. A complex comprising a sphingoid-polyalkylamine conjugate and a
biologically active molecule, the complex being capable of
enhancing or stimulating an immune response of a subject to provide
protection against an infection caused by an agent selected from
HBV, AIV, the bacterium Bacillus anthracis or the bacterium
Streptococcus pneumoniae.
32. The complex of claim 31, comprising N-palmitoyl D-erythro
sphingosyl carbamoyl spermine (CCS) associated with said
biologically active molecule.
33. A method for the treatment or prevention of a disease or
disorder comprising administering to a subject in need of the
treatment a composition comprising a biologically active molecule
and a sphingoid-polyalkylamine conjugate having the general formula
(I'): ##STR8## wherein R.sub.1 represents a hydrogen, a branched or
linear alkyl, saturated or unsaturated cycloalkyl, aryl,
hydroxyalkyl, alkylamine, or a group --C(O)R.sub.5; R.sub.2 and
R.sub.5 represent, independently, a branched or linear
C.sub.10-C.sub.24 alkyl, hydroxyalkyl, alkenyl, saturated or
unsaturated cycloalkyl, aryl or polyenyl groups; R.sub.3 is a group
--C(O)--NR.sub.6R.sub.7, R.sub.6 and R.sub.7 represent,
independently, a hydrogen, or a saturated or unsaturated branched
or linear polyalkylamine, wherein one or more amine units in said
polyalkylamine may be a quaternary ammonium; W represents a group
selected from --CH.dbd.CH--, --CH.sub.2--CH(OH)-- or
--CH.sub.2--CH.sub.2--.
34. The method of claim 33, for stimulating or enhancing an immune
response of a subject to provide protection against an
infection.
35. The method of claim 33, wherein said sphingoid-polyalkylamine
conjugate is N-palmitoyl D-erythro sphingosyl-3-carbamoyl
spermine.
36. The method of claim 33, wherein said composition comprises a
mixture of a first sphingoid-polyalkylamine conjugate of said
formula (I') and a second sphingoid-polyalkylamine conjugate of the
following formula (I): ##STR9## wherein R.sub.1 represents a
hydrogen, a branched or linear alkyl, saturated or unsaturated
cycloalkyl, aryl, hydroxyalkyl, alkylamine, or a group
--C(O)R.sub.5; R.sub.2 and R.sub.5 represent, independently, a
branched or linear C.sub.10-C.sub.24 alkyl, hydroxyalkyl, alkenyl,
saturated or unsaturated cycloalkyl, aryl or polyenyl groups;
R.sub.3 and R.sub.4 are independently a group
--C(O)--NR.sub.6R.sub.7, R.sub.6 and R.sub.7 being the same or
different for R.sub.3 and R.sub.4 and represent, independently, a
hydrogen, or a saturated or unsaturated branched or linear
polyalkylamine, wherein one or more amine units in said
polyalkylamine may be a quaternary ammonium; or R.sub.3 is a
hydrogen; or R.sub.3 and R.sub.4 form together with the oxygen
atoms to which they are bound a heterocyclic ring comprising
--C(O)--NR.sub.9--[R.sub.8--NR.sub.9].sub.m--C(O)--, R.sub.8
represents a saturated or unsaturated C.sub.1-C.sub.4 alkyl and
R.sub.9 represents a hydrogen or a polyalkylamine of the formula
--[R.sub.8--NR.sub.9].sub.n--, wherein said R.sub.9 or each
alkylamine unit R.sub.8NR.sub.9 may be the same or different in
said polyalkylamine; and n and m represent independently an integer
from 1 to 10; W represents a group selected from --CH.dbd.CH--,
--CH.sub.2--CH(OH)-- or --CH.sub.2--CH.sub.2--.
37. The method of claim 36, wherein said mixture comprises a first
sphingoid-polyalkylamine conjugate being
N-palmitoyl-D-erythro-sphingosyl-3-carbamoyl spermine and a second
sphingoid-polyalkylamine conjugate being
N-palmitoyl-D-erythro-sphingosyl-1-carbamoyl spermine.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns use of sphingolipids'
polyalkylamine conjugates for effective delivery of biologically
active materials, in particular, antigenic molecules.
LIST OF PRIOR ART
[0002] The following is a list of prior art which is considered to
be pertinent for describing the state of the art in the field of
the invention.
[0003] U.S. Pat. No. 5,334,761: "Cationic lipids";
[0004] US 2001/048939: "Cationic reagents of transfection";
[0005] U.S. Pat. No. 5,659,011: "Agents having high nitrogen
content and high cationic charge based on dicyanimide dicyandiamide
or guanidine and inorganic ammonium salts";
[0006] U.S. Pat. No. 5,674,908: "Highly packed polycationic
ammonium, sulfonium and phosphonium lipids";
[0007] U.S. Pat. No. 6,281,371: "Lipopolyamines, and the
preparation and use thereof";
[0008] U.S. Pat. No. 6,075,012: "Reagents for intracellular
delivery of macromolecules";
[0009] U.S. Pat. No. 5,783,565: "Cationic amphiphiles containing
spermine or spermidine cationic group for intracellular delivery of
therapeutic molecules";
[0010] Ilies M A. et al. Expert Opin. Ther. Patents.
11(11):1729-1752 (2001);
[0011] Miller A D. Chem. Int. Ed. Eng. 37:1768-1785 (1998);
[0012] Nakanichi T. et al. J. Control Release 61:233-240
(1999);
[0013] Brunel F. et al. Vaccine 17:2192-2193 (1999);
[0014] Guy B. et al. Vaccine 19:1794-1805 (2001);
[0015] Lima K M et al. Vaccine 19:3518-3525 (2001).
BACKGROUND OF THE INVENTION
[0016] Many natural biological molecules and their analogues,
including proteins and polynucleotides, foreign substances and
drugs, which are capable of influencing cell function at the
sub-cellular or molecular level are preferably incorporated within
the cell in order to produce their effect. For these agents the
cell membrane presents a selective barrier which is impermeable to
them. The complex composition of the cell membrane comprises
phospholipids, glycolipids, and cholesterol, as well as intrinsic
and extrinsic proteins, and its functions are influenced by
cytoplasmic components which include Ca.sup.++ and other metal
ions, anions, ATP, microfilaments, microtubules, enzymes, and
Ca.sup.++-binding proteins, also by the extracellular glycocalyx
(proteoglycans, glycose aminoglycans and glycoproteins).
Interactions among structural and cytoplasmic cell components and
their response to external signals make up transport processes
responsible for the membrane selectivity exhibited within and among
cell types.
[0017] Successful delivery of agents not naturally taken up by
cells into cells has also been investigated. The membrane barrier
can be overcome by associating agents in complexes with lipid
formulations closely resembling the lipid composition of natural
cell membranes. These formulations may fuse with the cell membranes
on contact, or what is more common, taken up by pinocytosis,
endocytosis and/or phagocytosis. In all these processes, the
associated substances are delivered into the cells.
[0018] Lipid complexes can facilitate intracellular transfers also
by overcoming charge repulsions between the cell surface, which in
most cases is negatively charged. The lipids of the formulations
comprise an amphipathic lipid, such as the phospholipids of cell
membranes, and form various layers or aggregates such as micelles
or hollow lipid vesicles (liposomes), in aqueous systems. The
liposomes can be used to entrap the substance to be delivered
within the liposomes; in other applications, the drug molecule of
interest can be incorporated into the lipid vesicle as an intrinsic
membrane component, rather than entrapped into the hollow aqueous
interior, or electrostatically attached to aggregate surface.
However, most phospholipids used are either zwiterionic (neutral)
or negatively charged.
[0019] An advance in the area of intracellular delivery was the
discovery that a positively charged synthetic cationic lipid,
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA), in the form of liposomes, or small vesicles, could
interact spontaneously with DNA to form lipid-DNA complexes which
are capable of adsorbing to cell membranes and being taken up by
the cells either by fusion or more probably by adsorptive
endocytosis, resulting in expression of the transgene [Felgner, P.
L. et al. Proc. Natl. Acad. Sci., USA 84:7413-7417 (1987) and U.S.
Pat. No. 4,897,355 to Eppstein, D. et al.]. Others have
successfully used a DOTMA analogue,
1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) in
combination with a phospholipid to form DNA-complexing vesicles.
The Lipofectin.TM. reagent (Bethesda Research Laboratories,
Gaithersburg, Md.), an effective agent for the delivery of highly
anionic polynucleotides into living tissue culture cells, comprises
positively charged liposomes composed of the positively charged
lipid DOTMA and a neutral lipid--dioleoyl-phosphatidyl-ethanolamine
(DOPE) referred to as a helper lipid. These liposomes interact
spontaneously with negatively charged nucleic acids to form
complexes, referred to as lipoplexes. When excess of positively
charged liposomes over DNA negative charges are used, the net
charge on the resulting complexes is also positive. Positively
charged complexes prepared in this way spontaneously attach to
negatively charged cell surfaces or introduced into the cells
either by adsorptive endocytosis or fuse with the plasma membrane,
both processes deliver functional polynucleotide into, for example,
tissue culture cells. DOTMA and DOTAP are good examples for
monocationic lipids. [Illis et al. 2001, ibid.]
[0020] Multivalent cations by themselves (including polyamines,
inorganic salts and complexes and dehydrating solvents) have also
been shown to facilitate delivery of macromolecules into cells. In
particular, multivalent cations provoke the collapse of oligo and
polyanions (nucleic acids molecules and some peptide molecules and
the like) to compact structural forms, and facilitate the packaging
of these polyanions into viruses, their incorporation into
liposomes, transfer into cells etc. [Thomas T. J. et al.
Biochemistry 38:3821-3830 (1999)]. The smallest natural polycations
able to compact DNA are the polyamines spermidine and spermine. By
attaching a hydrophobic anchor to these molecules via a linker, a
new class of transfection vectors, the polycationic lipids and
lipopolymers, has been developed.
[0021] Cationic lipids and cationic polymers interact
electrostatically with the anionic groups of DNA (or of any other
polyanionic macromolecule) forming DNA-lipid complexes (lipoplexes)
or DNA-polycation complexes (polyplexes). The formation of the
complex is associated with the release of counterions of the lipids
or polymer, which is thought to be the thermodynamic driving force
for lipoplex and polyplex spontaneous formation. The cationic
lipids can be divided into four classes: (i) quaternary ammonium
salt lipids (e.g. DOTMA (Lipofectin.TM.) and DOTAP) and
phosphonium/arsonium congeners; (ii) lipopolyamines; (iii) cationic
lipids bearing both quaternary ammonium and polyamine moieties and
(iv) amidinium, guanidinium and heterocyclic salt lipids.
SUMMARY OF THE INVENTION
[0022] According to a first of its aspects, the vaccination aspect,
the present invention concerns a method for stimulating or
enhancing an immune response of a subject to provide protection
against an infection, the method comprising administering to said
subject a combination of sphingoid-polyalkylamine conjugate and a
biologically active molecule, the combination being effective to
provide said stimulation or enhancement of the immune response,
wherein said infection is caused by an agent selected from
hepatitis B virus (HBV), avian influenza virus , the bacterium
Bacillus anthracis, or the bacterium Streptococcus pneumoniae.
[0023] According to a preferred embodiment, the
sphingoid-polyalkylamine conjugate comprises a sphingoid backbone
carrying, via a carbamoyl linkage, at least one polyalkylamine
chain.
[0024] The term sphingoid-polyalkylamine conjugate as used herein
denotes chemical conjugation (linkage) between a sphingoid base
(herein also referred to by the term "sphingoid backbone") and at
least one polyalkylamine chain. The conjugation between the
sphingoid base and the at least one polyalkylamine chain is via a
carbamoyl linkage, as further detailed hereinafter.
[0025] The sphingoid base/backbone, as used herein, includes, long
chain aliphatic aminoalcohols, containing two or three hydroxyl
groups, the aliphatic chain may be saturated or unsaturated. One
example of an unsaturated sphingoid base is that containing a
distinctive trans-double bond in position 4. It is noted that the
sphingoid backbone has at least two stereoisomeric centers and in
accordance with the invention, the stereoisomeric centers may be in
R- or S-configuration, as well as combinations of same. In other
words, the sphingoid backbone may have at least the following
configurations: D-erythro, L-erythro, L-threo, and D-threo.
[0026] The term "enhancing" or "stimulating" as used herein
includes increase in cellular response and/or humoral response of
the immune system upon infection caused by exposure to an agent
selected from hepatitis B virus (HBV), avian influenza virus (AIV),
the bacterium Bacillus anthracis, or the bacterium Streptococcus
pneumoniae as well as, at times, to exposure to a combination of
such agents, the increase in the immune response being as a result
of the administration of the sphingoid-polyalkylamine conjugate in
combination with a biologically active molecule.
[0027] The term biologically active molecule as used herein
generally denotes any substance which, when administered in
combination with the sphingoid-polyalkylamine conjugate, has a
protective effect or a therapeutic effect in a subject's body which
may be identified (in vitro or in vivo) by known biochemical
parameters. For example, the biologically active molecule may
include polynucleotides, oligonucleotides, proteins, peptides and
drugs having a biochemical effect within a subject's body. In
accordance with the vaccination aspect of the invention, the effect
may be an enhancing effect on the immune system of a subject. The
biologically active material in this case is preferably an
antigenic protein, antigenic peptide, antigenic polypeptide,
antigenic carbohydrate, or antigenic glyco-protein. The
biologically active molecule preferably has a net negative charge
or containing one or more regions or moieties carrying a (local)
negative charge, such that under suitable condition it interacts
with the net positive charge of the sphingoid-polyalkylamine
conjugate.
[0028] In the context of the vaccination aspect of the present
invention, the biologically active molecule may be one or more of
the following: [0029] (i) when said infection is caused by HBV,
said biologically active molecule is an Hepatitis B antigen; [0030]
(ii) when said infection is caused by avian influenza virus, said
biologically active molecule is an avian influenza antigen; [0031]
(iii) when said infection is caused by the bacterium Bacillus
anthracis, said biologically active molecule is an antigen of said
bacterium; and [0032] (iv) when said infection is caused by the
bacterium Streptococcus pneumoniae, said biologically active
molecule is an antigen of said bacterium.
[0033] The stimulation or enhancement is statistically significant
better (p<0.005), showing an increase by at least 20%, relative
to that elicited by the same biologically active molecule
administered without the conjugate.
[0034] The invention also concerns the increase of an immune
response in cases when the biologically active molecule
administered to the subject, without the conjugate, is
substantially ineffective in producing such a response.
[0035] The term "hepatitis B antigen" refers to any component that
is capable, either by itself, with an adjuvant, or in combination
with the sphingoid-polyalkylamine in accordance with the invention,
to produce an immune response which may be cellular, humoral or
both. The antigen may be the whole virus, attenuated, mutated,
deactivated or a dead virus. The antigen may also be virus
fragments in particular virus membrane fragments. The antigen may
further be a molecule or complex of molecules present in the virus
produced by isolation or by various biotechnological synthetic
technologies. Examples of such molecules are protein or protein
fragment, peptide or peptide fragment, nucleic acid molecule,
carbohydrate, glycol-protein or low molecular weight compound.
[0036] According to a preferred embodiment of the invention, the
antigen a particle comprising a hepatitis B surface antigen (HBsAg,
i.e. the S peptide), which may be in combination with the pre-S1 or
pre-S2 protein.
[0037] The term "avian influenza antigen" refers to any component
of the avian influenza virus that is capable, either by itself,
with an adjuvant, or in combination with the
sphingoid-polyalkylamine in accordance with the invention, to
produce an immune response which may be cellular, humoral or both.
The antigen may be the whole virus, attenuated, mutated,
deactivated or a dead virus. The antigen may also be virus
fragments. The antigen may further be a molecule or complex of
molecules present in the virus produced by isolation or by various
biotechnological synthetic technologies. Examples of such molecules
are protein or protein fragment, peptide or peptide fragment,
nucleic acid molecule, carbohydrate, glycol-protein or low
molecular weight compound.
[0038] In accordance with one embodiment of the invention, the
avian influenza antigen is an "inactivated purified whole avian
influenza virus". The term "inactivated purified whole avian
influenza virus" refers to any member of the type A influenza virus
which has been manipulated to be in its inactivated, non-pathogenic
form. The avian influenza viruses may be categorized into subtypes
according to the antigens of the haemagglutinin (H) and
neuraminidase (N) molecules expressed on their surfaces and
hitherto, and there are known 16 haemagglutinin subtypes and 9
neuraminidase subtypes of influenza A viruses. In the context of
the present invention, the inactivated purified whole avian
influenza virus preferably relates to those derived from the highly
pathogenic Al viruses comprising the H5 and/or the H7 subtypes, as
well as the less pathogenic H9 subtype. For example, the
inactivated purified whole avian influenza virus may be that
derived from the highly pathogenic H5N1 or H5N2 viruses. Additional
proteins that can serve as antigens for influenza vaccination
comprise the matrix proteins (e.g. M1 and M2 proteins) and the
nuclear proteins (e.g. NP).
[0039] In accordance with a further embodiment, the avian influenza
virus antigen is a virus clade. As known to those versed in the
art, phylogenetic analyses of the H5 HA genes from the 2004 and
2005 diseases outbreaks showed 2 different lineages of HA genes,
termed clades 1 and 2. A specific example for an avian influenza
virus antigen comprises the H5N1 clades.
[0040] In accordance with yet another embodiment, the avian
influenza antigen may be a whole virion, a split virion, or a virus
sub-unit.
[0041] The term "antigen of the bacterium Bacillus anthracis"
denotes the whole bacteria attenuated, mutated, deactivated or
dead, as well as non-encapsulated strains of the bacterium as well
as components of the bacterium elaborated by the bacteria that have
antigenic activity.
[0042] In accordance with one embodiment, the antigen may be a
component of the bacterium's capsule.
[0043] In accordance with another embodiment, the antigen is a
component of the bacterium's toxin. The term "antigen of a toxin
produced from the bacterium Bacillus anthracis" which may be used
interchangeably with the term "anthrax antigen" denotes any
component of the Bacillus anthracis toxin. The anthrax toxin
comprises three distinct proteins: lethal factor (LF), oedema
factor (OF), and the protective antigen (PA). The PA is a protein
that can insert into the membrane of a host cell to create a hole,
or pore, through the membrane. The pore then functions as a portal
to allow the other two components to get inside of the host cell.
The LF is a type of enzyme classified as a zinc protease which
attacks and breaks host proteins into smaller and nonfunctional
pieces. Destroying host cell proteins is lethal to the host cell,
hence the factor's name. The OF is a toxin; the destruction of the
host cells allows this toxin to enter the bloodstream, where it can
kill cells of the immune system. Disabling the host's immune
response allows the bacteria and the toxin to spread throughout the
body. In accordance with a preferred embodiment of the invention,
the antigen is the protective antigen (PA) moiety of the anthrax
toxin.
[0044] The term "antigen of Streptococcus pneumoniae" denotes any
component of the bacterium including attenuated or inactivated
(e.g. inactivated epizootic strains) forms of the bacterium. The
streptococci antigen may be a component isolated from the bacteria,
a fragment of any native component, a recombinant product
comprising a component of the bacterium, a fragment or variant
thereof (such as a recombinant protein, a fused protein, etc.), a
modified component (e.g. chemically modified). Preferably, the
Streptococci antigen comprises a polysaccharide or protein
conjugated antigen derived from the bacterium, the polysaccharide
antigen being preferably conjugated to a protein carrier. It is
noted that polysaccharide antigens being preferably conjugated to a
protein carrier are commonly used for polysaccharide-based
vaccines, although, it is to be understood that the invention is
not limited to such conjugations. Non-limiting commercial products
which may be used in accordance with this embodiment include
mixtures of several strains, such as 23-valent polysaccharide
vaccine Pneumovax.TM. (Merck NJ USA), and 7-valent conjugated
vaccine (for young children), (PrevNar.TM. or PCV7 (Wyeth, NJ,
USA).
[0045] The present invention concerns a vaccine comprising a
combination of a sphingoid-polyalkylamine conjugate and an amount
of a biologically active molecule, the amount of said biologically
active molecule, when combined with said sphingoid-polyalkylamine
conjugate, being effective to stimulate or enhance an immune
response of a subject against an infection, said infection being
caused by an agent selected from one or more of the following: HBV,
avian influenza virus, the bacterium Bacillus anthracis, and the
bacterium Streptococcus pneumoniae.
[0046] According to yet another embodiment, the invention provides
a complex comprising a sphingoid-polyalkylamine conjugate and a
biologically active molecule, the complex being capable of
enhancing or stimulating an immune response of a subject to provide
protection against an infection, said infection being caused by an
agent selected from one or more of the following: HBV, avian
influenza virus, the bacterium Bacillus anthracis, and the
bacterium Streptococcus pneumoniae.
[0047] In accordance with a second aspect, the present invention
relates to a method for the treatment or prevention of a disease or
disorder comprising administering to a subject in need of the
treatment a combination comprising a biologically active molecule
and a sphingoid-polyalkylamine conjugate having the general formula
(I'): ##STR1##
[0048] wherein
[0049] R.sub.1 represents a hydrogen, a branched or linear alkyl,
saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl,
alkylamine, or a group --C(O)R.sub.5;
[0050] R.sub.2 and R.sub.5 represent, independently, a branched or
linear C.sub.10-C.sub.24 alkyl, hydroxyalkyl, alkenyl, saturated or
unsaturated cycloalkyl, aryl or polyenyl groups;
[0051] R.sub.3 is a group --C(O)--NR.sub.6R.sub.7, R.sub.6 and
R.sub.7 represent, independently, a hydrogen, or a saturated or
unsaturated branched or linear polyalkylamine, wherein one or more
amine units in said polyalkylamine may be a quaternary
ammonium;
[0052] W represents a group selected from --CH.dbd.CH--,
--CH.sub.2--CH(OH)-- or --CH.sub.2--CH.sub.2--.
[0053] A preferred sphingoid-polyalkylamine conjugate of formula
(I') is N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine (C-3
CCS).
[0054] In accordance with this aspect of the invention, there is
also provided a pharmaceutical composition comprising a
biologically active molecule in combination with said
sphinogid-polyalkylamine conjugate of formula (I'). The composition
may include other sphinogid-polyalkylamine conjugates, such as
those included in general formula (I).
[0055] According to one embodiment, the sphinogid-polyalkylamine
conjugate of formula (I') is mixed with at least one additional
sphinogid-polyalkylamine conjugate, such as N-palmitoyl D-erythro
sphingosyl-1-carbamoyl spermine (C-1 CCS).
[0056] Further, according to an embodiment, the
sphinogid-polyalkylamine conjugate of formula (I') is used for the
preparation of vaccines.
BRIEF DESCRIPTION OF THE FIGURES
[0057] In order to understand the invention and to see how it may
be carried out in practice, some embodiments will now be described,
by way of non-limiting examples only, with reference to the
accompanying figures, in which:
[0058] FIGS. 1A-1E show several possible chemical structures,
"linear", "branched" or "cyclic" cationic lipid compounds which are
encompass under the general definition of sphingoid-polyalkylamine
conjugate of formula (I), wherein FIGS. 1A-1B shows a sphingoid
backbone (ceramide) linked to a single polyalkylamine chain, FIG.
1C and FIG. 1D show the same sphingoid backbone linked to two
polyalkylamine chains, FIG. 1E shows again the same backbone,
however, in which a single polyalkylamine chain is linked via the
two hydroxyl moieties to form a cyclic polyalkylamine
conjugate.
[0059] FIGS. 2A-2F show the bio-distribution and pharmacokinetics
of various fluorescently-labeled lipid formulations (liposomes),
with and without the influenza antigens (referred to as HN)
following intranasal (i.n.) administration to BALB/C mice: in the
nose --, GI--, lungs -.diamond-solid.- or spleen -- with
unrecovered --: FIG. 2A shows distribution of empty DMPC:DMPG (mole
ratio 9:1); FIG. 2B shows distribution of empty DOTAP:cholesterol;
FIG. 2C shows distribution of empty N-palmitoyl D-erythro
sphingosyl carbamoyl spermine (CCS):cholesterol; FIG. 2D shows
distribution of DMPC:DMPG:HN; FIG. 2E shows distribution of
DOTAP:cholesterol:HN; and FIG. 2F shows distribution of
CCS:cholesterol:HN,
[0060] FIGS. 3A-3D show bio-distribution of various .sup.125I-HN
loaded liposome formulations, administered as above, in the nose
--, GI--, lungs -- or spleen -- with unrecovered , and in
particular, FIG. 3A shows bio-distribution of free .sup.125I-HN;
FIG. 3B shows .sup.125I-HN loaded liposomes composed of
DOTAP:Cholesterol; FIG. 3C shows .sup.125I-HN loaded liposomes
composed of DMPC:DMPG and FIG. 3D shows .sup.125I-HN loaded
liposomes composed of CCS:Cholesterol.
[0061] FIG. 4 shows mean serum HI (haemagglutination inhibition)
antibody titers against A/New Caledonia virus following vaccination
of rats i.m., once, with 12 .mu.g total HA (trivalent vaccine),
with or without CCS/C (mole ratio 3:2), at a lipid:antigen w/w
ratio of 150:1, as determined by the HI assay.
[0062] FIG. 5 shows the mean sum of viral titer
(log.sub.10TCID.sub.50/ml) in nasal wash following i.m. vaccination
of ferrets with free HA (F-HA) or with CCS/C-HA and challenge with
the homologous influenza A/New Caledonia virus (H1N1) on day 28.
P<0.001 as compared to CCS/C only and F-HA.
[0063] FIG. 6 shows the hemagglutination inhibition (HI) antibody
levels in serum of BALB/C mice at weeks 2, 4, 8, 12 and 20 after a
single intramuscular (i.m.) immunization with avian influenza
(H5N1) vaccine at doses of 3 .mu.g or 6 .mu.g HA.
[0064] FIGS. 7A-7B shows serum anti-HBsAg antibody levels 5 and 12
weeks following intraperitoneal (i.p.) or intranasal (i.n.)
vaccination (FIG. 7A) and the specific isotypes against HBsAg
detected by ELISA 6 weeks post-vaccination (FIG. 7B) of BALB/C mice
with the free or liposomal (CCS/C) HBV vaccine.
[0065] FIG. 8A-8C show Anti-HBsAg (FIG. 8A), anti-HBsAg IgG1 (FIG.
8B) and anti-HBsAg IgG2a (FIG. 6C) titers in serum collected 14, 28
days, 2 months and 3 months following a single intraperitoneal
(i.p.) vaccination of BALB/C mice with free antigen (F-Ag), the
commercial vaccine Sci-B-Vac.TM. (Alum adsorbed Ag) or the
liposomal antigen (CCS/C-Ag).
[0066] FIG. 9A-9B are bar graphs showing serum anti-B. anthracis PA
IgG antibodies median levels in samples from BALB/C mice following
subcutaneous (s.c) or intranasal (i.n.) vaccination (a single or
double-dose vaccination, respectively) with free antigen (F-Ag),
Alum-Ag at two w/w ratios (7/1, 25/1), or liposomal
CCS/Cholesterol-antigen (CCS/C-Ag). FIG. 9A shows the O.D
(presenting levels) 2 and 4 weeks post-vaccination as determined
using a commercial kit: QuickELISA.TM. Anthrax-PA kit (Immunetics,
Inc, MA, USA) (% indicates percentage of responders. OD>0.186
was considered a positive response). FIG. 9B shows the titers as
determined by ELISA (as described herein below) where only i.n.
vaccination was tested also after 7 weeks (2 weeks post the second
vaccination), (%: percentage of responders (titer>30)).
[0067] FIG. 10 is a bar graph showing anti-PA antibodies median
levels in serum samples of Guinea pigs 28 days after a single
subcutaneous vaccination with free antigen (F-PA), liposomal
CCS/Cholesterol antigen (CCS/C-PA) or Sterne strain (STI,
commercial veterinary vaccine used as a positive control); as
determined by a commercial kit: QuickELISA.TM. Anthrax-PA kit
(Immunetics, Inc, MA, USA). (% indicates percentage of responders.
OD>0.186 was considered a positive response).
DETAILED DESCRIPTION
[0068] In accordance with a first of its aspects, the present
invention concerns the use of sphingoid-polyalkylamine conjugates
as adjuvants for biologically active molecules (e.g. antigens) for
enhancing the immune response of a subject, for the purpose of
treating the subject against an infection caused by an agent
selected from the group consisting of hepatitis B virus (HBV),
avian influenza virus (AIV), the bacterium Bacillus anthracis and
the bacterium Streptococcus pneumoniae.
[0069] The sphingoid-polyalkylamine conjugates are cationic lipids
compounds, which may be synthesized in the following manner.
N-substituted long-chain bases in particular, N-substituted
sphingoids or sphingoid bases are coupled together with different
polyalkylamines or their derivatives, to form a
polyalkylamine-sphingoid entity, which is used as is, or further
alkylated. Some sphingoid-polyalkylamine conjugates are also
commercially available.
[0070] Protonation at a suitable pH or alkylation of the formed
polyalkylamine-sphingoid entity attributes to the lipid-like
compounds a desired positive charge for interaction with
biologically active biological molecules to be delivered into
target cells and with the targeted cells. The
sphingoid-polyalkylamine conjugates may be efficiently associated
with the biologically active molecules by virtue of electrostatic
interactions between the anionic character of the biologically
active molecules and the polyalkylamine moieties of the conjugate
to form complexes (lipoplexes).
[0071] Thus, disclosed herein is a method for stimulating or
enhancing an immune response of a subject to provide protection
against an infection, the method comprising administering to said
subject a combination of at least one sphingoid-polyalkylamine
conjugate and a biologically active molecule, the combination being
effective to provide said stimulation or enhancement of the immune
response, wherein said infection is caused by an agent selected
from the group consisting of hepatitis B virus (HBV), avian
influenza virus (AIV), the bacterium Bacillus anthracis and the
bacterium Streptococcus pneumoniae, wherein said
sphingoid-polyalkylamine conjugate comprises a sphingoid backbone
carrying, via a carbamoyl linkage, one or two polyalkylamine
chains.
[0072] In accordance with a preferred embodiment, the
sphingoid-polyalkylamine conjugate includes a linkage between a
sphingoid backbone and at least one polyalkylamine chain, the
linkage is via corresponding carbamoyl linkage.
[0073] More preferably, the sphingoid-polyalkylamine conjugate has
the general formula (I): ##STR2##
[0074] wherein
[0075] R.sub.1 represents a hydrogen, a branched or linear alkyl,
saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl,
alkylamine, or a group --C(O)R.sub.5;
[0076] R.sub.2 and R.sub.5 represent, independently, a branched or
linear C.sub.10-C.sub.24 alkyl, hydroxyalkyl, alkenyl, saturated or
unsaturated cycloalkyl, aryl or polyenyl groups;
[0077] R.sub.3 and R.sub.4 are independently a hydrogen or a group
--C(O)--NR.sub.6 R.sub.7, R.sub.6 and R.sub.7 being the same or
different for R.sub.3 and R.sub.4 and represent, independently, a
hydrogen, or a saturated or unsaturated branched or linear
polyalkylamine, wherein one or more amine units in said
polyalkylamine may be a quaternary ammonium; wherein at least one
of said R.sub.3 or R.sub.4 are not simultaneously a hydrogen;
or
[0078] R.sub.3 and R.sub.4 form together with the oxygen atoms to
which they are bound a heterocyclic ring comprising
--C(O)--NR.sub.9--[R.sub.8--NR.sub.9].sub.m--C(O)--, R.sub.8
represents a saturated or unsaturated C.sub.1-C.sub.4 alkyl and
R.sub.9 represents a hydrogen or a polyalkylamine of the formula
--[R.sub.8--NR.sub.9].sub.n--, wherein said R.sub.9 or each
alkylamine unit R.sub.8NR.sub.9 may be the same or different in
said polyalkylamine; and
[0079] n and m are independently an integer from 1 to 10,
preferably 3 to 6;
[0080] W represents a group selected from --CH.dbd.CH--,
--CH.sub.2--CH(OH)-- or --CH.sub.2--CH.sub.2--.
[0081] According to one preferred embodiment the sphingoid backbone
is a ceramide having a carbamoyl moiety linked to one (FIG. 1A-1B)
or two (FIGS. 1C-1D) polyalkylamine chains, or linked via two
carbamoyl moieties to a single polyalkylamine chain, to form a
cyclic polyalkylamine conjugate (FIG. 1E).
[0082] When referring to the use of the sphingoid-polyalkylamine
conjugate for vaccination, the biologically active material is any
molecule which when administered with the sphingoid-polyalkylamine
conjugate has an effect on the immune system of a subject,
according to one embodiment, a stimulating or enhancing effect as
compared to the effect of the biologically active material when
provided without the conjugate. The effect is preferably by a
factor of two or more relative to the effect, if any, of the
biologically active molecule, when provided to a subject without
said conjugate. Thus, the sphingoid-polyalkylamine conjugate is to
be considered an efficacious adjuvant for vaccination.
[0083] According to one embodiment, the biologically active
material is a protein, polypeptide, peptide, glycol-protein or
carbohydrate, being derived from (either by isolation, modification
and/or production) HBV, avian influenza virus, the bacterium
Bacillus anthracis, or the bacterium Streptococcus pneumoniae.
Specifically, the biologically active molecule may be antigenic
protein, antigenic peptide, antigenic carbohydrate, antigenic
glycol-protein and immunostimulants. Antigenic proteins and
peptides and immunostimulants are all well known in the art.
Preferably, the biologically active protein or peptide or
carbohydrate has at a physiological pH either a net negative dipole
moment, a net negative charge or contains at least one negatively
charged region.
[0084] Preferred sphingoid-polyalkylamine conjugates according to
this aspect of the invention comprise the different structural and
stereoisomers of N-palmitoyl D-erythro sphingosyl
carbamoyl-spermine (CCS). This conjugate includes a ceramide linked
via a C-1 and/or C-3 carbamoyl linkage to spermine as well as to
mixtures of same. Further preferred sphingoid-polyalkylamine
conjugates according to the invention comprise N-palmitoyl
D-erythro sphingosyl-1-carbamoyl spermine (C-1 CCS), N-palmitoyl
D-erythro sphingosyl-3-carbamoyl spermine (C-3 CCS) as well as
mixtures of same and the various stereoisomers of same. In the
following text, the abbreviation CCS denotes either a single
conjugate or a mixture of same, unless otherwise specifically
stated.
[0085] According to one embodiment, the sphingoid-polyalkylamine
conjugate, and preferably the CCS, is used for the preparation of a
vaccine for hepatitis B virus (HBV). To this end, the conjugate is
formulated with a HBV antigen to form a vaccine (which may include
other constituents). A specifically preferred HBV derived antigenic
material is the HBsAg particle.
[0086] According to another embodiment, the
sphingoid-polyalkylamine conjugate, and preferably the CCS, is used
for the preparation of a vaccine for avian influenza virus (AIV).
In this particular embodiment, the biologically active material is
preferably an inactivated purified whole avian influenza virus or a
component thereof comprising at least one or a combination of the
haemagglutinin (H or HA) and neuraminidase (N or NA) antigens (the
combination referred to as HN, unless otherwise stated), or a
biologically active analog of a molecule derived from these
antigens, which is capable of eliciting an immune response against
the natural antigen(s) as defined hereinabove. A preferred
inactivated purified whole avian influenza virus in accordance with
the invention is the whole virus comprising the H5N1 antigens.
[0087] According to yet another embodiment, the
sphingoid-polyalkylamine conjugate, and preferably the CCS, is used
for the preparation of a vaccine for the bacterium Bacillus
anthracis (Anthrax). In this particular embodiment, the
biologically active material is preferably an antigen of a toxin
produced from the bacterium Bacillus anthracis or the immunogenic
component thereof. In accordance with a preferred embodiment, the
antigen comprises one or more of the bacterium toxin proteins
selected from the lethal factor (LF), oedema factor (OF), and the
protective antigen (PA), or a biologically active analog of the
protein, which is capable of eliciting an immune response against
the bacterium. A preferred antigen in accordance with this
embodiment of the invention is the protective antigen.
[0088] In accordance with yet a further embodiment, the
sphingoid-polyalkylamine conjugate, and preferably the CCS, is used
for the preparation of a vaccine for the bacterium Streptococcus
pneumoniae. In accordance with a preferred embodiment, the vaccine
comprises as the biologically active molecule an inactivated or
attenuated bacterium, an antigen, or combinations of antigens
derived from the Streptococcus pneumoniae bacterium.
[0089] Various Streptococcus pneumoniae antigens have already been
described in the art and the following is a non-limiting
description of some such antigens which may be utilized in
accordance some embodiments of the invention. Needless to say that
in the context of the present invention, one or more antigens
derived from Streptococcus pneumoniae may be utilized.
[0090] In accordance with one embodiment, there is disclosed the
use of a recombinant CIpP protein derived from Streptococcus
pneumoniae as an antigen. CIpP protein of Streptococcus pneumoniae
is serine protease having 21 kDa of molecular weight (Genebank
AE008443) which is one of heat shock proteins. Recombinant CIpP
protein can be prepared by large scale expression and isolation in
accordance with conventional genetic engineering techniques in the
art.
[0091] In accordance with a further embodiment, the antigen may be
a capsular saccharide antigen, which is preferably conjugated to a
carrier protein. The saccharide antigen may include mixtures of
polysaccharides from different serotypes (e.g. from the 90
different serotypes) from which there are 23 serotypes widely used
(1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F,
18C, 19A, 19F, 20, 22F, 23F, and 33). As known to those versed in
the art, PrevNar.TM., a commonly known vaccine, contains antigens
from seven serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) with each
saccharide individually conjugated to diphtheria CRM.sub.197
protein (a nontoxic variant of diphtheria toxin) isolated from
cultures of Corynebacterium diphtheriae strain C7 (.beta.197) by
reductive amination. Pneumovax.TM. is another polysaccharide-based
vaccine containing polysaccharides from the above mentioned 23
different strains.
[0092] In accordance with yet another embodiment, the antigen may
be a Streptococcus pneumoniae protein. Non-limiting examples of
Streptococcus pneumoniae proteins which may be used as
Streptococcus pneumoniae antigens, alone or in combination, e.g.
with a Streptococcus pneumoniae saccharide include Poly Histidine
Triad family (Pht; e.g. PhtA, PhtB, PhtD, or PhtE), Lyt family
(e.g. LytA, LytB, or LytC), SpsA, Sp128, Sp130, Sp125, Sp101 and
Sp133, or truncate or immunologically functional equivalent
thereof, optionally with a Th1 adjuvant (an adjuvant inducing a
predominantly Th1 immune response). Specific, however, non-limiting
Streptococcus pneumoniae proteins include pneumococcal surface
protein A (PspA [Nabors et al. Vaccine 18:1743-1754 (2000),
incorporated herein in its entirety, by reference]), pneumococcal
surface protein C (PspC), pneumococcal surface immunogenic protein
B (PsipB), pneumococcal adherence and virulence factor A (PavA),
pneumococcal glutamyl-tRNA synthetase, pneumolysin, cholin binding
protein A (CbpA), pneumococcal autolysin (LytA, mentioned above) as
well as pneumococcal surface adhesion A (PsaA) which is a
streptococcal common protein (Russell et al., J. Clin. Microbiol.,
28:2191-2195 (1990); and U.S. Pat. No. 5,422,427). The antigen may
also be any peptide comprising the epitope of the above mentioned
proteins. The complete genome sequence of a virulent isolate of
Streptococcus pneumoniae was determined and several surface exposed
proteins that may serve as potential vaccine candidates were
identified [Tettelin et al. Science 293:498-506 (2001); and Hoskins
et al. J Bacteriology, 183(19):5709-5717 (2001), both incorporated
herein in their entirety, by reference].
[0093] In a further embodiment, the antigen may comprise a
phosphocholine, which is an antigenic component (of the bacterial
cell wall) in a variety of pathogenic organisms.
[0094] Further, the antigen may be a Streptococcus pneumoniae
derived pili or a pilus subunit or subunits. Examples of three
pilus subunits are RrgA, RrgB and RrgC as described by P. Ruggiero
et al. [Infection and Immunity 75(2):1059-1062 (2007)]
[0095] The sphingoid-polyalkylamine conjugate may form part of a
kit for the preparation of a pharmaceutical composition for
stimulating or enhancing the immune response of a subject when
combined with a biologically active material. Specifically, the kit
may be for the preparation of a pharmaceutical composition for
stimulating or enhancing an immune response of a subject so as to
provide the same with protection against an infection caused by an
agent selected from hepatitis B virus (HBV), avian influenza virus
(AIV), the bacterium Bacillus anthracis and the bacterium.
Streptococcus pneumoniae. The kit comprises a combination of
sphingoid-polyalkylamine conjugate and the biologically active
material, the biologically active material being selected such
that: [0096] (i) when the infection is caused by HBV, said
biologically active material is an Hepatitis B antigen; [0097] (ii)
when the infection is caused by avian influenza virus, said
biologically active molecule comprises an avian influenza antigen;
[0098] (iii) when the infection is caused by the bacterium Bacillus
anthracis, said biologically active molecule comprises an antigen
of said bacterium; [0099] (iv) when the infection is caused by the
bacterium Streptococcus pneumoniae, said biologically active
molecule comprises an antigen of said bacterium.
[0100] The kit may comprise, in addition to said conjugate,
instructions for use of same in combination with one or more of the
above defined biologically active materials. The conjugate in the
kit may be in a dry form, in which case, the kit may also include a
suitable fluid with which the conjugate is mixed prior to use to
form a suspension, emulsion or solution, or it may already be in a
fluid (suspension, emulsion, solution, etc.) form.
[0101] Disclosed herein is also a complex comprising the
sphingoid-polyalkylamine conjugate as defined herein and preferably
N-palmitoyl-D-erythro-sphingosyl carbamoyl-spermine (CCS), being a
single isomer or mixture of CCS isomers, and the biologically
active molecule, the complex being capable of enhancing or
stimulating an immune response of a subject to provide protection
against an infection caused by an agent selected from HBV, AIV, the
bacterium Bacillus anthracis or the bacterium Streptococcus
pneumoniae. The biologically active molecule is as defined
hereinabove. The complex is preferably forms a lipid assembly, and
the lipid assembly is preferably a liposome, a vesicle or a
combination of same.
[0102] In accordance with a further aspect there is provided a
method for the treatment or prevention of a disease or disorder
comprising administering to a subject in need of the treatment a
composition comprising a biologically active molecule and a
sphingoid-polyalkylamine conjugate having the general formula (I'):
##STR3##
[0103] wherein
[0104] R.sub.1 represents a hydrogen, a branched or linear alkyl,
saturated or unsaturated cycloalkyl, aryl, hydroxyalkyl,
alkylamine, or a group --C(O)R.sub.5;
[0105] R.sub.2 and R.sub.5 represent, independently, a branched or
linear C.sub.10-C.sub.24 alkyl, hydroxyalkyl, alkenyl, saturated or
unsaturated cycloalkyl, aryl or polyenyl groups;
[0106] R.sub.3 is a group --C(O)--NR.sub.6 R.sub.7, R.sub.6 and
R.sub.7 represent, independently, a hydrogen, or a saturated or
unsaturated branched or linear polyalkylamine, wherein one or more
amine units in said polyalkylamine may be a quaternary
ammonium;
[0107] W represents a group selected from --CH.dbd.CH--,
--CH.sub.2--CH(OH)-- or --CH.sub.2--CH.sub.2--.
[0108] This aspect is at times referred to as the "C-3 conjugate
aspect" of the invention.
[0109] In accordance with the C-3 conjugate aspect of the
invention, the method is applicable for the treatment or prevention
of any disease or disorder, where the conjugate elicits or
stimulates the therapeutic effect of the biologically active
molecule. In accordance with one embodiment, the method is for
stimulating or enhancing an immune response of a subject to provide
protection against an infection.
[0110] The invention also provides pharmaceutical compositions
comprising at least the sphingoid-polyalkylamine conjugate of
formula (I') in combination with a biologically active
molecule.
[0111] The conjugate of general formula (I') is preferably
N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine.
[0112] The sphinogid-polyalkylamine conjugate of formula (I') may
be used as the only sphinogid-polyalkylamine conjugate or in
combination with other sphinogid-polyalkylamine conjugates such as
those defined by formula (I).
[0113] One preferred combination comprises, without being limited
thereto, a sphinogid-polyalkylamine conjugate mixture comprising a
first sphingoid-polyalkylamine conjugate being
N-palmitoyl-D-erythro-sphingosyl-3-carbamoyl spermine and a second
sphingoid-polyalkylamine conjugate being
N-palmitoyl-D-erythro-sphingosyl-1-carbamoyl spermine.
[0114] When using a mixture of sphinogid-polyalkylamine conjugates,
the mole:mole ratio between a first sphingoid-polyalkylamine
conjugate of general formula (I') and a second
sphingoid-polyalkylamine conjugate of formula (I) may be any ratio
as desired.
[0115] The conjugate of formula (I) or of formula (I') preferably
comprises one or more of the following:
[0116] R.sub.1 preferably represents a --C(O)R.sub.5 group;
[0117] R.sub.2 and R.sub.5 preferably represent, independently, a
linear or branched C.sub.12-C.sub.18 alkyl or alkenyl groups;
[0118] W preferably represents --CH.dbd.CH--;
[0119] In accordance with one preferred embodiment, the
sphinogid-polyalkylamine conjugate of formula (I) or (I') comprises
a polyalkylamine having the general formula (II): ##STR4##
[0120] wherein
[0121] R.sub.8 represent a C.sub.1-C.sub.4 alkyl, preferably
C.sub.3-C.sub.4 alkyl;
[0122] R.sub.9 represents a hydrogen or a polyalkylamine branch of
formula (II), said R.sub.8 and R.sub.9 may be the same or different
for each alkylamine unit, --R.sub.8NR.sub.9--, in the
polyalkylamine of formula (II); and
[0123] n represents an integer from 3 to 6.
[0124] A preferred group of polyalkylamine chains forming part of
the sphingoid-polyalkylamine conjugates comprises spermine,
spermidine, a polyalkylamine analog or a combination of same
thereof. The term polyalkylamine analog is used to denote any
polyalkylamine chain, and according to one embodiment denotes a
polyalkylamine comprising 1 to 10 amine groups, preferably from 3
to 6 and more preferably 3 or 4 amine groups. Each alkylamine
within the polyalkylamine chain may be the same or different and
may be a primary, secondary, tertiary amine or quaternary
ammonium.
[0125] The alkyl moiety, which may be the same or different within
the polyalkylamine chain, is preferably a C.sub.1-C.sub.6 aliphatic
repeating unit. Some non-limiting examples of polyalkylamines
include spermidine, N-(2-aminoethyl)-1,3-propane-diamine,
3,3'-iminobispropylamine, spermine and bis(ethyl) derivatives of
spermine, polyethyleneimine.
[0126] The sphingoid-polyalkylamine conjugates may be further
reacted with methylation agents in order to form quaternary
ammonium salts. The resulting compounds are positively charged to a
different degree depending on the ratio between the quaternary,
primary and/or secondary amines within the formed conjugates. As
such, the sphingoid-polyalkylamine conjugate exists as quaternized
nitrogen salt including, but not limited to, quaternary ammonium
chloride, a quaternary ammonium iodide, a quaternary ammonium
fluoride, a quaternary ammonium bromide, a quaternary ammonium
oxyanion and a combination thereof.
[0127] Non-limiting examples of the sphingoids or sphingoid bases
which may be used according to a more specific embodiment of the
invention, include, sphingosines, dihydrosphingosines,
phytosphingosines, dehydrophytosphingosine and derivatives thereof.
Non-limiting examples of such derivatives are acyl derivatives,
such as ceramide (N-acylsphingosine), dihydroceramides,
phytoceramides and dihydrophytoceramides, respectively, as well as
ceramines (N-alkylsphinogsine) and the corresponding derivatives
(e.g. dihydroceramine, phytoceramine etc.). The suitably
N-substituted sphingoids or sphingoid bases posses free hydroxyl
groups which are activated and subsequently reacted with the
polyalkylamines to form the polyalkylamine-sphingoid entity.
Non-limiting examples of activation agents are
N,N'-disuccinimidylcarbonate, di- or tri-phosgene or imidazole
derivatives. The reaction of these activation agents with the
sphingoids or the sphingoid bases yields a succinimidyloxycarbonyl,
chloroformate or imidazole carbamate, respectively, at one or both
hydroxyls. The reaction of the activated sphingoids with
polyalkylamines may yield mono-substituted, di-substituted,
branched, straight (unbranched) or cyclic conjugates.
[0128] When referring to all aspects of the invention, the
sphingoid-polyalkylamine conjugate (of formula (I) and/or of
formula (I')) may be in the form of free conjugate or as part of a
lipid assembly. One example of a suitable lipid assembly comprises
micelles or vesicles (liposomes). Other examples of assemblies
include the formation of micelles, inverted phases, cubic phases
and the like. Evidently, the sphingoid polyalkylamine conjugate may
be in combined vesicle/micelle form or any other combination of
assemblies. The assemblies may be loaded with the biologically
active molecules. It is noted that CCS, either the isolated isomer,
the mixture of isolated isomers, or the synthesized mixture of CCS,
do not form liposomes by themselves, i.e. they are not considered
as liposome-forming lipids and thus require a helper lipid (such as
cholesterol or DOPE). Lipid assemblies in the context of the
present invention comprise CCS in combination with at least one
helper lipid.
[0129] Lipid assembly as used herein denotes an organized
collection of lipid molecules forming inter alia, micelles,
liposomes and other forms of lipid assemblies. The lipid assemblies
are preferably stable lipid assemblies. Stable lipid assembly as
used herein denotes an assembly being chemically and physically
stable under storage conditions (4.degree. C., in physiological
medium) for at least one day, preferably one week and more
preferably more than a month.
[0130] When the assemblies are in the form of vesicles (e.g.
liposomes), the biologically active molecule may be encapsulated
within the vesicle, form part of its lipid bilayer, or be adsorbed
to the surface of the vesicle (or any combination of these three
options). When the assemblies are micelles, the biologically active
molecules may be inserted into the amphiphiles forming the micelles
and/or associated with it electrostatically, in a stable way.
[0131] According to one embodiment, the assemblies form liposomes.
The formed liposomes may be shaped as unsized heterogeneous and
heterolamellar vesicles (UHV) having a diameter of about 50-5000
nm. The formed UHV may be downsized and converted to (more
homogenous) unilamellar vesicles having a diameter of about 50-100
nm by further processing. The structure and dimensions of the
vesicles, e.g. their shape, lamellarity and size, may have
important implications on their efficiency as vehicles for delivery
of the active biological entities to the target, i.e. these
determine their delivery properties. It is noted that the
unilameller liposomes may be either small vesicles (equal or less
than 50 nm in diameter) as well as large unilamellar vesicles.
[0132] Thus, as used herein, the terms "encapsulated in",
"contained in", "loaded onto" or "associated with" indicate a
physical association between the lipid conjugate and the
biologically active molecule. The physical association may be
either containment or entrapment of the molecule within assemblies
(e.g. vesicles, micelles or other assemblies) formed from the
conjugate; non-covalent linkage of the biological molecule to the
surface of such assemblies, or embedment of the biological molecule
in between the sphingoid-polyalkylamine conjugates forming such
assemblies. Without being bound by theory, it is believed that
association of the sphingoid-polyalkylamine conjugate to the
biological molecule, under physiological conditions is related to
the positive charge or positive dipole of the conjugate.
[0133] The invention should not be limited by the particular type
of association formed between the sphingoid-polyalkylamine
conjugate and the biologically active molecule. Thus, association
means any interaction between the conjugate or the assembly formed
therefrom and the biologically active material which is capable of
achieving a desired therapeutic (as well as prophylactic)
effect.
[0134] The biologically active molecule and the conjugate may be
combined by any method known in the art. This includes, without
being limited thereto, post- or co-lyophilization of the conjugate
with the biologically active molecule, or by mere mixing of
preformed sphingoid-polyalkylamine conjugate with the biological
molecule. Method for co-lyophilization are described, inter alia,
in U.S. Pat. Nos. 6,156,337 and 6,066,331, while methods for
post-encapsulation are described, inter alia, in WO03/000227, all
incorporated herein by reference.
[0135] A preferred weight ratio between the
sphingoid-polyalkylamine conjugate and biologically active material
is 1000:1 to 1:1 weight ratio, with preferably 300:1 to 10:1 weight
ratio. It is noted that the biological activity of the composition
comprising the conjugate and the biologically active material does
not necessarily have to be linear with the increase in said ratio
and at times the "vaccination behavior" may have a bell shape
profile.
[0136] The sphingoid-polyalkylamine conjugate may be used as a sole
adjuvant or in combination with additional adjuvants, so as to
provide an increase in response to the biologically active molecule
such as antigenic molecules. Such substances include, for example,
immunostimulating agents (i.e. "adjuvants"), which when added to a
vaccine they improve the immune response to the antigen so that a
lower antigen dose can be used while producing a greater response
as compared to that without the CCS or other adjuvants. The
immunostimulating agent may be delivered together with the
conjugate/biologically active material, or within a specified time
interval (e.g. several hours or days before or after the
administration of the conjugate/biologically active molecule).
[0137] Preferred immunostimulating agents include, without being
limited thereto, cytokines, such as interleukins (IL-2, IL-12,
IL-15, IL-18), interferons (IFN alpha, beta, gamma), GM-CSF,
synthetic oligodeoxynucleotides (ODN, e.g. CpG), bacterial toxins
(e.g. cholera toxin [CT], staphylococcal enterotoxin B [SEB], heat
labile E. Coli enterotoxin [HLT]), as well as any other adjuvants
known to be used in the art for enhancing or stimulating the immune
response to an antigenic molecule.
[0138] Other pharmaceutically acceptable adjuvants which may be
used in combination with the sphingoid-polyalkylamine conjugate and
the biologically active material include, without being limited
thereto, aluminum hydroxide, alum, QS-21, monophosphoryl lipid A,
and 3-O-deacylated monophosphoryl lipid A (3D-MPL).
[0139] It is noted that the sphingoid-polyalkylamine conjugates of
the invention exhibited an activity of an adjuvant per se. The
results presented herein show that the sphingoid-polyalkylamine
conjugate of the invention and in particular, N-palmitoyl D-erythro
sphingosyl carbamoyl-spermine (including the C-1 and C-3 isomers of
CCS and their synthetic mixtures) which are discussed below, may
act as efficacious and safe adjuvants for a broad spectrum of
active substances, such as antigens. For example, it has been shown
that vaccines comprising an influenza antigens or hepatitis B
surface antigen (HBsAg), in combination with a single CCS isomer
(e.g. C-1 or C-3 isomer), or a mixture of isomers, induced a
faster, stronger and more durable immune response in mice, as
compared to these commercial vaccines administered without an
adjuvant or combined with other adjuvants, such as alum. CCS may
thus allow reduction of the dose and number and frequency of
administrations of a biologically active molecule, such as an
antigen.
[0140] The assemblies may include in addition to the
sphingoid-polyalkylamine conjugate (non-methylated or methylated)
other helper lipid substances. Such helper lipid substances may
include non-cationic lipids like DOPE, DOPC, DMPC, Cholesterol,
oleic acid or others at different mole ratios to the lipid-like
compound. Cholesterol is one preferred added substance for in vivo
application while DOPE may be a preferred helper lipid for in vitro
applications. In this particular embodiment the mole ratio of
cholesterol to the cationic lipid is within the range of 0.01-1.0
and preferably 0.1-0.4.
[0141] The assemblies may also include enhancers and co-factors (as
known in the art, such as CaCl.sub.2) and soluble
polyalkylamines.
[0142] Other components which may be included in the lipid
assembly, and which are known to be used in structures of the like,
are steric stabilizers. One example of a commonly used steric
stabilizer is the family of lipopolymers, e.g. polyethylene glycol
derivatized lipids (PEG-lipid conjugate). This family of compounds
are known, inter alia, to increase (extend) the blood circulation
time of lipids.
[0143] The methods disclosed herein comprises administration of the
sphingoid-polyalkylamine conjugate and biologically active material
either together, or within a predefined time interval, such as
several hours or several days (optionally in combination with an
immunostimulant). However, according to one embodiment, the
conjugate and biologically active material are mixed together prior
to administration to the subject.
[0144] Administration of the sphingoid-polyalkylamine conjugate
together with the biologically active material concerns another
aspect of the invention. Accordingly, there is provided a
pharmaceutical composition comprising a physiologically acceptable
carrier and an effective amount of the sphingoid-polyalkylamine
conjugate together with the biologically active material. The
sphingoid-polyalkylamine conjugate may be incorporated in the
composition as part of a carrier, as an adjuvant, and may be
combined with other pharmaceutically acceptable adjuvants, such as
immunostimulants.
[0145] The sphingoid-polyalkylamine conjugated in combination with
the biologically active material may be administered and dosed in
accordance with good medical practice, taking into account the
clinical condition of the individual subject, the site and method
of administration, scheduling of administration, subject age, sex,
body weight and other factors known to medical practitioners.
[0146] The "effective amount" for purposes herein denotes an amount
which is effective to achieve the desired therapeutic or
prophylactic effect. The effective amount is typically determined
in appropriately designed clinical trials (dose range studies) and
the person versed in the art will know how to properly conduct such
trials in order to determine the effective amount.
[0147] When referring to vaccination, the amount must be effective
to exhibit an enhancement or induction of a subject's immune
response relative to the effect obtained when the biologically
active material is provided to the subject without the
sphingoid-polyalkylamine conjugate(s).
[0148] Preferably, the amount would be sufficient to achieve
effective immunization of a subject against a specific disease or
disorder. It is noted that in accordance with some embodiments, the
amount of the active material may be in the range from 1 .mu.g to
125 .mu.g, with a lipid to active material w/w ratio in the range
of 10:1 to 1000:1 preferably in the range of 300:1 to 10:1 weight
ratio.
[0149] The sphingoid-polyalkylamine conjugate(s) associated with
the biologically active material may be administered in various
ways. Non-limiting examples of administration routes include oral,
subcutaneous (s.c.), intradermal (i.d.), intravenous (i.v.),
intra-arterial (i.a.), transdermal (t.d.), intramuscular (i.m.),
intraperitoneal (i.p.), intrarectal (i.r.), intra-vaginal, and
intranasal (i.n.) administration, as well as by infusion techniques
to the eye (intraocular).
[0150] Preferably modes of administration are the intranasal or
intramuscular administrations. Preferred administration in
accordance with the invention comprises intranasal or parenteral
(e.g. intramuscular or subcutaneous routes).
[0151] Intranasal preparations may include excipients, which do not
irritate the nasal mucosa, or do not inhibit severely the
mucociliary function, and diluents such as water, brine. The
intranasal preparations may include preservatives such as
chlorobutanol and benzalkonium chloride, and also include
surfactants for enhancing the absorption of biologically active
material by nasal mucosa. The intranasal preparation may be adapted
for administration by nasal spray, nasal drops, gel or powder etc.
Aerosol formulations can be placed into pressurized acceptable
propellants, such as propane, nitrogen, and the like. They also may
be formulated as pharmaceuticals for non-pressured preparations,
such as in a nebulizer or atomizer suitable carriers.
DESCRIPTION OF SOME NON LIMITING EXAMPLES
[0152] The following are experimental procedures and results
providing non-limiting examples for the use of a conjugate in
accordance with the invention.
Materials and Methods
Synthesis of N-palmitoyl D-erythro Sphingosyl Carbamoyl Spermine
(CCS)
[0153] (i) N-palmitoylsphingosine (1.61 g, 3 mmol) was dissolved in
dry tetrahydrofuran (THF) (100 ml) with heating. The clear solution
was brought to room temperature and N,N'-disuccinimidyl carbonate
(1.92 g, 7.5 mmol) was added. DMAP (4-dimethylamino pyridine) (0.81
g, 7.5 mmol) was added with stirring and the reaction further
stirred for 16 hours. The solvent was removed under reduced
pressure and the residue re-crystallized from n-heptane yielding
1.3 g (68%) of activated ceramide in the form of white powder m.p.
73-76.degree. C.
[0154] (ii) Spermine (0.5 g, 2.5 mmol) and the activated ceramide
(0.39 g, 0.5 mmol) were dissolved in dry dichloromethane with
stirring and then treated with catalytic amount of DMAP. The
solution was stirred at room temperature for 16 hours, the solvent
evaporated and the residue treated with water, filtered and dried
in vacuo, giving 0.4 .mu.g (82%) of crude material which was
further purified by column chromatography on Silica gel, using
60:20:20 Butanol:AcOH:H.sub.2O eluent.
[0155] Synthetic CCS was isolated as its tri-acetate salt. It
comprises a mixture of two isomers, the C-1 and C-3 isomers, as
confirmed by .sup.1H-NMR and .sup.13C-NMR spectrometry (see FIG. 9A
in co-pending WO 2004/110980, incorporated herein by reference in
its entirety).
[0156] The structure of the two isomers is illustrated in FIG. 1A
(N-palmitoyl-D-erythro-sphingosyl-1-carbamoyl spermine, where the
polyalkylamine (spermine) is attached via C-1, thus, at times
referred to be the abbreviated C-1 CCS) and FIG. 1B
(N-palmitoyl-D-erythro-sphingosyl-3-carbamoyl spermine, where the
polyalkylamine (spermine) is attached via C-3, thus, at times
referred to be N-palmitoyl D-erythro sphingosyl-3-carbamoyl
spermine, the abbreviated C-3 CCS).
[0157] In the above and below description, unless otherwise stated,
the product of the above synthetic procedure is used and
hereinafter it referred to as the general term CCS.
[0158] (iii) for obtaining a quaternary ammonium salt within the
compound, the product of step (ii) may be methylated with DMS or
CH.sub.3I.
Isolated Isomers N-palmitoyl D-erythro Sphingosyl Carbamoyl
spermine (C-1 CCS or C-3 CCS)
[0159] It has been found that the above synthetic method provides a
mixture of the two isomers, C-1 CCS and C-3 CCS. Isolated
N-palmitoyl D-erythro sphingosyl-1-carbamoyl spermine and
N-palmitoyl D-erythro sphingosyl-3-carbamoyl spermine were
purchased from Biolab Ltd., Jerusalem, Israel. The use of an
isolated isomer is specifically indicated, wherever
appropriate.
[0160] At times, a mixture of the isolated isomers was also used
and such a mixture is specifically referred to as the mixture of
the isolated C-1 CCS and C-3 CCS isomers.
Influenza Antigens
[0161] A monovalent subunit antigen preparation derived from
influenza A/New Caledonia/20/99-like (H1N1) strain was generously
provided by Drs. Gluck and Zurbriggen, Berna Biotech, Bern,
Switzerland. This preparation (designated herein HN) is comprised
of 80-90% wt % hemagglutinin (H), 5-10 wt % neuraminidase (N) and
trace amounts of NP and M1 proteins (this is referred to
hereinbelow by the abbreviation HN or HA). A commercial trivalent
subunit vaccine (Fluvirin.RTM.) for the 2003/2004 season containing
HN derived from A/New Caledonia/20/99 (H1N1), A/Panama/2007/99
(H3N2) and B/Shangdong/7/97 was obtained from Evans Vaccines Ltd.,
Liverpool, UK. This vaccine was concentrated .about..times.8
(Eppendorf Concentrator 5301, Eppendorf AG, Hamburg, Germany) prior
to encapsulation.
[0162] In addition, for assessing immunogenicity of isolated
isomers, the commercial trivalent vaccine Vaxigrip 2006-2007
(Aventis Pasteur) was used.
[0163] A whole inactivated virus was used in some experiments for
in vitro stimulation.
Lipids
[0164] The phospholipids (PL), dimyristoyl phosphatidylcholine
(DMPC), dimyristoyl phosphatidylglycerol (DMPG), and dioleoyl
phosphatidylethanolamine (DOPE) are from Lipoid GmbH, Ludwigshafen,
Germany or from Avanti Polar Lipids (Alabaster, Ala., USA). In
addition to DMPC (neutral) and DMPC/DMPG (9/1 mole ratio, anionic)
liposomes, 6 formulations of cationic liposomes/lipid assemblies
were prepared. The monocationic lipids dimethylaminoethane
carbamoyl cholesterol (DC-Chol),
1,2-distearoyl-3-trimethylammonium-propane (chloride salt) (DSTAP),
dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), and
dimyristoyl-3-trimethylammonium-propane (chloride salt) (DMTAP) are
from Avanti Polar Lipids (Alabaster, Ala., USA). The monocationic
lipid dimethyldioctadecylammonium bromide (DDAB) and cholesterol
(Chol) are from Sigma. The polycationic sphingolipid
N-palmitoyl-D-erythro-sphingosyl carbamoyl-spermine (acetate salt)
(ceramide carbamoyl-spermine, CCS, i.e. the C-1 and/or C3 isomers)
was produced as described above and the isolated isomers were
purchased from Biolab Ltd., Jerusalem, Israel. Where indicated, the
helper lipids (DOPE, Chol) were used at a lipid/helper lipid mole
ratio of 1/1 to 4/1.
Animals
[0165] Specific pathogen-free (SPF) female BALB/c mice, 6-8 weeks
old, and C57BL/6 mice, 18 month-old, were used (5-10 per
group).
[0166] In addition, Sprague Dawley rats (females, 9 weeks old) were
used (6 per group).
[0167] Ferrets (females, 6 months old) were used (8 per group).
[0168] Further, female Hartley strain guinea pigs, weighing about
300 grams each were used (from 5 to 7 per group)/
[0169] Animals were maintained under SPF conditions.
Preparation of Lipid Assemblies
[0170] For the formation of lipids assemblies, lipids (10-30 mg)
were dissolved in 1 ml tertiary butanol.
[0171] For CCS preparation, three procedures were used to form CCS
and/or cholesterol solutions: [0172] 1. an amount of CCS was
dissolved with DDW and the helper lipid, cholesterol, was dissolved
in tertiary butanol; [0173] 2. each of the lipids (CCS and the
helper lipid) were dissolved (separately) in tertiary butanol; or
[0174] 3. each of the lipids were dissolved in
Dichloromethane:Methanol (2:1).
[0175] The CCS solution and the cholesterol solution were mixed to
obtain a mixture with CCS/Cholesterol mole:mole ratio 3:2.
[0176] For all lipids, the lipid solutions were sterilized by
filtration (GF92, Glasforser, Vorfilter no. 421051, Schleicher
& Schuell, Dassel, Germany). The sterile lipid solution was
frozen at -70.degree. C. (for the CCS dissolved with
Dichloromethane:Methanol, the solution was evaporated with a gentle
stream of nitrogen) then lyophilized for 24 h to complete dryness.
The dried lipids could be stored at 4.degree. C. for >2 years
without significant (<10%) lipid degradation or loss of
"encapsulation" capability. Upon need, the lipid powder was
hydrated with the antigen solution (in PBS pH 7.2) at a
lipid:antigen (protein) w/w ratio of 3/1 to 800/1. The antigen
solution was added stepwise in small increments and vortexed
vigorously after each addition, up to a final volume of 0.5-1 ml.
In some experiments, the dried lipids were hydrated with PBS and
the preformed "empty" lipid assemblies were mixed with the antigen
solution. The mixture was vortexed for 1-2 min and used as is
within 30-60 min.
Encapsulation of Influenza Antigens in Liposomes/Lipid
Assemblies
[0177] HN antigens (see above) were encapsulated in large (mean
diameter 0.1-5 .mu.m) heterogeneous (unsized) vesicles.
[0178] To determine "encapsulation" efficiency, two procedures were
used, depending on the formulation, resulting in .gtoreq.80%
separation between the free antigen and the lipid-associated
antigen. For all vaccine formulations, except CCS, the following
separation technique was used. The lipid assemblies (1-30 mg lipid)
containing the HN antigen (50-100 .mu.g protein) were suspended in
0.5 ml PBS and carefully loaded over 0.5 ml of D.sub.2O (99.9%,
Aldrich Chemical Co., Milwaukee, Wis., USA). The sample was then
centrifuged for 1 h at 30.degree. C. at 45,000 rpm. The free,
non-encapsulated HN precipitates while the assembled (liposomal) HN
and protein-free lipid assemblies/liposomes remain in the
supernatant. The entire supernatant was collected and the
assemblies/liposomes were dissolved by adding 0.2 ml of warm 10%
Triton X-100 to both the supernatant and the pellet fractions.
Protein concentration in both fractions was determined by the
modified Lowry technique. For the CCS formulations, the CCS was
suspended in 0.5 ml of Ag solution and then carefully loaded over
0.5 ml D.sub.2O. The sample was then centrifuged for 10 min at
20.degree. C. at 10,000 rpm. The large liposomes were present in
the middle phase while the little ones may precipitate. The free HN
remains in the upper phase. The 3 fractions were collected
separately. Lipid dissolution was done using boiled 20% Triton
X-100 to give a final concentration of 10% Triton and protein
determination in the 3 fractions was carried out by the modified
Lowry technique. In both separation techniques, the overall
recovery of the HN antigens was >95%.
[0179] In order to test the immunogenicity of isolated CCS isomers
or of the mixture of the isolated isomers, a preparation of the
isolated CCS (or mixture thereof) and cholesterol (CCS/Cholesterol
mole:mole ratio 3:2) was prepared in two methods: DDW/T-Butanol and
Dichloromethane/methanol (described above).
Immunization
[0180] Immunization of Mice:
[0181] Free (F-HN) and assembled/liposomal (Lip-HN) vaccines,
0.25-4 .mu.g antigen/strain/dose and 0.075-1.2 mg lipid/dose, were
administered either once intramuscularly (i.m., in 30 .mu.l), once
or twice intranasally (i.n., in 5-50 .mu.l per nostril) spaced 3, 7
or 14 days apart, or twice orally (in 50 .mu.l) spaced 1 week
apart. When assessing the immunogenicity of the isolated isomers or
the mixture of isolated isomers (mole ratio of C-1 CCS to C-3 CCS
being 80:20), BALB/C mice (female 8 weeks) were vaccinated once
i.m. with 6 .mu.g total HA (Vaxigrip) with or without the isomer,
the isomers being mixed with cholesterol as described above. In the
latter experiment, the lipid/antigen ratio was 150:1; serum
antibody titer against A/New Calendonia virus was conducted 4 weeks
following vaccination using the HI assay as described below.
[0182] In all cases of intranasal (i.n.) administration mice were
lightly anesthetized with 0.15 ml of 4% chloral hydrate in PBS
given intraperitoneally. For oral vaccination mice were treated
orally with 0.5 ml of an antacid solution (8 parts Hanks' balanced
salt solution+2 parts 7.5% sodium bicarbonate) 30 min prior to
vaccination. Cholera toxin (CT, Sigma, USA), 1 .mu.g/dose, was used
in some experiments as a standard mucosal adjuvant for comparison.
In two experiments, CpG-ODN (ODN 1018, generously provided by Dr.
E. Raz, University of California, San Diego, Calif., USA), free and
liposomal, 10 .mu.g/dose, was used as an adjuvant.
[0183] Immunization of Rats:
[0184] Sprague Dawley rats (females, 9 weeks old) were vaccinated
intramuscularly (i.m.) once with 12 .mu.g total HA (Vaxigrip)
with/without CCS/C (3:2) [the CCS comprising the mixture of isomers
synthesized as described above]. Lipid:antigen ratio 150:1. Serum
antibody titer against A/New Caledonia virus was followed for 8
weeks by hemagglutination inhibition (HI) assay.
[0185] Immunization of Ferrets:
[0186] Ferrets (females, 6 months old) were vaccinated i.m. twice
on day 0 and day 14 with CCS/C alone (negative control), with free
antigen (F-HA) or with CCS/C-HA 15 .mu.g per strain (total 45
.mu.g, Vaxigrip). CCS/C-HA vaccine was also tested at a dose of 5
.mu.g per strain.
[0187] Immunization of Guinea Pigs:
[0188] Guinea pigs were vaccinated s.c. once on day 0 with free PA
antigen (F-PA) or with CCS/C-PA at an amount of 25 .mu.g PA antigen
(in 100 .mu.l) or with STI (positive control, 0.5 ml).
Assessment of Humoral Responses
[0189] Sera, lung homogenates and nasal washes were tested,
individually or pooled, 4-6 weeks post-vaccination, starting at
1/10 or 1/20 sample dilution. Hemagglutination inhibiting
antibodies were determined by the standard hemagglutination
inhibition (HI) assay. Mice with HI titer .gtoreq.40 (considered a
protective titer in humans) were defined as seroconverted.
Antigen-specific IgG1, IgG2a, IgA and IgE levels were measured by
ELISA. The highest sample dilution yielding absorbance of 0.2 OD
above the control (antigen+normal mouse serum, OD <0.1) was
considered the ELISA antibody titer [for ELISA protocol, see
materials and methods in Babai, S. Samira, Y. Barenholz, Z.
Zakay-Rones and E. Kedar, Vaccine 17 (1999) (9/10), pp.
1239-1250].
[0190] In some experiments (see below), the humoral response was
tested at other time points following vaccination.
[0191] In the description above and below, whenever referring to
ELISA and unless otherwise stated, the ELISA was performed as
described by Babai et al., mutatis mutandis.
Assessment of Cellular Responses
[0192] Splenocytes obtained at 5-6 weeks after vaccination were
tested for proliferative response, IFN.gamma. and IL-4 production,
and cytotoxic activity, following in vitro stimulation with the
antigen. Cultures were carried out at 37.degree. C. in enriched
RPMI 1640 or DMEM medium supplemented with 5% (for proliferation,
cytokines) or 10% (for cytotoxicity) fetal calf serum (FCS), with
(for cytotoxicity) or without 5.times.10.sup.-5M 2-mercaptoethanol.
Cell cultures were performed as follows: (i) Proliferation:
0.5.times.10.sup.6 cells per well were incubated in U-shaped
96-well plates, in triplicate, with or without the antigen (0.5-5
.mu.g per well), in a final volume of 0.2 ml. After 72-96 h,
cultures were pulsed with 1 .mu.Ci .sup.3H-thymidine for 16 h.
Results are expressed in .DELTA.cpm=(mean counts per minute of
cells cultured with antigen)-(mean counts per minute of cells
cultured without antigen). (ii) Cytokines: 2.5.times.10.sup.6 to
5.times.10.sup.6 cells per well were incubated in 24-well plates,
in duplicate, with or without the antigen (5-10 .mu.g per well), in
a final volume of 1 ml. Supernatants were collected after 48-72 h
and tested by ELISA for murine IFN.gamma. and IL-4 using the Opt
EIA Set (Pharmingen, USA). (iii) Cytotoxicity: Responding
splenocytes (2.5.times.10.sup.6) were incubated as in (ii) for 7
days together with an equal number of stimulating BALB/C
splenocytes that had been infected with the X/127(H1N1) influenza
virus (see below). For infection, the splenocytes were incubated,
with occasional stirring, for 3 h at 37.degree. C. in RPMI 1640
medium (without FCS) with 150 hemagglutination
units/1.times.10.sup.6 splenocytes of the virus, followed by
washing. Subsequently, the primed effector cells were restimulated
for 5 days with infected, irradiated (3,000 rad) splenocytes at an
effector/stimulator cell ratio of 1/4 in the presence of 10 IU/ml
of rhIL-2. Cytotoxicity was measured using the standard 4 h
.sup.51Cr release assay at an effector/target cell ratio of 100/1.
The labeled target cells used were unmodified P815 and P815 pulsed
for 90 min at 37.degree. C. with the HA2 189-199 peptide
(IYSTVASSLVL, 20 .mu.g/1.times.10.sup.6 cells).
Determination of Protective Immunity
[0193] Mice were anesthetized and 25 .mu.l of live virus suspension
per nostril, .about.10.sup.7 EID 50 (egg-infectious dose 50%), was
administered, using the reassortant virus X-127 (A/Beijing/262/95
(H1N1).times.X-31 (A/Hong Kong/1/68.times.A/PR/8/34), which is
infectious to mice and cross-reactive with A/New Caledonia. The
lungs were removed on day 4, washed thrice in cold PBS, and
homogenized in PBS (1.5 ml per lungs per mouse, referred to as 1/10
dilution). Homogenates of each group were pooled and centrifuged at
2000 rpm for 30 min at 4.degree. C. and the supernatants collected.
Serial 10-fold dilutions were performed and 0.2 ml of each dilution
was injected, in duplicate, into the allantoic sac of 11-day-old
embryonated chicken eggs. After 48 h at 37.degree. C. and 16 h at
4.degree. C., 0.1 ml of allantoic fluid was removed and checked for
viral presence by hemagglutination (30 min at room temperature)
with chicken erythrocytes (0.5 wt. %, 0.1 ml). The lung virus titer
is determined as the highest dilution of lung homogenate producing
virus in the allantoic fluid (positive hemagglutination).
[0194] The ferrets were challenged intranasally with the homologous
Influenza A/New Caledonia/20/99 virus [H1N1] (4.93 TCID.sub.50/ml
diluted 1/100 v/v) on day 28 post-immunization. Following
challenge, nasal washes were performed daily for 9 days
post-infection, for analysis of virus shedding from the nasal
mucosa. Viral shedding from nasal washes was determined by
titration on MDCK cells.
[0195] MDCK cells were plated and grown for 1-2 days until they
were observed to be between 50% and 70% confluent. Samples were
added to the cells in duplicates, and diluted at a 1 in 10 (v/v)
serial dilution, to give a dilution range of 10-1 to 10-6 (v/v). A
known titre of virus was used as a control in the assay. The cells
were incubated at 37.degree. C. with 5% CO2. Once the control
titration was observed to have cytopathic effect (CPE) in the
expected virus control wells (approximately 3-4 days), the
hemagglutination assay was performed on a sample of the
supernatants.
Biodistribution and Pharmacokinetics of Various
Fluorescently-Labeled Lipid Formulations and Radioactively-Labeled
HN Antigen
[0196] Mice were vaccinated once intranasally (i.n.) with
lissamine-rhodamine labeled liposomes, either empty or associated
with trivalent subunit influenza vaccine (HN) in a volume of 20
.mu.l. After 1, 5 or 24 hours, mice were sacrificed and various
organs were removed. The organs were stored at -20 deg overnight,
and the next morning homogenized in lysis buffer. 0.2 ml the
subsequent homogenate was transferred to Eppendorf tubes, 0.8 mL of
isopropanol was added, and spun for 15 minutes to release
fluorescent probe into the supernatant. 50 uL of the supernatant
was loaded onto a 384 black plate and the fluorescence was read
(Em: 545, Ex: 596).
[0197] In a further assay, 450 .mu.g of trivalent HN vaccine (in 5
mL) were dialysed against DDW (to remove salt) and then
concentrated .times.1000 to 5 .mu.L. The protein was then diluted
in 0.1M borate buffer (pH 8.5) to a stock solution of 450 .mu.g in
15 .mu.L. The protein was then labeled with .sup.125I using the
Bolton Hunter reagent, according to the manufacturer's
instructions. Mice were immunized i.n. with the .sup.125I-labeled
HN (2 .mu.g), either free or liposome encapsulated, and at 1, 5 and
24 hrs the mice were sacrificed, and various organs (see FIG. 3)
were removed into vials and read in a .gamma.-counter calibrated
for .sup.125I.
Results
Characterization of HN Antigen-Loaded Cationic Liposomes
[0198] Efficiency of encapsulation of HN (a commercial preparation
of hemagglutinin and neuraminidase derived from influenza viruses)
loaded onto various cationic liposomal formulations, at different
lipid/protein w/w ratios (3/1-300/1), and with or without
cholesterol (Chol) was tested. Table 1 shows the results of such an
experiment, using the cationic lipids DOTAP and CCS. TABLE-US-00001
TABLE 1 The effect of the lipid (DOTAP, CCS)/protein ratio and
cholesterol (Chol) on HN encapsulation efficiency DOTAP/HN
DOTAP/Chol % HN CCS/HN CCS/Chol % HN w/w ratio mole ratio
encapsulation w/w ratio mole ratio encapsulation 300/1 1/1 93 300/1
1/0 73 100/1 1/1 90 100/1 1/0 64 50/1 1/1 90 30/1 1/0 38 30/1 1/1
88 10/1 1/0 1 10/1 1/1 79 3/1 1/1 35 100/1 1/0 90 300/1 3/2 71
100/1 1/1 92 100/1 3/2 64 100/1 2/1 89 30/1 3/2 41 100/1 4/1 80
10/1 3/2 0 A monovalent vaccine was used for DOTAP and a trivalent
vaccine (concentrated) for CCS.
[0199] The percentage of antigen loading for DOTAP was 80-90% using
a lipid/protein w/w ratio of 30/1 to 300/1, with and without Chol,
decreasing to 79% and 35% at 10/1 and 3/1 w/w ratios, respectively.
The addition of Chol to the formulation did not affect loading at
DOTAP/Chol mole ratios of 1/1 and 2/1, with slightly lower
encapsulation (80%) at a ratio of 4/1. For CCS, with or without
Chol, the loading efficiency was lower (64-73% at w/w ratio of
100/1-300/1).
[0200] HN association with the liposomes upon simple mixing of the
soluble antigen with preformed empty liposomes was also determined.
In such cases, 40-60% of the antigen was associated with the
liposomes using a lipid/protein w/w ratio of 100/ to 300/1,
regardless of the formulation.
[0201] These finding, collectively, indicate very high loading
efficiency (>60%) using a simple and fast (5 min.) procedure in
all formulations. Furthermore, even preformed liposomes in aqueous
suspension were capable of effectively associating with the
influenza virus surface antigens.
[0202] The immunogenicity of the various lipid/antigen w/w ratios
(with or without the addition of cholesterol) was also
evaluated.
[0203] In a first experiment sera levels of HI, IgG1 and IgG2a
antibodies following i.n. vaccination of young (2-month-old) BALB/c
mice with HN-loaded neutral, anionic or cationic liposomes were
determined (Table 2A). The HN antigen was a monovalent subunit
vaccine derived from the A/New Caledonia (H1N1) strain. In the same
experiment, lung and nasal levels of HI, IgG1, IgG2a and IgA
antibodies, and INF.gamma. levels produced by spleen cells, were
also tested (Tables 2B and 2C). TABLE-US-00002 TABLE 2A Sera levels
of HI, IgG1 and IgG2a antibodies (i.n. administration) Group Serum
(n = 5) Vaccine HI (mean .+-. SD) IgG1 IgG2a 1 PBS 0 0 0 2 F-HN 0
55 0 3 Lip (DMPC)-HN 3 .+-. 7 (0) 150 0 (Neutral) 4 Lip
(DMPC/DMPG)- 6 .+-. 13 (20) 500 0 HN (Anionic) 5 Lip
(DC-Chol:DOPE)- 18 .+-. 7 (0) 0 0 HN 6 Lip (DSTAP:Chol)- 28 .+-. 29
(40) 20 0 HN 7 Lip (DDAB:Chol)-HN 136 .+-. 32 (100) 100 0 8 Lip
(DOTAP:Chol)- 576 .+-. 128 (100) 15000 730 HN 9 Lip (DMTAP:Chol)-
672 .+-. 212 (100) 30000 470 HN 10 Lip (CCS:Chol)-HN 2368 .+-. 1805
(100) 30000 9000 11 F-HN + CT (1 .mu.g) 1664 .+-. 572 (100) 55000
7000 F-HN, free antigen; Chol, cholesterol; CT, cholera toxin.
Groups 5-10 are cationic liposomes. In parentheses, %
seroconversion - % of mice with HI titer .gtoreq.40.
[0204] In particular, a comparison was made between neutral (DMPC),
anionic (DMPC/DMPG, 9/1 mole ratio) and cationic (6 formulations)
assemblies (Lip) encapsulating the HN antigens to induce local and
systemic responses following two i.n. administrations. For all
formulations, the lipid/HN w/w ratio was 300/1, and the cationic
lipid/Chol or cationic lipid/DOPE mole ratio was 1/1. Free antigen
(F-HN) and F-HN co-administered with cholera toxin (CT, 1 .mu.g) as
an adjuvant were tested in parallel. The vaccine was given on days
0 and 7, 3 .mu.g/dose (10 .mu.l per nostril), and the responses
were determined 4 (HI) and 6 (Elisa) weeks after the second vaccine
dose. TABLE-US-00003 TABLE 2B lung and nasal wash levels of IgG1,
IgG2a and IgA antibodies Group Lung Nasal (n = 5) Vaccine IgG1
IgG2a IgA IgG1 IgG2a IgA 1 PBS 0 0 0 0 0 0 2 F-HN 0 0 0 0 0 0 3 Lip
(DMPC)-HN (Neutral) 30 0 0 0 0 0 4 Lip (DMPC/DMPG)-HN (Anionic) 40
0 0 0 0 0 5 Lip (DC-Chol:DOPE)-HN 0 0 0 0 0 0 6 Lip (DSTAP:Chol)-HN
0 20 0 0 80 0 7 Lip (DDAB:Chol)-HN 0 80 30 0 30 0 8 Lip
(DOTAP:Chol)-HN 730 1050 170 40 180 30 9 Lip (DMTAP:Chol)-HN 470
3000 30 15 80 70 10 Lip (CCS:Chol)-HN 9000 30000 1900 15000 30 300
11 F-HN + CT (1 .mu.g) 7000 10000 1800 40 120 30
[0205] TABLE-US-00004 TABLE 2C Spleen INF.gamma. levels (pg/ml)
Group Spleen (n = 5) Vaccine IFN.gamma. (pg/ml) 1 PBS 1800 2 F-HN
1400 3 Lip (DMPC)-HN (Neutral) 4200 4 Lip (DMPC/DMPG)-HN (Anionic)
4000 5 Lip (DC-Chol:DOPE)-HN 4900 6 Lip (DSTAP:Chol)-HN 2300 7 Lip
(DDAB:Chol)-HN 3100 8 Lip (DOTAP:Chol)-HN 8000 9 Lip
(DMTAP:Chol)-HN 7800 10 Lip (CCS:Chol)-HN 10200 11 F-HN + CT (1
.mu.g) 5200
[0206] As shown in Tables 2A-2C, the free antigen, as well as the
neutral and anionic Lip-HN were virtually ineffective intranasal
vaccines. In contrast, the cationic Lip-HN, particularly those
designated DOTAP-HN, DMTAP-HN and CCS-HN, evoked a robust systemic
and mucosal humoral response, with high levels of IgG1, IgG2a and
IgA antibodies, namely a mixed Th1+Th2 response. No IgE antibodies
were defected. The cationic liposomal vaccines comprising DOTAP-HN,
DMTAP-HN and CCS-HN also induced high levels of IFN.gamma. (but not
IL-4) in antigen-stimulated spleen cells. The responses produced by
CCS-HN were even stronger than those induced by F-HN adjuvanted
with CT. Based on these findings, only the cationic liposomal
formulations: DOTAP-HN, DMTAP-HN and CCS-HN were further used.
[0207] In a second experiment, the effect of lipid/HN w/w ratio on
the immunogenicity of HN-loaded cationic liposomes and of preformed
liposomes, simply mixed with the soluble antigen, was determined.
The data shown in Tables 3A-3C indicate that all three formulations
induced a strong systemic (serum) and local (lung) response, and
that lowering the lipid HN w/w ratio below 100/1 markedly reduced
the antibody response. TABLE-US-00005 TABLE 3A Serum levels of HI,
IgG1, IgG2a and IgA antibodies Lipid/HN Serum No. Vaccine (n = 5)
w/w ratio HI IgG1 IgG2a IgA 1 F-HN 0 0 0 0 2 Lip (DOTAP)-HN 300/1
496 .+-. 495 (100) 15000 450 0 3 100/1 196 .+-. 119 (100) 5000 280
0 4 30/1 36 .+-. 50 (80) 1000 200 0 5 10/1 28 .+-. 18 (60) 600 30 0
6 3/1 0 20 0 0 7 Lip (DMTAP)-HN 300/1 388 .+-. 260 (100) 2500 250 0
8 100/1 208 .+-. 107 (100) 2200 600 0 9 50/1 130 .+-. 118 (80) 850
150 0 10 30/1 48 .+-. 71 (40) 450 0 0 11 10/1 24 .+-. 35 (40) 120 0
0 12 Lip (CCS)-HN 300/1 560 .+-. 480 (100) 2000 1800 200 13 100/1
752 .+-. 504 (100) 6500 6000 0 14 50/1 272 .+-. 156 (100) 1900 700
0 15 30/1 112 .+-. 125 (80) 650 400 0 16 10/1 52 .+-. 68 (40) 275
440 0 17 F-HN + CT (1 .mu.g) -- 896 .+-. 350 (100) 30000 8000 120
18 F-HN + Lip (DOTAP) 300/1 864 .+-. 446 (100) 5000 1500 0 19 F-HN
+ Lip (DMTAP) 300/1 320 .+-. 226 (100) 1900 400 0 20 F-HN + Lip
(CCS) 300/1 704 .+-. 525 (100) 30000 5000 500 In groups 18-20
preformed liposomes were mixed with the soluble antigen.
[0208] TABLE-US-00006 TABLE 3B Lung levels of HI, IgG1, IgG2a and
IgA Lipid/ HN w/w Lung No. Vaccine (n = 5) ratio HI IgG1 IgG2a IgA
1 F-HN 0 0 0 0 2 Lip (DOTAP)-HN 300/1 40 600 85 30 3 100/1 40 500
20 0 4 30/1 30 250 35 0 5 10/1 20 250 0 0 6 3/1 10 20 0 0 7 Lip
(DMTAP)-HN 300/1 0 5500 200 1200 8 100/1 0 7000 350 0 9 50/1 0 4500
250 0 10 30/1 0 1500 110 0 11 10/1 0 500 0 0 12 Lip (CCS)-HN 300/1
80 12500 3000 20000 13 100/1 80 7000 5500 65000 14 50/1 40 5500 900
20000 15 30/1 0 1500 200 0 16 10/1 0 500 200 0 17 F-HN + CT (1
.mu.g) -- 80 45000 2250 3000 18 F-HN + Lip 300/1 0 6000 500 1200
(DOTAP) 19 F-HN + Lip 300/1 0 3750 225 1500 (DMTAP) 20 F-HN + Lip
(CCS) 300/1 80 35000 3000 80000
[0209] TABLE-US-00007 TABLE 3C Spleen INF.gamma. levels (pg/ml)
Lipid/HN Spleen No. Vaccine (n = 5) w/w ratio IFN.gamma. (pg/ml) 1
F-HN 4500 2 Lip (DOTAP)-HN 300/1 9780 3 100/1 42220 4 30/1 20440 5
10/1 20400 6 3/1 27780 7 Lip (DMTAP)-HN 300/1 3500 8 100/1 5850 9
50/1 3400 10 30/1 3050 11 10/1 Not done 12 Lip (CCS)-HN 300/1 8000
13 100/1 8250 14 50/1 10650 15 30/1 3500 16 10/1 Not done 17 F-HN +
CT (1 .mu.g) -- 22800 18 F-HN + Lip (DOTAP) 300/1 3400 19 F-HN +
Lip (DMTAP) 300/1 5700 20 F-HN + Lip (CCS) 300/1 4100
[0210] The superiority of Lip CCS-HN vaccine over the other vaccine
formulations is again seen as reflected by the high levels of serum
and lung IgG2a and IgA antibodies (groups 12-16). Interestingly,
simple mixing of soluble antigen with preformed liposomes generated
very potent vaccines (groups 18-20) that are equal to liposomes
encapsulating the antigen. This suggests that real encapsulation of
the antigen may not be necessary for the adjuvanticity of the
cationic assemblies/liposomes.
[0211] In a further experiment the effect of cholesterol on the
immunogenicity of the HN-loaded liposomes was tested. Tables 4A-4C
show the results of this experiment, indicating that the addition
of Cholesterol slightly reduced the systemic HI response to
DOTAP-HN at 2/1 and 4/1 mole ratios (groups 4, 5), but not at a 1/1
mole ratio (group 3), and moderately enhances the overall response
to DMTAP-HN at all ratios (groups 7-9) and the local (lung)
response CCS-HN at a 1/1 ratio (group 11). TABLE-US-00008 TABLE 4A
Serum levels of HI, IgG1, IgG2a and IgA antibodies Vaccine Cat
lipid/Chol Serum No. (n = 5) mole ratio HI IgG1 IgG2a IgA 1 F-HN --
0 0 0 0 2 Lip (DOTAP)-HN 1/0 320 .+-. 0 (100) 15000 450 0 3 Lip
(DOTAP:Chol)- 1/1 496 .+-. 295 (100) 15000 450 0 HN 4 2/1 168 .+-.
216 (100) 7000 800 0 5 4/1 195 .+-. 111 (100) 15000 250 0 6 Lip
(DMTAP)-HN 1/0 320 .+-. 188 (100) 20000 290 0 7 Lip (DMTAP:Chol)-
1/1 672 .+-. 419 (100) 30000 300 0 HN 8 2/1 576 .+-. 368 (100)
25000 650 0 9 4/1 608 .+-. 382 (100) 30000 600 0 10 Lip (CCS)-HN
1/0 2560 .+-. 1568 (100) 30000 7000 100 11 Lip (CCS:Chol)-HN 1/1
2368 .+-. 1805 (100) 30000 9000 100 12 F-HN + CT (1 .mu.g) -- 1664
.+-. 572 (100) 55000 7000 20
[0212] TABLE-US-00009 TABLE 4B Lung levels of HI, IgG1, IgG2a and
IgA antibodies Cat lipid/ Chol Vaccine mole Lung No. (n = 5) ratio
HI IgG1 IgG2a IgA 1 F-HN -- 0 0 0 0 2 Lip (DOTAP)-HN 1/0 40 900 85
25 3 Lip (DOTAP:Chol)- 1/1 40 600 80 30 HN 4 2/1 40 680 180 22 5
4/1 60 720 50 60 6 Lip (DMTAP)-HN 1/0 60 1000 40 0 7 Lip
(DMTAP:Chol)- 1/1 120 3000 30 15 HN 8 2/1 160 2500 160 200 9 4/1 80
4000 100 150 10 Lip (CCS)-HN 1/0 640 30000 1500 9000 11 Lip
(CCS:Chol)-HN 3/2 1280 30000 1900 15000 12 F-HN + CT (1 .mu.g) --
20 10000 1800 1000
[0213] TABLE-US-00010 TABLE 4C Spleen INF.gamma. levels (pg/ml)
Vaccine Cat lipid/Chol Spleen No. (n = 5) mole ratio IFN.gamma.
(pg/ml) 1 F-HN -- 7430 2 Lip (DOTAP)-HN 1/0 7480 3 Lip
(DOTAP:Chol)-HN 1/1 9780 4 2/1 12870 5 4/1 9330 6 Lip (DMTAP)-HN
1/0 8520 7 Lip (DMTAP:Chol)-HN 1/1 10900 8 2/1 8560 9 4/1 7490 10
Lip (CCS)-HN 1/0 15550 11 Lip (CCS:Chol)-HN 3/2 13780 12 F-HN + CT
(1 .mu.g) -- 11110
[0214] The immunogenicity of CCS-HN vaccine was also evaluated in
aged (18 month) C57BL/6 mice following intramuscular (once on day
0) or intranasal (twice, days 0 and 7) administration of 1 .mu.g
and 2 kg, respectively, of subunit (HN) vaccine (derived from
A/Panama [H3N2] virus). The lipid assemblies were composed of
CCS/cholesterol (3:2 molar ratio) and the lipid/HN w/w ratio was
200:1. As opposed to zero activity of the commercial vaccine, the
CCS-HN vaccine evoked high levels of serum HI and IgG2a antibodies
(tested at 4 weeks post vaccination) and lung (tested at 6 weeks
post vaccination) IgG2a and IgA antibodies, as can be seen in
Tables 5A and 5B (the data show mean titers). TABLE-US-00011 TABLE
5A Serum levels of HI, IgG1, IgG2a and IgA in aged mice
Vaccine.sup.a Serum No. (n = 5) HI IgG1 IgG2a 1 PBS i.n. .times. 2
0 0 0 2 F-HN i.m. .times. 1 0 15 0 3 F-HN i.n. .times. 2 0 0 0 4
Lip (CCS)-HN i.n. .times. 2 80 130 350
[0215] TABLE-US-00012 TABLE 5B Lung levels of IgG1, IgG2a and IgA
in aged mice Vaccine Lung No. (n = 5) IgG1 IgG2a IgA 1 PBS i.n.
.times. 2 0 0 0 2 F-HN i.m. .times. 1 0 0 0 3 F-HN i.n. .times. 2 0
0 0 4 CCS-HN i.n. .times. 2 0 180 840
[0216] In addition, the induction of cellular responses by the
various vaccine formulations was tested. In particular, young mice
were immunized i.n. (days 0, 7) with various cationic liposomal
formulations and the splenocyte cellular responses--cytotoxicity,
proliferation and IFN.gamma. production--were measured 6 weeks
after vaccination. In the experiment, the results of which are
shown in Table 6, a comparison was made between HN-loaded liposomes
(groups 3-10) and free antigen (F-HN) given alone (group 2) or
admixed with preformed empty liposomes (groups 11-13). The
immunogenicity of Lip (DMTAP)-HN and Lip (CCS)-HN prepared at
varying lipid/HN w/w ratios (30/1-300/1) was also determined.
TABLE-US-00013 TABLE 6 Induction of cellular responses by cationic
liposomes administered i.n. Lipid/HN % cytotoxicity Proliferation
IFN.gamma. No. Vaccine w/w ratio P815 + peptide P815 .DELTA.cpm
(mean) (pg/ml) 1 PBS -- 6 4 7010 1900 2 F-HN -- 8 5 7700 4500 3 Lip
(DMTAP)-HN 300/1 16 13 10960 3500 4 100/1 9 9 12870 5850 5 50/1 3 2
17670 3400 6 30/1 3 2 17920 3050 7 Lip (CCS)-HN 300/1 4 2 20370
8000 8 100/1 21 7 24870 8250 9 50/1 6 3 20980 10650 10 30/1 8 5
11510 3500 11 F-HN + Lip (DOTAP) 300/1 17 4 19390 3400 12 F-HN +
Lip (DMTAP) 300/1 17 7 11850 5700 13 F-HN + Lip (CCS) 300/1 16 8
19270 4100
[0217] Preferential cytotoxicity against the specific target cells
(P815 pulsed with the influenza peptide) was obtained only with
CCS-HN at a lipid/HN w/w ratio of 100/1 (group 8) and with all the
three preformed liposomes (DOTAP, DMTAP and CCS) co-administered
with free antigen. The maximum proliferative response was observed
with DMTAP-HN at lipid/HN w/w ratios of 50/1 and 30/1 and with
CCS-HN at 300/1, 100/1 and 50/1 ratios. The proliferative and
cytotoxic responses elicited by the most efficacious liposomal
formulations were 2-3 times greater than those induced by free
antigen.
[0218] These findings suggest that as compared with the humoral
response (Table 3), where the highest levels of all types of
antibodies measured were obtained at lipid/HN w/w ratios of
100/1-300/1, lower w/w ratios (e.g. 30/1-100/1) may be optimal for
the cellular responses. Moreover, whereas DMTAP-HN elicits a strong
humoral response, this formulation is a poor inducer of cytotoxic
activity, as compared with CCS-HN. Interestingly, vaccination with
mixtures of free antigen with preformed cationic liposomes (all
three formulations) in suspension evokes good cellular responses
that are similar in magnitude to those induced by the encapsulated
antigen. Thus, simple mixing of free antigen with preformed
cationic liposomes may be sufficient to induce both strong humoral
(Table 3A-3C) and cellular (Table 6) responses.
[0219] In yet a further experiment, the results of which are shown
in Tables 7A-7C, a comparison was made between 1 i.m. dose, 1 or 2
i.n. doses and 2 oral doses of a monovalent HN-loaded cationic
liposomes comprising DOTAP, DMTAP or CCS with regard to
immunogenicity and induction of protective immunity to live virus
challenge. In this experiment, the lipid/HN w/w ratio was 300/1 and
the cationic lipid/Chol ratio was 1/1 for DOTAP and DMTAP systems
and 3/2 for CCS system. Of the three routes, i.n. administration
twice generates the strongest humoral (systemic and mucosal) and
cellular response and protective immunity. Of the 3 formulations,
CCS induces the highest response, particularly with regard to IgG2a
and IgA antibodies. TABLE-US-00014 TABLE 7A Serum levels of HI,
IgG1, IgG2a and IgA Vaccine Serum No. (n = 10) Route HI IgG1 IgG2a
IgA 1 PBS 0 0 0 0 2 F-HN i.m. .times. 1 60 .+-. 37 (70) 1000 40 0 3
oral .times. 2 0 0 0 0 4 i.n. .times. 1 0 0 0 0 5 i.n. .times. 2 0
55 0 0 6 Lip (DOTAP/Chol)-HN i.m. .times. 1 424 .+-. 141 (100)
21000 5500 0 7 oral .times. 2 0 0 0 0 8 i.n. .times. 1 40 .+-. 28
(50) 450 80 0 9 i.n. .times. 2 409 .+-. 172 (100) 25000 1300 60 10
Lip (DMTAP/Chol)-HN i.m. .times. 1 768 .+-. 211 (100) 24000 8000 0
11 oral .times. 2 0 0 0 0 12 i.n. .times. 1 10 .+-. 10 (0) 300 60 0
13 i.n. .times. 2 532 .+-. 763(100) 10500 380 50 14 Lip
(CCS/Chol)-HN i.m. .times. 1 864 .+-. 1100 (100) 25000 10000 0 15
oral .times. 2 0 0 16 i.n. .times. 1 34 .+-. 50 (20) 1000 30 0 17
i.n. .times. 2 2289 .+-. 1576 25000 20000 400 (100) 18 F-HN + CT (1
.mu.g) i.n. .times. 2 756 .+-. 650 (100) 21000 15000 20
[0220] TABLE-US-00015 TABLE 7B Lung antibodies Vaccine Lung No. (n
= 5) Route HI IgG1 IgG2a IgA 1 PBS 0 0 0 0 2 F-HN i.m. .times. 1 0
80 0 0 3 oral .times. 2 0 0 0 0 4 i.n. .times. 1 0 0 0 0 5 i.n.
.times. 2 0 70 20 0 6 Lip (DOTAP/Chol)- i.m. .times. 1 40 900 500 0
HN 7 oral .times. 2 0 0 0 0 8 i.n. .times. 1 0 50 20 0 9 i.n.
.times. 2 120 10000 1000 350 10 Lip (DMTAP/Chol)- i.m. .times. 1 20
900 150 0 HN 11 oral .times. 2 0 0 0 0 12 i.n. .times. 1 0 35 20 0
13 i.n. .times. 2 240 20000 700 2200 14 Lip (CCS/Chol)-HN i.m.
.times. 1 60 3500 900 0 15 oral .times. 2 0 0 0 0 16 i.n. .times. 1
0 120 0 35 17 i.n. .times. 2 360 30000 5000 20000 18 F-HN + CT (1
.mu.g) i.n. .times. 2 240 22000 2500 1800
[0221] TABLE-US-00016 TABLE 7C Cellular response and protective
immunity Spleen Lung Vaccine .DELTA.cpm IFN.gamma. Virus titer No.
(n = 5) Route (mean) (pg/ml) (log 10) 1 PBS 1641 0 7 2 F-HN i.m.
.times. 1 1909 0 4 3 oral .times. 2 2253 0 ND 4 i.n. .times. 1 669
0 ND 5 i.n. .times. 2 2813 0 5 6 Lip (DOTAP/Chol)-HN i.m. .times. 1
3452 3300 0 7 oral .times. 2 0 1150 ND 8 i.n. .times. 1 482 1900 ND
9 i.n. .times. 2 8391 3200 0 10 Lip (DMTAP/Chol)-HN i.m. .times. 1
5632 0 1 11 oral .times. 2 553 0 ND 12 i.n. .times. 1 1277 0 ND 13
i.n. .times. 2 7331 3150 0 14 Lip (CCS/Chol)-HN i.m. .times. 1 6196
5750 0 15 oral .times. 2 476 550 ND 16 i.n. .times. 1 1705 6250 ND
17 i.n. .times. 2 4912 15500 0 18 F-HN + CT (1 .mu.g) i.n. .times.
2 1933 5650 0
[0222] In the experiment described in Tables 8-10, a commercial
trivalent vaccine was tested and a comparison was made between a
single intranasal (i.n.) CCS-based vaccine dose (using 2 or 4 .mu.g
of antigen [HN] of each viral strain) and two i.n. vaccine doses (2
.mu.g/strain/dose), given at 3, 7 or 14 day intervals between
administrations. The lipid assemblies were composed of CCS/Chol
(cholesterol) at a 3/2 mole ratio, and the lipid/HN w/w ratio was
100/1 for all formulations. As controls, the standard trivalent
commercial vaccine (HN) was administered either alone or combined
with 1 .mu.g cholera toxin (CT), used as a mucosal adjuvant. Sera,
lung homogenates and nasal washes were tested 5-6 weeks after the
first vaccine dose for HI antibodies (Table 8), as well as for
antigen-specific IgG1, IgG2a, IgA and IgE antibodies (Table 9). In
addition, 5 mice from selected groups were challenged i.n. with
live virus (using the mouse adapted reassortant X-127 virus) and
protection was assessed by quantifying lung virus titer 4 days
later (Table 10).
[0223] As opposed to the poor or no immunogenicity of the
commercial flu vaccine (HN) (groups 2-6), CCS/Chol-flu vaccine
induced high titers of all types of antibodies tested (except for
IgE which was undetected), especially against the two A virus
strains (groups 8-11; Tables 8, 9). For the 2-dose regimen, a
1-week interval appears to be the optimal (gr. 10). For the single
dose regimen, 4 .mu.g antigen, but not 2 .mu.g (gr. 8 vs. gr. 7),
induced high titers of serum HI, IgG1 and IgG2a antibodies and lung
IgG1 antibodies. However, in comparison with the 2-dose regimen,
the 1-dose regimen did not elicit lung IgG2a and IgA antibodies or
nasal antibodies (Table 9).
[0224] In the protection assay (Table 10), the CCS-flu vaccine
administered i.n. either once (4 .mu.g) or twice (2 .mu.g/dose)
afforded full protection against viral infection (6 log reduction
in lung virus titer) whereas the standard vaccine reduced virus
titer by only 0.5-1 log. Thus, although the single dose regimen
with the CCS-flu vaccine is inferior to the two-dose regimen for
certain antibody isotypes, the two regimens provide a similar
degree of protection.
[0225] In this experiment, we also compared CCS alone to CCS/Chol
as the vaccine carrier (administered on days 0 and 7) and found no
difference in immunogenicity between the two formulations (data not
shown). Another formulation modification was the reduction of the
size of the CCS/Chol lipid assemblies (diameter 0.05-5 .mu.m) by
extrusion (diameter .ltoreq.0.02 .mu.m). Antibody titers induced by
the extruded vaccine were 50-80% lower than those produced by the
non-extruded vaccine (data not shown). Thus, unsized CCS lipid
assemblies, with or without cholesterol, are highly efficient as a
vaccine carrier for trivalent flu vaccine. TABLE-US-00017 TABLE 8
Elicitation of hemagglutination inhibition (HI) antibodies
following intranasal vaccination with trivalent influenza vaccine,
free and in CCS lipid assemblies, administered once or twice at
various time intervals to young (2 mo.) BALB/C mice Mean HI titer
(% seroconversion).sup.b Dosing A/New Caledonia A/Panama
B/Yamanashi No. Vaccine.sup.a (n = 5) days serum lung serum lung
serum lung 1 None (PBS) .times.2 0, 7 0 0 0 0 0 0 2 F-HN 2 .mu.g
.times. 1 0 0 0 0 0 0 0 3 4 .mu.g .times. 1 0 0 0 0 0 0 0 4 2 .mu.g
.times. 2 0, 3 0 0 0 0 0 0 5 2 .mu.g .times. 2 0, 7 0 0 0 0 0 0 6 2
.mu.g .times. 2 0, 14 0 0 0 0 0 0 7 Lip (CCS/Chol)-HN 2 .mu.g
.times. 1 0 0 0 0 0 0 0 8 4 .mu.g .times. 1 0 336 (100) 40 328
(100) 40 52 (80) 0 9 2 .mu.g .times. 2 0, 3 544 (100) 80 408 (100)
40 52 (80) 0 10 2 .mu.g .times. 2 0, 7 544 (100) 80 544 (100) 120
88 (100) 0 11 2 .mu.g .times. 2 0, 14 480 (100) 60 368 (100) 40 80
(80) 0 12 F-HN + CT (1 .mu.g) 2 .mu.g .times. 2 0, 7 608 (100) 80
664 (100) 120 84 (80) 0 .sup.aMice were immunized with Fluvirin
.RTM. 2003/2004 trivalent subunit vaccine preparation consisting of
A/New Caledonia/20/99 (H1N1)-like, A/Moscow/10/99 (H3N2)-like and
B/Hong Kong/330/2001-like, either free (F-HN) or incorporated into
CCS/Chol (3/2 mole ratio) lipid assemblies (0.6 mg for groups 7, 9,
10, 11; 1.2 mg for group 8). .sup.bSerum HI titer was determined on
individual mice 35 days after the first vaccine dose. Lung (pooled)
HI titer was tested on day 42. In parentheses - % of mice with HI
titer .gtoreq.40.0 denotes HI titer <20.
[0226] TABLE-US-00018 TABLE 9 Elicitation of serum, lung and nasal
antigen-specific IgG1, IgG2a and IgA antibodies following
intranasal vaccination with trivalent influenza vaccine, free and
in CCS lipid assemblies, administered once or twice at various
intervals to young (2 mo.) BALB/c mice Mean antibody titer Dosing
Serum Lung Homogenate Nasal wash No. Vaccine.sup.a (n = 5) days
IgG1 IgG2a IgG1 IgG2a IgA IgG1 IgG2a IgA 1 None (PBS) .times.2 0, 7
0 0 0 0 0 0 0 0 2 F-HN 2 .mu.g .times. 1 0 0 0 0 0 0 0 0 0 3 4
.mu.g .times. 1 0 320 90 1500 0 0 0 0 0 4 2 .mu.g .times. 2 0, 3 0
0 0 0 0 0 0 0 5 2 .mu.g .times. 2 0, 7 0 0 0 0 0 0 0 0 6 2 .mu.g
.times. 2 0, 14 40 0 0 0 0 0 0 0 7 Lip (CCS/Chol)-HN 2 .mu.g
.times. 1 0 300 0 600 0 0 0 0 0 8 4 .mu.g .times. 1 0 12000 4500
13000 0 0 0 0 0 9 2 .mu.g .times. 2 0, 3 15000 10000 15000 2500
3500 0 10 0 10 2 .mu.g .times. 2 0, 7 15000 12000 14000 2500 9000
200 30 100 11 2 .mu.g .times. 2 0, 14 13000 5500 12000 1800 3000 50
0 0 12 F-HN + CT (1 .mu.g) 2 .mu.g .times. 2 0, 7 21000 15000 20000
2500 2000 250 30 45 .sup.aSee Table 8 for experimental details.
Samples were pooled and tested by ELISA against the 3 viral strains
(pooled HN) 42 days after the first vaccine dose. 0 denotes titer
<10.
[0227] TABLE-US-00019 TABLE 10 Protection of young BALB/c mice
against viral challenge following intranasal vaccination with
trivalent influenza vaccine, free and in CCS lipid assemblies
Dosing No. Vaccine.sup.a (n = 5) days Lung virus titer (log
10).sup.b 1 None -- 6 2 F-HN 4 .mu.g .times. 1 0 5.5 3 F-HN 2 .mu.g
.times. 2 0, 7 5 4 Lip (CCS/Chol)-HN 4 .mu.g .times. 1 0 0 5 Lip
(CCS/Chol)-HN 2 .mu.g .times. 2 0, 7 0 6 F-HN 2 .mu.g + CT (1
.mu.g) .times. 2 0, 7 0 .sup.aSee table 8 for experimental details.
In groups 4, 5 the lipid/HN w/w ratio was 100/1. .sup.bThe mice
were infected intranasally 42 days after the first vaccine dose,
using .about.10.sup.6 egg infectious dose 50% (EID 50) of the
mouse-adapted reassortant X-127 virus (A/Beijing/262/95 [H1N1]
.times. X-31 [A/Hong Kong/1/68 .times. A/PR/8/34). Lungs were
harvested 4 days later, homogenized, serially diluted, and injected
into the allantoic sac of 10 d. fertilized chicken eggs. After 48 h
at 37.degree. C. and 16 h at 4.degree. C., 0.1 mL of # allantoic
fluid was removed and checked for viral presence by
hemagglutination.
[0228] In the experiment described in Tables 11 and 12, the
trivalent-flu vaccine was formulated with the CCS/Chol lipid
assemblies using varying amounts of the HN antigens and the lipid.
In this experiment the vaccines were prepared with: (a) varying
amounts of the antigen (0.25-2 .mu.g per viral strain) and of the
lipid (0.075-0.6 mg), keeping the lipid/HN w/w ratio constant at
100/1; (b) graded amounts of the antigen (0.25-2 .mu.g) and a
constant amount of the lipid (0.6 mg) thereby varying the lipid/HN
w/w ratio from 100/1 to 800/1. As can be seen in Table 11 (HI
titer) and Table 12 (isotype titers) vaccines prepared at a 100/1
lipid/HN w/w ratio using 2 or 1 .mu.g antigen of each strain and
0.6 or 0.3 mg lipid, respectively, produced high and similar levels
of antibodies against the 3 viral strains (groups 2, 3). At lower
antigen (0.5, 0.25 .mu.g/strain) and lipid (0.15, 0.075 mg) doses
the response decreased markedly (groups 4, 5), particularly the
mucosal response (lung, nasal) (Table 12). When a constant dose of
lipid was used (0.6 mg), high levels of antibodies were obtained
even with the two lower doses of antigen (0.25, 0.5 .mu.g/strain)
(groups 6-8). Thus, the amount of the CCS lipid is critical, and
with the appropriate lipid dose the antigen dose can be reduced 4-8
fold (from 1-2 .mu.g to 0.25-0.5 .mu.g) namely a clear dose-sparing
effect. TABLE-US-00020 TABLE 11 Effect of the antigen dose and
lipid dose on the induction of HI antibodies following intranasal
vaccination with trivalent influenza vaccine formulated with CCS
lipid assemblies, administered twice (at 1 week interval) to young
(2 mo.) BALB/c mice Mean HI titer (% seroconversion) A/New Lipid/HN
Caledonia A/Panama B/Yamanashi No. Vaccine.sup.a (n = 5) HN (.mu.g)
Lipid (mg) w/w ratio Serum Lung Serum Lung Serum Lung 1 F-HN 2 --
-- 0 0 0 0 0 0 2 Lip (CCS/Chol)-HN 2 0.6 100/1 544 (100) 80 544
(100) 120 88 (100) 0 3 1 0.3 100/1 320 (100) 80 544 (100) 160 40
(100) 0 4 0.5 0.15 100/1 416 (100) 20 448 (100) 40 32 (100) 0 5
0.25 0.075 100/1 180 (100) 0 100 (100) 20 0 0 6 1 0.6 200/1 672
(100) 80 736 (100) 160 104 (100) 0 7 0.5 0.6 400/1 560 (100) 80 608
(100 160 104 (100) 0 8 0.25 0.6 800/1 512 (100) 80 512 (100) 120 48
(100) 0 .sup.aSee Table 8 for experimental details.
[0229] TABLE-US-00021 TABLE 12 Effect of the antigen dose and lipid
dose on the induction of serum, lung and nasal antigen-specific
IgG1, IgG2a and IgA antibodies following intranasal vaccination
with trivalent influenza vaccine formulated with CCS lipid
assemblies, administered twice (at 1 week interval) to young BALB/c
mice Lipid/HN Mean antibody titer Vaccine.sup.a HN Lipid w/w Serum
Lung homogenate Nasal wash (n = 5) (.mu.g) (mg) ratio IgG1 IgG2a
IgG1 IgG2a IgA IgG1 IgG2a IgA 1 F-HN 2 -- -- 0 0 0 0 0 0 0 0 2 Lip
(CCS/Chol)-HN 2 0.6 100/1 15000 12000 14000 2500 9000 200 30 100 3
1 0.3 100/1 14000 2500 10000 1000 8000 100 0 80 4 0.5 0.15 100/1
15000 1300 8000 1500 4000 0 0 0 5 0.25 0.075 100/1 12000 400 3500
400 2500 0 0 0 6 1 0.6 200/1 20000 15000 12000 2500 8000 200 15 80
7 0.5 0.6 400/1 15000 14000 15000 5000 15000 150 35 100 8 0.25 0.6
800/1 15000 9000 21000 2500 13000 250 25 90 .sup.aSee Tables 8, 9
for experimental details.
[0230] In a further experiment, the subunit flu vaccine, either
free (HN) or associated with the CCS/Chol lipid assemblies (Lip
HN), was tested for its ability to induce HI antibodies
cross-reacting with various influenza A and B substrains that were
not included in the vaccine. The data shown in Table 13 indicate
that intranasal (i.n.) and intramuscular (i.m.) vaccination,
administered once or twice, with either a monovalent or trivalent
CCS-based influenza vaccine, elicits high serum titers of HI
antibodies directed against the immunizing strains, as well as HI
antibodies cross-reacting with several A/H1N1, A4H3N2 and B strains
that were circulating in the years 1986-1999 and were not included
in the vaccine. Slightly lower HI titer was found after a single
i.n. vaccine dose (gr. 6 vs. gr. 7). Lung homogenate HI titers (gr.
4, 8) were lower than the corresponding serum titers. Thus,
parenteral or intranasal vaccination with the CCS-based vaccine may
afford protection against a wide spectrum of A and B viral strains.
Such antigenic variants may emerge during a flu epidemic/pandemic
as a result of antigenic drift. In contrast, the standard
commercial vaccine administered i.n. (gr. 1, 5) was totally
ineffective in inducing antibodies against both the homologous and
the heterologous strains. TABLE-US-00022 TABLE 13 Induction of
strain cross-reactive HI antibodies following intranasal or
intramuscular vaccination of young BALB/c mice with CCS-based
monovalent and trivalent influenza vaccine Mean HI titer against:
A/H1N1 New A/H3N2 Cale- Tex- Singa- Pana- Johan- B Vaccine Sample
donia/ Beijing/ as/ pore/ ma/ Sydney/ Nanchang/ nesburg/ Yamanashi/
Harbin/ No Vaccine.sup.a strains tested 20/99 262/95 36/91 6/86
2007/99 5/97 333/95 33/94 166/98 07/94 1 HN A/New serum 0 0 0 0 0 0
0 0 0 0 2 .mu.g .times. 2 i.n. Caledonia 2 Lip HN serum 1280 1280
1280 240 0 0 0 0 0 0 2 .mu.g .times. 2 i.n. 3 Lip HN serum 640 640
320 40 0 0 0 0 0 0 1 .mu.g .times. 1 i.m. 4 Lip HN lung 320 240 240
20 0 0 0 0 0 0 2 .mu.g .times. 2 i.n. homogenate 5 HN A/New serum 0
0 0 0 0 0 0 0 0 0 2 .mu.g .times. 2 i.n. Caledonia, 6 Lip HN
A/Panama, serum 320 80 120 0 320 320 120 120 60 120 4 .mu.g .times.
1 i.n. B/Hong 7 Lip HN Kong serum 480 120 240 20 640 640 120 120 80
320 2 .mu.g .times. 2 i.n. 8 Lip HN lung 80 80 40 0 120 80 0 0 0 40
2 .mu.g .times. 2 i.n. homogenate 9 HN 2 .mu.g + serum 480 240 120
40 480 480 120 120 80 240 CT 1 .mu.g .times. 2 i.n. .sup.aPooled
sera and lung homogenate obtained 5 weeks after vaccination were
tested for HI antibodies. For experimental details, see Table 8.
The lipid (Lip) assemblies were composed of CCS/Chol (3/2 mole
ratio) and the lipid/HN w/w ratio was 300/1 in groups 2-4 and 100/1
in groups 6-8. Except for groups 3 and 6, the two vaccine doses
were spaced 1 week apart. In bold, antibody titers against the
immunizing strains. 0 denotes HI titer <10.
Biodistribution of Anionic and Cationic Liposomes Loaded with HN
and Administered Intranasally
[0231] In a biodistribution experiment, 3 formulations of lipid
assemblies: DMPC/DMPG (anionic), DOTAP/Chol (cationic) and CCS/Chol
(cationic), either empty or loaded with the influenza HN antigens,
were administered intranasally (200 kg lipid, 2 .mu.g antigen per
mouse) into BALB/c mice. The fluorescently labeled lipid was then
traced in the homogenates of various tissues (nose, lungs,
gastrointestinal tract, brain, liver, kidneys, heart and spleen)
over a period of 24 h (at 1, 5, and 24 hours post
administration).
[0232] As can be seen in the following Table 14 and in FIG. 2A-2F,
after 1 and 5 hours there was 75-100% recovery of total lipid
administered (% from the administered dose) of all the three
formulations tested. This recovery however dropped significantly at
24 hours in all formulations except for the CCS formulation. The
CCS formulation containing the HN antigens displayed the longest
retention (>24h.) in the 3 target organs (nose, lungs, GI tract)
while there was no lipid accumulation in the brain and no
significant accumulation in the other organs tested (liver,
kidneys, heart, spleen). TABLE-US-00023 TABLE 14 Recovery at 1, 5,
and 24 hours of fluorescently labeled lipid assemblies administered
intranasal % Recovery (of total lipid administered) in nose, lungs,
gastrointestinal tract, brain, liver, kidneys, heart and spleen
Lipid assembly formulation 1 hour 5 hours 24 hours DMPC/DMPG
(empty) 100.2 99.3 26.9 DMPC/DMPG:HN 100.2 99.9 8.3 DOTAP/Chol
(empty) 107.0 75.1 8.1 DOTAP/Chol:HN 99.9 106.4 6.7 CCS/Chol
(empty) 99.6 96.9 74.2 CCS/Chol:HN 101.1 101.5 94.5 *The values
show % recovery of the lipid as related to total dose
administered.
[0233] When .sup.125I-labeled HN was used, its biodistribution
resembled that of the fluorescent lipid. This long retention of the
CCS vaccine components in the respiratory and GI tracts may
explain, in part, its superior immunogenicity over the other
liposomal formulations. This is exhibited in the following study in
which the antigen component of the vaccine was traced. HN proteins
were labeled with .sup.125I and administered intranasally either
free or associated with one of the lipid formulations used in the
fluorescent biodistribution experiments. Radioactivity of the
various tissues was determined at 1, 5 and 24 h post
instillation.
[0234] Table 15 teaches that recovery of the antigen was high in
this experiment as well. As can be seen in FIGS. 3A-3D, the
biodistribution pattern of the .sup.125I-labeled HN is similar to
that of the lipid (FIGS. 2A-2F), further establishing that: (a)
there is indeed an in vivo association between the HN-proteins and
the lipid assemblies, and (b) the prolonged retention in the nose
of the antigen when associated with the cationic lipid assemblies
may be due to the cationic lipid assemblies and not an inherent
property of the HN proteins, since there is no HN retention when
the protein is administered by itself in soluble form.
[0235] Also this experiment may teach that there is no HN protein
accumulation in the brain when administered alone or associated
with lipid-assemblies (a major safety concern with intranasal
vaccination). Since the radioactive tracing method is much more
sensitive than the fluorescent method, this result is more
confidently based. TABLE-US-00024 TABLE 15 Recovery of .sup.125I
labeled HN administered intranasally either alone or associated
with lipid assemblies at 1, 5 and 24 hours Recovery (% of total
administered) nose, lungs, gastrointestinal tract, brain, liver,
kidneys, Lipid heart and spleen) assembly formulation 1 hour 5
hours 24 hours HN 77 48 17 Lip (DMPC:DMPG) HN 88 50 26 Lip
(DOTAP:Chol) HN 105 58 32 Lip (CCS:Chol) HN 100 74 41 *The values
show % recovery of the HN antigen as related to total dose
administered.
[0236] In an attempt to test if the protein and lipid are retained
and/or cleared by similar or different kinetics in the various
tissues, another analysis of the data was performed, where the
ratio between the % antigen retention (of the total dose
administered) and % lipid retention in the various tissues at
various time points was determined. When the ratio is constant and
.apprxeq.1, it means that both components were similarly retained
in the same organ, while when this ratio is either larger or
smaller than 1 it suggests that the clearance kinetics of each
component was different, and one component was cleared faster than
the other.
[0237] As can be seen in Table 16 below, the only ratio that
remained constant with time was that of CCS/Chol-HN in the nose
(ratio=.about.0.45). This suggests that: (a) the high retention of
the antigen in the nose with CCS and DOTAP is in correlation with
the level of association and due to the binding of these
formulations to the nasal mucosa, in contrast to DMPC/DMPG; and (b)
while the other formulations' components dissociate in the body and
are cleared at different rates, the CCS-HN based formulation was
stable, especially in the nose, and this may contribute to the
enhanced immunogenicity seen with the CCS-based vaccines.
TABLE-US-00025 TABLE 16 Time-dependent biodistribution of vaccine
component (lipid and HN antigen) after i.n. administration Lip
(DMPC:DMPG) Lip (DOTAP:Chol) HN HN Lip (CCS:Chol) HN 1 h 5 h 24 h 1
h 5 h 24 h 1 h 5 h 24 h HN nose 9% 4% 2% 38% 16% 2% 41% 14% 12%
lungs 30% 3% 4% 19% 4% 12% 24% 21% 11% GI 35% 32% 11% 33% 27% 10%
22% 28% 8% recovery: 88% 50% 26% 105% 58% 32% 100% 74% 41% lipid
nose 0% 0% 0% 46% 56% 0% 88% 30% 25% lungs 67% 80% 5% 38% 3% 3% 12%
35% 14% GI 33% 20% 4% 16% 47% 3% 1% 37% 55% recovery: 100% 100% 8%
100% 106% 7% 101% 102% 95% HN/lipid nose -- -- -- 0.82 0.29 -- 0.47
0.45 0.47 ratio lungs 0.44 0.03 0.85 0.50 1.29 3.56 2.01 0.60 0.80
GI 1.07 1.56 2.96 2.12 0.57 2.86 16.08 0.76 0.15 recovery: 0.88
0.50 3.12 1.05 0.55 4.78 0.99 0.73 0.44 The values show % recovery
of the lipid or HN protein as related to total dose
administered.
Preliminary Safety Study of the Intranasal Flu Vaccine
[0238] Toxicity (local, systemic) is a major concern with both i.m.
and i.n. vaccines and therefore a pilot toxicity study was studied.
Cationic lipid formulations (DMTAP, DOTAP, CCS-based) loaded with
the influenza antigens hemagglutinin+neuraminidase (HN) were
administered i.n. (twice, spaced 1 week apart) to mice (n=4/group),
and blood counts (total, differential), blood chemistry and
histological examination (nose, lung sections) were performed 72
hours later. The mice showed no apparent signs of any toxicity.
Blood counts and blood chemistry were within the normal range, and,
as expected, minimal-mild inflammatory response was seen in the
nose and lungs of mice treated with the cationic formulations. A
similar, albeit less pronounced, inflammatory response was also
seen in some mice treated with saline alone or with the
non-encapsulated antigen.
Immunomodulatory Activity of CCS-Flu Vaccine in Mice
[0239] In these experiments, mice were injected i.p. with various
liposomal formulations (composed of DMPC, DMPC/DMPG, DOTAP/Chol,
CCS/Chol), 0.5-1 mg lipid, with or without the HN antigens. The
mice were either untreated or i.p. injected with thioglycollate
(TG, to increase macrophage production) 2 days before the injection
of the liposomal formulations. Peritoneal cells were harvested
24-48 h. after administration of the liposomes and used as such or
after 4 h. adsorption at 37.degree. C. to plastic dishes and
removal of the non-adherent cells. In other experiments, peritoneal
cells were harvested from TG treated mice and incubated with the
liposomal formulations for 24-48 h. The cells were tested by flow
cytometry for the expression of MHC II and the co-stimulatory
molecules CD40 and B7. The supernatants were tested for the
cytokines interferon .gamma. (IFN .gamma.), tumor necrosis factor
.alpha. (TNF .alpha.) and interleukin 12 (IL-12), and for nitric
oxide (NO).
[0240] All the cationic formulations (CCS/Chol, DOTAP/Chol,
DMTAP/Chol) upregulated the expression of B7 and CD40 more than the
other formulations (DMPC [neutral], DMPC/DMPG [anionic]) and
induced higher levels of IFN .gamma. and IL-12. In some cases the
CCS/Chol formulation was more effective than the other cationic
formulations. No significant levels of TNF .alpha. and NO were
induced by any of the formulations. The enhanced expression of
co-stimulatory molecules on antigen presenting cells and the
induction of IL-12 and IFN.gamma. by the cationic formulations can
explain, in part, the greater adjuvant activity of these
formulations. These findings combined with the long retention of
the CCS-flu vaccine in the respiratory tract (FIGS. 2C and 2F and
FIGS. 3A-3D) after intranasal administration may explain why CCS is
such an efficient mucosal vaccine carrier/adjuvant.
Immunogenicity of Isolated CCS Isomers or their Synthetic Mixture
(CCS)
[0241] As indicated above, in order to assess the immunogenicity of
the isolated CCS isomers, i.e. N-palmitoyl D-erythro
sphingosyl-1-carbamoyl spermine (C-1 CCS) or (N-palmitoyl D-erythro
sphingosyl-3-carbamoyl spermine (C-3 CCS), the following different
formulations were evaluated:
[0242] CCS/C-Ag: a formulation of CCS, cholesterol and the antigen,
where the CCS is obtained by the synthetic procedure described
above;
[0243] C-1-CCS/C-Ag: a formulation of the isolated C-1 CCS isomer,
cholesterol and the antigen;
[0244] C-3-CCS/C-Ag: a formulation of the isolated C-3 CCS isomer,
cholesterol and the antigen;
[0245] C-1-CCS/C-3-CCS/C-Ag: a formulation of a mixture of the
isolated C-1 CCS isomer, C-3 CCS isomer (mole:mole ratio of 80:20),
cholesterol and the antigen.
[0246] As control, free Ag was used (F-Ag).
[0247] The anti A/New Caledonia titers were determined and are
presented in Table 17: TABLE-US-00026 TABLE 17 Serum HI titers 4
weeks post immunization (i.m. administration) anti A/New Caledonia
titers Vaccine average SD Median Free-Ag 70 30 80 Lipids prepared
by the dichloromethane/methanol method CCS/C-Ag 1550 870 1280 C-1
CCS/C-Ag 1040 880 960 C-3 CCS/C-Ag 2240 1610 1920 C-1CCS/C-3
CCS/C-Ag 590 260 640 Lipids prepared by DDW/T-Butanol and
lyophilization method CCS/C-Ag 910 710 960 C-1 CCS/C-Ag 560 130 640
C-3 CCS/C-Ag 2190 1900 1600 C-1CCS/C-3 CCS/C-Ag 1000 830 640
[0248] The results presented in Table 17 show that both C-1 and C-3
CCS isomers effectively enhance the response to the antigen, with a
significantly greater effect exhibited when using as an adjuvant
the isolated C-3 CCS isomer as compared to the C-1 isomer.
Immunogenicity of CCS in Rat Model
[0249] The HI antibody levels of CCS/C-HA vaccinated rats was
significantly higher than that of mice vaccinated with the
commercial vaccine alone (p<0.05) at all tested time points as
shown in FIG. 4.
Protective Efficacy of CCS/C in Ferret Model
[0250] FIG. 5 shows the mean sum of virus titer in nasal wash
following infection of ferrets with the antigen. The results show
that the CCS/C vaccinated group, both the 15 kg and 5 .mu.g HA
dose, had significant reduction in mean sum virus titer following
infection as compared to the CCS/C only group and also as compared
to Free-HA group (P<0.001).
Avian Influenza Vaccine
[0251] The feasibility of improving the anti-influenza vaccine, as
compared to an Alum-adsorbed Ag, by using a single dose of
inactivated whole virus with the polycationic sphingolipid, CCS, in
mice was also examined by determining serum anti-hemagglutinin (HA)
antibodies.
Materials and Methods
[0252] Antigen (Ag): H5N1--A/Vietnam/1194/2004, inactivated
purified whole virus (NIBSC, Hertforshire, UK). .about.60 .mu.g/ml
HA, .about.160 .mu.g/ml total protein.
[0253] Mice: BALB/C females aged 9 weeks, n=5-6/group.
Treatments
[0254] Doses: a single-dose vaccination as follows:
[0255] Free Ag: 3 or 6 .mu.g per animal
[0256] Liposomal formulation: The liposomes were composed of CCS
and Cholesterol (CCS/C, [Biolab, Jerusalem] and Cholesterol
[Minakem, France]) at mole:mole ratio of 3:2. lipid:HA w/w ratio
250: 1, lipid:total protein w/w ratio 75:1.
[0257] Alum-Ag: Aluminium hydroxide (Alhydrogel 2%, Sigma Israel).
Alum:HA 25:1 w/w. Alum adsorption: 2 hours at RT with shaking.
[0258] Administration: intramuscularly (i.m.) into the hind leg, 50
.mu.l to one leg for 3 .mu.g and 50 .mu.l/leg to 2 legs for 6
.mu.g. TABLE-US-00027 TABLE 18 Treatment group assignment for
animals Group Formulation Dose administration number 1 F--Ag 2
Alum-Ag 3 .mu.g HA + 0.75 6 mg lipid 3 CCS/C-Ag i.m. .times. 1 4
F--Ag 5 5 Alum-Ag 6 .mu.g + 1.5 mg lipid 6 6 CCS/C-Ag 6
[0259] Serum samples were collected at weeks 2, 4, 8, 12 and 20
after immunization.
[0260] Antibody level determination: Serum anti-hemagglutinin titer
was determined by hemagglutination inhibition assay (HI).
[0261] Statistical analysis: The difference between the test groups
was done by student t-test.
Results
[0262] The results are presented in FIG. 6. Specifically shown are
the serum HI Ab titers against H5N1 virus (A/Vietnam/1194/2004)
with the different agents: free Antigen (F-Ag); Alum-antigen
(alum-Ag); and CCS/cholesterol-Antigen (CCS/C-Ag), at two different
Ag dose levels (3 .mu.g and 6 .mu.g). It is clear that when using
liposomal CCS/cholesterol as the carrier, a much higher HI antibody
titer is obtained (*p=0.04 as compared to Alum-Ag i.m. 3 .mu.g at 8
weeks).
Hepatitis A Virus (HAV)
[0263] In addition to influenza, the immune enhancing potential of
CCS lipid assemblies was also tested for HAV vaccine administered
by the intranasal (i.n.) and the intrarectal (i.r.) routes in
BALB/C mice.
[0264] HAV vaccine (Aventis Pasteur), 10 EU (.about.1.5 .mu.g
protein), was administered twice at a 2-week interval and the
response was tested by the ELISPOT technique 3 weeks after the
second vaccine dose. CpG-ODN, used as a mucosal adjuvant, was given
at 10 .mu.g/dose. The HAV-CCS lipid assemblies were prepared as
described above for the influenza vaccine (Table 1).
[0265] The data presented in Table 19 show that whereas the
commercial HAV vaccine failed to induce an IgA response in both
tissues (lamina propria, Peyer's patches) tested, and by both
administration routes (i.n., i.r.), the vaccine formulated with
either CCS or CpG-ODN generated a significant response in most
cases. The combination of HAV-CCS lipid assemblies and CpG-ODN
resulted in a synergistic response in all cases. Thus, CCS lipid
assemblies alone, and particularly in combination with CpG-ODN, are
also effective as a carrier/adjuvant for mucosal vaccination
against HAV. TABLE-US-00028 TABLE 19 Induction of IgA antibodies
following intranasal (i.n.) or intrarectal (i.r.) vaccination of
BALB/c mice with hepatitis A virus (HAV) vaccine, alone and in
combination with CCS lipid assemblies and/or CpG-ODN Mean no. of
IgA AFC.sup.a/ 10.sup.6 cells in: Lamina Peyer's propria patches
Vaccine i.n. i.r. i.n. i.r. HAV alone 0 0 0 0 HAV-CCS 12 27 0 1 HAV
+ CpG-ODN 16 22 0 14 HAV-CCS + CpG-ODN 139 68 28 23
.sup.aAFC--antibody-forming-cells
C. Botulinum
[0266] In a further experiment, Mice were immunized i.n. with 0.4
.mu.g dose of a commercial C. botulinum toxoid (CBT, as a model for
bioterror agent, Uruguay, alum free) and antibody titers were
tested by ELISA 4 weeks after the second vaccine dose.
[0267] The results of an experiment with C. botulinum toxoid are
summarized in Table 20, which shows the superiority of the
CCS-toxoid formulation over the standard vaccine following i.n.
instillation, particularly with regard to the TgA levels in the
small intestine and feces. Such Antibodies are expected to
neutralize the toxin upon oral exposure. Mice immunized i.n. with
the vaccine alone did not produce IgA. TABLE-US-00029 TABLE 20
Induction of IgG1, IgG2a and IgA antibodies in BALB/c mice
vaccinated intranasally (twice, 1 week apart) with free or
CCS-associated Clostridium botulinum toxoid (CBT) Mean antibody
titer Vaccine.sup.a Serum Small intestine Feces n = 10 IgG1 IgG2a
IgG1 IgG2a IgA IgA CBT 0 0 1000 180 0 0 CCS-CBT 400 24 1600 0 1800
1800
Hepatitis B Virus (HBV)
[0268] In addition, the immune enhancing potential of CCS lipid
assemblies was also tested for vaccination of mice against
Hepatitis B.
[0269] Hepatitis B surface antigen particles derived from CHO cells
were characterized as described before [Diminsky, D., et al.
"Comparison between hepatitis B surface antigen (HBsAg) particles
derived from mammalian cells (CHO) and yeast cells (Hansenula
polymorpha): composition, structure and immunogenicity" Vaccine
15:637-647 (1997); Diminsky, D., et al. "Physical, chemical and
immunological stability of CHO-derived hepatitis B surface antigen
(HBsAg) particles". Vaccine 18:3-17 (1999)]. In addition, the
electrostatic properties of the recombinant HBsAg particles were
characterized for their Zeta Potential using Zetasizer 3000 HAS,
Malvern Instruments, Malvern, UK, and as also described by
Garbuzenko O. et al. [Garbuzenko O. et al. "Electrostatics of
PEGylated micelles and liposomes containing charged and neutral
lipopolymers" Langmuir 21:2560-8 (2005)]. HBsAg particles has a
negative zeta potential -26.7 mV. This suggests that these
particles bind with cationic CCS/Chol assemblies.
Experiment 1 : Intraperitoneal and Intranasal Vaccination
[0270] The liposomal formulation was prepared at a lipid/protein
antigen w/w ratio of 600/1 (CCS/Chol mole:mole ratio of 3:2).
BALB/C mice (females, 8 weeks old, n=5-6) were vaccinated once i.p.
or i.n. with purified CHO-derived recombinant HBsAg particles
(S+preS1+preS2 particles), Scigen, Yavne, Israel) either alone
(referred to as naked particles, F-Ag) or with CCS/Chol liposomal
formulation and serum antibody levels were determined 5 and 12
weeks following vaccination. The vaccine dose was 1 .mu.g HBsAg
protein with 0.6 mg lipid for i.p. administration and 2 .mu.g HBsAg
protein with 1.2 mg lipid for i.n. administration.
[0271] The vaccination results are presented in FIGS. 7A-7B.
Specific antibodies against HBsAg were detected by Microparticle
Enzyme Immunoassay (MEIA Diminsky, D., et al. (1997) ibid.;
Diminsky, D., et al. (1999) ibid.]). Specifically, FIG. 7A shows
that vaccination with the liposomal CCS/Chol formulation resulted
in higher serum levels of anti-HBsAg antibodies as compared to the
naked antigen following both i.p. and i.n. administration.
[0272] Moreover, measurement of specific isotypes against HBsAg
showed that only mice immunized with the CCS/Chol vaccine, both
i.p. or i.n., produced IgG2a antibodies while those immunized with
naked HBsAg did not (FIG. 7B).
Experiment 2: Intraperitoneal Vaccination
Materials and Methods
Antigens
[0273] Ag: Scigen HBsAg (S+preS1+preS2) without Alum (Scigen,
Yavne, Israel) batch 6P-01-001-09.
[0274] Positive control: Sci-B-Vac.TM., Lot 03870101, (Scigen Ltd,
Singapore), manufactured by BTG Israel, Alum-adsorbed Ag.
[0275] Animals: BALB/C mice, females aged 8 weeks, n=8 animals per
group.
Treatment Schedule
Doses: a single-dose vaccination as follows:
[0276] Free Ag: 0.5 .mu.g per animal
[0277] Liposomal formulation: The liposomes were composed of CCS
and Cholesterol (CCS/C, batch no. NV/010905, MediWound, Yavne) at
mole:mole ratio of 3:2. Lipid:Antigen weight ratio 1200:1.
Therefore, 0.5 .mu.Ag with 0.6 mg total lipid per animal.
[0278] Sci-B-Vac.TM.: 0.5 .mu.g per animal
[0279] Administration: intraperitoneal (i.p.) 100 .mu.l.
TABLE-US-00030 TABLE 21 Treatment group assignment for animals
Formulation dose administration 1 Scigen F-HBsAg 0.5 .mu.g Ag 2
Sci-B-Vac .TM. 0.5 .mu.g Ag + 75 .mu.g Alum i.p. .times. 1 3 Scigen
CCS/C-HBsAg 0.5 .mu.g Ag + 0.6 mg lipid
[0280] Blood collection for antibody detection: Blood was collected
14, 28 days, 2 months and 3 months post-vaccination, from the
orbital sinus vein.
[0281] Antibody level determination: Serum total anti-HBsAg titer
was determined by the AxSYM AUSAB MEIA (Abbot). IgG1, IgG2a were
determined by ELISA.
[0282] Statistical analysis: The difference between mice vaccinated
with Sci-B-Vac.TM. and CCS/C-Ag was done by Student t-test.
Results
[0283] The results are presented in FIGS. 8A-8C.
[0284] Specifically, FIG. 8A shows that a statistically significant
higher anti-HBsAg titer is obtained with the composition comprising
the antigen carried by liposomes composed of CCS in combination
with cholesterol (*p<0.05 between CCS/C-Ag and Sci-B-Vac 8 weeks
post-vaccination).
[0285] Further, FIG. 8B shows that a statistically significant
higher anti-HBsAg IgG1 titer is obtained with the composition
comprising the antigen carried by liposomes composed of CCS in
combination with cholesterol (*p<0.05 between CCS/C-Ag and
Sci-B-Vaccine at 4, 8 and 12 weeks post-vaccination, 0 denotes
<40).
[0286] Yet further, FIG. 8C shows that a statistically significant
higher anti-HBsAg IgG2a titer is obtained with the composition
comprising the antigen carried by liposomes composed of CCS in
combination with cholesterol (**p<0.01 between CCS/C-Ag and
Sci-B-Vaccine, at 4, 8 and 12 weeks post-vaccination 0 denotes
<40).
Anthrax
[0287] The immunogenicity of CCS/C-Anthrax vaccine was also
examined in a mouse model (A) and in a guinea pig model (B).
Specifically, the human anti-Bacillus anthracis vaccine comprising
the bacterium's Protective Antigen (PA) was used to assess the
feasibility of improving the anti-B. anthracis vaccine by using a
single or double vaccination with the commercial PA in combination
with CCS-based liposomes.
(A) Mouse Model
Materials and Methods
[0288] Ag: recombinant Anthrax Protective Antigen (PA) from B.
anthracis List biological laboratories (INC, California, USA), lot
no. 17111A1B.
[0289] Animals: BALB/C mice, females aged 8-9 weeks, n=4-6 per
group.
Treatments
[0290] Doses: a single or double-dose vaccination as follows:
[0291] Free Ag: 10 .mu.g or 20 .mu.g per animal
[0292] Alum-Ag: Alhydrogel 2% (Sigma). Alum adsorption: 2 hours at
RT with shaking. Two Alum:Ag w/w ratios: 7:1 and 25:1.
[0293] Liposomal formulation: The liposomes were composed of CCS
and Cholesterol (CCS/C, batch no. NV/010905 [MediWound, Yavne]) at
molar ratio of 3:2. Lipid:Antigen weight ratio 150:1 for s.c, 113:1
for i.n.
[0294] Administration: single subcutaneous (s.c) administration: 50
.mu.l for 10 .mu.g PA, 100 .mu.l for 20 .mu.g PA.
[0295] Double intranasal (i.n) administration: 30 .mu.l, 20 .mu.g
per dose, at weeks 0 and 5. TABLE-US-00031 TABLE 22 Treatment group
assignment for animals admin- Group Formulation Dose istration
number 1 Free-Ag 10 .mu.g Ag 4 2 Free-Ag 20 .mu.g Ag 4 3 Alum-Ag 10
.mu.g Ag + 70 .mu.g Alum 6 7:1 (w/w) 4 Alum-Ag 10 .mu.g Ag + 250
.mu.g Alum s.c. .times. 1 6 25:1 (w/w) 5 Alum-Ag 20 .mu.g Ag + 500
.mu.g Alum 6 25:1 (w/w) 6 CCS/C-Ag 10 .mu.g Ag + 1.5 mg Lipid 6 7
CCS/C-Ag 20 .mu.g Ag + 3 mg Lipid 6 8 CCS/C-Ag 20 .mu.g Ag + 2.25
mg i.n .times. 2 6 Lipid (0 and 5 w)
[0296] Blood collection for antibody detection: Blood was collected
from the orbital sinus vein at weeks 2, 4 (for s.c. and i.n.), and
7 (only for i.n.) post-vaccination.
[0297] Antibody level determination: Individual serum samples
analyzed for anti-PA antibodies titers by ELISA using a commercial
kit: QuickELISA.TM. Anthrax-PA kit (Immunetics, Inc, MA, USA)
(Veterinary Institute).
Results
[0298] The results are presented in FIGS. 9A-9B.
[0299] Specifically, FIG. 9A shows anti-PA IgG antibodies median
levels (data presented as O.D) as determined using a commercial
kit: QuickELISA.TM. Anthrax-PA kit (Immunetics, Inc, MA, USA).
O.D>0.186 is considered as positive response. FIG. 9B shows the
anti-PA antibodies median levels determined by ELISA. %--percentage
of responders.
[0300] As shown in FIG. 9A, serum anti-PA levels (represented by
their respective O.D.) and percentage of responding mice, 2 and 4
weeks post a single s.c. vaccination with the PA+CCS/C liposomes
are much higher than those exhibited with Alum-Ag or the free
antigen vaccination. A single i.n. dose of CCS/C-PA did not induce
a significant antibody development. However, a second i.n. dose
with CCS/C-PA on week 5 induced a robust antibody development 2
weeks later (FIG. 9B).
(B) Guinea Pig Model
Materials and Methods:
Antigens
[0301] PA: A commercial preparation of a recombinant anthrax
protective antigen (List Biological laboratories, INC, California,
USA) was used.
[0302] Positive control: Sterne strain (STI) live attenuated
vaccine (Onderstepoort, Namibia), commonly used in veterinary
medicine.
[0303] Animals: Guinea pigs are the laboratory animals most
commonly used in experiments with B. anthracis. Hartley strain
guinea pigs, weighing about 300 grams each.
Treatments:
[0304] Doses: a single-dose vaccination as follows:
[0305] Free PA: 25 .mu.g
[0306] Liposomal formulation: The liposomes were composed of CCS
and Cholesterol (CCS/C, batch MediWound #NV/010905) at mole:mole
ratio of 3:2. Lipid:Antigen weight ratio 150:1. Therefore, 25 .mu.g
PA with 3.75 mg total lipid per animal
[0307] STI vaccine: 0.5 ml per animal
[0308] Administration: PA vaccine: subcutaneous (s.c) 100 .mu.l, or
STI vaccine: s.c. 0.5 ml. TABLE-US-00032 TABLE 23 Treatment group
assignment for animals Group Formulation dose Administration 1
Free-Ag 25 .mu.g Ag 2 CCS/C-Ag 25 .mu.g Ag + 3.75 mg s.c. .times. 1
Lipid 3 Sterne strain (STI) 0.5 ml virus
[0309] Blood collection for antibody detection: Blood was collected
28 days post-vaccination, from the heart.
[0310] Antibody level determination: by Immunetics.RTM.
QuickELISA.TM. Anthrax-PA kit (Immunetics, Inc, MA, USA).
[0311] Statistical analysis: The difference between guinea pigs
vaccinated with PA and CCS/C-PA subcutaneously was done by the
Kruskal-Wallis non-parametric ANOVA test (Statistix.RTM. package,
version 7 (Analytical Software, USA).
Results
[0312] The results are presented in FIG. 10. Specifically, the
figure shows anti-PA antibodies median levels (presented as O.D) in
Guinea pigs as determined by a commercial kit: QuickELISA.TM.
Anthrax-PA kit (Immunetics, Inc, MA, USA) (O.D>0.186 is
considered as positive response) and that the anti-PA antibody
levels are statistically higher with CCS/C-PA vaccine (*
Significant statistical difference between the groups of CCS/C-PA
vaccine and F-PA vaccine (p<0.001)).
Streptococcus pneumoniae
Proposed Preclinical Studies
Intranasal Challenge of Mice Immunized with rPsaA/CCS
[0313] Groups of 10 mice 3- to 5-week-old CBA/NCAHNXID mice
(Jackson Laboratories, Barr Harbor, Me.) will be immunized
intranasally (i.n.) using purified rPsaA at the following doses:
150 ng or 500 ng per animal each dose adjuvated with CCS (at an
antigen:CCS ratio of between 1:10 to 1:600). For intranasal
administration, 10 .mu.l of each dose with or without CCS will be
prepared freshly with 0.85% physiological saline using a stock
solution of 1 mg/ml of purified rPsaA. Mice will be boosted twice
with the same dose of rPsaA on day 7 and day 14 post-initial
dose.
[0314] On day 14 following the final booster, saliva (60
.mu.l/mouse) and blood (100 .mu.l) from the tail vein will be
collected from each mouse and analyzed for an IgG response by
ELISA. Six weeks after final boosting (day 38), mice will be
challenged with 10.sup.6 colony forming units of PLN D39 suspended
in 10 .mu.l of 0.85% saline. On day 7 post-challenge, mice will be
euthanized and intranasal wash (6 drops) and blood (100 .mu.l) will
be collected. Both blood and intranasal wash samples will be
serially diluted and plated to 5% sheep blood agar plate
supplemented with 0.3 .mu.g/ml erythromycin. Bacterial cultures
will be incubated at 37.degree. C. in a 5% CO.sub.2 incubator, and
colonies will be counted following 24 h incubation [De B. K. et al.
Purification and characterization of Streptococcus pneumoniae
palmitoylated pneumococcal surface adhesin A expressed in
Escherichia coli. Vaccine; (2000) 18(17):1811-1821]
Immunization of Mice with Recombinant 6PGD and CCS
[0315] Six-week-old BALB/c female mice (Harlan Laboratories,
Israel) will be immunized intraperitoneally with 25 .mu.g of r6PGD
and adjuvanted with 75 .mu.l of CCS (at an antigen:CCS ratio of
between 1:10 to 1:600) on days 0 (primary immunization), 7, 14 and
21 (booster). Control mice were sham immunized with CCS adjuvant
only and another control group with r6PGD adsorbed to Alum. Blood
samples will be collected from mice 1 week prior to immunization
and 1 week after booster immunization. The sera will be pooled for
immunological assays. For inhibition of adhesion experiments S.
pneumoniae (10.sup.6 CFU) will be added to A549 cells, as described
[D. Danieliy et al. Pneumococcal 6-phosphogluconate-dehydrogenase,
a putative adhesin, induces protective immune response in mice.
Clinical & Experimental Immunology Volume 144 Issue 2 Page
254--(2006)] prior to or after 30 min incubation with serum
obtained from r6PGD immunized mice. The bacteria will be spun and
resuspended in culture media prior to their addition to the
cultured A549 cells. Incubation and results evaluation will be
performed, again, as described by D. Danieliy et al. [Clinical
& Experimental Immunology (2006) ibid.].
Respiratory Challenge with S. pneumoniae Strain WU2
[0316] For respiratory challenge r6PGD/CCS immunized (n=29) and
control (n=14) mice will be anaesthetized and inoculated
intranasally with 1.times.10.sup.8 CFU of S. pneumoniae strain WU2
(in 25 .mu.l PBS). This inoculum's size is used as it was found to
be the lowest that causes 100% mortality in our mouse model system
within 96 h. Survival was monitored daily. The experiments will be
conducted on three different occasions and the results will be
pooled. Bacterial load will be determined in r6PGD/CCS immunized
(n=3) and control mice (n=3). The nasopharynx and lungs will be
excised and homogenized and blood will be withdrawn. Bacterial load
in the nasopharynx, lungs and the blood is determined 48 h
following intranasal challenge with S. pneumoniae strain WU2.
[0317] In a similar assay, immunization and challenge will be
performed using CW-T, CW-L or CW-NL as antigens [M. Portnoi et al.
The vaccine potential of Streptococcus pneumoniae surface lectin-
and non-lectin proteins. Vaccine 24 (2006) 1868-1873] in
combination with CCS at different antigen:adjuvant ratios between
1:10 to 1:600. Specifically, six week old BALB/c and C57BL/6 female
mice; Harlan Laboratories, Israel) will be intramuscularly
immunized with 25 .mu.g of CW-T, CW-L or CW-NL adjuvanted with CCS
at day 0 (primary immunization) and CCS (about 1.5 mg), days 7 and
14 (booster immunizations). Control mice will be sham immunized
with CCS adjuvant alone and another control groups with CW-T, CW-L
or CW-NL adsorbed to Alum. Mice will be bled 1 week prior to
immunization and 1 week after second booster. The sera will be
pooled. For challenge, with virulent S. pneumoniae strain WU2 (LD50
for intranasally challenge was 1.times.10.sup.7 CFU and for
intraperitoneally challenge was 1.times.10.sup.6 CFU), mice were
anaesthetized with pentobarbital sodium (0.6 mg/kg), and inoculated
intranasally with 5.times.10.sup.8 CFU of S. pneumoniae (in 25
.mu.l PBS) or intraperitoneally with 5.times.10.sup.7 CFU of S.
pneumoniae (in 100 .mu.l PBS). Mortality will be monitored
daily.
Proposed Clinical Trials
Immunization of Human Subjects with Recombinant Truncated Rx1
Strain rPspA in Combination with CCS
Human Study
[0318] Patients aged 18.+-.45 are immunized with recombinant
truncated Rx1 strain rPspA (1.+-.314) [G. S. Nabors et al. Vaccine
18 (2000) 1743-1754] adjuvanted with CCS prepared as described
above. Groups of 30 adults will be immunized with rRx1 alone or
with CCS (at a rRx1:CCS ratio of between 1:10 to 1:600) on day 0,
Day 7 and day 14, and sera will be collected at various times after
immunization. The control group will include immunization with Alum
(ahydrogell Al(OH).sub.3 gel, superfos biosector a/s Denmark).
[0319] Human sera will be assayed for their ability to bind to
rPspAs representing each of the six recognized clades using ELISAs.
For each assay, the ELISA method will be essentially the same.
Briefly, plates will be coated overnight at 48.degree. C. with
0.5.+-.1 mg/ml rPspA antigen in a volume of 100 ml/well. Plates
will be washed with PBS containing 0.05% Tween-20, and test sera
will be serially diluted across plates. After a 2 h incubation at
room temperature, plates will be washed, and a 1:500 dilution of
goat anti-human IgG Fc (Kirkegaard and Perry, Gaithersburg, Md.)
will be added.
[0320] The invention will now be defined by the appended claims,
the contents of which are to be read as included within the
disclosure of the specification.
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