U.S. patent application number 12/369983 was filed with the patent office on 2009-08-13 for polymyxin b analogs for lps detoxification.
This patent application is currently assigned to SANOFI PASTEUR. Invention is credited to Tino Krell, Noelle Mistretta, Monique Moreau, Massimo Porro, Alessandro Rustici, Massimo Velucchi.
Application Number | 20090203881 12/369983 |
Document ID | / |
Family ID | 37524819 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090203881 |
Kind Code |
A1 |
Porro; Massimo ; et
al. |
August 13, 2009 |
Polymyxin B Analogs for LPS Detoxification
Abstract
The invention relates to SAEP II peptide dimers that mimic
polymyxin B i.a. in its ability to bind non-covalently the
lipopolysaccharide (LPS) of Gram-negative bacteria with high
affinity, and therefore to detoxify LPS as polymyxin B does. The
dimeric structure is maintained by a pair of disulphide bonds
involving the two cystein residues present in the peptide sequence,
which does not exceed 17 amino acids and essentially comprises
cationic and hydrophobic amino acid residues. In the dimers of the
invention, peptides may have a parallel or anti-parallel
orientation. As a matter of example, a dimer of the invention is
constituted by a peptide of formula
NH2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH, either in a
parallel or antiparallel dimeric form. SAEP II dimers are useful
for treating or preventing septic shock and related disorders
generated by Gram-negative bacteria infection. The invention also
relates to LPS-peptide complexes in which LPS and SAEP II dimers
are non-covalently bound together. These complexes are useful as
vaccinal agents against Gram-negative bacteria infection.
Inventors: |
Porro; Massimo; (Rapolano
Terme, IT) ; Velucchi; Massimo; (Cortona, IT)
; Rustici; Alessandro; (Sovicille, IT) ; Moreau;
Monique; (Lyon, FR) ; Mistretta; Noelle; (Sain
Bel, FR) ; Krell; Tino; (Granada, ES) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE, 32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
SANOFI PASTEUR
LYON CEDEX 07
FR
BIOSYNTH SRL
RAPOLANO TERME (SIENA)
IT
|
Family ID: |
37524819 |
Appl. No.: |
12/369983 |
Filed: |
February 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11398915 |
Apr 6, 2006 |
7507718 |
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12369983 |
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60697121 |
Jul 7, 2005 |
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Current U.S.
Class: |
530/344 |
Current CPC
Class: |
C07K 7/06 20130101; A61K
38/00 20130101; C07K 7/08 20130101; Y02A 50/483 20180101; Y02A
50/478 20180101; Y02A 50/471 20180101; Y02A 50/475 20180101; Y02A
50/30 20180101; C07K 7/54 20130101; Y02A 50/473 20180101 |
Class at
Publication: |
530/344 |
International
Class: |
C07K 1/14 20060101
C07K001/14 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 11, 2005 |
EP |
EP 05300270.5 |
Claims
1-28. (canceled)
29. A method of preparing an SAEP II peptide dimer of formula (I),
NH.sub.2-A-Cys1-B-Cys2-C-COOH NH.sub.2-A'-Cys1-B'-Cys2-C'-COOH
wherein Cys1 and Cys2 are each a cysteine amino acid; wherein the
two Cys1 residues are linked together through an intermolecular
disulphide bond and the two Cys2 residues are linked together
through an intermolecular disulphide bond; wherein A and A'
independently are a peptide moiety of from 2 to 5 amino acid
residues, in which at least 2 amino acid residues, are
independently selected from Lys, Hyl (hydroxy-Lysine), Arg and His;
wherein B and B' independently are a peptide moiety of from 3 to 7
amino acid residues, which comprise at least two amino acid
residues independently selected from Val, Leu, Ile, Phe, Tyr and
Trp; and wherein C and C' are optional and are independently an
amino acid residue or a peptide moiety of from 2 to 3 amino acid
residues; provided that the cationic amino acid
residues/hydrophobic amino acid residues ratio (cat/hydroph ratio)
is from 0.4 to 2; the method comprising separating the dimer of
formula (I) from (a) corresponding dimers of formula (II),
NH.sub.2-A-Cys1-B-Cys2-C-COOH HOOC-C'-Cys2-B'-Cys1-A'-NH.sub.2
wherein the Cys1 residues are linked to the Cys2 residues through
intermolecular disulphide bonds; and/or (b) corresponding monomers
of formulae NH.sub.2-A-Cys1-B-Cys2-C-COOH and
NH.sub.2-A'-Cys1-B'-Cys2-C'-COOH, and/or (c) cyclic counterparts of
the corresponding monomers.
30. The method according to claim 29, wherein the SAEP II peptide
dimer of formula (I) has a cat/hydroph ratio is from 0.5 to
1.5.
31. The method according to claim 30, wherein the cat/hydroph ratio
is from 0.6 to 1.
32. The method according to claim 31, wherein the cat/hydroph ratio
is from 0.6 to 0.8.
33. The method according to claim 29, wherein the B and B' peptide
moieties comprise the sequence -X1-X2-X3-, in which X1 and X2; X2
and X3; or X1, X2 and X3 are independently selected from Val, Leu,
Ile, Phe, Tyr and Trp.
34. The method according to claim 33 wherein the B and B' peptide
moieties comprise: (i) the sequence -X1-X2-X3-, in which: X1 is
Lys, Hyl, His or Arg; X2 is Phe, Leu, Ile, Tyr, Trp or Val; and X3
is Phe, Leu, Ile, Tyr, Trp or Val; and (ii) amino acid residues, if
any, each being independently selected from the group consisting of
Val, Leu, Ile, Phe, Tyr, Trp, Lys, Hyl, Arg and His.
35. The method according to claim 29, wherein the SAEP II peptide
dimer of formula (I) is of formula (III)
NH.sub.2-A-Cys1-B-Cys2-COOH NH.sub.2-A'-Cys1-B'-Cys2-COOH wherein
the two Cys1 residues are linked together through a disulphide bond
and the two Cys2 residues are linked together through a disulphide
bond; and the SAEP II peptide dimer of formula (II) is of formula
(IV) NH.sub.2-A-Cys1-B-Cys2-COOH HOOC-Cys2-B'-Cys1-A'-NH.sub.2,
wherein the Cys1 residues are linked to the Cys2 residues through a
disulphide bond, and the monomers are of formulae
NH.sub.2-A-Cys1-B-Cys2-COOH or NH.sub.2-A'-Cys1-B'-Cys2-COOH.
36. The method of claim 29 wherein the SAEP II peptide dimer of
formulae I is a homologous peptide dimer.
37. The method of claim 29 wherein the SAEP II peptide dimer of
formula I is a parallel dimer of formula (VII)
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH wherein the
two Cys1 residues are linked together through a disulphide bond and
the two Cys2 residues are linked together through a disulphide
bond, and the SAEP II peptide dimer of formula II is an
antiparallel dimer form of formula (VI)
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH
COOH-Cys2-Leu-Leu-Leu-Phe-Lys-Cys1-Lys-Thr-Lys-NH.sub.2, wherein
the Cys1 residues are linked to the Cys2 residues through a
disulphide bond, and and the monomers are of formulae
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH and
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH.
38. A method of preparing an SAEP II peptide dimer of formula (II),
NH.sub.2-A-Cys1-B-Cys2-C-COOH HOOC-C'-Cys2-B'-Cys1-A'-NH.sub.2
wherein Cys1 and Cys2 are each a cysteine amino acid; wherein the
Cys1 residues are linked to the Cys2 residues through
intermolecular disulphide bonds; wherein the two Cys1 residues are
linked together through an intermolecular disulphide bond and the
two Cys2 residues are linked together through an intermolecular
disulphide bond; wherein A and A' independently are a peptide
moiety of from 2 to 5 amino acid residues, in which at least 2
amino acid residues, are independently selected from Lys, Hyl
(hydroxy-Lysine), Arg and His; wherein B and B' independently are a
peptide moiety of from 3 to 7 amino acid residues, which comprise
at least two amino acid residues independently selected from Val,
Leu, Ile, Phe, Tyr and Trp; and wherein C and C' are optional and
are independently an amino acid residue or a peptide moiety of from
2 to 3 amino acid residues; provided that the cationic amino acid
residues/hydrophobic amino acid residues ratio (cat/hydroph ratio)
is from 0.4 to 2; the method comprising separating the dimer of
formula (I) from (a) corresponding dimers of formula (I),
NH.sub.2-A-Cys1-B-Cys2-C-COOH NH.sub.2-A'-Cys1-B'-Cys2-C'-COOH
wherein Cys1 and Cys2 are each a cysteine amino acid; and/or (b)
corresponding monomers of formulae NH.sub.2-A-Cys1-B-Cys2-C-COOH
and NH.sub.2-A'-Cys1-B'-Cys2-C'-COOH, and/or (c) cyclic
counterparts of the corresponding monomers.
39. The method according to claim 38, wherein the SAEP II peptide
dimer of formula (II) has a cat/hydroph ratio is from 0.5 to
1.5.
40. The method according to claim 39, wherein the cat/hydroph ratio
is from 0.6 to 1.
41. The method according to claim 40, wherein the cat/hydroph ratio
is from 0.6 to 0.8.
42. The method according to claim 38, wherein the B and B' peptide
moieties comprise the sequence -X1-X2-X3-, in which X1 and X2; X2
and X3; or X1, X2 and X3 are independently selected from Val, Leu,
Ile, Phe, Tyr and Trp.
43. The method according to claim 42 wherein the B and B' peptide
moieties comprise: (i) the sequence -X1-X2-X3-, in which: X1 is
Lys, Hyl, His or Arg; X2 is Phe, Leu, Ile, Tyr, Trp or Val; and X3
is Phe, Leu, Ile, Tyr, Trp or Val; and (ii) amino acid residues, if
any, each being independently selected from the group consisting of
Val, Leu, Ile, Phe, Tyr, Trp, Lys, Hyl, Arg and His.
44. The method according to claim 38, wherein the SAEP II peptide
dimer of formula (I) is of formula (III)
NH.sub.2-A-Cys1-B-Cys2-COOH NH.sub.2-A'-Cys1-B'-Cys2-COOH wherein
the two Cys1 residues are linked together through a disulphide bond
and the two Cys2 residues are linked together through a disulphide
bond; and the SAEP II peptide dimer of formula (II) is of formula
(IV) NH.sub.2-A-Cys1-B-Cys2-COOH HOOC-Cys2-B'-Cys1-A'-NH.sub.2,
wherein the Cys1 residues are linked to the Cys2 residues through a
disulphide bond, and the monomers are of formulae
NH.sub.2-A-Cys1-B-Cys2-COOH or NH.sub.2-A'-Cys1-B'-Cys2-COOH.
45. The method of claim 38 wherein the SAEP II peptide dimer of
formulae II is a homologous peptide dimer.
46. The method of claim 38 wherein the SAEP II peptide dimer of
formula II is an antiparallel dimer form of formula (VI)
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH
COOH-Cys2-Leu-Leu-Leu-Phe-Lys-Cys1-Lys-Thr-Lys-NH.sub.2, wherein
the Cys1 residues are linked to the Cys2 residues through a
disulphide bond, and the SAEP II peptide dimer of formula I is a
parallel dimer of formula (VII)
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH wherein the
two Cys1 residues are linked together through a disulphide bond and
the two Cys2 residues are linked together through a disulphide
bond, and the monomers are of formulae
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH and
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH.
Description
[0001] The present invention relates to peptide analogs of
polymyxin B that are useful for LPS detoxification. In the
pharmaceutical field, they may be used (i) as such i.a. to treat
fatal disorders, such as septic shock, caused by Gram-negative
bacteria infection; or (ii) non-covalently bound to LPS which is
therefore detoxified; the complex thereof being useful as vaccinal
agent against Gram-negative bacteria infection.
[0002] Lipopolysaccharide (LPS) is a major constituent of the outer
membrane of the cell wall of Gram-negative bacteria. LPS is highly
toxic in mammals, particularly humans and with regard of its
biological activity has been called endotoxin. It is responsible
for the effects deriving from endotoxicosis in septic shock, a
life-threatening event that occurs upon acute infection (sepsis) by
Gram-negative bacteria.
[0003] LPS structure is constituted by a lipid moiety, called Lipid
A, covalently linked to a polysaccharide moiety.
[0004] Lipid A is responsible for the toxic effect of LPS, in
particular through interaction with B-cells and macrophages. This
interaction induces the secretion of pro-inflammatory cytokines.
The inflammatory condition may reach the fatal state of endotoxic
shock.
[0005] Lipid A is highly hydrophobic and anchors LPS in the outer
layer of the bacterial cell wall. Lipid A is composed of (i) a
conserved bis-phosphorylated disaccharide region (most frequently,
N,O-acyl beta-1,6-D-glucosamine 1,4'-bisphosphate) with (ii) fatty
acids, that substitute various hydrogen atoms pertaining to the
disaccharide hydroxyls. The number of the fatty acids and their
composition are interspecies variable. As a matter of example, each
of the two symmetric glucosamines (GlcN1 and GlcN2) of Neisseria
meningitidis lipid A carries the following fatty acids:
2N--C14,3OH; C12; and 3O--C12,3OH.
[0006] The LPS polysaccharide moiety is constituted by carbohydrate
chains, responsible for antigenicity. The carbohydrate chain
structure is itself composed of (i) a conserved inner core called
the KDO (2-keto, 3-desoxy octulosonic acid) region bound to lipid A
and (ii) a variable outer core bound to the KDO region, that is
commonly defined as including various saccharides, and the first
repeat unit (that may comprised up to ten saccharides) of (iii) the
external O-specific chains
[0007] In Gram-negative, non-enteric bacteria such as Neisserias,
Bordetellas, Haemophilus and Moraxellas, the O-specific chains do
not exist (what is defined as the first repeat unit is in fact not
repeated). Therefore, the LPS of these bacteria are often referred
to as lipooligosaccharide (LOS).
[0008] LPS is not only toxic but also highly immunogenic. In
mammals, anti-LPS antibodies are induced during infection and
carriage, and may be protective. In view of this, it has been
already proposed to detoxify LPS and to use the detoxified form
thereof in prophylaxis of Gram-negative bacterial infections and
related diseases.
[0009] Several detoxification methods are already known. In
particular, it is possible to detoxify LPS while using polymyxin B
or more appropriately, peptide analogs thereof.
[0010] Polymyxin B is a molecule that binds Lipid A with high
affinity so that LPS is significantly detoxified. When given
therapeutically in animal models, polymyxin B can prevent septic
shock. However, polymyxin B is a polycationic antibiotic that may
be somewhat toxic to humans because of its non-biodegradability and
the consequent tendency to accumulate in the kidneys. Therefore, it
is not recommended for use in prophylactic or therapeutic
products.
[0011] To overcome this limitation, peptide analogs to polymyxin B
have been developed. They do not retain the polymyxin B toxicity
but merely mimic the primary and secondary structures of polymyxin
B and bind lipid A at the same site as polymyxin B does, so that a
LPS-peptide complex is formed. As a result, LPS is detoxified.
Peptide analogs are in particular described in U.S. Pat. No.
5,358,933, WO 93/14115, WO 95/03327, WO 96/38163, EP 842 666 and EP
976 402. One of them, the cyclic monomer SAEP2 (synthetic
anti-endotoxin peptide 2) of formula KTKCKFLKKC has been more
particularly studied (Rustici et al, 1993, Science 259: 361 and
Velucchi et al, 1997, J. Endotox. Res. 4(4): 261).
[0012] It has now been found that the SAEP2 peptide as well as
similar peptides including in their sequences a number of uncharged
polar amino acids surrounded by two adjacent cysteine residues and
counter-balanced by a short external tail made of cationic amino
acids (hereinafter generically referred to as SAEP II peptides) are
of particular interest when they are in dimeric form; the dimer
being conformationally made and maintained by a pair of disulphide
bonds between the cysteine residues. Indeed, SAEP II peptide dimers
exhibit enhanced detoxification properties over the corresponding
monomers.
[0013] Therefore, the invention relates to a SAEP II peptide dimer
of formula (I)
NH2-A-Cys1-B-Cys2-C-COOH
NH2-A'-Cys1-B'-Cys2-C'-COOH [0014] wherein the two Cys1 residues
are linked together through a disulphide bond and the two Cys2
residues are linked together through a disulphide bond; or formula
(II)
[0014] NH2-A-Cys1-B-Cys2-C-COOH
COOH-C'-Cys2-B'-Cys1-A'-NH2 [0015] wherein the Cys1 residues are
linked to the Cys2 residues through a disulphide bond; wherein A
and A' independently are a peptide moiety of from 2 to 5,
preferably 3 or 4 amino acid residues, in which at least 2 amino
acid residues, are independently selected from Lys, Hyl
(hydroxy-Lysine), Arg and His; wherein B and B' independently are a
peptide moiety of from 3 to 7, preferably 4 or 5 amino acid
residues, which comprise at least two, preferably three amino acid
residues independently selected from Val, Leu, Ile, Phe, Tyr and
Trp; and wherein C and C' are optional (these positions may be
empty or not) and are independently an amino acid residue or a
peptide moiety of from 2 to 3 amino acid residues;
[0016] provided that the cationic amino acid residues/hydrophobic
amino acid residues ratio (cat/hydroph ratio) is from 0.4 to 2,
advantageously from 0.5 to 1.2 or 1.5, preferably from 0.6 to 1;
most preferably from 0.6 to 0.8; e.g. 0.75.
[0017] Advantageously, A and A' independently are a peptide moiety
of from 2 to 5, preferably 3 or 4 amino acid residues, in which at
least one, preferably 2 amino acid residues, are independently
selected from Lys, Hyl, Arg and His; those that are not selected
from Lys, Hyl, Arg and His ("the remaining amino acid residues"),
if any, being selected from the group consisting of uncharged polar
or nonpolar amino acids residues; preferably Thr, Ser and Gly; most
preferably Thr.
[0018] When the A and A' peptide moieties comprise 3 amino acid
residues, each of them can be a cationic residue; or alternatively,
two out of three residues are cationic amino acids, whereas the
remaining residue is selected from the group consisting of
uncharged polar or nonpolar amino acids residues; preferably Thr,
Ser and Gly; most preferably Thr.
[0019] When the A and A' peptide moieties comprise 4 amino acid
residues, it is preferred that two or three out of four residues be
selected from the groups of cationic amino acid residues as defined
above, whereas the remaining residue(s) is (are) selected from the
group consisting of uncharged polar or non-polar amino acids
residues as defined above.
[0020] When the A and A' peptide moieties comprise 5 amino acid
residues, it is preferred that three or four out of five residues
be selected from the groups of cationic amino acid residues as
defined above, whereas the remaining residue (s) is (are) selected
from the group consisting of uncharged polar or non-polar amino
acids residues as defined above.
[0021] Advantageously, B and B' independently are a peptide moiety
of from 3 to 7, preferably 4 or 5 amino acid residues, which
comprises at least two, preferably three amino acid residues
independently selected from Val, Leu, Ile, Phe, Tyr and Trp;
preferably from Leu, Ile and Phe; those that are not selected from
Val, Leu, Ile, Phe, Tyr and Trp ("the remaining amino acid
residues"), if any, being independently selected from the group
consisting of Lys, Hyl, Arg and His. As may be easily understood,
the B and B' peptide moieties may comprise up to 7 amino acid
residues independently selected from Val, Leu, Ile, Phe, Tyr and
Trp.
[0022] Advantageously, the B and B' peptide moieties comprise the
sequence -X1-X2-X3-, in which X1 and X2; X2 and X3; or X1, X2 and
X3 are independently selected from Val, Leu, Ile, Phe, Tyr and Trp;
preferably from Leu, Ile and Phe. In a preferred embodiment, the
sequence -X1-X2-X3- comprises the Phe-Leu motif.
[0023] Particular embodiments of peptide moieties B and B'
include:
(i) the -X1-2-X3- sequence in which: [0024] X1 is Lys, Hyl, His or
Arg, preferably Lys or Arg; more preferably Lys; [0025] X2 is Phe,
Leu, Ile, Tyr, Trp or Val; preferably Phe or Leu; more preferably
Phe; and [0026] X3 is Phe, Leu, Ile, Tyr, Trp or Val; preferably
Phe or Leu; more preferably Leu; and (ii) amino acid residues, if
any, each being independently selected from the group consisting of
Val, Leu, Ile, Phe, Tyr, Trp, Lys, Hyl, Arg and His; preferably
Val, Leu, Ile, Phe, Tyr and Trp; more preferably Leu, Ile and
Phe.
[0027] When B and B' comprise more than 4 nonpolar amino acid
residues, A and A' preferably comprises at least 3 positively
charged amino acid residues.
[0028] In the C and C' peptides moieties, the amino acid residue(s)
may be any amino acid residues provided that the cationic amino
acid residues/hydrophobic amino acid residues ratio remains within
the specified range. Advantageously, they are independently
selected from uncharged amino acid residues polar or nonpolar,
these latter being preferred. However, in a preferred manner, C and
C' are empty positions.
[0029] Therefore, a preferred class of dimers are of formula
(III)
NH2-A-Cys1-B-Cys2-COOH
NH2-A'-Cys1-B'-Cys2-COOH
or formula (IV)
NH2-A-Cys1-B-Cys2-COOH
HOOC-Cys2-B'-Cys1-A'-NH2
wherein A, A', B and B' are as described above; provided that the
cationic amino acid residues/hydrophobic amino acid residues ratio
is from 0.4 to 2, advantageously from 0.5 to 1.2 or 1.5, preferably
from 0.6 to 1; most preferably from 0.6 to 0.8; e.g. 0.75.
[0030] Dimers of formula (I) or (III), that is with peptides in the
parallel orientation, are referred to as parallel dimers. Dimers of
formula (II) or (IV), that is with peptides in the anti-parallel
orientation, are referred to as antiparallel dimers.
[0031] In formulas (I) to (IV), A and A' are preferably identical.
The same holds true for B and B'; and C and C'. A peptide dimer of
formula (I), (II), (III) or (IV), in which A and A'; B and B'; and
C and C' are two-by-two identical, is referred to as homologous
dimer. Indeed, in this case, the peptide subunits included in the
dimer are identical.
[0032] As a matter of example, the following peptides are cited as
being suitable for use in dimers of the invention:
NH.sub.2-Lys-Arg-His-Hyl-Cys-Lys-Arg-Ile-Val-Leu-Cys-COOH;
NH.sub.2-Lys-Arg-His-Cys-Val-Leu-Ile-Trp-Tyr-Phe-Cys-COOH;
NH.sub.2-Lys-Thr-Lys-Cys-Lys-Phe-Leu-Leu-Leu-Cys-COOH; and
NH.sub.2-Hyl-Arg-His-Lys-Cys-Phe-Tyr-Trp-Val-Ile-Leu-Cys-COOH.
[0033] The respective cat/hydroph ratio of the corresponding
homologous dimers are 2.00, 0.50, 0.75 and 0.67.
[0034] A particular example of the dimers described above, is
constituted by a peptide of formula (V)
NH2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH. This peptide is
hereinafter referred to as the SAEP2-L2 peptide. As described
above, it can also be in parallel or anti-parallel dimeric
form.
[0035] Peptides involved in the or dimers of the invention can be
conventionally synthesized by classical methods using e.g. a
computer-driven automatic synthesizer. It is within the skills of
professional practitioners in the art of peptide synthesis to know
how to design procedures so that a particular peptide is obtained.
It goes without saying that during the synthesis phase, the
cysteine thiol groups can be protected. Once the synthesis is
completed, they are de-protected and oxidation of the thiol groups
is achieved in order to generate the cyclic monomer, the parallel
or anti-parallel dimer.
[0036] When both cysteine residues present in the peptide are
de-protected simultaneously, it is theoretically possible to
generate each of the three forms upon oxidation. Then each of the
three forms can be separated from each other by conventional
biochemical purification methods. Preparative reverse-phase high
performance liquid chromatography (RP-HPLC) is cited as a suitable
example. Indeed, one may expect that each of the three forms elutes
at a different retention time. Therefore, a preparation containing
the purified cyclic monomer, or the purified parallel and
anti-parallel dimers can be simply obtained by pooling together the
respective peak fractions.
[0037] The respective proportions of each of the three forms
generated upon oxidation depend on i.a. the specific amino acid
sequence and importantly, the concentration of the peptide. It may
happen that one or two of the three forms be predominantly created
and indeed, the prevalence of one or two forms may be such that the
other(s) are not formed at all.
[0038] As a matter of example, the SAEP2-L2 peptide spontaneously
oxidises into cyclic monomer and anti-parallel dimer, in
proportions, which depend from the concentration of the peptide in
solution. The internal steric hindrance of the "side-chains" (the
NH2-Lys-Thr-Lys-portion) of the anti-parallel dimer is obviously
lower than that of the parallel dimer and one may expect that a
lower minimal energy be responsible for the privileged formation of
the anti-parallel dimer in aqueous solvents by comparison with the
parallel dimer. As a direct consequence of this
concentration-driven process, the formation of the anti-parallel
dimer and to a lesser extent the cyclic monomer is favoured up to
the exclusion of the parallel dimer from the equilibrium.
[0039] When the parallel dimer cannot be spontaneously generated
upon oxidation, it is necessary to adopt particular measures to
make the peptide associate within the parallel orientation. These
measures are within the skills of the professional practitioners in
the art of peptide synthesis. Nevertheless and as a matter of
example only, it is indicated that differential protection of the
Cys1 and Cys2 amino acids followed by selective de-protection is a
convenient way to achieve dimerisation with the parallel
orientation. Then the dimer may be purified by conventional
methods, including RP-HPLC.
[0040] Peptides that are chemically synthesized and purified are
commonly obtained in salt form due to the fact that acids and salts
are used during the chemical synthesis and purification steps.
Acetate is a salt commonly used. Therefore, it shall be understood
that the term "peptide" as used in the present description
encompasses the salt form as well.
[0041] Peptides for use in the dimers of the invention can be
characterized by various techniques, including i.a. Ion Cyclotron
Resonance (ICR), Mass Assisted Laser Desorption Ionisation-Time of
Flights (MALDI-ToF) spectrometry and Nuclear Magnetic Resonance
(NMR) spectrophotometry. In particular, it is possible to
discriminate each of the three forms (cyclic monomer, parallel and
anti-parallel dimer) by NMR analysis. MALDI-ToF mass spectrometry
allows discriminating between monomer and dimers only.
[0042] The purity of compounds of the invention can be evaluated by
RP-HPLC. Briefly, a preparation of compound is submitted to
RP-HPLC. The relative purity degree is calculated by integrating
the peak surfaces. It is expressed as the compound peak
surface/surfaces of the whole peaks. It is usual to prepare
compounds of the invention that each exhibits a purity degree of at
least 95%, frequently of at least 97%.
[0043] The invention also relates to compositions comprising:
[0044] A SAEP II peptide, wherein the peptide is essentially in
dimeric parallel form; [0045] A SAEP II peptide, wherein the
peptide is essentially in dimeric anti-parallel form; or [0046]
mixtures thereof.
[0047] By "essentially" it is meant that in the compositions, a
particular form is at least 95%, preferably at least 97%, more
preferably 98% pure.
[0048] Mixed compositions in which the SAEP II peptide is present
under several forms (dimeric parallel, dimeric anti-parallel and/or
monomeric forms) may spontaneous result from the evolution of a
composition comprising a single entity, e.g. the dimeric parallel
form, kept at an appropriate temperature over a certain period of
time. This may be revealed by e.g. RP-HPLC analysis. The respective
amounts of the various peptide forms may be quantified by the same
token.
[0049] The SAEP II dimers are useful as such as a detoxifying agent
of Gram-negative bacterial LPS in vitro as well as in vivo.
Accordingly, they may be used to prevent or treat pathological
conditions due to the release of LPS into the systemic circulation,
e.g. into blood, as a result of Gram-negative bacteria infections.
These conditions include i.a. endotoxicosis, bacterial sepsis and
septic shock.
[0050] Therefore, the invention encompasses: [0051] The
pharmaceutical use of a compound or composition of the invention;
[0052] A pharmaceutical composition comprising a compound or a
composition of the invention together with a pharmaceutically
acceptable diluent or carrier; [0053] The use of a compound or
composition of the invention in the preparation of a medicament for
treating or preventing septic shock; and [0054] A method for
treating or preventing septic shock, which comprises administering
a therapeutically or prophylactically effective amount of a
compound or composition of the invention, to an individual in
need.
[0055] A compound or composition of the invention may be
administered to mammals, i.e. humans, when a Gram-negative bacteria
infection is diagnosed that may lead to endotoxicosis, bacterial
sepsis and/or septic shock. Gram-negative bacteria that may be
responsible for these fatal disorders include i.a., N.
meningitidis, E. coli, Salmonella typhi, Bordetella pertussis and
Pseudomonas aeruginosa. A compound or composition of the invention
may be administered to an individual in need by a systemic route,
preferably the intravenous route. The dose to be administered
depends on various factors including i.a. the age, weight,
physiological condition of the patient as well as the infection
status. It may be administered once or several times until the risk
of fatal event is avoided.
[0056] Since the SAEP II dimers and the SAEP2-L2 peptide are also
able to detoxify LPS in vitro, the invention also relates to a
LPS-peptide complex comprising (i) a LPS moiety of Gram-negative
bacteria, and (ii) a SAEP II peptide dimer or the SAEP2-L2 peptide;
wherein the LPS moiety and the SAEP II peptide dimer or the
SAEP2-L2 peptide are non-covalently bound to each other.
[0057] LPS detoxification may be assessed in a number of assays
referred to in the European Pharmacopeia They include the Limulus
Amebocyte Lysate (LAL) assay; the pyrogen test in rabbits and the
acute toxicity assay in D-galactosamine sensitized mice. These
assays are illustrated hereinafter in the examples. In each of the
assays the effect of LPS and that of the LPS-peptide complex are
measured in parallel so that a detoxification ratio be
established.
[0058] In the LAL assay, the detoxification ratio is expressed by
the LPS/LPS-peptide complex ratio. In the pyrogen test and the
acute toxicity assay, the detoxification ratio is expressed by the
LPS-peptide complex/LPS ratio.
[0059] Significant detoxification is achieved, when the
detoxification ratio measured in: [0060] (i) the LAL assay is at
least of 100, preferably 500, more preferably 1000; [0061] (ii) the
pyrogen test is at least of 50, preferably of 100, more preferably
500; or [0062] (iii) D-galactosamine mice is at least of 50,
preferably of 100, more preferably of 200.
[0063] Detoxification may also be evaluated while comparing the
effect of LPS and a LPS-peptide complex on the release of
pro-inflammatory cytokines such as IL6, IL8 and TNF.alpha., in in
vitro or in vivo assays. These assays are illustrated hereinafter
in the examples. Significant detoxification is achieved, when the
LPS-peptide complex allows for at least 25-fold decrease,
preferably at least 50-fold, more preferably at least 75-fold, most
preferably at least 100-fold decrease in IL6 secretion in the in
vivo assay as described in the examples, section 5.4.1.
[0064] LPS-peptide complex of the invention is advantageously
characterized by a molar LPS:peptide ratio of from 1:1.5 to 1:0.5,
preferably 1:1.2 to 1:0.8, more preferably of 1:1.1 to 1:0.9, most
preferably 1:1.
[0065] For use in the complex of the invention the LPS is
advantageously a LPS of N. meningitidis; E. coli; Salmonella typhi;
Salmonella paratyphi; Shigella flexneri Haemophilus influenzae;
Helicobacter pylori; Chlamydia trachomatis; Bordetella pertussis;
Brucella; Legionella pneumophia; Vibrio cholera; Moraxella
catharralis; Pseudomonas aeruginosa; Yersinia; aid Kiebsiella
pneumonia.
[0066] As mentioned in the introduction, detoxified LPS may be
useful as vaccinal agent against Gram-negative bacteria
infection.
[0067] Meningitis is a life-threatening disease of either viral or
bacterial origin. H. influenzae and N. meningitidis are
respectively responsible for about 40 and 50% of bacterial
meningitis. While a vaccine against H. influenzae has been on the
market for more than 10 years, there is still a need for a vaccine
against N. meningitidis.
[0068] Meningococcal invasive diseases may manifest as either an
inflammation of the meninges of the brain and spinal cord
(meningitis) or a systemic infection of the blood (meningococcal
sepsis or meningoccaemia).
[0069] Meningococci are classified using serological methods based
on the structure of the polysaccharide capsule. Thirteen
antigenically and chemically distinct polysaccharides capsules have
been described. Almost all the invasive meningococcal diseases are
caused by five serogroups: A, B, C, Y and W-135. The relative
importance of each serogroup depends on the geographic location.
Serogroup B is responsible for the majority of meningococcal
diseases in temperate countries.
[0070] While conjugated polysaccharide vaccines already exist
against serogroup A, C, Y and W-135, there is currently no vaccine
available against the serogroup that is prevalent in the USA and
Europe. Indeed, the use of capsular polysaccharide as a vaccinal
agent for preventing menB diseases has been problematic.
[0071] Therefore, the use of N. meningitidis LPS as vaccinal agent,
in a fully antigenic and ad hoc detoxified form, is a promising
alternative that may offer a desirable vaccinal coverage, in
particular to serogroup B.
[0072] As mentioned hereinabove in the introduction, the major
constituent of the cell wall of Gram-negative, non-enteric bacteria
such as Neisserias, Bordetellas, Haemophilus and Moraxellas, is a
lipooligosaccharide (LOS) rather than a true LPS. Nevertheless, for
the purpose of this application, the term LPS shall be understood
as encompassing LOS. LOSs constitute a particular sub-class of LPS.
The terms "meningococcal LPS" and "meningococcal LOS" are used
hereinafter interchangeably.
[0073] FIG. 1 shows a scheme of the structure of a N. meningitidis
LOS. LOS is constituted by a branched oligosaccharide composed of 5
to 10 monosaccharides linked to lipid A by a KDO. Lipid A and the
inner core constituted by two KDO, two heptoses (Hep I and II) and
a N-acetylated glucosamine (GlcNAc), are conserved intraspecies.
The remaining of the oligosaccharide chains that constitutes the
outer core .alpha.-chain attached to HepI; .beta.-chain attached to
position 3 of HepII; and .gamma.-chain attached to position 2 of
HepII) is variable according to the immunotypes (ITs).
[0074] N. meningitidis LPS can be classified into 13 immunotypes,
based on their reactivity with a series of monoclonal antibodies
(Achtman et al, 1992, J Infect. Dis. 165: 53-68). Differences
between immunotypes come from variation in the composition and
conformation of the oligosaccharides chains. This is to be seen in
the table hereinafter.
TABLE-US-00001 Additional HepII substituents in IT .alpha.-chain
.beta.-chain position 6 or 7 .gamma.-chain L1
NeuNAc.alpha.2-6Gal.alpha.1-4Gal.beta.1-4Glc.beta.1-4 PEA (1-3)
None GlcNAc.alpha.1-2 L2
NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4
Glc.beta.1-4 Glc.alpha. (1-3) PEA (1-6) ou
(Ac.sub.0.4)-GlcNAc.alpha.1-2 PEA (1-7) L3
NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4
Glc.beta.1-4 PEA (1-3) None GlcNAc.alpha.1-2 L4
NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4
Glc.beta.1-4 H (3) PEA (1-6) Ac.sub.0.5-GlcNAc.alpha.1-2 L5
NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4Glc.beta.1-4Glc.-
beta.1-4 Glc.alpha. (1-3) None (Ac.sub.0.6-0.4)-GlcNAc.alpha.1-2 L6
GlcNAc.beta.1-3Gal.beta.1-4 Glc.beta.1-4 H (3) PEA (1-6) ou
GlcNAc.alpha.1-2 PEA (1-7) L7
NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4
Glc.beta.1-4 PEA (1-3) None GlcNAc.alpha.1-2 L8 Gal.beta.1-4
Glc.beta.1-4 PEA (1-3) None GlcNAc.alpha.1-2 L9
Gal.beta.1-4GlcNAc.beta.1-3 Gal.beta.1-4 Glc.beta.1-4 PEA (n.e.)
n.e. GlcNAc.alpha.1-2 L10 Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4
Glc.beta.1-4 PEA (n.e.) n.e. (n.e.)-GlcNAc.alpha.1-2 L11
Gal.alpha.1-4Gal.beta.1-4Glc.beta.1-4 PEA (n.e.) n.e.
(n.e.)-GlcNAc.alpha.1-2 L12 n.e. PEA (n.e.) n.e.
(n.e.)-GlcNAc.alpha.1-2 L13 n.e. n.e. n.e. n.e.
[0075] As indicated in the above table, a phospho ethanol amine
(PEA) replaces the Glc of the .beta.-chain at position 3 of HepII
in LOS L1, L3, L7 and L8. A PEA is attached in position 6 or 7 in
LOS L2, L4 and L6. LOS L2, L3, L4, L5, L5, L7 may also be
sialylated with N-acetyl neuraminidic acid, on the terminal
galactose (Gal) of the .alpha.-chain.
[0076] Immunotypes L1-L8 are essentially associated with serogroups
B and C, while immuno-types L9-L12 are found predominantly within
serogroup A.
[0077] While any LOS can be equally detoxified, it may be
advantageous to employ LOS L8 in the complexes of the invention as
these latter are further intended to vaccinal use. Indeed, the
complete structure of the LOS L8 .alpha.-chain is common to all the
immunotypes for which the structure has been identified so far
(Kahler & Stephens, 1988, Crit. Rev. Microbiol. 24: 281).
[0078] Meningococcal strains frequently express several
immunotypes, the presence of which may be influenced by the culture
conditions. If there is a special interest in LOS L8, it may be
desirable to extract this LOS from a strain known to predominantly
express the L8 immunotype, or even better, to exclusively express
it. Strain A1 (also called 2E) of serogroup A, strain M978 of
serogroup B (Mandrell & Zollinger, 1977, Infect. Immmun. 16:
471; Gu et al, 1992, J. Clin. Microbiol. 30: 2047-2053; Zhu et al,
2001, FEMS Microbiol. Lett. 203: 173), strain 8680 of serogroup B
(Dominique Caugeant collection) and strain 8532 (U.S. Pat. No.
6,476,201) are suitable to this end. These strains are obtainable
from the scientific community (U.S. Pat. No. 6,531,131).
[0079] Monoclonals that are specific for LOS L8 include Mab 2-1-18
(Moran et al, 1994 Infect Immun. 62: 5290-5295; Mandrell et al,
1986, Infect Immun. 54: 63-69) Mab 6E7-10 (Braun et al, 2004,
Vaccine 22: 898-908) Mab 4387A5 and 4385G7 (Andersen et al, 1995,
Microb. Pathog. 19: 159-168; Gu et al (supra)).
[0080] For use in the complexes of the invention, LPS may be
obtained by conventional means; in particular it may be extracted
from a Gram-negative bacterial culture and then purified according
to classical procedures. Numerous descriptions of such procedures
may be found in the literature. This includes i.a. Gu & Tsai,
1993, Infect. Immun. 61 (5): 1873, Wu et al, 1987, Anal. Biochem.
160: 281 and U.S. Pat. No. 6,531,131 all cited by way of
illustration only. An LPS preparation may also be quantified
according to procedures well-known in the art. A convenient method
is the KDO dosage with high performance anion exchange
chromatography (HPAEC) PAD.
[0081] LPS may be complexed to the compounds of the invention as
such or in a conjugated form. LPS conjugates can be conventionally
prepared by covalently linking LPS to a carrier molecules, e.g. a
polypeptide or a peptide; either through a direct covalent link or
using chemical spacer/linker molecules. Examples of carrier
molecules include the pertussis, diphtheria or tetanus toxoid and
outer membrane proteins (OMP) such as the OMP1 or OMP2/3 of N.
meningitidis. Numerous descriptions of such conjugation processes
may be found in the literature. U.S. Pat. No. 6,531,131 is cited by
way of illustration only.
[0082] When used in a conjugate form, the LPS is advantageously
conjugated before being complexed to the compounds of the
invention. This being said, non-conjugated LPS is suitable as
well.
[0083] The invention also relates to: [0084] A process for
detoxifying Gram-negative bacteria LPS, which comprises mixing
together (i) a LPS of Gram-negative bacteria and (ii) a compound of
the invention; and [0085] A process for preparing a LPS-peptide
complex, which comprises mixing together (i) a LPS of Gram-negative
bacteria and (ii) a compound of the invention.
[0086] For use in the processes of the invention, both constituents
are advantageously in a liquid medium, suitably water. LPS and
compound solutions are advantageously sterilized before mixing. The
preparation process is advantageously achieved under sterile
conditions. Upon mixing, a precipitate containing the complex is
formed. It can be recovered i.a. by centrifugation, and submitted
to one or several washing steps, if necessary.
[0087] As mentioned above, LPS-peptides complexes of the invention
are useful in that they can be safely administered to mammals.
Indeed, LPS is detoxified to such an extent that adverse events
shall not occur upon administration. As a matter of example, a
LPS-peptide complex that exhibits a pyrogenic threshold superior to
1, preferably 10 ng/mL/kg IV dose in the rabbit pyrogen assay, is
suitable. Alternatively or additionally, one may refer to the LAL
assay. As vaccines containing LPS amounting 3,000-5,000 LAL
endotoxin units have already been authorized for human
administration (Frederiksen et al, 1991, NIPH Annals 14 (2): 67),
it is possible to predict that a dose of the vaccine of the
invention may safely exhibit 5,000 LAL endotoxin units or less,
e.g. less than 3,000, 2,000, 1,000 or 500 LAL endotoxin units.
[0088] As a matter of example, a complex that exhibits e.g. 100
endotoxin units (EU)/.mu.g in the LAL assay, may be therefore
acceptable for administration at a dose of 20 .mu.g. This is
achievable with the complexes of the invention as they may exhibit
an LAL activity inferior to 50 EU/.mu.g, frequently inferior to 20
EU/.mu.g.
[0089] Further, LPS-peptides complexes of the invention are stable,
even in physiological conditions. By "stable" it is meant that the
detoxification status of LPS in the complexes remains constant over
time, at least 3, 6, 12 or 18 months. This can be monitored by
evaluating the detoxification ratio at intervals, i.e. in at least
one of the assays listed above. No significant difference is
observed in the detoxification ratio over time.
[0090] LPS-peptides complexes of the invention are also useful in
that they are able to induce an immune response against
Gram-negative bacteria. This may be shown upon administration of
complexes to mammals, e.g. rabbits, mice or humans, followed by
ELISA analysis of the sera to reveal the presence of antibodies
(i.a. immunoglobulins G or M) specific for LPS. Advantageously, the
immune response (antibodies induced) may have bactericidal and/or
opsonic activity.
[0091] The ability of the immune response induced by the complexes
of the invention to protect against Gram-negative bacteria
infection may be evaluated in appropriate animal models that are
currently specific for a bacterial species or disease. It is within
the skills of the professionals in the art of vaccines to select a
known animal model with regard to a particular bacteria or
disease.
[0092] As a matter of example, the ability of the immune response
induced by the complexes of the invention to protect against N.
meningitidis may be evaluated in the mouse intraperitoneal
infection model (Schryvers et al, 1989, Infect. Immun. 57 (8): 2425
and Danve et al, 1993, Vaccine 11 (12): 1214). It may be also
evaluated in humans by measuring the bactericidal activity of the
human serum after a complex is administered. Indeed, this test has
been proposed to serve as a surrogate test of protection at least
for N. meningitidis serogroup B (Holst et al, 2003, Vaccine, 21:
734). A human serum bactericidal activity (SBA) titer superior or
equal to 4 has been shown to correlate with protection.
[0093] In view of this, the invention also relates to: [0094] (i)
The use of a LPS-peptide complex of the invention, for treating or
preventing a Gram-negative bacterial infection; [0095] (ii) A
pharmaceutical (vaccinal) composition comprising a LPS-peptide
complex of the invention and a pharmaceutically acceptable diluent
or carrier; [0096] (iii) The use of a LPS-peptide complex of the
invention, in the preparation of a medicament for treating or
preventing a Gram-negative bacterial infection; [0097] (iv) A
method for inducing an immune response in a mammal against a
Gram-negative bacteria LPS or a Gram-negative bacteria, which
comprises administering an effective amount of a LPS-peptide
complex of the invention, to the mammal; and [0098] (v) A method
for treating or preventing a Gram-negative bacterial infection,
which comprises administering a therapeutically effective amount of
a LPS-peptide complex of the invention, to an individual in
need.
[0099] A vaccinal composition of the invention can be administered
by any conventional route, in particular by systemic or
intramuscular route; as a single dose or as a dose repeated once or
several times, e.g. two or three times at intervals, e.g. at 1, 2,
3, 6, 10, 12 month-interval. A vaccinal composition of the
invention can be conventionally formulated, advantageously in
liquid form. If necessary, an adjuvant can be added to the vaccinal
composition of the invention; however, it is indicated that
complexes of the invention can be sufficiently immunogenic so that
the presence of adjuvant in the vaccinal compositions is not
required.
[0100] The appropriate dosage depends on various parameters, for
example the individual treated (adult or child), the mode and
frequency of administration and the LPS detoxification status, as
can be determined by persons skilled in the art. In general, it is
indicated that a dose for administration to a human adult should
not excess 10,000; advantageously 8,000; preferably 5,000; more
preferably 1,000; most preferably 500 LAL Endotoxin Unit. In the
LAL assay, the value measured for a complex of the invention may
commonly be as low as 10-20 EU/.mu.g. Therefore, a dose can contain
from 1 to 500, advantageously from 2.5 to 100, preferably from 10
to 50, more preferably from 15 to 30 .mu.g.
[0101] It is reminded that, by convention, amounts of complex are
always expressed as LPS content. Accordingly and by way of example
only, "50 .mu.g of complex" actually means 50 .mu.g of LPS in the
complex preparation.
[0102] The Examples reported hereinafter further illustrate the
invention by reference to the following figures.
[0103] FIG. 1A shows the structure of the LPS L8 of N.
meningitidis. Kdo stands for 2-keto, 3-desoxy octulosonic acid; Hep
stands for heptose; Glc stands for glucose; Gal stands for
galactose; and GlcNAc stands for N-acetylated glucosamine.
[0104] FIG. 1B shows the reaction that occurs upon LPS treatment
with acetic acid.
[0105] FIGS. 2A-2C show the HPLC chromatogram obtained at 214 nm
with a composition essentially comprising the SAEP2-L2 peptide in
monomeric form (2A), in parallel dimeric form (2B) and
anti-parallel dimeric form (2C). Coordinates are: times (min) and
absorbance unit (AU).
[0106] FIG. 3 shows the HPLC chromatogram obtained at 214 nm with a
composition comprising the SAEP2-L2 peptide in monomeric form,
parallel dimeric form and anti-parallel dimeric form.
[0107] FIGS. 4A-4C show the .sup.1H NMR spectra obtained with a
composition essentially comprising the SAEP2-L2 peptide in
monomeric form (4A), in parallel dimeric form (4B) and
anti-parallel dimeric form (3C). In all of them, a peak at 1.9 ppm
indicates that the peptide is in an acetate salt form.
[0108] FIGS. 5A-5C show an enlargement of the region of the .sup.1H
NMR spectra of FIGS. 4A-4C comprised between 6.5 and 7.5 ppm.
[0109] FIG. 6 shows the 6.5-7.5 ppm region of the .sup.1H NMR
spectrum obtained with a composition comprising the SAEP2-L2
peptide in monomeric form, parallel dimeric form and anti-parallel
dimeric form.
[0110] FIGS. 7A-7C show the MALDI-ToF spectra of the calibration
standard (7A), the parallel dimer (7B) and the anti-parallel dimer
(7C).
[0111] FIG. 8 shows the HPEAC-PAD chromatogram of LPS hydrolysed by
acetic acid treatment.
EXAMPLE 1
Preparation of the SAEP2-L2 Parallel Dimer
1.1. Synthesis
[0112] The synthesis of the corresponding linear monomer is
achieved on solid phase using a computer-driven automatic
synthesizer Milligen 9050 (Millipore Inc.) operating with columns
containing resin supports e.g. polyoxyethylene glycol-activated
polystyrene, or activated polyacrylamide, which are appropriately
activated according to the choice of the first amino acid of the
selected peptide sequence as reported by Atheron & Shepard: in
Solid phase peptide synthesis, 1989, IRL press, Oxford U.
[0113] The synthesis cycle proceeds step-by-step, according to the
reported linear sequence. It is performed in pure solvent
dimethylformamide (DM). Side-protected, activated amino acids are
used.
[0114] The thiol group of the Cys residue in position 10 (Cys-10)
is protected with the acid-labile group Trityl (triphenyl-methyl
derivative, Trt). The thiol group of the Cys residue in position 4
(Cys-10) is protected with the acid-resistant group
S-acetamido-methyl (Acm).
[0115] All the amino acids are activated at the --COOH side by
O-penta-fluorophenyl-phosphate esters (O-Pfp-derivatives). They are
temporarily protected at the --NH.sub.2 side by
9-fluorenyl-methyloxy-carbonyl esters (Fmoc-derivatives).
[0116] Once synthesized, the protected peptide is cleaved from the
resin support using TFA 95% in the presence of the scavenger
ethandithiol at 2-5% (v/v). In these conditions, the thiol group of
the Cys-10 is de-protected while the thiol group of the Cys-4
remains Acm-protected. The free, Acm-protected peptide is
concentrated by vacuum-evaporation and then recovered by
precipitation with ether at 80% (v/v) final concentration.
[0117] The Cys-4 protected, Cys-10 de-protected peptide is dried
under vacuum, then solubilized in water at the concentration of 1
to 10 mg/mL and adjusted at pH 7.50 with 0.1 M aqueous ammonia. In
order to achieve dimerization through the Cys 10 residues,
oxidation is then performed by vigorous stirring of the aqueous
solution at 4.degree. C., under a pressure of 1 Atm, for 18-24
hours. Complete oxidation of the thiol groups is determined by the
Elman colorimetric assay.
[0118] The partly oxidized peptide in solution at the concentration
of 1 to 10 mg/mL is then processed for de-protection of the
remaining Cys-4 S-Acm functions. To this end, the peptide solution
is added with mercuric acetate at a final concentration of 0.1 M,
using phenol at 2-5% (v/v) as scavenger. The solution is again
vigorously stirred at 20.degree. C., under a pressure of 1 Atm, for
18-24 hours. Complete oxidation of the thiol groups is determined
by the Elman colorimetric assay.
1.2. Purification
[0119] In order to remove the low-MW molecules contained in the
peptide preparation (scavenger, mercuric acetate etc.), this latter
is applied on a reverse-phase column Sep-Pack (Millipore) operated
under pressure of 1 Atm. In an aqueous solvent, the peptide is
retained on the column by hydrophobic forces, while all the
hydro-soluble, low-MW molecules go with the flow-through. The
peptide is then eluted by a mixture of methanol-water 50-70% (v/v).
The peptide eluted in the alcoholic solvent, is recovered by vacuum
concentration and solubilized again in water at the desired
concentration.
[0120] Final purification is achieved on HPLC-operated
reverse-phase C18 column (dimensions=250.times.4 mm) using a linear
gradient 0-100% of Solvent A (0.1% TFA (trifluoroacetic acid) in
water) and Solvent B (nitryl acetate 80% in water). In these
conditions, the parallel dimer elutes as a single sharp peak. Peak
fractions are recovered.
[0121] The preparation is kept in lyophilized form, at
+2-+6.degree. C., under a neutral gas, argon or nitrogen.
1.3. Characterization of the Purified Peptide
1.3.1. Amino Acid Composition
[0122] The amino acid composition is analysed by the Pico-Tag
method (Millipore). Results are reported in the table
hereinafter.
TABLE-US-00002 Amino acid Theoretical (moles/mole) Found
(moles/mole) Lysine 6.0 5.90 Threonine 2.0 2.00 Phenylalanine 2.0
2.05 Leucine 6.0 6.10 Cysteine 4.0 3.85
1.3.2. Molecular Mass
[0123] The molecular mass is measured by Ion Cyclotron Resonance
(ICR). The value found is 2,387.33.+-.0.3 AMU, a value coherent
with the elementary structure
C.sub.110H.sub.190O.sub.24N.sub.26S.sub.4 of the peptide
formula.
EXAMPLE 2
Preparation of the SAEP2-L2 Monomer and Anti Parallel Dimer
2.1. Synthesis
[0124] The synthesis of the linear monomer is performed as in
Example 1, except that a the different methodology is used for
protecting the thiol groups of the cysteine residues: Both Cys-4
and -10 are protected at their --SH group by the acid-labile group
Trityl (triphenil-methyl, Trt).
[0125] The protected peptide is cleaved from the resin support by
TFA 95%, in the presence of the scavenger Ethandithiol at 2-5%
(v/v). In these conditions, the thiol groups of both Cys-4 and 10
residues are de-protected. The cleaved and de-protected peptide is
then concentrated under vacuum-evaporation and recovered by
precipitation with ether 80% (v/v).
[0126] The de-protected peptide is solubilized in water at the
concentration 1 to 10 mg/mL and the pH is adjusted to 7.50 with 0.1
M aqueous ammonia.
[0127] Oxidation is then performed by vigorous stirring of the
aqueous solution for 18-24 hours, at 4.degree. C., under pressure
of 1 Atm. Complete oxidation of the thiol groups is determined by
the Elman colorimetric assay.
2.2. Purification of the Peptides
[0128] The peptides in solution actually constitute a mixture of
cyclic monomer (about 40%) and anti-parallel dimer (about 60%).
Each form is purified by preparative Reverse-phase HPLC
chromatography. Indeed, it is possible to separate the cyclic
monomer from the anti-parallel dimer since these forms elute, each
as a single sharp peak, at different retention times. The
anti-parallel dimer elutes at a lower retention time. This is
consistent with the different molecular symmetry of the two dimers.
The anti-parallel peptide may assume a lower minimal energy in
aqueous solvents by virtue of its lower internal steric hindrance
of the side-chains, similarly to the "trans" vs "cis" conformation
of any other isomeric entities.
[0129] All preparations are kept in lyophilized form, at
+2-+6.degree. C., under a neutral gas, argon or nitrogen.
2.3. Characterization of the Antiparallel Dimer
2.3.1. Amino Acid Composition
[0130] The amino acid composition is analysed by the Pico-Tag
method (Millipore). Results are reported in the table
hereinafter.
TABLE-US-00003 Amino acid Theoretical (moles/mole) Found
(moles/mole) Lysine 6.0 6.10 Threonine 2.0 1.95 Phenylalanine 2.0
1.90 Leucine 6.0 6.05 Cysteine 4.0 3.90
2.3.2. Molecular Mass
[0131] The molecular mass is measured by Ion Cyclotron Resonance
(ICR). The value found is 2,387.30.+-.0.3 AMU, a value coherent
with the elementary structure
C.sub.110H.sub.190O.sub.24N.sub.26S.sub.4 of the peptide
formula.
EXAMPLE 3
Further Characterization of the Monomer, Parallel and Antiparallel
Dimers by HPLC-Reverse Phase, NMR and MALDI-ToF Mass
Spectrometry
[0132] The dimeric parallel peptide as prepared in Example 1 and
the monomeric and dimeric antiparallel peptides as prepared in
Example 2 are characterized by HPLC-reverse phase (FIGS. 2A-2C) and
NMR (FIGS. 4A-4C and 5A-5C).
3.1. Characterization by HPLC-Reverse Phase
Experimental Conditions
[0133] This technique is carried out on a HPLC chain (Waters.TM.),
using the Millennium software 32 V30501 (Waters.TM.) for data
acquisition. The analytical column Macherey Nagel.TM. ref 720014.6
(Nucleosil 5 .mu.m C18 100 Angstrom 250.times.4.6 mm) is operated
at 25.degree. C.
[0134] 30-40 .mu.g of each lyophilised peptide are diluted first in
30 .mu.l water; to which is added 30 .mu.l of trifluoroacetic acid
(TFA) 0.1% in water.
[0135] A mixture of the monomeric, dimeric parallel and
antiparallel peptides is also prepared by mixing 40 .mu.g of a
powdered preparation of each peptide in 60 .mu.l water; to which is
added 60 .mu.l of TFA 0.1% in water.
[0136] The column is equilibrated using 20% phase mobile B (TFA
0.1%, CH.sub.3CN 80% in water). Once samples are applied to the
equilibrated column, the phase B gradient runs from 20 to 60%
within 40 min (1% B/min), at a flow rate of 1 mL/min.
[0137] Detection is achieved at 214 nm. Results are to be seen in
FIGS. 2A-2C.
Results
[0138] Each peptide is eluted at a different retention time In the
experimental conditions described above, elution occurs at the
following retention time (RT): [0139] monomer: RT=28.283 min [0140]
parallel diner: RT=29.708 min [0141] antiparallel dimer: RT=22.059
min
[0142] The HLC-RP technique is used to verify the purity of each
peptide preparation. The relative purity degree of each peptide is
calculated by integrating the peak surfaces. It is expressed as the
peptide peak surface/surfaces of the whole peaks.
[0143] In FIG. 2A-2C, it can be seen that the monomer and parallel
and antiparallel dimer preparations exhibit a purity degree of 98,
96.9 and 97% respectively.
[0144] FIG. 3 shows the HPLC chromatogram of the mixture.
3.2. Characterization by NMR
Experimental Conditions
[0145] .sup.1H NMR analysis (500 MHz, 25.degree. C., HOD
presaturation) is carried out using samples of peptides diluted in
a H.sub.2O/D.sub.2O mixture (90/10 v/v). A Bruker.TM. DRX500
spectrometer and associated software for data acquisition are
used.
[0146] In more details, peptides preparation kept at -70.degree. C.
are used for analysis. Dimeric peptide solutions 0.5 mM are
prepared while diluting 1.33 g in 1 mL H.sub.2O. 144 .mu.l of the
solutions are mixed with 16 .mu.l of D.sub.2O 99.9% D in 3 mm NMR
tubes. For calibration, an external solution of TSP-d4
(3-(trimethylsilyl)propionic-2,2,3,3,-d4 acid sodium salt; Aldrich
ref 29304-0) 0.075% (w/w) in H.sub.2O/D.sub.2O mixture (90/10 v/v)
is used. The spectrometer is calibrated so that the unique
resonance signal of TSP-d4 be at 0 ppm.
Results
[0147] In the experimental conditions used, .sup.1H NMR spectra of
the monomer and dimers cover a range from 0 to 9.5 ppm and are
composed of 3 main regions: [0148] from 6.5 to 7.5 ppm; [0149] from
5.5 to 2.5 ppm; and [0150] from 2 to 0.3 ppm. This is to be seen in
FIG. 4A-4C.
[0151] .sup.1H NMR spectrum of the monomer is characterized by a
NMR pattern of 5 aromatic protons that are expected between 7.25
and 7.45 ppm, in the experimental conditions reported hereinabove.
In the experiment reported in FIG. 5A, this NMR pattern is itself
composed of a first multiplet from 7.25 to 7.35 ppm with an
integral curve corresponding to 3H and a second multiplet
(pseudo-triplet), centered at 7.39 ppm with an integral curve of
2H. This latter signal is characteristic of the monomer only.
[0152] .sup.1H NMR spectrum of the parallel dimer is characterized
by a doublet signal between 7.10 and 7.25 ppm corresponding to 4
aromatic protons and a multiplet between 7.25 and 7.40 ppm with an
integral curve of 6H. In the experiment reported in FIG. 5B, the 4H
doublet is found centered at 7.185 ppm (pics at 7.18 and 7.19
ppm).
[0153] .sup.1H NMR spectrum of the antiparallel dimer is
characterized by a doublet signal 4 aromatic protons between 6.95
and 7.10 ppm and a multiplet between 7.10 and 7.30 ppm with an
integral curve of 6H. In the experiment reported in FIG. 5C, the 4H
doublet is found centered at 7.025 ppm (pics at 7.02 and 7.03
ppm).
[0154] As shown in FIG. 4C, the .sup.1H NMR spectrum of the
antiparallel dimer is also characterized by two upfield methylic
resonances that are expected between (i) 0.40 and 0.65 (doublet)
and (ii) 0.70 and 0.85 ppm (doublet). In one experiment, these
doublets are found centered at 0.42 and 0.68 ppm. They are observed
neither in the monomer, nor in the parallel dimer.
3.3. Identification by MALDI-ToF Mass Spectrometry
[0155] Analysis by MALDI-ToF (Mass Assisted Laser Desorption
Ionisation-Time of Flight) mass spectrometry allows determining the
monoisotopic mass of the peptide. This technique does not
discriminate the antiparallel and parallel dimers.
Experimental Conditions
[0156] MALDI-ToF analysis is achieved using the Biflex III mass
spectrometer (Bruker.TM.) and associated softwares, in a positive
reflector mode. Peptides are mixed with a matrix (alpha
cyano-4hydroxy cinnamic acid) that absorbs laser energy.
[0157] The spectrometer is externally calibrated with a mixture of
synthetic peptides (ACTH 18-39 (adenocorticotropic fragment 18-39)
bombesine, and somatostatine 28.
[0158] A saturated HCCA matrix solution is prepared while diluting
50 mg HCCA in 300 .mu.l 70% ACN (acetonitril) 0.1% TFA
(trifluoroacetic acid) in water.
[0159] A 1/2 saturated HCCA solution is further prepared while
diluting vol:vol with 30% ACN, 0.1% TFA in water.
[0160] For calibration, primary standard solutions are first
prepared in 0.1% TFA. They are as follows: [0161]
Adenocorticotropic fragment 18-39 (ACTH 18-39): 100 pmoles/.mu.l
(0.247 mg/mL); [0162] Bombesine: 100 pmoles/.mu.l (0.160 mg/mL);
and [0163] Somatostatine 28: 100 pmoles/.mu.l (0.31 mg/mL).
[0164] A secondary standard solution is prepared as follows:
TABLE-US-00004 ACTH 100 pmoles/.mu.l 2 .mu.l Bombesine 100
pmoles/.mu.l 4 .mu.l Somatostatine 100 pmoles/.mu.l 4 .mu.l ACN
30%, TFA 0.1% 50 .mu.l
[0165] Peptide solutions at 1 mg/mL in water are diluted down to
0.02 mg/mL with 30% ACN, 0.1% TFA in water.
[0166] Calibration and peptide samples are diluted vol:vol with the
1/2 saturated HCCA solution. Droplets of about 1 .mu.l are
deposited on a steel target (Bruker.TM.) and dried by
evaporation.
Results
[0167] Results are to be seen in FIGS. 7A-7C.
[0168] The theoretical monoisotopic masses calculated by the
software based on the amino acid sequences are:
ACTH 28 M+H.sup.+=2465.199 Da
Bombesine M+H.sup.+=1619.823 Da
Somatostatine 28 M+H.sup.+=3147.471 Da
SAEP2-L2 M+H.sup.+=2388.35 Da.
[0169] La norme retenue pour le controle est fixee a.+-.2 Da par
rapport a la mass theorique.
[0170] As shown in FIG. 7A, the experimental values found for the
calibration peptides are 2465.225, 1619.814 and 3147.454 Da
respectively. L'ecart de mesure interne est donc
(0.026+0.009+0.017)/7232.493=7.2 ppm. (authorized <50 parts per
million).
[0171] As shown in FIGS. 7B and 7C, the experimental values found
for the parallel and antiparallel dimer preparations are 2388.449
and 2388.532 Da. These values are within the identity range (+2 Da)
centered on the theoretical values range. This means that the
samples contain what is expected.
EXAMPLE 4
Preparation of a LPS L8/Peptide I'' Complex/Aggregate
4.1. Preparation of LPS L8
4.1.1. Meninge Culture
[0172] Preculture: Two mL frozen samples of working seed from a N.
meningitidis A strain known to express LPS exclusively under the L8
form, are used to inoculate in a 2 l erlen containing 200 mL of
Mueller-Hinton broth (Merck) complemented with 4 mL of a glucose
solution in water (500g/l). This operation is repeated 4 times.
Erlens are incubated at 36.+-.1.degree. C. for 10.+-.1 hrs while
stirring (100 rpm).
[0173] Culture: The erlen contents are gathered together and the
preculture is complemented with 400 mL of a glucose solution in
water (500 g/l) and 800 mL of an amino acid solution. This
preparation is used to inoculate the Mueller-Hinton broth, in a 30
l fermentor (B. Braun.TM.) at an initial OD.sub.600nm close to
0.05. Fermentation is performed overnight at 36.degree. C., pH
6.8.+-.0.2, 100 rpm, pO.sub.2 30%, and initial flow rate of the air
0.75 l/min/L culture. After 7.+-.1 hrs, (OD.sub.600nm about to 3),
the culture is feeded by MH broth at a flow rate of 440 g/h. When
the glucose concentration is lower than 5 g/l, the fermentation is
stopped. Usually, the final OD.sub.600nm is comprised between 20
and 40. Cells are collected by centrifugation for 1 h 30 at 7000 g
at 4.degree. C. Pellets are kept frozen at -35.degree. C.
4.1.2. Purification of LPS
First Phenol Extraction
[0174] Pellets are defrosted and suspended with 3-volume phenol
4.5% (v/v) and stirred vigorously for 4 hrs minimum at about
5.degree. C.
[0175] The bacterial suspension is heated at 65.degree. C. and then
mixed v/v with phenol 90% at 65.degree. C. The suspension is
stirred vigorously, at 65.degree. C. for 50-70 min and then cooled
down to about 20.degree. C.
[0176] The suspension is centrifuged for 20 min, at 11 000 g, at
about 20.degree. C. The aqueous phase is collected and kept. The
phenol phase and the interphase are recovered and submitted to a
second extraction.
Second Phenol Extraction
[0177] The phenol phase and the interphase are heated at 65.degree.
C. and mixed with a volume of water equivalent to the volume of the
aqueous phase that was previously collected. The mixture is stirred
vigorously for 50-70 min at 65.degree. C. and then cooled down to
about 20.degree. C. The mixture is centrifuged for 20 min, at 11
000 g, at about 20.degree. C. The aqueous phase is collected and
kept. The phenol phase and the interphase are recovered and
submitted to a third extraction.
Third Phenol Extraction
[0178] Procedure for the second extraction is repeated.
Dialysis
[0179] The 3 aqueous phases are dialysed overnight and separately
against 40 l of water. The dialysates are pooled together. The
dialysate pool is adjusted with Tris 20 mM, MgCl.sub.2 2 mM (one
volume per 9 volumes of the dialysate pool). pH is adjusted to
8.0.+-.0.2 with NaOH 4 N.
DNAse Treatment
[0180] 250 UI of DNAse is added per gram of treated bacterial
pellet (wet weight). The preparation is stirred at 37.+-.2.degree.
C. for 55-65 min. pH is adjusted at 6.8.+-.0.2. The preparation is
filtered on 0.22 .mu.m membranes.
[0181] Gel filtration: The preparation is purified on a Sephacryl
S-300 column (5.0.times.90 cm; Pharmacia.TM.).
First Alcoholic Precipitation
[0182] Powder of MgCl.sub.2, 6H.sub.2O is added to the
LPS-containing fractions pooled together, to reach an MgCl.sub.2
concentration of 0.5 M and dissolved while stirring.
[0183] While stirring at 5.+-.3.degree. C., dehydrated absolute
alcohol is added to a final concentration of 55% (v/v). Stirring is
performed overnight at 5.+-.3.degree. C., followed by
centrifugation at 5,000 g for 30 min at 5.+-.3.degree. C. The
supernatants are discarded and the pellets are submitted to a
second extraction.
Second Alcoholic Precipitation
[0184] The pellets are resuspended with at least 100 mL MgCl.sub.2
0.5 M, while stirring. The previous procedure is repeated. Pellets
are resuspended with at least 150 mL water.
[0185] Final step: Gel filtration is repeated and the
LPS-containing fractions pooled together are finally sterilised by
filtration (0.8-0.22 .mu.m) and kept at 5+3.degree. C.
[0186] As a preliminary control, the LPS preparation is analyzed by
SDS-PAGE electrophoresis. Upon silver nitrate staining, a single
large band is revealed This indicates at least that the preparation
does not contain any entity other than LPS L8.
[0187] The purification process as described allows obtaining about
150 mg LPS L8 per culture liter (yield about 50%).
4.1.3. LPS L8 Quantification: KDO Dosage with HPAEC-PAD
[0188] The bibliographic reference for this technique is Kiang et
al, (1997) Determination of 2-keto-3-deoxyoctulosonic acid (KDO)
with high performance anion exchange chromatography (HPAEC): Survey
of stability of KDO and optimal hydrolytic conditions Anal.
Biochem. 245: 7.
[0189] As shown in FIGS. 1A-1B, LPS comprises in its structure 2
KDO units, one being in a lateral position.
[0190] LPS quantification is achieved through dosage of the lateral
KDO unit liberated upon soft acid hydrolysis (See FIG. 1B).
Acid Hydrolysis
[0191] Samples of the LPS preparation obtained after the last
diafiltration of section 4.1.2. are recovered and diluted with
water under a final volume of 400 .mu.l in Dionex.TM. 1.5 mL flasks
so that LPS concentration of the samples falls under the etalon
range (1.4-72.1 .mu.g/mL).
[0192] Samples to be quantified as well as the KDO etalon range are
proceeded as follows 100 .mu.l of the hydrolysis solution (acetic
acid 5%; glucuronic acid (GlcA) 20 .mu.g/mL) are added. Hydrolysis
is performed for 1 h at 100.degree. C. Flasks are then dried at
40.degree. C. under nitrogen and filled with 400 .mu.l water.
Dosage
[0193] This technique is carried out on a HPAEC chain (Dionex.TM.),
using the Chromeleon Dionex.TM. software for data acquisition. The
analytical column Carbopac PA1 4.times.250 mm (Dionex.TM.) is
operated at 30.degree. C.
[0194] The column is equilibrated with the elution solution (NaOH
75 mM, AcONa 90 mM). 100 .mu.l of sample are injected into the
column. Then the column is submitted to an elution flow rate of 1
mL/min for 22 min.
[0195] Chromatogram of LPS sample is to be seen in FIG. 8. The KDO
amount present in the sample is determined by integration of the
KDO peak. As one KDO mole liberated by hydrolysis corresponds to
one LPS mole, it is possible to determine the LPS concentration of
the initial preparation.
4.2. Preparation of Peptides
[0196] Peptides are prepared according to the processes described
in Examples 1 and 2 above.
4.3. Preparation of the LPS L8/Peptide I'' Complex/Aggregate
[0197] Purified LPS is used as pseudo-solution at 1 mg/mL in
sterile, pyrogen free water (Milli Q quality, adjusted to pH 7.2
Limulus negative). The translucid pseudo-solution is sterilized by
filtration using a 0.22 .mu.m membrane.
[0198] A solution of peptide SAEP2-L2 at 1 mg/mL in sterile,
pyrogen-free water (Milli Q quality, adjusted to pH 7.2, Limulus
negative) is also sterilized by filtration on 0.22 .mu.m
membrane.
[0199] All the next steps are achieved under sterile
conditions.
[0200] One volume of the LPS pseudo-solution is added to one volume
of the solution of peptide SAEP2-L2. A precipitate (endotoxoid
complex) immediately appears. Stirring is carried out for 5 min at
room temperature. The preparation is left to stand at +4.degree. C.
overnight.
[0201] The precipitate (Endotoxoid) is then recovered by
centrifugation at 3000 rpm for 10 min. The supernatant is
discarded.
[0202] The pellet is washed with one volume of sterile, pyrogen
free water (Milli Q quality, adjusted to pH 7.2, Limulus negative).
Centrifugation/washing steps are repeated five times.
[0203] At last, the pellet is resuspended in sterile, pyrogen free
water (milli Q quality) pH=7.2, at about 1 mg/mL concentration,
based on the wet weight of the precipitate. The suspension is
stored at +4.degree. C. A KDO dosage is achieved to determine the
LPS content and the suspension is adjusted to e.g. 0.50 mg/mL of
complex (expressed as LPS content). [0204] The LPS-peptide complex
tested in the following examples is the LPS-antiparallel dimer
complex as obtained in section 4.3, unless otherwise indicated.
Therefore, this specific complex is simply referred to as
LPS-peptide complex. [0205] In a similar manner, the LPS as
obtained in section 4-2. is simply referred to as LPS. [0206]
Comparison of LPS and LPS-peptide complex is achieved using the LPS
lot also used for the preparation of the complex.
EXAMPLE 5
Evaluation of the Detoxification of the LPS-Peptide Complex
[0207] Several assays are used to evaluate the detoxification.
5.1. Limulus Amebocyte Lysate (LAL) Assay
[0208] In this assay, the ability of the SAEP2-L2 anti-parallel and
parallel dimers and the SAEP2-L2 cyclic monomer to detoxify LPS is
compared. To this end, the LPS-peptide complexes involving the
parallel dimer or the monomer are prepared exactly as it is
reported in Example 4 for the LPS-antiparallel peptide complex.
[0209] LAL is a very sensitive test used to detect and quantify
endotoxins of gram-negative bacteria. The test is based on the
property of the amoebocyte lysate protein from horseshoe crab
(Limulus polyphemus) to induce coagulation in the presence of
endotoxin.
[0210] The evaluation of the LPS endotoxin activity is performed by
using the end-point-chromogenic technique, in accordance with the
European Pharmacopeia [as described in the European Pharmacopeia
techniques (Edition 5.0, paragraph 2.6.14)]. To this end, the kit
QCL-1000 ref 50-647 U (Cambrex-BioWhittaker.TM.) is used (linear
zone of the kit: 0.1 to 1 UI/mL) as well as a positive control (E.
coli endotoxin, 4 10.sup.3 EU/mL, Sigma).
[0211] Dilution of (i) samples to be tested, (ii) standard and
(iii) positive control are achieved with dilution buffer
(Cambrex-BioWhittaker.TM.) to cover the respective ranges: 1/10 to
1/10.sup.5; 0.5 to 0.031 EU/mL and 1/10.sup.4 to 1.8 10.sup.4.
[0212] 50 .mu.l of sample, standard and positive control dilutions
are dispensed per well of 96 flat-bottom well ELISA plate. Fifty
.mu.l of lysate are added per well. Incubation is pursued for 10
min at 37.degree. C. Then 100 .mu.l of the p-nitroaniline
chromogenic substrate are added. Incubation is pursued for 6 min at
37.degree. C. The chromogenic reaction is stopped while adding 100
.mu.l freezed acetic acid 25% in water. Plate is read by
spectrophotometry at 405 nm.
[0213] The results are expressed in Endotoxin Unit (EU)/.mu.g of
complex. They are shown in the table hereinafter. The
detoxification ratio can be established by the LPS/LPS-peptide
complex ratio and expressed in log unit.
TABLE-US-00005 Mean Detoxification ratio Range value Expressed
EU/.mu.g EU/.mu.g in log LPS (6 assays) 1-12 10.sup.4 25,000
LPS-antiparallel peptide 5-32 12-20 1,250-2,000 3.5 complex (13
assays) LPS-parallel peptide 30-40 30-40 600-800 3 complex
LPS-monomer peptide >2000 >2000 <12.5 <1 complex
[0214] As may be seen, the dimeric forms of the SAEP2-L2 peptide
are more effective in detoxifying LPS than the cyclic monomer
form.
5.2. Pyrogen Test in Rabbits
[0215] Rabbit is known to be the animal specie with sensitivity to
pyrogenic effects of LPS equivalent to humans. The pyrogen test
consists in measuring the rise in body temperature evoked in three
rabbits by the intravenous (IV) injection of a sterile solution of
the substances to be examined. The test, reading and calculations
are performed in accordance with the European Pharmacopoeia,
(Edition 5.0, paragraph 2.6.8). The temperature rise is interpreted
depending the summed response of the temperatures: conformity is
met when the summed response does not exceed 1.15.degree. C. and
non-conformity, when the summed response exceeds 2.65.degree. C. In
the present case, the pyrogenic threshold is set up below, between
1.15.degree. C. and 2.65.degree. C.
[0216] As found, the limit pyrogen dose (IV) in rabbit corresponds
to 0.025 ng/kg (LPS), and 10-25 ng/kg (LPS-peptide complex). These
results show that the LPS-peptide complex is less pyrogen than LPS,
when given by the intravenous route. As measured in this test, the
detoxification ratio (LPS-peptide complex/LPS) is between 400 and
1,000.
5.3. Acute Toxicity Assay: LD50 in D-Galactosamine Sensitized Mice
References for this assay include i.a. Galanos et al, 1979, PNAS
76: 5939 Baumgartner et al, 1990, J. Exp. Med. 171 (3): 889 and
U.S. Pat. No. 6,531,131.
[0217] Groups of eight-week old female inbred mice are injected by
the intraperitoneal (IP) route (0.5 mL) with escalating doses of
LPS or LPS-peptide complex, just after being treated with
D-galactosamine (15 mg/0.2 mL) by the IP route (the toxicity of LPS
is increased of around 1,000 fold with the D-galactosamine
treatment which renders the model very sensitive). The death rate
is then followed during four days.
[0218] The LD50 observed with the LPS is 3.6 ng/mouse (1.91-6.70
ng/mouse); whereas that observed with the LPS-peptide complex is 1
.mu.g/mouse (0.2-5 .mu.g/mouse), indicating that the detoxification
ratio (LPS-peptide complex/LPS) is about 250 (100-1000).
5.4. Attenuation of the Pro-Inflammatory Effects of LPS when
Completed with Peptide
[0219] In order to evaluate to which extent the LPS-peptide complex
can attenuate LPS-induced toxic effects, the effect of the
LPS-peptide complex on the release of pro-inflammatory cytokines is
monitored (assessed) in in vitro and in vivo assays. [0220] In
vivo: cytokine (IL6 and TNF.alpha.) releases in the sera of mice
immunized either with LPS or LPS-peptide complex are compared by
ELISA. Blood samples are recovered 90 min after SC immunization,
which is the optimal time for the release of those cytokines.
C3H/HeOuJ, TLR4- -/- -, C3H/HeN and CD1 mouse strains are tested
The two first are sensitive neither to LPS nor LPS-peptide complex.
The third and fourth are both found LPS-sensitive. CD1 mice are
found more LPS-peptide complex-sensitive than the others and
therefore selected for further experiments retaining the most
severe conditions. [0221] In vitro: cytokine (IL6, IL8 and
TNF.alpha.) releases from human whole blood cell cultures
stimulated for 24 h at 37.degree. C., with different concentrations
of LPS or LPS-peptide complex are compared.
5.4.1. In Vivo Assay
[0222] CD1 mice are administered subcutaneously (SC) (i) either 10
.mu.g of LPS or (ii) 10 .mu.g of LPS-peptide complex. They are bled
90 minutes after injection. IL6 and TNF.alpha. releases are
measured in the sera by ELISA.
ELISA Detection of Cytokine Secretion
[0223] ELISAs are carried out using the OptEIA mouse IL6 and
TNF.alpha. sets (Pharmingen), each including the capture antibody
(anti-mouse cytokine), the detection antibody (biotinylated
anti-mouse cytokine), avidin-horseradish peroxidase conjugate and
the standard (recombinant cytokine), all from Pharmingen.
[0224] Anti-mouse IL6 and TNF.alpha. antibodies are 1/250 diluted
in 0.1 M carbonate buffer pH 9.5 (Sigma). For each assay, 100 .mu.l
of an antibody dilution are distributed per well of a Maxisorp NUNC
96 flat-bottom well ELISA plate. Plates are incubated overnight at
+4.degree. C.
[0225] Plates are washed in PBS 0.05% Tween 20. 200 .mu.l of PBS,
0.5% bovine serum albumin (BSA) saturation buffer are then added
per well. Incubation is pursued for one hr at room temperature.
Plates are washed in PBS 0.05% Tween 20.
[0226] Recombinant IL6 or TNF.alpha. cytokine dilutions are
prepared in the RPMI medium 1% FCS 10%, within the range of (i)
4,000 pg/mL-62.5 pg/mL standard. 100 .mu.l of each dilution are
distributed per well, to establish the standard curve.
[0227] Serum dilutions are prepared in the RPMI medium P.S. glu 1%
FCS 10%. Sera of mice injected with LPS are 1/25 and 1/125 diluted.
Sera of mice injected with LPS-peptide complex are 1/5 and 1/25
diluted. 100 .mu.l of each dilution are distributed per well.
[0228] Incubation is pursued for 2 hrs at room temperature.
[0229] Plates are washed in PBS 0.05% Tween 20. Biotinylated
anti-mouse cytokine antibodies and the enzyme are each 1/250
diluted in PBS 10% fetal calf serum. 100 .mu.l of each dilution are
added per well. Incubation is pursued for one hr at room
temperature.
[0230] Plates are washed in PBS 0.05% Tween 20. 100 .mu.l of
tetramethylbenzidine (TMB) substrate (TMB solutions A and B (KPL)
mixed vol/vol) are distributed in wells. Incubation is pursued for
10-30 min at room temperature.
[0231] The reaction is stopped by adding 100 .mu.l of 1 M
H.sub.3PO.sub.4 per well. Plates are read at 450 nm. Results are to
be seen in the table hereinafter.
TABLE-US-00006 IL6 release TNF.alpha. release Product Mean Mean
injected to (pg/mL) Detoxification (pg/mL) Detoxification mice n =
6 (log) ratio (log unit) n = 6 (log) ratio (log unit) LPS 4.7 4.1
LPS-peptide 2.2 2.5 <1 >3.1 complex Peptide <1 <1
[0232] The peptide alone does not induce IL6 or TNF.alpha.. The
LPS-peptide complex allows for about 100-fold of detoxification
(100-fold decrease in IL6 secretion).
5.4.2. In Vitro Assay
Preparation of the Test Substances
[0233] LPS preparation (1 mg/mL) and LPS-peptide complex (500
.mu.g/mL) are each diluted in 10 mM Tris, NaCl 150 mM, 0.05% Tween
20, 5% sucrose to a concentration of 50 .mu.g/mL. They are further
diluted in physiological saline to a concentration of 5
.mu.g/mL.
[0234] Serial 1/5 dilutions are performed in AIM-V medium (Gibco
(Invitrogen)) for each test substance down to a concentration of
2.56 10.sup.-3 pg/mL
Stimulation
[0235] Human blood collected on sodium heparin (25,000 U/5 mL
sanofi-synthelabo) is diluted 1:4 (vol:vol) in AIM-V medium and
distributed in Micronics.TM. tubes (400 .mu.l/tube). 100 .mu.l of a
dilution of the test substances are added. Peptide and buffer
controls are tested at 1/20 dilution. Tubes are incubated for 24
hrs at 37.degree. C., in a wet atmosphere at 5% CO.sub.2.
Plasma Recovery
[0236] Tubes are then centrifuged for 10 min at 500 g. At least 200
.mu.l of supernatant are recovered from each tube and kept frozen
at -80.degree. C. until titration.
ELISA Detection of Cytokine Secretion
[0237] ELISAs are carried out using the OptEIA human IL6, IL8 and
TNF.alpha. sets from Pharmingen, each including the capture
antibody (mouse anti human cytokine), the detection antibody
(biotinylated mouse anti-human cytokine), avidin-horseradish
peroxidase conjugate and the standard (recombinant cytokine).
[0238] Anti-human IL6, IL8 and TNF.alpha. antibodies are 1/250
diluted in 0.1 M carbonate buffer pH 9.5 (Sigma). For each assay,
100 .mu.l of an antibody dilution are distributed per well of a
Maxisorp NUNC 96 flat-bottom well ELISA plate. Plates are incubated
overnight at +4.degree. C.
[0239] Plates are washed in PBS 0.05% Tween 20. 200 .mu.l of PBS,
0.5% bovine serum albumin (BSA) saturation buffer are then added
per well. Incubation is pursued for one hr at room temperature.
Plates are washed in PBS 0.05% Tween 20.
[0240] Recombinant IL6, IL8 or TNF.alpha. cytokine dilutions are
prepared in AIM-V medium within respective range of (i) 1,200
pg/mL-18.75 pg/mL; (ii) 800 pg/mL-12.5 pg/mL; and (iii) 1,000
pg/mL-15.87 pg/mL standard. 100 .mu.l of each dilution are
distributed per well, to establish the standard curve.
[0241] Plasma dilutions are prepared in the AIM-V. Plasmas
recovered from blood stimulated with LPS are 1/25 and 1/125
diluted. Those recovered from blood in contact with the LPS-peptide
complex are 1/5 and 1/25 diluted. 100 .mu.l of each dilution are
distributed per well.
[0242] Incubation is pursued for 2 hrs at room temperature.
[0243] Plates are washed in PBS 0.05% Tween 20. Biotinylated
anti-human cytokine antibodies and the enzyme are each 1/250
diluted in PBS 10% fetal calf serum. 100 .mu.l of each dilution are
added per well. Incubation is pursued for one hr at room
temperature.
Plates are washed in PBS 0.05% Tween 20. 100 .mu.l of
tetramethylbenzidine (TMB) substrate (TMB solutions A and B (KPL)
mixed vol/vol) are distributed in wells. Incubation is pursued for
10-30 min at room temperature.
[0244] The reaction is stopped by adding 100 .mu.l of 1 M
H.sub.3PO.sub.4 per well. Plates are read at 450 nm.
Results
[0245] The raw results and the cytokine release curves=f (LPS or
complex concentrations) do not allow comparison of different
samples. Calculating the detoxification ratio can eliminate
inter-blood donor and inter-test variability. Only the linear parts
of the curves are taken into account for calculation of the
detoxification ratio. The maximum IL6 release beyond which a linear
progression is no longer observed is determined and then, the
amount of substance required to induced 50% of that maximum is
calculated by linear regression.
[0246] The detoxification ratio is expressed as the ratio of the
concentration of the LPS-peptide complex inducing 50% of maximum
IL6 release (ED.sub.50 expressed in pg/mL) in over that observed
with LPS. Higher the ratio, stronger the detoxification is. As the
detoxification ratio is systematically measured using whole blood
of several independent donors, results are averaged.
[0247] The detoxification ratio observed with the LPS-peptide
complex is measured several times. Mean data out of six values
obtained in the IL6 release assay: 64.+-.20.
[0248] The IL6 release correlates with the TNF.alpha. and IL8
secretions. Therefore, the IL6 release assay is selected to
routinely evaluate the inflammation decrease observed with the
LPS-peptide complex.
5.5. Conclusion
[0249] The detoxification ratio is measured between 10.sup.2 and
10.sup.3, depending on the test. The detoxification values are
summarized in the following table.
TABLE-US-00007 LPS-peptide Detoxification Assays LPS L8 complex
ratio LAL 25,000 EU/.mu.g 12-20 EU/.mu.g 1,250-2,000 Limit pyrogen
dose (IV) in rabbit 0.025 ng/kg 10-25 ng/kg 400-1,000 Cytokine
release test in mice IL6 = 25,000 pg/mL IL6 = 270 pg/mL 100 IL6 =
10,000 pg/mL IL6 = 100 pg/mL In vitro assay of IL6 release by
ED.sub.50 = 2 pg/mL ED.sub.50 = 880 pg/mL 64 human PBMC (ED.sub.50:
concentration of product inducing 50% of maximum IL6 release) LD50
in galactosamine-sensitized 4 ng/souris 1 .mu.g/souris (0.2-5) 250
mice
EXAMPLE 6
LPS Peptide Complex Stability Study
[0250] The stability of the LPS-peptide complex is studied for 6
months and evaluated by measuring the detoxification ratio in two
assays (LAL and in vitro IL-6 release by huPBMC). Pyrogen test in
rabbits may also be achieved.
6.1. In Vitro Stability of the Formulated LPS Peptide Complex
[0251] The stability of the formulated LPS-peptide complex is
followed at 5.degree. C., for 6 months. Measurements are made at
day=0, 90 and 180 (6 months). Results are as follows.
TABLE-US-00008 Pyrogen test IL6 release from LAL assay Pyrogenic
threshold, human blood cells endotoxin as chosen: (detoxification
ratio) (EU/.mu.g) 10 ng/mL/kg IV 3 6 3 6 3 6 0 months months 0
months months 0 months months 125 40 163 14 58 10 C* C C C*:
conform
[0252] The detoxification ratio in IL6 release test are not
significantly different after 3 and 6 months, indicating the
stability of the LPS-peptide complex: LPS complexed with peptide
remains detoxified after 6 months at 5.degree. C.
6.2. In Vitro Stability of the LPS Peptide Complex in Physiological
Liquid
[0253] The aim of the experiment is to verify that LPS is not
released when the complex is administered and that the
detoxification rate does not decrease after a contact with a
physiological liquid.
[0254] One mL of the LPS-peptide complex, mixed with 1 mL of human
serum, is incubated at 37.degree. C. The detoxification rate is
evaluated after 1 and 24 hours. Human serum and the LPS-peptide
complex as prepared in section 4.3. are also tested in
parallel.
[0255] No significant difference of the detoxification evaluated by
both assays is observed after a 1-hour and 24-hour contact of the
LPS-peptide complex with human serum at 37.degree. C. and results
are similar to the LPS-peptide complex control.
EXAMPLE 7
Immunogenicity of the LPS-Peptide Complex
7.1. Bactericidal Activity of Anti LPS Antibodies Induced in
Rabbits by the LPS-Peptide Complex
[0256] Immunization of three adult New-Zealand rabbits is performed
with 100 .mu.g of LPS-peptide complex by intramuscular (IM) and
subcutaneous (SC) routes (2.times.0.5 mL and 5.times.0.2 mL
respectively) in the presence of adjuvant. They receive three
injections at 3-week interval; the first one with complete Freund
adjuvant (FA), the second and third ones with incomplete Freund
adjuvant. They are bled two weeks after the last injection. A
control group is immunized with the peptide with adjuvant (71
.mu.g, equivalent to the amount of the peptide in 100 .mu.g of
LPS-peptide complex) using the same protocol.
[0257] The bactericidal activity of the serum (SBA) samples is
evaluated against the N. meningitidis strain used for LPS
production as described in Example 5 in the presence of baby rabbit
serum as exogenous source of complement.
SBA Assay
[0258] Sera are heat-inactivated during 30 min at 56.degree. C. In
the wells of a 96-well microplate, heat-inactivated sera are then
twofold serially diluted (10 times) in Dulbecco's phosphate
buffered saline containing Ca.sup.++ and Mg.sup.++ (volume per
well: 50 .mu.l).
[0259] Twenty five .mu.l of a log phase culture of N. meningitidis
grown in Mueller-Hinton broth (4.10.sup.3 CFU/mL) and 25 .mu.l of
baby rabbit serum are added to each well. The plate is incubated
one hour at 37.degree. C., under shaking.
[0260] Fifty .mu.l of the mixture from each well are plated onto
Mueller-Hinton agar. Petri dishes are incubated overnight at
+37.degree. C. in a 10% CO.sub.2 atmosphere.
[0261] In each experiment, controls include (i) bacteria and the
complement source without antibodies (complement control), (ii)
bacteria and heat-inactivated complement, and (iii) bacteria and
heat-inactivated complement, in the presence of antibodies.
[0262] Bactericidal titre is reported as the highest reciprocal
serum dilution at which .gtoreq.50% killing of bacteria is observed
as compared to the complement control.
SBA Results
[0263] Results are to be seen in the table hereinafter. High SBA
titers are obtained with the complex. The specificity of the SBA
response is confirmed with the extinction of the response, when the
sera (post-dose 3) are adsorbed on LPS.
TABLE-US-00009 Post-dose 3 Post-dose 3 Rabbit Pre-immunized
immunized immunized sera # sera sera adsorbed on LPS LPS-peptide A
16 512 4 complex B 16 1,024 8 C 4 128 <4 Peptide D 4 16 4 E 16
16 8
7.2. Immune Response Induced in Mice with the LPS-Peptide
Complex
[0264] Ten six-week old female outbred CD1 mice are immunized with
a 10 .mu.g dose of LPS-peptide complex by the subcutaneous route
(0.2 mL). They receive two injections at 3-week interval. They are
bled before each injection and exsanguinated 14 days after the last
injection. A control group is injected with buffer.
[0265] In a first experiment, the antibody response is evaluated by
ELISA and the bactericidal activity of the post-dose 2 serum
samples is evaluated against the N. meningitidis stain used for LPS
production as described in Example 4 (homologous strain) and a
heterologous N. meningitidis strain [N. meningitidis group B strain
RH873 (L4, 7, 8 immunotype)].
[0266] In a second experiment, the antibody response is evaluated
by ELISA and the opsonic activity of the post-dose 2 serum samples
is evaluated by FACS.
7.2.1. Immunogenicity of LPS-Peptide Complex in Mice
ELISA Titration of Anti-LPS Antibodies
[0267] Wells of a 96-well microplate are coated with 100 .mu.l of a
10 .mu.g/mL LPS solution in buffer 1 (PBS+10 mM MgCl.sub.2). The
plate is incubated 2 hours at +37.degree. C.; then overnight at
+5.degree. C.
[0268] The LPS solution is removed from the plate and wells are
saturated with 150 .mu.l of buffer 2 (PBS+milk 1%+Tween 20 0.05%).
The plate is incubated one hour at 37.degree. C.; then washed with
buffer 3 (PBS+Tween 20 0.05%).
[0269] Sera are serially diluted 12-fold, directly in the wells
using buffer 2 (volume: 100 .mu.l per well). The plate is incubated
for 90 min at +37.degree. C.; then washed with buffer 3.
[0270] Hundred .mu.l of a diluted goat anti-mouse IgG (.gamma.
chain specific) or IgM (.mu. chain specific) peroxydase conjugate
are added in each well. The plate is incubated 90 min at 37.degree.
C. and then washed with buffer 3.
[0271] The reaction is developed by adding 100 .mu.l of a
tetramethylbenzidine substrate solution in each well. The plate is
incubated 20 min at 37.degree. C. The reaction is stopped by adding
1 M HCl and absorbance is measured at 450 nm.
ELISA Results
[0272] Results are expressed in arbitrary ELISA Unit/mL (EU/mL) by
comparison to a reference serum.
[0273] In a preliminary immunization experiment, the ELISA assay is
achieved using a pool of 10 sera. As shown in the following table,
the LPS-peptide complex is able to induce high anti-LPS IgG titers
in mice and anti-LPS IgM after one injection (ELISA). A significant
IgG booster is observed after the second injection, whereas no
significant IgM increase is observed.
TABLE-US-00010 Anti-LPS IgG (EU/mL) Anti-LPS IgM (EU/mL) Post dose
1 Post dose 2 Post dose 1 Post dose 2 LPS-peptide 1,800 22,000 280
550 complex Buffer <40 <40 <40 <40
[0274] In a further immunisation experiment, the ELISA assay is
achieved individually. After the second injection, seven out of the
ten mice exhibits high IgG and IgM titers. Global mean titers
expressed in log are about 3.7 and 2.8 respectively.
7.2.2. Bactericidal Activity of Mouse Sera
[0275] Bactericidal activity is measured as described in section
7.1.
[0276] Forty % of the post-dose 2 sera exhibit a bactericidal
activity (SBA titre.gtoreq.16) against the homologous N.
meningitidis strain. Four are bactericidal against the heterologous
strain.
7.2.3. Opsonic Activity of Mouse Sera
Opsonisation Assay
[0277] The opsonic activity is measured by flow cytometry
technology (FACS) using human promyelocytic differentiated HL60
cells as effector and LPS coated latex fluorescent beads as
target.
[0278] Effector cells are differentiated into granulocytes after
treatment with 100 mM dimethylformamide. The resulting cells are
washed, resuspended in Hanks' balanced salt solution and their
concentration is adjusted to 2.5.times.10.sup.7 cells/mL.
[0279] Sera are heat inactivated during 30 min at 56.degree. C. In
a 96 deep-well microplate, heat-inactivated sera are serially
fivefold diluted (3 times) in Hanks balanced salt buffer containing
Ca.sup.++ and Mg.sup.++ (volume per well: 300 .mu.l).
[0280] Twenty .mu.l of LPS coated latex fluorescent beads and 10
.mu.l of baby rabbit serum as exogenous complement source are added
to each well. The plate is incubated 30 min at +37.degree. C.,
under shaking.
[0281] Fifty .mu.l of the effector cell suspension are added to
each well. The plate is incubated 30 min at +37.degree. C., under
shaking.
[0282] One hundred fifty .mu.l from each well are transferred in a
second deep well and the reaction is stopped by adding 400 .mu.l of
PBS+0.02% EDTA. The plate is centrifuged and washed twice with
PBS+BSA buffer.
[0283] The phagocytosis of LPS coated beads by effector cells, in
the presence of antiserum and exogenous complement source is
measured by FACS.
[0284] Opsonic activity is expressed as the inverse of serum
dilution giving a phagocytosis product (PP)=200. PP is measured as
the ratio number of beads/phagocytic cells.times.number of
fluorescent cells.
[0285] Controls wells lacking antiserum and a positive monoclonal
antiserum are included in each experiment.
Opsonisation Results
[0286] Eight out of ten mice exhibit high opsonic activity
(.gtoreq.350).
* * * * *