Note: Descriptions are shown in the official language in which they were submitted.
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Polymyxin B analogs for LPS detoxification
The present invention relates to peptide analogs of polymyxin B that are
useful for LPS
detoxification. In the phannaceutical 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.
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.
LPS structure is constituted by a lipid moiety, called Lipid A, covalently
linked to a
polysaccharide moiety.
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.
Lipid A is highly liydrophobic and anchors LPS in the outer layer of the
bacterial cell
wall. Lipid A is composed of (i) a conserved bis-phosphorylated dissacharide
region
(most frequently, N,O-acyl beta-l,6-D-glucosamine 1,4'-bisphosphate) with (ii)
fatty
acids, that substitute various hydr'ogen 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 (GlcNl and G1cN2) of
Neisseria meningitidis lipid A carries the following fatty acids : 2N-C14,30H
; C12
and 30-C12,30H.
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
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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 0-specific chains
In Gram-negative, non-enteric bacteria such as Neisserias, Bordetellas,
Haefnophilus
and Moraxellas, the 0-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).
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.
Several detoxification methods are already known. In particular, it is
possible to
detoxify LPS while using polyrnyxin B or more appropriately, peptide analogs
thereof.
Polyinyxin 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.
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 US 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).
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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 confonnationally 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.
Therefore, the invention relates to a SAEP II peptide dimer of formula (I)
NH2-A-Cys 1-B-Cys2-C-COOH
NH2-A'-Cys 1-B'-Cys2-C'-COOH
wherein the two Cysl residues are linked together through a disulphide bond
and the two Cys2 residues are linked together through a disulphide bond ;
or formula (II)
NH2-A-Cysl-B-Cys2-C-COOH
COOH-C'-Cys2-B'-Cys 1-A'-NH2
wherein the Cysl 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
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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 ;
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.
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.
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.
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.
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.
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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.
Advantageously, the B and B' peptide moieties comprise the sequence - Xl - X2 -
X3
-, in which XI and X2 ; X2 and X3 ; or Xl, 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.
Particular embodiments of peptide moieties B and B' include :
(i) the - X1 - X2 - X3 - sequence in which :
Xl is Lys, Hyl, His or Arg, preferably Lys or Arg ; more preferably Lys ;
X2 is Phe, Leu, Ile, Tyr, Trp or Val ; preferably Phe or Leu ; more preferably
Phe ; and
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.
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.
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
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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.
Therefore, a preferred class of dimers are of formula (III)
NH2-A-Cys 1-B-Cys2-COOH
NH2-A'-Cys 1-B'-Cys2-COOH
or formula (IV)
NH2-A-Cys 1-B-Cys2-COOH
HOOC-Cys2-B'-Cys 1-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.
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.
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.
As a matter of example, the following peptides are cited as being suitable for
use in
dimers of the invention :
NH2-Lys-Arg-His-Hyl-Cys-Lys-Arg-Ile-Val-Leu-Cys-COOH ;
NH2-Lys-Arg-His-Cys-Val-Leu-Ile-Trp-Tyr-Phe-Cys-COOH ;
NH2-Lys-Thr-Lys-Cys-Lys-Phe-Leu-Leu-Leu-Cys-COOH ; and
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NH2-Hyl-Arg-His-Lys-Cys-Phe-Tyr-Trp-V al-Ile-Leu-Cys-COOH.
The respective cat/hydroph ratio of the corresponding homologous dimers are
2.00,
0.50, 0.75 and 0.67.
A particular example of the dimers described above, is constituted by a
peptide of
formula (V) NH2-Lys-Thr-Lys-Cysl-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.
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.
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.
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
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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.
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.
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 Cysl 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.
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.
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)
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by NMR analysis. MALDI-ToF mass spectrometry allows discriminating between
monomer and dimers only.
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
%.
The invention also relates to compositions comprising :
- A SAEP II peptide, wherein the peptide is essentially in dimeric parallel
form ;
- A SAEP II peptide, wherein the peptide is essentially in dimeric anti-
parallel
form ; or
- mixtures thereof.
By "essentially" it is meant that in the compositions, a particular form is at
least 95 %,
preferably at least 97 %, more preferably 98 % pure.
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.
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.
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into blood, as a result of Gram-negative bacteria infections. These conditions
include
i.a. endotoxicosis, bacterial sepsis and septic shock.
Therefore, the invention encompasses :
- The pharmaceutical use of a compound or composition of the invention ;
- A pharmaceutical composition comprising a compound or a composition of
the invention together with a pharmaceutically acceptable diluent or carrier ;
- The use of a compound or composition of the invention in the preparation of
a medicament for treating or preventing septic shock ; and
- 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.
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 Pseudonzonas 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.
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.
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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.
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.
Significant detoxification is achieved, when the detoxification ratio measured
in :
(i) the LAL assay is at least of 100, preferably 500, more preferably 1000 ;
(ii) the pyrogen test is at least of 50, preferably of 100, more preferably
500 ; or
(iii) D-galactosamine mice is at least of 50, preferably of 100, more
preferably
of 200.
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
TNFa, 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.
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.
For use in the complex of the invention the LPS is advantageously a LPS of N.
nzeningitidis ; E. coli ; Salmorzella typhi ; Salrnonella par=atyphi ;
Slaigella flexneri ;
Haemophilus influenzae ; Helicobacter pylor i ; Clzlamydia tr achonaatis ; Bor
detella
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pertussis ; Brucella ; Legionella pneumopjiia ; Vibrio cholera ; Moraxella
catharYalis ;
Pseudomonas aeruginosa ; Yersinia ; and Kiebsiella pneumonia.
As mentioned in the introduction, detoxified LPS may be useful as vaccinal
agent
against Gram-negative bacteria infection.
Meningitis is a life-threatening disease of either viral or bacterial origin.
H. influenzae
and N. ineningitidis 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.
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).
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 ofineningococcal diseases in temperate countries.
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.
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.
As mentioned hereinabove in the introduction, the major constituent of the
cell wall of
Gram-negative, non-enteric bacteria such as Neisserias, Bordetellas,
Haeynophilus and
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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.
Figure 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 (G1cNAc), are conserved intraspecies.
The
remaining of the oligosaccharide chains that constitutes the outer core (a-
chain
attached to Hepl ;(3-chain attached to position 3 of HepII ; and y-chain
attached to
position 2 of HepII) is variable according to the immunotypes (ITs).
N. meningitidis LPS can be classified into 13 immunotypes, based on their
reactivity
with a series of monoclonal antibodies (Achtman et al, 1992, Jlnfect. Dis.
165: 53-68).
Differences between immunotypes, come from variation in the composition and
conforination of the oligosaccharides chains. This is to be seen in the table
hereinafter.
f~-chain Additional HepII
IT a-chain substituents in y-chain
position 6 or7
Ll NeuNAca2-6Ga1a1-4Ga1(31-4G1c(i1-4 PEA (1-3) None G1cNAca1-2
L2 NeuNAca2-3Ga1(31-4G1cNAc(31-3Ga1(31-4 Glcol-4 Gica (1-3) PEA (1-6) ou
(Aco,4)-G1cNAcal-2
PEA (1-7)
L3 NeuNAca2-3Ga1(31-4G1cNAc(31-3Ga1(31-4 Glcpl-4 PEA (1-3) None G1cNAca1-2
L4 NeuNAca2-3Ga1(31-4G1cNAc(31-3Ga1(31-4 Glcpl-4 H(3) PEA (1-6) Aco,5-G1cNAca1-
2
L5 NeuNAca2-3Ga1(31-4GIcNAc(31-3Ga1(31-4G1c(31-4G1c(31-4 Glca (1-3) None
(Aco.e-o.4)-G1cNAca1-2
L6 GIcNAc(31-3Ga1(31-4 Glcpl-4 H(3) PEA (1-6) ou G1cNAca1-2
PEA (1-7)
L7 NeuNAca2-3Ga1P1-4G1cNAc(31-3Ga1(31-4 G1c(31-4 PEA (1-3) None G1cNAca1-2
L8 Gal(31-4 Glcpl-4 PEA (1-3) None G1cNAca1-2
L9 Ga1(31-4G1cNAc(31-3Ga1(31-4 Glcpl-4 PEA (n.e.) n.e. GlcNAcal-2
L10 Ga1(31-4G1cNAc(31-3Gal(i1-4 Glcpl-4 PEA (n.e.) n.e. (n.e.)-G1cNAca1-2
L11 Galal-4Ga1(il-4Glc(31-4 PEA (n.e.) n.e. (n.e.)-GIcNAcal-2
L12 n.e. PEA (n.e.) n.e. (n.e.)-G1eNAca1-2
L13 n.e. n.e. n.e. n.e.
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As indicated in the above table, a phospho ethanol amine (PEA) replaces the
Glc of the
0-chain at position 3 of HepII in LOS Ll, 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 a-chain.
Immunotypes Ll-L8 are essentially associated with serogroups B and C, while
immuno-types L9-L12 are found predominantly within serogroup A.
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 a-chain is common to all the
immunotypes for which the structure has been identified so far (Kahler &
Stephens,
1988, Crit. Rev. Microbiol. 24 : 281).
Meningococcal strains frequently express several immunotypes, the presence of
wliich
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 Al (also
called 2E) of
serogroup A, strain M978 of serogroup B (Mandrell & Zollinger, 1977, Infect.
Immun.
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 (US 6,476,201) are suitable to this end. These
strains are
obtainable from the scientific community (US 6,531,131).
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)).
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 US 6,531,131 all cited
by way
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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.
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 cheinical 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. rneningitidis. Numerous descriptions of such conjugation
processes may be found in the literature. US 6,531,131 is cited by way of
illustration
only.
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.
The invention also relates to :
- 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
- 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.
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 inixing. The preparation process is advantageously achieved
under
sterile conditions. Upon mixing, a precipitate containing the complex is
formed. It can
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be recovered i.a. by centrifugation, and submitted to one or several washing
steps, if
necessary.
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 coinplex 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.
As a matter of example, a complex that exhibits e.g. 100 endotoxin units (EU)
/ g in
the LAL assay, may be therefore acceptable for administration at a dose of 20
gg. This
is achievable with the complexes of the invention as they may exhibit an LAL
activity
inferior to 50 EU/ g, frequently inferior to 20 EU/ g.
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.
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 mainmals, 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.
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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.
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.
In view of this, the invention also relates to :
(i) The use of a LPS-peptide complex of the invention, for treating or
preventing a
Gram-negative bacterial infection ;
(ii) A pharmaceutical (vaccinal) composition comprising a LPS-peptide complex
of
the invention and a pharmaceutically acceptable diluent or carrier ;
(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 ;
(iv) A method for inducing an immune response in a mammal against a Gram-
negative bacteria LPS or a Gram-negative bacteria, which coinprises
administering an effective amount of a LPS-peptide complex of the invention,
to the mammal ; and
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(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.
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.
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/ 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 g.
It is reminded that, by convention, amounts of complex are always expressed as
LPS
content. Accordingly and by way of example only, "50 g of complex" actually
means
50 g of LPS in the complex preparation.
The Examples reported hereinafter further illustrate the invention by
reference to the
following figures.
Figure lA shows the structure of the LPS LS of N. rneningitidis. Kdo stands
for 2-keto,
3-desoxy octulosonic acid ; Hep stands for heptose ; Glc stands for glucose ;
Gal stands
for galactose ; and G1cNAc stands for N-acetylated glucosamine.
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Figure 1B shows the reaction that occurs upon LPS treatment with acetic acid.
Figures 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).
Figure 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.
Figures 4A-4C show the 'H 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 theni, a peak at 1.9 ppm
indicates
that the peptide is in an acetate salt form.
Figures 5A-5C show an enlargement of the region of the 1H NMR spectra of
Figures
4A-4C comprised between 6.5 and 7.5 ppm.
Figure 6 shows the 6.5-7.5 ppm region of the 1H NMR spectrum obtained with a
composition comprising the SAEP2-L2 peptide in monomeric form, parallel
dimeric
form and anti-parallel dimeric form.
Figures 7A-7C show the MALDI-ToF spectra of the calibration standard (7A), the
parallel dimer (7B) and the anti-parallel dimer (7C).
Figure 8 shows the HPEAC-PAD chromatogram of LPS hydrolysed by acetic acid
treatment.
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Example 1: Preparation of the SAEP2-L2 parallel dimer
1.1. Synthesis
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.
The synthesis cycle proceeds step-by-step, according to the reported linear
sequence. It
is performed in pure solvent dimethylformamide (DMF). Side-protected,
activated
amino acids are used.
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).
All the amino acids are activated at the -COOH side by 0-penta-fluorophenyl-
phosphate esters (O-Pfp-derivatives). They are temporarily protected at the -
NH2 side
by 9-fluorenyl-methyloxy-carbonyl esters (Fmoc-derivatives).
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.
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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
C, under a
pressure of 1 Atm, for 18-24 hours. Complete oxidation of the thiol groups is
determined by the Elman colorimetric assay.
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 rnercuric 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 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
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.
Final purification is achieved on HPLC-operated reverse-phase C18 column
(dimensions = 250 x 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.
The preparation is kept in lyophilized form, at +2 -+6 C, under a neutral gas,
argon or
nitrogen.
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1.3. Characterization of the purified peptide
1.3.1. Amino acid composition
The a.inino acid composition is analysed by the Pico-Tag method (Millipore).
Results
are reported in the table hereinafter.
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
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 Clro
Hi9o 024
N26 S4 of the peptide formula.
Example 2: Preparation of the SAEP2-L2 monomer and anti parallel dimer
2.1. Synthesis
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).
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
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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).
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.
Oxidation is then performed by vigorous stirring of the aqueous solution for
18-24
hours, at 4 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
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.
All preparations are kept in lyophilized form, at +2 -+6 C, under a neutral
gas, argon
or nitrogen.
2.3. Characterization of the antiparallel dimer
2.3.1. Amino acid composition
The amino acid composition is analysed by the Pico-Tag method (Millipore).
Results
are reported in the table hereinafter.
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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
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 CliO
H19o 024
N26 S4 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
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 (Figures 2A-2C) and NMR (Figures 4A-4C and 5A-5C).
3.1. Characterization by I3PLC-reverse phase
Experimental conditions
This technique is carried out on a HPLC chain (WatersTM), using the Millenium
software 32 V30501 (WatersTm) for data acquisition. The analytical column
Macherey
NagelTm ref 720014.6 (Nucleosil 5 m C18 100Angstrom 250 x 4.6 mm) is operated
at
C.
30-40 [Eg of each lyophilised peptide are diluted first in 30 gl water ; to
which is added
gl of trifluoroacetic acid (TFA) 0.1 % in water.
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A mixture of the monomeric, dimeric parallel and antiparallel peptides is also
prepared
by mixing 40 g of a powdered preparation of each peptide in 60 l water ; to
which is
added 60 1 of TFA 0.1 % in water.
The column is equilibrated using 20 % phase mobile B (TFA 0.1 %, CH3CN 80 % in
water). Once samples are applied to the equilibrated column, the phase B
gradient runs
from 20 to 60 % within 40 inin (1 % B/ min), at a flow rate of 1 mL/min.
Detection is achieved at 214 mu. Results are to be seen in Figures 2A-2C.
Results
Each peptide is eluted at a different retention time. In the experimental
conditions
described above, elution occurs at the following retention time (RT) :
- monomer : RT = 28.283 min
- parallel dimer : RT = 29.708 min
- antiparallel dimer : RT = 22.059 min
The HPLC-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.
In Figure 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.
Figure 3 shows the HPLC chromatogram of the mixture.
3.2. Characterization by NMR
Experimental conditions
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'H NMR analysis (500 MHz, 25 C, HOD presaturation) is carried out using
samples of
peptides diluted in a H20/D20 mixture (90/10 v/v). A Bruleerrm DRX500
spectrometer
and associated sofware for data acquisition are used.
In more details, peptides preparation kept at -70 C are used for analysis.
Dimeric
peptide solutions 0.5 mM are prepared while diluting 1.33 g in 1 mL H20. 144
1 of the
solutions are mixed with 16 l of D2O 99.9 % D in 3mm 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 H2O/D20 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
In the experimental conditions used,1H NMR spectra of the monomer and dimers
cover
a range from 0 to 9.5 ppm and are composed of 3 main regions :
- from6.5to7.5ppm;
- from 5.5 to 2.5 ppm ; and
- from 2 to 0.3 ppm.
This is to be seen in Figure4A-4C.
'H 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 Figure 5A, this NMR
pattern is
itself composed of a first multiplet from 7.25 to 7.35 ppm with an integral
curvr
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.
'H 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
Figure 5B, the
4H doublet is found centered at 7.185 ppm (pics at 7.18 and 7.19 ppm).
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'H 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 Figure 5C, the
4H
doublet is found centered at 7.025 ppm (pics at 7.02 and 7.03 ppm).
As shown in figure 4C, the 'H 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
Analysis by MALDI-ToF (Mass Assisted Laser Desorption lonisation - 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
MALDI-ToF analysis is achieved using the Biflex III mass spectrometer
(BrukerTM)
and associated softwares, in a positive reflector mode. Peptides are mixed
with a matrix
(alpha cyano-4-hydroxy cinnamic acid) that absorbs laser energy.
The spectrometer is externally calibrated with a mixture of synthetic peptides
(ACTH
18-39 (adenocorticotropic fragment 18-39) bombesine, and somatostatine 28.
A saturated HCCA matrix solution is prepared while diluting 50 mg HCCA in 300
l
70 % ACN (acetonitril) 0.1 % TFA (trifluoroacetic acid) in water.
A%2 saturated HCCA solution is further prepared while diluting vol : vol with
30 %
ACN, 0.1 % TFA in water.
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For calibration, primary standard solutions are first prepared in 0.1 % TFA.
They are as
follows :
- Adenocorticotropic fragment 18-39 (ACTH 18-39) : 100 pmoles / 1 (0.247
mg/mL) ;
- Bombesine : 100 pmoles / l (0.160 mg/mL) ; and
- Somatostatine 28 : 100 pmoles / l (0.31 mg/mL).
A secondary standard solution is prepared as follows :
- ACTH 100 pmoles / l 2 1
- Bombesine 100 pmoles / .l 4 l
- Somatostatine 100 pmoles / l 4 1
- ACN30 1o,TFA0.1% 50 l
Peptide solutions at 1 mg/mL in water are diluted down to 0.02 mg/mL with 30 %
ACN, 0.1 % TFA in water.
Calibration and peptide samples are diluted vol : vol with the 1/Z saturated
HCCA
solution. Droplets of about 1 l are deposited on a steel target (BrukerTM)
and dried by
evaporation.
Results
Results are to be seen in Figures 7A-7C.
The theoretical monoisotopic masses calculated by the software based on the
amino
acid sequences are :
ACTH 28 M+H+ = 2465.199 Da
Bombesine M+H+ =1619.823 Da
Somatostatine 28 M+H+ = 3147.471 Da
SAEP2-L2 M+H+ = 2388.35 Da.
La norme retenue pour le contr6le est flxee a+ 2 Da par rapport a la mass
theorique.
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As shown in Figure 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).
As shown in Figures 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
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 1
erlen containing 200 mL of Mueller-Hinton broth (Merck) complemented with 4 mL
of
a glucose solution in water (500 g/1). This operation is repeated 4 times.
Erlens are
incubated at 36 + 1 C for 10 + 1 hrs while stirring (100 rpm).
Culture : The erlen contents are gathered together and the preculture is
complemented
with 400 mL of a glucose solution in water (500 g/1) and 800 mL of an amino
acid
solution. This preparation is used to inoculate the Mueller-Hinton broth, in a
30 1
fermentor (B. BraunTM) at an initial OD6ooõm close to 0.05. Fermentation is
performed
overnight at 36 C, pH 6.8 + 0.2, 100 rpin, P02 30 %, and initial flow rate of
the air 0.75
1/min/L culture. After 7 1 hrs, (OD60o,,,,, 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/1,
the
fermentation is stopped. Usually, the final OD600,,,n is comprised between 20
and 40.
Cells are collected by centrifugation for 1 h 30 at 7000 g at 4 C. Pellets are
kept frozen
at - 35 C
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4.1.2. Purification of LPS
First phenol extraction
Pellets are defrosted and suspended with 3-volume phenol 4.5 %(v/v) and
stirred
vigorously for 4 hrs minimum at about 5 C.
The bacterial suspension is heated at 65 C and then mixed v/v with phenol 90 %
at
65 C. The suspension is stirred vigorously, at 65 C for 50-70 min and then
cooled
down to about 20 C.
The suspension is centrifuged for 20 min, at 11 000 g, at about 20 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
The phenol phase and the interphase are heated at 65 C and mixed with a volume
of
water equivalent to the volume of the aqueous phase that was previously
collected. The
rnixture is stirred vigorously for 50-70 min at 65 C and then cooled down to
about
20 C. The mixture is centrifuged for 20 min, at 11 000 g, at about 20 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 : Procedure for the second extraction is repeated.
Dialysis
The 3 aqueous phases are dialysed overnight and separately against 40 1 of
water. The
dialysates are pooled together. The dialysate pool is adjusted with Tris 20
mM, MgCla
2 mM (one volume per 9 volumes of the dialysate pool). pH is adjusted to 8.0 +
0.2
with NaOH 4 N.
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DNAse treatment
250 UI of DNAse is added per gram of treated bacterial pellet (wet weight).
The
preparation is stirred at 37 + 2 C for 55-65 min. pH is adjusted at 6.8 + 0.2.
The
preparation is filtered on 0.22 m membranes.
Gel filtration : The preparation is purified on a Sephacryl S-300 column (5.0
x 90 cm ;
PharmaciaTm).
First alcoholic precinitation
Powder of MgC12, 6H20 is added to the LPS-containing fractions pooled
together, to
reach an MgCl2 concentration of 0.5 M and dissolved while stirring.
While stirring at 5+ 3 C, dehydrated absolute alcohol is added to a final
concentration
of 55 %(v/v). Stirring is performed overnight at 5+ 3 C, followed by
centrifugation at
5,000 g for 30 min at 5+ 3 C. The supematants are discarded and the pellets
are
submitted to a second extraction.
Second alcoholic precipitation
The pellets are resuspended with at least 100 mL MgCl2 0.5 M, while stirring.
The previous procedure is repeated. Pellets are resuspended with at least 150
mL water.
Final stpv : Gel filtration is repeated and the LPS-containing fractions
pooled together
are finally sterilised by filtration (0.8-0.22 m) and kept at 5 + 3 C.
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.
The purification process as described allows obtaining about 150 mg LPS L8 per
culture liter (yield about 50 %).
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4.1.3. LPS L8 quantiffcation : KDO dosage with HPAEC-PAD
The bibliographic reference for this teclznique 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.
As shown in Figures 1A-1B, LPS comprises in its structure 2 KDO units, one
being in
a lateral position.
LPS quantification is achieved through dosage of the lateral KDO unit
liberated upon
soft acid hydrolysis (See Figure 1B).
Acid hydrolysis
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 l in DionexT"'
1.5 mL
flasks so that LPS concentration of the samples falls under the etalon range
(1.4 - 72.1
g/mL).
Samples to be quantified as well as the KDO etalon range are proceeded as
follows
100 l of the hydrolysis solution (acetic acid 5 % ; glucuronic acid (G1cA) 20
g/mL)
are added. Hydrolysis is performed for 1 h at 100 C. Flasks are then dried at
40 C
under nitrogen and filled with 400 l water.
Dosage
This technique is carried out on a HPAEC chain (DionexTM), using the
Chromeleon
DionexTm software for data acquisition. The analytical column Carbopac PAl 4 x
250
mm (DionexTm) is operated at 30 C.
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The column is equilibrated with the elution solution (NaOH 75 mM, AcONa 90
mM).
100 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.
Chromatogram of LPS sample is to be seen in Figure S. 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 : 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
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 m membrane.
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 m
membrane.
All the next steps are achieved under sterile conditions.
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 C
overnight.
The precipitate (Endotoxoid) is then recovered by centrifugation at 3000 rpm
for 10
min. The supernatant is discarded.
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The pellet is washed with one volume of sterile, pyrogen free water (Milli Q
quality,
adjusted to pH 7.2, Limulus negative). Centrifu.gation/washing steps are
repeated five
times.
5. 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 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).
The LPS-peptide complex tested in the following examples is the LPS-
antiparallel dimer conaplex as obtained in section 4.3, unless otlierwise
indicated. Therefore, this specific complex is simply referred to as LPS-
peptide
complex.
In a similar manner, the LPS as obtained in section 4.2. is simply referred to
as
LPS.
Comparison ofLPS and LPS-peptide complex is achieved using the LPS lot also
used for the preparation of the conzplex.
Example 5: Evaluation of the detoxification of the LPS-peptide complex
Several assays are used to evaluate the detoxification.
5.1. Limulus Amebocyte Lysate (LAL) assay
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.
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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 polyphernus) to induce coagulation in the presence of
endotoxin.
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-BioWhittakerTm) is used (linear zone of
the
kit : 0.1 to 1 UT/mL) as well as a positive control (E. coli endotoxin, 4 103
EU/mL,
Sigma).
Dilution of (i) samples to be tested, (ii) standard and (iii) positive control
are achieved
with dilution buffer (Cambrex-BioWhittakerTM) to cover the respective ranges :
1/10 to
1/105 ; 0.5 to 0.031 EU/mL and 1/104 to 1.8 104.
50 l of sample, standard and positive control dilutions are dispensed per
well of 96
flat-bottom well ELISA plate. Fifty l of lysate are added per well.
Incubation is
pursued for 10 min at 37 C. Then 100 l of the p-nitroaniline chromogenic
substrate
are added. Incubation is pursued for 6 min at 37 C. The chromogenic reaction
is
stopped while adding 100 gl freezed acetic acid 25 % in water. Plate is read
by
spectrophotometry at 405 nm.
The results are expressed in Endotoxin Unit (EU)/ 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.
Range EU/ g Mean value Detoxification ratio
EU/ g Expressed in log
LPS (6 assays) 1-12 104 25,000
LPS-antiparallel peptide 5- 12-20 1,250-2,000 3.5
32
complex (13 assays)
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LPS-parallel peptide 30-40 30-40 600-800 3
complex
LPS-monomer peptide > 2000 > 2000 <12.5 <1
complex
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
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
C ;
and non-conformity, when the summed response exceeds 2.65 C. In the present
case,
the pyrogenic threshold is set up below, between 1.15 C and 2.65 C.
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 US 6,531,131.
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
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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.
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 I g / mouse (0.2-5 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 complexed with
peptide
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.
~ In vivo : cytokine (IL6 and TNFa) releases in the sera of mice immunized
either with LPS or LPS-peptide coinplex are compared by ELISA. Blood
samples are recovered 90 min after SC iminunization, 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.
~ In vitro : cytokine (IL6, IL8 and TNFa) releases from human whole blood cell
cultures stimulated for 24h at 37 C, with different concentrations of LPS or
LPS-peptide complex are compared.
5.4.1. In vivo assay
CD1 mice are administered subcutaneously (SC) (i) either 10 g of LPS or (ii)
10 g of
LPS-peptide conlplex. They are bled 90 minutes after injection. IL6 and TNFa
releases
are measured in the sera by ELISA.
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ELISA detection of cytokine secretion
ELISAs are carried out using the OptEIA mouse IL6 and TNFa 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.
Anti-mouse IL6 and TNFa antibodies are 1/250 diluted in 0.1 M carbonate buffer
pH
9.5 (Sigma). For each assay, 100 l of an antibody dilution are distributed
per well of a
Maxisorp NLTNC 96 flat-bottom well ELISA plate. Plates are incubated
overni.ght at
+4 C.
Plates are washed in PBS 0.05 % Tween 20. 200 l of PBS, 0.5 % bovine seruin
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.
Recombinant IL6 or TNFa cytokine dilutions are prepared in the RPMI medium I %
FCS 10 %, within the range of (i) 4,000 pg/mL - 62.5 pg/mL standard. 100 1 of
each
dilution are distributed per well, to establish the standard curve.
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 gl of each dilution are
distributed per
well.
Incubation is pursued for 2 hrs at room temperature.
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 l
of each dilution are added per well. Incubation is pursued for one hr at room
temperature.
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Plates are washed in PBS 0.05 % Tween 20. 100 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.
The reaction is stopped by adding 100 l of 1 M H3P04 per well. Plates are
read at 450
nm. Results are to be seen in the table hereinafter.
Product injected to IL6 release TNFa release
mice Mean (pg/mL) Detoxification Mean (pg/mL) Detoxification
n=6 (log) ratio (log unit) n=6 (log) ratio (log unit)
LPS 4.7 4.1
LPS-peptide complex 2.2 2.5 <1 >3.1
Peptide <1 <1
The peptide alone does not induce IL6 or TNFa. 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
LPS preparation (lmg/mL) and LPS-peptide complex (500 gg/inL) are each diluted
in
10 mM Tris, NaCl 150 mM, 0.05 % Tween 20, 5 % sucrose to a concentration of 50
g/mL. They are further diluted in physiological saline to a concentration of 5
gg/mL.
Serial 1/5 dilutions are performed in AIM-V medium (Gibco (Invitrogen)) for
each test
substance down to a concentration of 2.56 10"3 pg/mL.
Stimulation
Human blood collected on sodium heparin (25,000 U/5mL ; sanofi-synthelabo) is
diluted 1: 4 (vol : vol) in AIM-V medium and distributed in MicronicsTM tubes
(400 l
/ tube). 100 l of a dilution of the test substances are added. Peptide and
buffer controls
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are tested at 1/20 dilution. Tubes are incubated for 24 hrs at 37 C, in a wet
atmosphere
at5%C02.
Plasma recovery
Tubes are then centrifuged for 10 min at 500 g. At least 200 l of supernatant
are
recovered from each tube and kept frozen at -80 C until titration.
ELISA detection of cytokine secretion
ELISAs are carried out using the OptEIA human IL6, IL8 and TNFa 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).
Anti-human IL6, IL8 and TNFa antibodies are 1/250 diluted in 0.1 M carbonate
buffer
pH 9.5 (Sigma). For each assay, 100 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 C
Plates are washed in PBS 0.05 % Tween 20. 200 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.
Recombinant IL6, IL8 or TNFa 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 l of each dilution
are
distributed per well, to establish the standard curve.
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 l of each dilution are
distributed
per well.
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Incubation is pursued for 2 hrs at room temperature.
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 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 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.
The reaction is stopped by adding 100 1 of 1 M H3PO4 per well. Plates are
read at
450 nm.
Results
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.
The detoxification ratio is expressed as the ratio of the concentration of the
LPS-
peptide complex inducing 50 % of maximunz IL6 release (ED50 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.
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.
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The IL6 release correlates with the TNFa 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
The detoxification ratio is measured between 102 and 103, depending on the
test. The
detoxification values are summarized in the following table.
Assays LPS L8 LPS-peptide Detoxification
complex ratio
LAL 25,000 EU/ g 12-20 EU/ 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/m.L 100
IL6 = 10,000 pg/mL IL6 = 100 pg/mL
In vitro assay of IL6 release by ED50 = 2 pg/mL ED50 = 880 pg/mL 64
human PBMC
(EDSo: concentration of product inducing
50% of maximum IL6 release)
LD50 in galactosami.ne-sensitized 4 ng/souris 1 g/souris (0.2-5) 250
mice
Example 6: LPS peptide complex stabifity study
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 viti=o stability of the formulated LPS peptide complex
The stability of the formulated LPS-peptide complex is followed at 5 C, for 6
months.
Measurements are made at day = 0, 90 and 180 (6 months). Results are as
follows.
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IL6 release from human LAL assay Pyrogen test
blood cells (detoxification endotoxin (EU/ g) Pyrogenic threshold, as chosen :
ratio) 10 ng/mL/kg IV
0 3 months 6 months 0 3 months 6 months 0 3 months 6 months
125 40 163 14 58 10 C* C C
C*: conform
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 C.
6.2. In vitro stability of the LPS peptide complex in physiological liquid
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.
One mL of the LPS-peptide complex, mixed with 1 mL of human serum, is
incubated
at 37 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.
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 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
Immunization of three adult New-Zealand rabbits is performed with 100 g of
LPS-
peptide complex by intramuscular (IM) and subcutaneous (SC) routes (2 x 0.5 mL
and
5 x 0.2 mL respectively) in the presence of adjuvant. They receive three
injections at 3-
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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 g, equivalent
to the
amount of the peptide in 100 p.g of LPS-peptide complex) using the same
protocol.
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
Sera are heat-inactivated during 30 min at 56 C. Tn 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 and Mg~+ (volume per well : 50 l).
Twenty five gl of a log phase culture of N. meningitidis grown in Mueller-
Hinton broth
(4.103 CFU/mL) and 25 l of baby rabbit serum are added to each well. The
plate is
incubated one hour at 37 C, under shaking.
Fifty gl of the mixture from each well are plated onto Mueller-Hinton agar.
Petri dishes
are incubated overnight at +37 C in a 10% CO2 atinosphere.
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.
Bactericidal titre is reported as the highest reciprocal serum dilution at
which _ 50 %
killing of bacteria is observed as compared to the complement control.
SBA results
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
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response, when the sera (post-dose 3) are adsorbed on LPS.
Rabbit # Pre-immunized Post-dose 3 Post-dose 3
sera immunized sera immunized 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
Ten six-week old female outbred CD1 mice are immunized with a 10 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.
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 strain
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)].
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
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Wells of a 96-well microplate are coated with 100 l of a 10 .g/mL LPS
solution in
buffer 1 (PBS + 10 mM MgC12). The plate is incubated 2 hours at +37 C ; then
overnight at +5 C.
The LPS solution is removed from the plate and wells are saturated with 150 l
of
buffer 2 (PBS + milk 1 % + Tween 20 0.05 %). The plate is incubated one hour
at 37 C
; then washed with buffer 3 (PBS + Tween 20 0.05 %).
Sera are serially diluted 12-fold, directly in the wells using buffer 2
(volume : 100 l
per well). The plate is incubated for 90 inin at +37 C ; then washed with
buffer 3.
Hundred l of a diluted goat anti-mouse IgG (y chain specific) or IgM ( chain
specific) peroxydase conjugate are added in each well. The plate is incubated
90 min at
37 C and then washed with buffer 3.
The reaction is developed by adding 100 gl of a tetramethylbenzidine substrate
solution
in each well. The plate is incubated 20 min at 37 C. The reaction is stopped
by adding
1 M HCl and absorbance is measured at 450 nm.
ELISA results
Results are expressed in arbitrary ELISA Unit/mL (EU/mL) by comparison to a
reference serum.
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.
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Anti-LPS IgG (EU/mL) Anti-LPS IgM (EU/mL)
Post dose 1 Post dose 2 Post dose 1 Post dose 2
LPS-peptide complex 1,800 22,000 280 550
Buffer <40 <40 <40 <40
In a further imunisation 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
Bactericidal activity is measured as described in section 7.1.
Fourty % of the post-dose 2 sera exhibit a bactericidal activity (SBA titre >_
16) against
the homologous N. meningitidis strain. Four are bactericidal against the
heterologous
strain.
7.2.3. Opsonic activity of mouse sera
Opsonisation assay
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.
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 2.5x107 cells/mL.
Sera are heat inactivated during 30 min at 56 C. In a 96 deep-well microplate,
heat-
inactivated sera are serially fivefold diluted (3 times) in Hanks balanced
salt buffer
containing Ca and Mg~+ (volume per well : 300 l).
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Twenty l of LPS coated latex fluorescent beads and 10 l of baby rabbit serum
as
exogenous complement source are added to each well. The plate is incubated 30
min at
+37 C, under shaking.
Fifty l of the effector cell suspension are added to each well. The plate is
incubated 30
min at +37 C, under shaking.
One hundred fifty l from each well are transferred in a second deep well and
the
reaction is stopped by adding 400 l of PBS + 0.02 % EDTA. The plate is
centrifuged
and washed twice with PBS+BSA buffer.
The phagocytosis of LPS coated beads by effector cells, in the presence of
antiserum
and exogenous complement source is measured by FACS.
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 x
number of fluorescent cells.
Controls wells lacking antiserum and a positive monoclonal antiserum are
included in
each experiment.
Opsonisation results
Eight out of ten mice exhibit high opsonic activity (>_ 350).
