U.S. patent application number 16/177738 was filed with the patent office on 2019-04-25 for meningococcal vaccine based on lipooligosaccharide (los) originating from modified neisseria meningitidis strains of immunotype l6.
This patent application is currently assigned to Sanofi Pasteur SA. The applicant listed for this patent is Sanofi Pasteur SA. Invention is credited to Noelle Mistretta, Monique Moreau, Genevieve Renauld-Mongenie, Bachra Rokbi.
Application Number | 20190117684 16/177738 |
Document ID | / |
Family ID | 41213428 |
Filed Date | 2019-04-25 |
United States Patent
Application |
20190117684 |
Kind Code |
A1 |
Mistretta; Noelle ; et
al. |
April 25, 2019 |
Meningococcal vaccine based on lipooligosaccharide (LOS)
originating from modified Neisseria meningitidis strains of
immunotype L6
Abstract
The invention especially relates to multivalent vaccine
compositions that can treat or prevent at least 60, preferably 75%
of infections caused by Neisseria meningitidis especially of
serogroup B. To this end, the invention in particular provides a
lipooligosaccharide (LOS) of N. meningitidis in particular
constituted by a lipid A, an inner core, an .alpha. chain of L6 or
L8 type, in which the heptose II residue of the inner core bears in
position O-3 and in position O-6 or O-7 a phosphoethanolamine (PEA)
substituent, and also to the construction of the strain of N.
meningitidis that is capable of expressing such an LOS. The
invention also relates to a strain of N. meningitidis of serogroup
A that bears a lipooligosaccharide (LOS) in particular constituted
by a lipid A, an inner core, an .alpha. chain of L6 type, in which
the heptose II residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7. The LOSs cited or originating
from the mentioned strains may be used as vaccine antigens,
especially in multivalent, e.g. divalent compositions, so as to
offer protection against the major epidemiological complexes of N.
meningitidis, especially of serogroup B.
Inventors: |
Mistretta; Noelle; (Sain
Bel, FR) ; Moreau; Monique; (Lyon, FR) ;
Renauld-Mongenie; Genevieve; (Chaponost, FR) ; Rokbi;
Bachra; (Lyon, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sanofi Pasteur SA |
Lyon |
|
FR |
|
|
Assignee: |
Sanofi Pasteur SA
Lyon
FR
|
Family ID: |
41213428 |
Appl. No.: |
16/177738 |
Filed: |
November 1, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
15584619 |
May 2, 2017 |
10137145 |
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16177738 |
|
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|
14756143 |
Aug 5, 2015 |
9669052 |
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15584619 |
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12800454 |
May 14, 2010 |
9132181 |
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14756143 |
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61271986 |
Jul 29, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/127 20130101;
A61P 31/04 20180101; A61K 39/095 20130101; A61K 31/739
20130101 |
International
Class: |
A61K 31/739 20060101
A61K031/739; A61K 39/095 20060101 A61K039/095; A61K 9/127 20060101
A61K009/127 |
Foreign Application Data
Date |
Code |
Application Number |
May 14, 2009 |
FR |
0902333 |
Claims
1. A N. meningitidis strain that exhibits a lipooligosaccharide
(LOS) comprising a lipid A, an inner core, an .alpha. chain of L6
type, in which the heptose II residue of the inner core bears in
position O-3 and in position O-6 or O-7 a PEA substituent.
2. The N. meningitidis strain as claimed in claim 1, wherein the
strain is of serogroup A.
3. The N. meningitidis strain as claimed in claim 2, wherein the
strain is of immunotype L6.
4. The N. meningitidis strain as claimed in claim 1, wherein the
strain exhibits an active msbB gene.
5. The N. meningitidis strain as claimed in claim 1, wherein the
lipid A is a non-detoxified lipid A.
6. The N. meningitidis strain as claimed in claim 1, wherein the
strain expresses its lpt3 gene or the lpt3 gene of a heterologous
N. meningitidis strain.
7. The N. meningitidis strain as claimed in claim 6, wherein the
strain is strain C708 deposited as strain CNCM 1-3942 modified to
restore the functionality of its lpt3 gene.
8. A lipooligosaccharide (LOS) exhibited by the N. meningitidis
strain according to claim 1.
9. A lipooligosaccharide (LOS) exhibited by the N. meningitidis
strain according to claim 2.
10. A lipooligosaccharide (LOS) exhibited by the N. meningitidis
strain according to claim 3.
11. A lipooligosaccharide (LOS) exhibited by the N. meningitidis
strain according to claim 4.
12. A lipooligosaccharide (LOS) exhibited by the N. meningitidis
strain according to claim 5.
13. A lipooligosaccharide (LOS) exhibited by the N. meningitidis
strain according to claim 6.
14. A lipooligosaccharide (LOS) exhibited by the N. meningitidis
strain according to claim 7.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/584,619, filed on May 2, 2017, which is a continuation of
U.S. application Ser. No. 14/756143, filed on Aug. 5, 2015, now
U.S. Pat. No. 9, 669,052, which is a divisional of U.S. application
Ser. No. 12/800454, filed on May 14, 2010, now U.S. Pat. No.
9,132,181, which claims priority to U.S. Provisional Patent
Application Ser. No. 61/271,986 filed on Jul. 29, 2009, the
contents of each of which are incorporated herein by reference in
their entirety.
[0002] The invention relates to the field of vaccines for combating
infections caused by Neisseria meningitidis and especially proposes
a vaccine composition comprising two types of lipooligosaccharide
(LOS), each of the two types originating from a particular strain
of N. meningitidis. The invention also proposes intermediate means
contributing toward the preparation of the vaccine composition
mentioned above.
[0003] In the field of vaccines, one of the major challenges in the
near future will especially be the marketing of a vaccine intended
for preventing all the infections caused by N. meningitidis
serogroup B. This bacterium is responsible for a certain number of
pathologies, the dominant ones of which are meningitis and
meningococcia, but also arthritis and pericarditis. Meningococcia
may be complicated with purpura fulminans and fatal septic
shock.
[0004] In general, meningitis is either of viral origin or of
bacterial origin. In developed countries, the bacteria mainly
responsible are: N. meningitidis and Streptococcus pneumoniae,
which are involved, respectively, in about 40% and 50% of the cases
of bacterial meningitis. In developing countries, Haemophilus
influenzae also remains a significant source of meningitis.
[0005] In France, about 600 to 800 cases of N.
meningitides-mediated meningitis are reported per year. In the
United States, the number of cases rises to about 2500 to 3000 per
year.
[0006] The species N. meningitidis is subdivided into serogroups
according to the nature of the capsule polysaccharides. Although
there are about a dozen serogroups, 90% of the cases of meningitis
are attributable to the serogroups: A, B, C, Y and W135.
[0007] Efficient vaccines based on capsule polysaccharides exist
for preventing the N. meningitidis-mediated meningitis of the
serogroups A, C, Y and W135. These polysaccharides per se are not
or are only sparingly immunogenic in children under 2 years old and
do not induce any immune memory. However, these drawbacks may be
overcome by combining these polysaccharides with a carrier
protein.
[0008] However, the capsule polysaccharide of N. meningitidis
serogroup B is not or only sparingly immunogenic in man, whether or
not it is in conjugated form (Bruge et al, Vaccine (2004) 22:
1087). Moreover, this polysaccharide bears an epitope that might
potentially undergo a cross reaction with human tissues. Thus, it
appears highly desirable to search for a vaccine for combating
meningitis induced by N. meningitidis especially of the serogroup B
other than a vaccine based on capsule polysaccharide.
[0009] The liposaccharide (LPS) is a major constituent of the outer
membrane of the wall of Gram-negative bacteria. LPS is toxic at
high doses to mammals and, in view of this biological activity, has
been called an endotoxin. It is responsible for septic shock, a
fatal pathology which develops following acute infection with a
Gram-negative bacterium.
[0010] Nevertheless, LPS is not only toxic, it is also immunogenic.
In mammals, anti-LPS antibodies are generated during carrying and
infection and can be protective. Thus, the use of LPS has already
been envisioned in the prophylaxis of infections due to
Gram-negative bacteria and associated diseases.
[0011] The structure of LPS is in particular constituted of a lipid
portion, called lipid A, covalently bonded to a polysaccharide
portion.
[0012] Lipid A is responsible for the toxicity of LPS. It is highly
hydrophobic and enables the LPS to be anchored in the outer
membrane of the wall. Lipid A is composed of a disaccharide
structure substituted with fatty acid chains. The number and the
composition of the fatty acid chains vary from one species to the
other.
[0013] The polysaccharide portion is in particular constituted of
carbohydrate chains which are responsible for the antigenicity. At
least 3 major regions can be distinguished in this polysaccharide
portion: [0014] (i) an inner core in particular constituted of
monosaccharides [one or more KDO (2-keto-3-deoxyoctulosonic acid)
and one or more heptoses (Hep)] which are invariant within the same
bacterial species; [0015] (ii) an outer core bonded to heptose and
in particular constituted of various monosaccharides; and [0016]
(iii) an O-specific outer chain in particular constituted of a
series of repeating units--these repeating units themselves being
composed of one or more different monosaccharides.
[0017] The structure of LPS varies from one species to another.
This is why, for example, in a certain number of non-enteric
Gram-negative bacteria such as Neisseriae, Bordetellae,
Branhamellas, Haemophilus and Moraxellae, the O-specific chain does
not exist. The LPS saccharide portion of these bacteria is in
particular constituted only of the oligosaccharide core.
Consequently, the LPS from these bacteria is often called
lipooligosaccharide (LOS).
[0018] The structure of the LPS varies not only from one species to
another, but also within the same species.
[0019] Thus, not all the strains of N. meningitidis have an LOS of
the same structure, although all the LOSs of meningococcus share
the basic structure, which is represented schematically in the
following structural formula:
##STR00001##
[0020] The outer core (or .alpha. chain) is variable as a function
of the type of oligosaccharide (substituent R1) attached to the
glucose residue borne by the heptose I.
[0021] Whereas lipid A is essentially invariable, the inner core,
which itself also is formed in an invariant manner from two KDO
(2-keto-3-deoxyoctulosonic acid) and two heptoses (HepO and HepII),
bears various substituents (i) on heptose II (substituents R2 and
R3); and (ii) on the .gamma. chain, formed from an N-acetyl
glucosamine (GlcNAc), which may or may not be O-acetylated. In the
literature, the R2 residue is commonly referred to as the .beta.
chain, when R2 is a glucose residue.
[0022] Originally, the LOS from N. meningitidis was listed as 13
immunotypes (IT L1 to L13), as a function of its reactivity with a
series of antibodies (Achtman et al, 1992, J. Infect. Dis. 165:
53-68). The majority of invasive strains with N. meningitidis
serogroup B is of immunotype L3,7 as demonstrated by the reactivity
of these strains with a monoclonal antibody called L3,7,9. This
monoclonal antibody is capable of recognizing each of the
immunotypes L3, L7 and L9 (Gu et al, J. Clin. Microbiol. (1992) 30:
2047; Moran et al, Infect. Immun. (1994) 62 (12): 5290; et Scholten
et al, J. Med. Microbiol. (1994) 41: 236). The differences between
immunotypes originate from variations in the composition and in the
conformation of the oligosaccharide chains. This shows in the table
below (Table I), derived from Table 2 of Braun et al, Vaccine
(2004) 22: 898, supplemented with data obtained subsequently and
relating to immunotypes L9 (Schoudhury et al, Carbohydr. Res.
(2008) 343: 2771) and L11 (Mistretta et al, (2008) Poster at the
16th International Pathogenic Neisseria Conference, Rotterdam):
TABLE-US-00001 TABLE I IT R1 (.alpha. chain) R2 R3 L1
NeuNAc.alpha.2-6Gal.alpha.1-4Gal.beta.1-4 PEA-3 -- L2
NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4 Glc.alpha.
PEA-6 or (1-3) PEA-7 L3
NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4 PEA-3 -- L4
NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4 -- PEA-6 L5
NeuNAc.alpha.2-3Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1- Glc.alpha.
-- 4Glc.beta.1-4 (1-3) L6 GlcNAc.beta.1-3Gal.beta.1-4 -- PEA-6 or
PEA-7 L7 Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4 PEA-3 -- L8
Gal.beta.1-4 PEA-3 -- L9 Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4 --
PEA-6 L10 n.d. n.d. n.d. L11 Glc.beta.1-4 PEA-3 PEA-6. L12 n.d n.d.
n.d. L13 n.d n.d. n.d. n.d.: not determined.
[0023] It may be noted, inter alia, that certain LOSs may be
sialylated (presence of N-acetylneuraminic acid on the terminal
galactose residue (Gal) of the .alpha. chain). Thus, immunotypes L3
and L7 differ only by the respective presence/absence of this
sialylation.
[0024] Moreover, most LOSs are substituted with an O-acetyl group
on the glucosamine residue (.alpha.-GlcNAc) of the inner core
(Wakarchuk et al. (1998) Eur. J. Biochem. 254: 626; Gamian et al.
(1992) J. Biol. Chem. 267: 922; Kogan et al (1997) Carbohydr. Res.
298: 191; Di Fabio et al. (1990) Can. J. Chem. 68: 1029; Michon et
al. (1990) J. Biol. Chem. 275: 9716; Choudhury et al. (above); and
Mistretta et al. (above)).
[0025] The other variations in the structure of the LOS are due to
different genetic factors, including: [0026] (i) the
presence/absence of certain genes involved in the LOS biosynthetic
pathway and in possible mutual associations of the genes; [0027]
(ii) the phase variation to which certain genes are subjected;
[0028] (iii) homologous recombination [since certain genes have
conserved regions (lgtB, lgtE and lgtH) and other genes are hybrid
(lgtZ is the hybrid of the genes lgtA and lgtB), rearrangements may
take place]; and [0029] (iv) mutations.
[0030] The genes involved in the LOS biosynthetic pathway (with the
exception of two) are divided into three loci (lgt-1, lgt-2 and
lgt-3). The description of these genes and their function is given
later, illustrated schematically by FIG. 1, which shows the
structure of the LOS of N. meningitidis, the various sites at which
the variability is expressed and also the levels of intervention of
the genes.
[0031] The off-locus genes are lpt3 and lot3. The gene lpt3 codes
for a PEA transferase. This enzyme has the capacity to attach a
phosphoethanolamine (PEA) residue in the O-3 position of heptose
II. The gene lot3 codes for an LOS O-acetyltransferase that has the
capacity to O-acetylate the .gamma. chain. It is subject to a phase
variation.
[0032] The lgt-1 locus comprises 7 genes: lgtA, lgtB, lgtC, lgtD,
lgtE, lgtH and lgtZ, each coding for a particular glycosyl
transferase. Among these genes, lgtA and lgtC are subject to a
phase variation. lgtE and lgtH have an allelic variation: the codon
that determines the amino acid in position 153 codes either for a
threonine residue (and in this case the resulting enzyme is a
Gal-transferase) or for a methionine residue (and in this case the
resulting enzyme is a Glc-transferase).
[0033] The lgt-1 locus is classed into 8 genetic types (Zhu et al,
Microbiology (2002) 148: 1833).
[0034] The lgt-2 locus comprises 2 genes: lgtF and lgtK coding for
glycosylases. The product of the lgtF gene intervenes in the
construction of the .alpha. chain by enabling the binding of the
glucose residue to heptose I, and therefore does not intervene in
the nature of the immunotype; nor, for that matter, does the gene
lgtK.
[0035] The lgt-3 locus comprises 2 genes: lgtG and lpt6. The gene
lgtG codes for a Glc synthetase that has the capacity to attach a
glucose residue in the O-3 position of heptose II. The gene lpt6
codes for a PEA transferase that has the capacity to attach a
phosphoethanolamine (PEA) substituent in the O-6 or O-7 position of
heptose II. The gene lgtG is subject to a phase variation. When it
is "On" and accompanied by a functional lpt3 gene, the attachment
of the glucose residue always takes place at the expense of PEA
(whose attachment is mediated by lpt3).
[0036] The lgt-3 locus is classed into 5 genetic types (Wright et
al., J. Bact. (October 2004): 6970).
[0037] The Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4Glc.beta.1-4
carbohydrate unit or lacto-N-neotetraose unit which is present in
the .alpha. chain of certain N. meningitidis LOS immunotypes
constitutes an epitope which can potentially crossreact with human
erythrocytes. Thus, with a view to producing a vaccine for use in
humans, it is advisable to choose an LOS which does not possess
this unit. It may therefore be particularly advantageous to use an
LOS originating from strains of immunotype L6 or L8.
[0038] However, a genotype study of epidemiological strains made it
possible to discover that the strains of immunotype L6 or L8 needed
to be optimized in order to modify the structure of their original
LOS; this being in order to manufacture an improved LOS-based
vaccine.
[0039] The genotype study in question was performed in two
stages.
[0040] First, about twenty strains of N. meningitidis were analyzed
both by genotyping and by biochemical analysis (mass spectrometry
and nuclear magnetic resonance). In a first stage, the immunotype
of these strains was predicted from the genotyping results. The
results of the biochemical analysis revealed an excellent
correlation between the structure effectively determined and the
immunotype predicted by genotyping. Given the close parallel
between the genotyping results and the biochemical analysis
results, it was possible in a second stage to validly continue the
analysis of a much broader collection of strains, solely by
genotyping.
[0041] Thus, a collection of 163 strains was gathered, most of
which were provided by the laboratories of Drs D. A. Caugant (NIPH,
Oslo, Norway), D. Martin (ESR, Porirua, New Zealand) and M. Diggle
(SMPRL, Glasgow, United Kingdom). The strains of this collection
are sourced worldwide. About half of them were isolated in Europe.
They are in the very large majority of serogroup B and are divided
into the 6 major epidemiological complexes found in Europe in the
invasive strains of the serogroup B: i.e. the MLST (multilocus
sequence type) ST-8, ST-11, ST-18, ST 32, ST-41/44 and ST-269
complexes.
[0042] Specifically, epidemiologically, the most recent European
data indicate that 64% of the invasive strains of meningo B are
divided among these 6 complexes, whereas, at the present time, 50
complexes have been described (19% of the European strains not
having been assigned to a determined complex).
[0043] The table below (Table II) provides further details
concerning these 163 strains and indicates in parentheses the
previous nomenclature or names of the corresponding MLEE (multi
electrophoretic enzyme) complexes or electrophoretic (ET)
complexes:
TABLE-US-00002 TABLE II Origin Number of (number of Period of
Complex strains countries) isolation Main source ST-41/44 34 9
1963-1994 D. Caugant/ (lineage III) D. Martin ST-32 53 10 1981-1996
D. Caugant (ET-5) ST-269 14 2 1988-2007 D. Caugant ST-18 7 2
1985-2005 D. Caugant ST-8 28 12 1967-1994 D. Caugant/ (cluster A4)
M. Diggle ST-11 27 7 1969-1988 D. Caugant/ (ET-37) M. Diggle
[0044] The genes participating in the biosynthesis of the LPS that
were analyzed by genotyping are the following: lgtA, lgtB, lgtC,
lgtE and lgtH; lgtF; lgtG and lpt6; lpt3 and lot3. This analysis
made it possible to predict the structure of the LOS in the 6 major
epidemiological complexes of meningococcus B. The results are given
in FIG. 2. It will be noted that the genotyping results for certain
strains are such that it had to be deduced that such a strain is
capable of manufacturing several types of LOS. The LOS of such a
strain may thus be classed into several categories. To conclude,
this explains, for example, why a strain can be compatibilized in
several categories of .alpha. chain.
[0045] The results of the genotyping relating to 3 of the 4 genes
involved in the biosynthesis of the inner core and acting on its
variability (i.e. lgtG, lpt6 and lpt3) and also the structure
deduced therefrom are presented in detail in Table III below:
TABLE-US-00003 TABLE III Combination between clonal complexes and
phenotype/genotype of the inner core ST complexes 41/44 32 8 11 Sum
of (lineage III) (ET-5) 269 18 (cluster A4) (ET-37) the strains
Distribution of the 6 major epidemiological complexes in the
invasive strains of serogroup B 25% 20% 7% 4% 4% 4% 64% No lpt3-
lgtG- lpt6- 6 7 No substitution lpt3+ lgtG Off lpt6- 1 4.3%
substitution deleted lpt3- Glu-3 lpt3- lgtG On lpt6- 10 Glu-3 lpt3+
lgtG On lpt6- 9 1 6.13% PEA-3 lpt3+ lgtG- lpt6- 33 14 92 PEA-3
lpt3+ lgtG Off lpt6- 44 1 56.44% PEA-3 lpt3+ lgtG- lpt6+ 32 PEA-3
PEA-6 lpt3+ lgtG Off lpt6+ 13 19 19.63% PEA-6 Glu-3 lpt3- lgtG On
lpt6+ 21 Glu-3 PEA-6 lpt3+ lgtG On lpt6+ 13 8 12.88% PEA-6 PEA-6
lpt3- lgtG- lpt6+ 1 PEA-6 lpt3- lgtG Off lpt6+ 1 0.60% Number of
strains tested 34 53 14 7 28 27 163
[0046] Moreover, the genotyping study relating to the lot3 gene
reveals that the vast majority of the strains tested O-acetylate
their LOS (lot3 gene present and "On").
BRIEF DESCRIPTION OF THE FIGURES
[0047] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0048] FIG. 1 shows the structure of the LOS of N. meningitidis,
the various sites at which the variability is expressed and also
the levels of intervention of the genes.
[0049] FIG. 2 shows the results of the genotyping analysis of the
lgtA, lgtB, lgtC, lgtE and lgtH; lgtF; lgtG and lpt6; lpt3 and lot3
genes that participate in the biosynthesis of LPS.
[0050] FIG. 3 shows the ELISA titers expressed as log.sub.10 of the
anti-LOS IgGs of the rabbit sera of groups A, B, C, D, E and F in
the Immunogenicity study No. 1 in rabbits.
[0051] In coherence with the results of the genotyping study
relating to the lgtG, lpt6 and lpt3 genes, a vaccine composition
intended to prevent or treat infections caused by N. meningitidis
is proposed, which comprises one or more LOSs of N. meningitidis;
this being (i) so as to treat or prevent at least 70%,
advantageously at least 75% and preferably at least 80% of
infections caused by N. meningitidis, especially caused by N.
meningitidis serogroup B or (ii) to vaccinate against 75 to 90%,
advantageously from 75 to 100%, preferably from 80 to 90% and most
preferably particularly from 80 to 100% of infections caused by N.
meningitidis, especially of serogroup B.
[0052] To this end, the choice of an LOS bearing an .alpha. chain
of L6 type and a heptose II residue of the inner core substituted
in the O-3 position with a phosphoethanolamine residue proves to be
particularly advantageous. Now, as reported previously in Table I
above, the L6 immunotype strains bear only one PEA substituent in
position 6/7.
[0053] Thus, when the vaccine composition comprises only one LOS,
the latter has to be necessarily optimized. When the vaccine
composition comprises at least two LOSs, one of the at least two
LOSs must necessarily have been optimized; the second may be a
natural (non-optimized) LOS.
[0054] In order to be able to manufacture a vaccine according to
the invention, three strain manufacturing processes are first
proposed, listed as follows: [0055] (i) a process of making a
strain of N. meningitidis exhibiting a lipooligosaccharide (LOS) in
particular constituted by (comprising) a lipid A, an inner core, an
.alpha. chain of L6 type, in which the heptose II residue of the
inner core bears in position O-3 and in position O-6 or O-7 a
phosphoethanolamine (PEA) substituent; wherein an N. meningitidis
strain of immunotype L6 is modified such that it expresses an lpt3
gene; [0056] (ii) a process of making an N. meningitidis strain
exhibiting a lipooligosaccharide (LOS) in particular constituted by
(comprising) a lipid A, an inner core, an .alpha. chain of L6 type,
in which the heptose II residue of the inner core bears in position
O-3 a phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7; wherein an N. meningitidis
strain of immunotype L6 is modified such that it expresses an lpt3
gene and such that it no longer expresses the lpt6 gene; and [0057]
(iii) a process of making an N. meningitidis strain exhibiting a
lipooligosaccharide (LOS) in particular constituted by a lipid A,
an inner core, an .alpha. chain of L8 type, in which the heptose II
residue of the inner core bears in position O-3 and in position O-6
or O-7 a phosphoethanolamine (PEA) substituent; wherein an N.
meningitidis strain of immunotype L8 is modified such that it
expresses an lpt6 gene.
[0058] The N. meningitidis serogroup against which it is imperative
to propose a vaccine in priority is the serogroup B (vaccines
against the other prevalent serogroups A, C, Y and W135 are already
available). Purification of the LOS from a strain of serogroup B
may lead to a product containing the undesirable residual B capsule
in the vaccine. To overcome these difficulties, it has now been
found that an ad hoc LOS derived from strains of serogroup A can
satisfy the needs in terms of vaccination against the serogroup B.
This is why, in the manufacturing processes (i) and (ii) according
to the invention, an N. meningitidis strain of immunotype L6,
serogroup A is preferably used as starting strain.
[0059] A strain that may be used in the manufacturing processes may
express an LOS bearing a non-detoxified lipid A. In particular, the
msbB gene may be functional.
[0060] A strain of this type is the strain C708 filed on 11 March
2008 at the Collection Nationale de Culture de Microorganisme, 25
rue du Dr Roux 75015 Paris, according to the terms of the treaty of
Budapest. This strain bears the order number CNCM I-3942. This
strain bears, inter alia, an active lgtA gene (gene switched "ON");
an lgtB gene--(non-functional gene); an lgtG gene (switched "Off");
a truncated lpt3 gene; an active lpt6 gene; an active lot3 gene;
and an active msbB gene.
[0061] The strain C708 comprises a truncated lpt3 gene. To modify
it such that the LOS bears a PEA substituent in position O3, the
functionality of the lpt3 gene can be restored, especially by
homologous recombination using a complete (full-length) lpt3 gene.
When this strain is used in process (ii) according to the
invention, it is also appropriate to deactivate the lpt6 gene so as
to obtain a strain that no longer expresses this gene. The
deactivation of this gene may especially be achieved by total or
partial deletion of the lpt6 gene or alternatively by insertion of
a non-pertinent sequence into the gene, for example an
antibiotic-resistant gene.
[0062] According to another aspect, a subject of the invention is
also: [0063] (i) an N. meningitidis strain, especially of serogroup
A, exhibiting a lipooligosaccharide (LOS) in particular constituted
by a lipid A, an inner core, an .alpha. chain of L6 type, in which
the heptose II residue of the inner core bears in position O-3 and
in position O-6 or O-7 a phosphoethanolamine (PEA) substituent;
[0064] (ii) an N. meningitidis strain of serogroup A, which
exhibits a lipooligosaccharide (LOS) in particular constituted by a
lipid A, an inner core, an .alpha. chain of L6 type, in which the
heptose II residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7; [0065] (iii) a
lipooligosaccharide (LOS) of N. meningitidis in particular
constituted by a lipid A that may or may not be detoxified, an
inner core, an .alpha. chain of L6 type, in which the heptose II
residue of the inner core bears in position O-3 and in position O-6
or O-7 a phosphoethanolamine (PEA) substituent; and [0066] (iv) a
lipooligosaccharide (LOS) of N. meningitidis in particular
constituted by a non-detoxified lipid A, an inner core, an .alpha.
chain of L6 type, in which the heptose II residue of the inner core
bears in position O-3 a phosphoethanolamine (PEA) substituent and
does not bear a PEA substituent in positions O-6 and O-7.
[0067] Advantageously, the strains according to the invention have
a functional lot3 gene, in "On" phase variation such that the LOS
that they produce is O-acetylated, at least partially, on their
.gamma. chain. The strains according to the invention may bear a
functional msbB gene and/or express an LOS in particular
constituted by a non-detoxified lipid A.
[0068] Advantageously, an LOS according to the invention bears a
.gamma. chain that is O-acetylated, at least partially.
[0069] An LOS included in the composition of a vaccine according to
the invention may be prepared according to one of the following
three processes: [0070] (i) a process for preparing a
lipooligosaccharide (LOS) of N. meningitidis in particular
constituted by a lipid A, an inner core, an .alpha. chain of L6
type, in which the heptose II residue of the inner core bears in
position O-3 and in position O-6 or O-7 a phosphoethanolamine (PEA)
substituent; wherein (a) a strain obtained from the manufacturing
process (i) according to the invention or the strain (i) according
to the invention is cultured, and (b) the LOS is harvested from the
culture obtained in step (a); [0071] (ii) a process for preparing a
lipooligosaccharide (LOS) of N. meningitidis in particular
constituted by a lipid A, an inner core, an .alpha. chain of L6
type, in which the heptose II residue of the inner core bears in
position O-3 a phosphoethanolamine (PEA) substituent and does not
bear a PEA substituent in positions O-6 and O-7; wherein (a) a
strain obtained from the manufacturing process (ii) or the strain
(ii) according to the invention is cultured, and (b) the LOS is
harvested from the culture obtained in step (a); and [0072] (iii) a
process for preparing a lipooligosaccharide (LOS) of N.
meningitidis in particular constituted by a lipid A, an inner core,
an .alpha. chain of L8 type, in which the heptose II residue of the
inner core bears in position O-3 and in position O-6 or O-7 a
phosphoethanolamine (PEA) substituent; wherein (a) a strain
obtained from the manufacturing process (iii) is cultured and (b)
the LOS is harvested from the culture obtained in step (a).
[0073] In each of the three preparation processes, the LOS may be
harvested either in a form combined with outer membrane vesicles
(OMVs), or extracted so as to be purified thereafter.
[0074] The first case amounts to harvesting OMVs that contain LOS.
Such OMVs may be isolated according to many techniques. See, for
example, WO 04/014417, Fredriksen et al., NIPH Annals (1991) 14:
67-79; Zollinger et al., J. Clin Invest (1979) 63: 836-848;
Saunders et al., Infect. Immun. (1999) 67: 113-119; and Drabick et
al., Vaccine (1999) 18: 160-172.
[0075] These techniques may be divided into two major groups; those
using deoxycholate (DOC) at a concentration of about 0.5%; and
those using lower concentrations or no DOC at all. The techniques
that dispense with DOC have the advantage of concentrating the LOS
in the OMVs (about 10-fold more LOS than in the OMVs extracted via
a technique using DOC). However, the techniques using low doses of
DOC may also be advantageous in that they can reduce the residual
toxicity of the LOS while at the same time making it possible to
obtain advantageous concentrations of LOS. Once extracted, the OMVs
can be purified by ultrafiltration or diafiltration techniques
known to those skilled in the art and described, for example, in
Frasch et al. "Outer membrane protein vesicle vaccines for
meningococcal disease" in Methods in Molecular Medicine, vol. 66,
Meningococcal Vaccines: Methods and Protocols (2001): 81-107
(Edited by A J. Pollard and M. C. Maiden, Humana Press Totowa,
N.J.).
[0076] In the second case, the LOS is extracted from a bacterial
culture and then purified according to standard methods. Many
purification processes are described in the literature. By way of
example, mention is made of Gu & Tsai, 1993, Infect. Immun. 61
(5): 1873, Wu et al., 1987, Anal. Biochem.160: 281 and U.S. Pat.
No. 6,531,131. An LPS preparation can also be quantified by using
known techniques. A practical method consists in assaying the KDO
by HPAEC-PAD chromatography (high performance anion exchange
chromatography).
Detoxification of the LOS
[0077] For incorporation into a vaccine, the LOS needs to be
detoxified. The toxicity of the LOS is due to its lipid A. However,
it is neither imperative to remove the lipid A in its entirety; nor
to modify it, for example by mutation (e.g. msbB minus mutation).
In fact, since the toxicity is more particularly linked to a
supramolecular conformation conferred by all the fatty acid chains
borne by the disaccharide nucleus of the lipid, according to one
advantageous embodiment, it is sufficient to act on these
chains.
[0078] The detoxification can be obtained according to various
approaches: chemical, enzymatic or genetic or alternatively by
complexation with a peptide analog of polymyxin B or alternatively
by incorporation/formulation into liposomes.
[0079] The level of detoxification of the LOS can be assessed inter
alia according to one of the two following standard tests: [0080]
the pyrogenic test in rabbits. This test, the calculations and the
reading thereof have been implemented according to the principles
set out in the European Pharmacopoeia (Edition 6.0, paragraph
2.6.8.). [0081] the LAL test (Limulus Amebocyte Lysate) implemented
according to the principles set out in the European Pharmacopoeia
(Edition 6.0, paragraph 2.6.14.).
Detoxification via the Chemical Route
[0082] The chemical approach consists in treating the LOS with a
chemical agent. According to one particular embodiment, the LOS is
subjected to mild acid hydrolysis with acetic acid which removes
the lipid A and also the branched KDO(s) when it (they) is (are)
present in the LOS structure. Such a treatment is, for example,
described in Gu & Tsai Infect. Immun. (1993) 61: 1873.
According to an alternative and preferred embodiment, the LOS is
subjected to a de-O-acylation, preferably a primary de-O-acylation,
i.a. by treatment with hydrazine, which hydrolyzes the esterified
primary fatty acid chains of the lipid A. Such a treatment is, for
example, described in U.S. Pat. No. 6,531,131, Gupta et al, Infect.
Immun. (1992) 60 (8): 3201 and Gu et al, Infect. Immun. (1996) 64
(10): 4047.
Detoxification via the Enzymatic Route
[0083] The enzymatic approach consists in placing the LOS in the
presence of lipases capable of digesting the esterified fatty acid
chains of the lipid A. Such lipases are produced by the amoeba
Dictyostelium discoideum. According to one particularly
advantageous embodiment, the amoeba and a Gram-negative bacterium
that can be phagocytosed by the amoeba, such as N. meningitidis,
are cultured together (coculture). The supernatant is then
recovered and the LOS is extracted from the supernatant which is
then free of fatty acid chains. It may also be an acyloxyacyl
hydrolase produced by certain human cells (patent WO 87/07297
Munford R.) or by Salmonella typhimurium (Trent et al 2001 J. Biol.
Chem. 276: 9083-9092; Reynolds et al. 2006 J. Biol. Chem. 281:
21974-21987) (enzyme encoded by the PagL or LpxR genes in the
latter case).
Detoxification via the Genetic Route
[0084] The genetic approach consists in using an LOS produced by a
bacterial strain of which the genotype is such that the entity of
the LOS normally responsible for its toxicity (lipid A, and more
particularly the lipid tails of lipid A) has a greatly reduced or
even nonexistent degree of toxicity. Such a bacterial strain can be
conveniently obtained by mutation. Starting from a wild-type strain
(i.e. a strain producing a toxic LOS), this then involves
inactivating, by mutation, certain genes involved in the
biosynthesis of the fatty acid chains, or in the attachment thereof
to the disaccharide nucleus of lipid A. Thus, it is possible to
envision inactivating the lpxL1 or lpxL2 genes (also called
htrB1/htrB2) of N. meningitidis or equivalents thereof in other
species (for example, the equivalents of the meningococcal lpxL1
and lpxL2 genes are respectively called msbB or 1pxM and htrB or
lpxL in E. coli.). A mutation that inactivates one of these genes
results in an LOS devoid of one or of two secondary acyl chains.
lpxL1 or L2 mutants of N. meningitidis or of Haemophilus influenzae
are in particular described in patent applications WO 00/26384, US
2004/0171133 and WO 97/019688. In N. meningitidis, the endogenous
lpxA gene can also be replaced with the homologous gene originating
from E. coli or Pseudomonas aeruginosa. The fatty acid chains
thereof are modified, resulting in a less toxic lipid A (Steeghs et
al, Cell. Microbiol. (2002) 4 (9): 599). The genetic approach is
favored when the LOS is purified in the form of OMVs.
LOS Detoxified by Complexation with a Peptide Analog of Polymyxin
B
[0085] A fourth approach consists in complexing the LOS with a
peptide analog of polymyxin B, as is, for example, described in
patent application WO 06/108586. The LOS that is complexed and
consequently detoxified is called endotoxoid.
[0086] The polymyxin B analog included in the composition of an
endotoxoid that is useful for the purposes of the present invention
may be any peptide that is capable of detoxifying the LPS by simple
complexation. Such peptides are especially described in patent or
patent applications U.S. Pat. No. 6,951,652, EP 976 402 and WO
06/108 586.
[0087] Thus, an advantageous peptide may be the peptide of formula
(I) NH.sub.2-A-Cys1-B-Cys2-C--COOH (SEQ ID NO: 1), in which: [0088]
A is a peptide of 2 to 5 and preferably 3 or 4 amino acid residues,
in which at least 2 amino acid residues are independently chosen
from Lys, Hyl (hydroxylysine), Arg and His; [0089] B is a peptide
of 3 to 7 and preferably 4 or 5 amino acid residues, which
comprises at least two and preferably three amino acid residues
chosen from Val, Leu, Ile, Phe, Tyr and Trp; and [0090] C is
optional (this position may or may not be empty) and is an amino
acid residue or a peptide formed from 2 to 3 amino acid residues;
on condition that the cationic amino acid/hydrophobic amino acid
ratio in the peptide of formula I is from 0.4 to 2, advantageously
from 0.5 to 1.2 or 1.5, preferably from 0.6 to 1; better still from
0.6 to 0.8; for example 0.75.
[0091] Preferably, in the peptide of formula (I), position C is an
empty position.
[0092] Particular examples of the peptide of formula (I) are the
following peptides:
TABLE-US-00004 (SEQ ID NO: 2)
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Lys-Lys-Cys2-COOH (peptide
SAEP2); (SEQ ID NO: 3)
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH (peptide
SAEP2-L2);. (SEQ ID NO: 4)
NH.sub.2-Lys-Arg-His-Hy1-Cys1-Lys-Arg-Ile-Val-Leu-Cys2- COOH; (SEQ
ID NO: 5) NH.sub.2-Lys-Arg-His-Cys1-Val-Leu-Ile-Trp-Tyr-Phe-Cys2-
COOH; (SEQ ID NO: 6)
NH.sub.2-Lys-Thr-Lys-Cys1-Lys-Phe-Leu-Leu-Leu-Cys2-COOH; and (SEQ
ID NO: 7) NH.sub.2-Hy1-Arg-His-Lys-Cys1-Phe-Tyr-Trp-Val-Ile-Leu-
Cys2-COOH.
[0093] The peptides of formula (I) may be in monomer form or,
preferably, in parallel or antiparallel dimer form.
[0094] In general, use may also be made of a dimeric peptide of
formula (II)
##STR00002##
in which the two Cys1 residues are linked together via a disulfide
bridge and the two Cys2 residues are linked together via a
disulfide bridge; or of formula (III)
##STR00003##
in which the Cys1 residues are linked to the Cys2 residues via
peptide inter-chain disulfide bridges; in which formulae (II) and
(III): [0095] A and A' are, independently, a peptide of 2 to 5 and
preferably 3 or 4 amino acid residues, in which at least 2 amino
acid residues are independently chosen from Lys, Hyl
(hydroxylysine), Arg and His; [0096] B and B' are, independently, a
peptide of 3 to 7 and preferably 4 or 5 amino acid residues, which
comprise at least two and preferably three amino acid residues
independently chosen from Val, Leu, Ile, Phe, Tyr and Trp; and
[0097] C and C' are optional (these positions may or may not be
empty) and are, independently, an amino acid residue or a peptide
of 2 to 3 amino acid residues; on condition that the cationic amino
acid/hydrophobic amino acid ratio in the dimer of formula (II) or
(III) is from 0.4 to 2, advantageously from 0.5 to 1.2 or 1.5,
preferably from 0.6 to 1 and better still from 0.6 to 0.8; for
example. 0.75.
[0098] Advantageously, A and A' are, independently, a peptide of 2
to 5 and preferably 3 or 4 amino acid residues, in which at least
one and preferably 2 amino acid residues are independently chosen
from Lys, Hyl, Arg and His; and, where appropriate, those that are
not chosen from Lys, Hyl, Arg and His ("the remaining amino acid
residues") being chosen from the group of uncharged, polar or
nonpolar amino acid residues; preferably Thr, Ser and Gly; most
particularly preferably Thr.
[0099] When A and A' comprise 3 amino acid residues, each of them
may be a cationic residue; or alternatively, two of the three
residues are cationic amino acids, whereas the remaining residue is
chosen from the group of uncharged, polar or nonpolar amino acid
residues; preferably Thr, Ser and Gly; most particularly preferably
Thr.
[0100] When A and A' comprise 4 amino acid residues, it is
preferable for two or three of the four residues to be chosen from
the groups of cationic amino acid residues as defined above,
whereas the remaining residue(s) is(are) chosen from the group of
uncharged, polar or nonpolar amino acid residues as defined
above.
[0101] When A and A' comprise 5 amino acid residues, it is
preferred for three or four of the five residues to be chosen from
the groups of cationic amino acid residues as defined above,
whereas the remaining residue(s) is(are) chosen from the group of
uncharged, polar or nonpolar amino acid residues as defined
above.
[0102] Advantageously, B and B' are, independently, a peptide of 3
to 7 and preferably 4 or 5 amino acid residues, which comprises at
least two and preferably three amino acid residues independently
chosen from Val, Leu, Ile, Phe, Tyr and Trp; preferably Leu, Ile
and Phe; and, where appropriate, those that are not chosen from
Val, Leu, Ile, Phe, Tyr and Trp ("the remaining amino acid
residues") being chosen independently from the group formed by Lys,
Hyl, Arg and His. As may readily be understood, B and B' may
comprise up to 7 amino acid residues independently chosen from Val,
Leu, Ile, Phe, Tyr and Trp.
[0103] Advantageously, B and B' comprise the sequence -X1-X2-X3-,
in which X1 and X2; X2 and X3; or X1, X2 and X3 are independently
chosen from Val, Leu, Ile, Phe, Tyr and Trp; preferably from Leu,
Ile and Phe. In one preferred embodiment, the sequence -X1-X2-X3-
comprises the Phe-Leu unit.
[0104] The particular embodiments of B and B' include: [0105] (i)
the sequence -X1-X-X3- in which: [0106] X1 is Lys, Hyl, His or Arg,
preferably Lys or Arg; preferably Lys; [0107] X2 is Phe, Leu, Ile,
Tyr, Trp or Val; preferably Phe or Leu; more particularly
preferably Phe; and [0108] X3 is Phe, Leu, Ile, Tyr, Trp or Val;
preferably Phe or Leu; more particularly preferably Leu; and [0109]
(ii) where appropriate, the amino acid residues are each
independently chosen from the group formed by Val, Leu, Ile, Phe,
Tyr, Trp, Lys, Hyl, Arg and His; preferably Val, Leu, Ile, Phe, Tyr
and Trp; more particularly preferably Leu, Ile and Phe.
[0110] When B and B' comprise more than 4 nonpolar amino acid
residues, A and A' preferably comprise at least 3 positively
charged amino acid residues.
[0111] In C and C', the amino acid residues may be any amino acid
residue, on condition that the cationic amino acid
residues/hydrophobic amino acid residues ratio remains in the
indicated range. Advantageously, they are independently chosen from
uncharged, polar or nonpolar amino acid residues, the latter being
preferred. However, preferably, the positions C and C' are empty
positions.
[0112] Consequently, a preferred class of the dimers is of formula
(IV)
##STR00004##
in which the two Cys1 residues are linked together via a disulfide
bridge and the two Cys2 residues are linked together via a
disulfide bridge; or of formula (V)
##STR00005##
in which the Cys1 residues are linked to the Cys2 residues via
peptide inter-chain disulfide bridges; in which formulae (IV) and
(V), in which A, A', B and B' are as described above; on condition
that the cationic amino acid/hydrophobic amino acid ratio in the
dimer of formula (IV) or (V), is from 0.4 to 2, advantageously from
0.5 to 1.2 or 1.5, preferably from 0.6 to 1 and better still from
0.6 to 0.8; for example 0.75.
[0113] In formulae (II) to (V), A and A' are preferably identical.
This is likewise the case as regards B and B', on the one hand, and
C and C', on the other hand. A dimer of formula
[0114] (II) to (V), in which A and A'; B and B'; and C and C' are
identical in pairs, is designated as a homologous dimer.
[0115] For these dimers, mention may be made, for example, of the
parallel and antiparallel dimers formed from the peptide
SAEP2-L2:
##STR00006##
[0116] The endotoxoid that is useful for the purposes of the
present invention may advantageously be characterized by an
LOS/peptide mole ratio from 1/1.5 to 1/0.5, preferably from 1/1.2
to 1/0.8, and most particularly preferably from 1/1.1 to 1/0.9,
e.g. 1/1.
LOS in Detoxified Liposomes
[0117] When the LOS is formulated in liposomes, it does not appear
to be necessarily required to detoxify it beforehand. This is
because LOS in liposomes--i.e. associated with the lipid bilayer
forming the liposomes--may experience a very substantial decrease
in toxicity. The size of this decrease, which can be as much as a
substantial loss, depends partly on the nature of the components
forming the liposome. Thus, when positively charged components
(components of cationic nature) are used, the loss of toxicity may
be greater than with uncharged (neutral) or anionic components.
[0118] The term "liposomes" is intended to mean a synthetic entity,
preferably a synthetic vesicle, formed of at least one lipid
bilayer membrane (or matrix) enclosing an aqueous compartment. For
the purposes of the present invention, the liposomes may be
uni-lamellar (a single bilayer membrane) or multi-lamellar (several
membranes layered like an onion). The lipids constituting the
bilayer membrane comprise a non-polar region which, typically, is
made of chain(s) of fatty acids or of cholesterol, and a polar
region, typically made of a phosphate group and/or of tertiary or
quaternary ammonium salts. Depending on its composition, the polar
region may, in particular at physiological pH (pH.apprxeq.7) carry
either a negative (anionic lipid) or positive (cationic lipid) net
(overall) surface charge, or not carry a net charge (neutral
lipid).
[0119] For the purposes of detoxifying the LOS, the liposomes may
be liposomes of any type; in particular, they may be in particular
constituted of any lipid known to be of use in the production of
liposomes. The lipid(s) that go(es) to make up the composition of
the liposomes may be neutral, anionic or cationic lipid(s); the
latter being preferred. These lipids may be of natural origin
(plant or egg extraction products, for example) or synthetic
origin; the latter being preferred. The liposomes may also be in
particular constituted of a mixture of these lipids; for example,
of a cationic or anionic lipid and of a neutral lipid, as a
mixture. In the latter two cases, the neutral lipid is often
referred to as co-lipid. According to one advantageous mixture
embodiment, the charged (cationic or anionic) lipid: neutral lipid
mole ratio is between 10:1 and 1:10, advantageously between 4:1 and
1:4, preferably between 3:1 and 1:3, limits included.
[0120] With regard to the neutral lipids, mention is made, by way
of example, of: (i) cholesterol; (ii) phosphatidylcholines such as,
for example, 1,2-diacyl-sn-glycero-3-phosphocholines, e.g.
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and also
1-acyl-2-acyl-sn-glycero-3-phosphocholines of which the acyl chains
are different than one another (mixed acyl chains); and (iii)
phosphatidylethanolamines such as, for example,
1,2-diacyl-sn-glycero-3 -phosphoethanolamines, e.g.
1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), and also
1-acyl-2-acyl-sn-glycero-3-phosphoethanolamines bearing mixed acyl
chains.
[0121] With regard to the anionic lipids, mention is made, by way
of example, of: (i) cholesteryl hemisuccinate (CHEMS); (ii)
phosphatidylserines such as
1,2-diacyl-sn-glycero-3-[phospho-L-serine]s, e.g.
1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), and
1-acyl-2-acyl-sn-glycero-3-[phospho-L-serine]s bearing mixed acyl
chains; (iii) phosphatidylglycerols such as
1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)]s, e.g.
1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG), and
1-acyl-2-acyl-sn-glycero-3-[phospho-rac-(1-glycerol)]s bearing
mixed acyl chains; (iv) phosphatidic acids such as
1,2-diacyl-sn-glycero-3-phosphates, e.g.
1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), and
1-acyl-2-acyl-sn-glycero-3-phosphates bearing mixed acyl chains;
and (v) phosphatidylinositols such as
1,2-diacyl-sn-glycero-3-(phosphoinositol)s, e.g.
1,2-dioleoyl-sn-glycero-3 -(phosphoinositol) (DOPI), and
1-acyl-2-acyl-sn-glycero-3-(phosphoinositol)s bearing mixed acyl
chains.
[0122] With regard to the cationic lipids, mention is made, by way
of example, of: [0123] (i) lipophilic amines or alkylamines such
as, for example, dimethyldioctadecylammonium (DDA),
trimethyldioctadecylammonium (DTA) or structural homologs of DDA
and of DTA [these alkylamines are advantageously used in the form
of a salt; mention is made, for example, of
dimethyldioctadecylammonium bromide (DDAB)]; [0124] (ii)
octadecenoyloxy(ethyl-2-heptadecenyl-3-hydroxyethyl)imidazolinium
(DOTIM) and structural homologs thereof; [0125] (iii) lipospermines
such as N-palmitoyl-D-erythrosphingosyl-1-O-carbamoylspermine (CCS)
and dioctadecylamidoglycylspermine (DOGS, transfectam); [0126] (iv)
lipids incorporating an ethylphosphocholine structure, such as
cationic derivatives of phospholipids, in particular phosphoric
ester derivatives of phosphatidylcholine, for example those
described in patent application WO 05/049080 and including, in
particular: [0127]
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, [0128]
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, [0129]
1,2-palmitoyloleoyl-sn-glycero-3-ethylphosphocholine, [0130]
1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSPC), [0131]
1,2-dioleyl-sn-glycero-3-ethylphosphocholine (DOEPC or EDOPC or
ethyl-DOPC or ethyl PC), [0132] and also structural homologs
thereof; [0133] (v) lipids incorporating a trimethylammonium
structure, such as
N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium (DOTMA) and
structural homologs thereof and those incorporating a
trimethylammonium propane structure, such as
1,2-dioleyl-3-trimethylammonium propane (DOTAP) and structural
homologs thereof; and also lipids incorporating a dimethylammonium
structure, such as 1,2-dioleyl-3-dimethylammonium propane (DODAP)
and structural homologs thereof; and [0134] (vi) cationic
derivatives of cholesterol, such as
3.beta.-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol
(DC-Chol) or other cationic derivatives of cholesterol, such as
those described in U.S. Pat. No. 5,283,185, and in particular
cholesteryl-3.beta.-carboxamidoethylenetrimethylammonium iodide,
cholesteryl-3.beta.-carboxyamidoethyleneamine,
cholesteryl-3.beta.-oxysuccinamidoethylenetrimethylammonium iodide
and 3.beta.-[N-(polyethyleneimine)carbamoyl] cholesterol.
[0135] The term "structural homologs" signifies lipids which have
the characteristic structure of the reference lipid while at the
same time differing therefrom by virtue of secondary modifications,
especially in the nonpolar region, in particular of the number of
carbon atoms and of double bonds in the fatty acid chains.
[0136] These fatty acids, which are also found in the neutral and
anionic phospholipids, are, for example, dodecanoic or lauric acid
(C12:0), tetradecanoic or myristic acid (C14:0), hexadecanoic or
palmitic acid (C16:0), cis-9-hexadecanoic or palmitoleic acid
(C16:1), octadecanoic or stearic acid (C18:0), cis-9-octadecanoic
or oleic acid (C18:1), cis, cis-9,12-octadecadienoic or linoleic
acid (C18:2), cis-cis-6,9-octadecadienoic acid (C18:2),
all-cis-9,12,15-octadecatrienoic or a-linolenic acid (C18:3),
all-cis-6,9, 12-octadecatrienoic or .gamma.-linolenic acid (C18:3),
eicosanoic or arachidic acid (C20:0), cis-9-eicosenoic or gadoleic
acid (C20:1), all-cis-8,11,14-eicosatrienoic acid (C20:3),
all-cis-5,8,11,14-eicosatetraenoic or arachidonic acid (C20:4),
all-cis-5,8,11,14, 17-eicosapentaneoic acid (C20:5), docosanoic or
behenic acid (C22:0), all-cis-7,10,13,16, 19-docosapentaenoic acid
(C22:5), all-cis-4,7,10,13,16,19-docosahexaenoic acid (C22:6) and
tetracosanoic or lignoceric acid (C24:0).
[0137] According to one particular embodiment, a mixture of
cationic lipid and neutral lipid is used. By way of example,
mention is made of: [0138] a mixture of DC-chol and DOPE, in
particular in a DC-chol:DOPE mole ratio ranging from 10:1 to 1:10,
advantageously from 4:1 to 1:4, preferably from approximately 3:1
to 1:3; [0139] a mixture of ethyl-DOPC and cholesterol, in
particular in an ethyl-DOPC:cholesterol mole ratio ranging from
10:1 to 1:10, advantageously from 4:1 to 1:4, preferably from
approximately 3:1 to 1:3; and [0140] a mixture of ethyl-DOPC and
DOPE, in particular in an ethyl-DOPC:DOPE mole ratio ranging from
10:1 to 1:10, advantageously from 4:1 to 1:4, preferably from
approximately 3:1 to 1:3.
[0141] According to one advantageous method of preparation, in an
initial step, a dry lipid film is prepared with all the compounds
that go to make up the composition of the liposomes. The lipid film
is then rein particular constituted in an aqueous medium, in the
presence of LOS, for example in a lipid:LOS mole ratio of 100 to
500, advantageously of 100 to 400, preferably of 200 to 300, most
particularly preferably of approximately 250. In general, it is
considered that this same mole ratio should not substantially vary
at the end of the method of preparing the LOS liposomes.
[0142] In general, the reconstitution step in an aqueous medium
results in the spontaneous formation of multilamellar vesicles, the
size of which is subsequently homogenized by gradually decreasing
the number of lamellae by extrusion, for example using an extruder,
by passing the lipid suspension, under a nitrogen pressure, through
polycarbonate membranes having decreasing pore diameters (0.8, 0.4,
0.2 .mu.m). The extrusion process can also be replaced with another
process using a detergent (surfactant) which disperses lipids. This
detergent is subsequently removed by dialysis or by adsorption onto
porous polystyrene microbeads with a particular affinity for
detergent (BioBeads). When the surfactant is removed from the lipid
dispersion, the lipids reorganize in a double layer.
[0143] At the end of the incorporation of the LOS into liposomes, a
mixture in particular constituted of ad hoc liposomes and of LOS in
free form may commonly be obtained. Advantageously, the liposomes
are then purified in order to be rid of the non-detoxified LPS in
free form.
Conjugation of LOS
[0144] In a vaccine according to the invention, the LOS is
advantageously in the form of a LOS/polypeptide carrier conjugate,
in particular when it is not in the form of OMVs or liposomes.
[0145] The carrier polypeptide can be any carrier polypeptide,
oligopeptide or protein in use in the conjugated vaccines field;
and in particular pertussis, diphtheria or tetanus toxoid, the
diphtheria toxin mutant named CRM197, a bacterial OMP or a
bacterial protein complex (for example, N. meningitidis OMPC
(outer-membrane protein Complex)), Pseudomonas exotoxin A,
Haemophilus influenzae lipoprotein D, Streptococcus pneumoniae
pneumolysine, Bordetella pertussis filamentous hemagglutinin and
the subunit B of the human transferrin receptor of N. meningitidis
(TbpB).
[0146] Many methods of conjugation exist in the technical field.
Some are listed, for example, in patent applications EP 941 738 and
WO 98/31393.
[0147] In general, the reactive groups of the LOS involved in the
conjugation are those of the inner core or of lipid A. It may
involve, inter alia, the acid function of the KDO, or else an
aldehyde generated subsequent to an appropriate treatment on the
disaccharide of lipid A. For example, a phosphatase treatment
generates an aldehyde on the structure of the second glucosamine of
lipid A from N. meningitidis (Brade H. (2002) J. Endotoxin Res. 8
(4): 295 Mieszala et al, (2003) Carbohydrate Res. 338: 167 and Cox
et al, (2005) Vaccine 23 (5): 5054).
[0148] Advantageously, the method of conjugation makes use (i) of a
bifunctional linking agent (linker) or (ii) of a spacer and of a
linker.
[0149] For example, in the first case, the LOS is activated with a
bifunctional coupling agent (linker) of formula R1-A-R2, such that
the R2 radical reacts with a reactive group of the KDO or of the
lipid A in order to obtain an activated LOS; the activated LOS is
then conjugated with the polypeptide such that the R1 substituent
reacts with a functional group borne by the polypeptide, in order
to obtain a conjugate.
[0150] For example, in the second case, the LOS is derivatized with
a spacer of formula R3-B-R4 such that the R3 radical reacts with a
reactive group of the KDO or of the lipid A in order to obtain a
derivatized LOS; the derivatized LOS is then activated with a
bifunctional coupling agent (linker) of formula R1-A-R2 such that
the R2 radical reacts with the R4 radical in order to obtain a
derivatized and activated LOS; finally, the derivatized and
activated LOS is conjugated with the polypeptide such that the R1
radical reacts with a functional group borne by the polypeptide in
order to obtain a conjugate.
[0151] In the second case, the process can also be carried out in
the following way: the protein is derivatized with a spacer of
formula R3-B-R4 such that the R4 radical reacts with a functional
group borne by the polypeptide; the LOS is activated with a
bifunctional linker of formula R1-A-R2 such that the R2 radical
reacts with a reactive group of the KDO or of the lipid A, in order
to obtain an activated LOS; and then the activated LOS is
conjugated with the derivatized protein such that the R1 radical of
the activated LOS reacts with the R3 radical of the derivatized
polypeptide, in order to obtain a conjugate.
[0152] In the formula of the spacer, B may be a carbon chain,
preferably carbonyl, alkyl or alkylene, for example C1 to C12. R3
and R4 may independently be a thiol or amine group or a residue
bearing same, for example a hydrazide group, i.e.
NH.sub.2--NH--CO--. Compounds that may be used as a spacer have,
for example, the formula NH.sub.2--B--NH.sub.2, or preferably
NH.sub.2--B--SH and NH.sub.2--B--S--S--B'--NH.sub.2. By way of
particular example, mention is made of: cysteamine, cysteine,
diamines, e.g. diaminohexane, adipic acid dihydrazide (ADH), urea
and cystamine.
[0153] In the formula of the linker, A may be an aromatic or
preferably aliphatic chain which is substituted or unsubstituted
and which advantageously contains from 1 to 12 carbon atoms,
preferably 3 to 8 carbon atoms. For example, A may be a C2 to C8
alkylene, a phenylene, a C7 to C12 aralkylene, a C2 to C8 alkyl, a
phenyl, a C7 to C12 aralkyl, a C6 alkanoyloxy or a
benzylcarbonyloxy, which may be substituted or unsubstituted.
[0154] The R2 radical is the functional group of the linker which
creates the link with the LOS or with the derivatized LOS. Thus, R2
is a functional group which can react with a carboxyl, hydroxyl,
aldehyde or amine group. If the linker must react with a hydroxyl,
carboxyl or aldehyde group, R2 is preferably an amine group or a
residue carrying an amine group, for example a hydrazide group,
i.e. NH.sub.2--NH--CO--. If the linker must react with an amine
group, R2 is preferably a carboxyl, succinimidyl (e.g.
N-hydroxy-succinimidyl) or sulfosuccinimidyl (e.g.
N-hydroxysulfosuccinimidyl) group.
[0155] Thus, compounds that can be used as a linker may be chosen
from adipic acid dihydrazide (ADH); sulfosuccinimidyl
6-(3-[2-pyridyldithio]propionamido)hexanoate (Sulfo-LC-SPDP);
succinimidyl 6-(3[2-pyridyldithio]propionamido)hexanoate (LC-SPDP);
N-succinimidyl-S-acetyl thioacetate (SATA); N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP), succinimidyl
acetylthiopropionate (SATP);
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC);
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl
(4-iodoacetyl)aminobenzoate (SIAB); succinimidyl
4-(p-maleimidophenyl)butyrate (SMPB); bromoacetic
acid-N-hydroxysuccinimide (BANS) ester;
dithiobis(succinimidylpropionate) (DTSSP);
H-(.gamma.-maleimidobutyryloxy)succinimide ester (GMBS);
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate;
N-succinimidyl-4-(4-maleimidophenyl) butyrate;
N-[.beta.-maleimidocaproic acid] hydrazide (BMCH);
N-succinimidyl-4-maleimidobutyrate; and
N-succinimidyl-3-maleiimidobenzoate.
[0156] By way of example, it is proposed to use the acid function
of the KDO in order to derivatize the LOS with ADH in the presence
of a carbodiimide [e.g. 3-ethyl-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDAC)]. The amine function thus
introduced is then reacted with the carboxyl functions of the
polypeptide, in the presence of EDAC, after having protected the
amine functions of the latter (Wu et al (2005) Vaccine 23: 5177) or
having converted them to acid functions (succinylation of the
protein; Pavliakova et al, Infect. Immun. (1999) 67 (10):
5526).
[0157] Alternatively, it is proposed to use the acid function of
the KDO in order to derivatize the LOS with cysteamine or cysteine
in the presence of EDAC. The thiol function thus introduced is then
reacted with the maleimide function of a homobifunctional linker,
such as bismaleimidohexane; or a heterobifunctional linker, such as
GMBS. In the first case, the maleimide function thus introduced is
then reacted with the thiol functions of the polypeptide. In the
second case, the succinimidyl function of the derivatized and
activated LOS is reacted with the amine functions of the
polypeptide.
[0158] Depending on the method of conjugation selected, the LOS and
the polypeptide can be conjugated to one another in an
LOS:polypeptide mole ratio of from 10.sup.-1 to 10.sup.2,
advantageously from 1 to 10.sup.2, preferably from 1 to 50; most
particularly preferably of approximately 20.
[0159] As stated previously, a subject of the invention is a
pharmaceutical composition for combating infections caused by N.
meningitidis, especially of serogroup B, which comprises: [0160]
(i) an N. meningitidis LOS, in particular constituted by a
non-detoxified lipid A, an inner core, an .alpha. chain of L6 type,
in which the heptose II residue of the inner core bears in position
O-3 a phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7 or an LOS obtained according
to the preparation process (ii); and, optionally, [0161] (ii) an N.
meningitidis LOS in particular constituted by a lipid A, an inner
core, an .alpha. chain of L6 type, in which the heptose II residue
of the inner core bears in position O-3 and in position O-6 or O-7
a phosphoethanolamine (PEA) substituent or an LOS obtained
according to the preparation process (i); and, optionally, [0162]
(iii) an N. meningitidis LOS in particular constituted by a lipid
A, an inner core, an .alpha. chain of L6 or L8 type, in which the
heptose II residue of the inner core bears in position O-6 or O-7 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in position O-3.
[0163] A subject of the invention is also a pharmaceutical
composition for combating infections caused by N. meningitidis,
especially of serogroup B, which comprises an N. meningitidis LOS
in particular constituted by a lipid A, an inner core, an .alpha.
chain of L6 type, in which the heptose II residue of the inner core
bears in position O-3 and in position O-6 or O-7 a
phosphoethanolamine (PEA) substituent; or obtained according to
preparation process (i).
[0164] As indicated previously, the latter composition may also
comprise an LOS obtained according to preparation process (ii) or
an N. meningitidis LOS in particular constituted by a lipid A, an
inner core, an .alpha. chain of L6 type, in which the heptose II
residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7; and also an N. meningitidis
LOS in particular constituted by a lipid A, an inner core, an
.alpha. chain of L6 type, in which the heptose II residue of the
inner core bears in position O-6 or O-7 a phosphoethanolamine (PEA)
substituent and does not bear a PEA substituent in position
O-3.
[0165] A subject of the invention is also a pharmaceutical
composition for combating infections caused by N. meningitidis,
especially of serogroup B, which comprises: [0166] (i) an LOS of N.
meningitidis, preferably of serogroup A, in particular constituted
by a lipid A, an inner core, an .alpha. chain of L8 type, in which
the heptose II residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7; and [0167] (ii) an N.
meningitidis LOS, in particular constituted by a non-detoxified
lipid A, an inner core, an .alpha. chain of L6 type, in which the
heptose II residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7 or an LOS obtained according
to preparation process [0168] (ii); or an N. meningitidis LOS in
particular constituted by a lipid A, an inner core, an .alpha.
chain of L6 type, in which the heptose II residue of the inner core
bears in position O-3 and in position O-6 or O-7 a
phosphoethanolamine (PEA) substituent; or obtained according to
preparation process (i).
[0169] In the latter composition, the LOS of point (i) may
advantageously be the LOS of a strain of immunotype L8.
[0170] The invention also relates to a pharmaceutical composition
against infections caused by N. meningitidis, especially of
serogroup B, which comprises: [0171] (i) an LOS of N. meningitidis,
preferably of serogroup A, in particular constituted by a lipid A,
an inner core, an .alpha. chain of L8 type, in which the heptose II
residue of the inner core bears in position O-3 and in position O-6
or O-7 a phosphoethanolamine (PEA) substituent; and [0172] (ii) an
N. meningitidis LOS, in particular constituted by a non-detoxified
lipid A, an inner core, an .alpha. chain of L6 type, in which the
heptose II residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7 or an LOS obtained according
to preparation process (ii); or an N. meningitidis LOS in
particular constituted by a lipid A, an inner core, an .alpha.
chain of L6 type, in which the heptose II residue of the inner core
bears in position O-3 and in position O-6 or O-7 a
phosphoethanolamine (PEA) substituent; or obtained according to
preparation process (i).
[0173] In the latter composition, the LOS of point (i) may
advantageously be the LOS of the strain C708 lpt3FL lpt6TR
lgtA::erm or the LOS of the strain A1 (Zhu, Klutch & Tsai, FEMS
Microbiology Letters (2001) 203: 173 and Gu, Tsai & Karpas, J.
Clin. Microbiol. (August 1992) 30 (8):2047).
[0174] Thus, a divalent vaccine composition i.a. according to the
invention may be made up in various ways. The following 6 examples
are mentioned: [0175] 1) A vaccine composition according to the
invention, which comprises: [0176] (i) an LOS obtained according to
preparation process (i) or an N. meningitidis LOS in particular
constituted by a lipid A, an inner core, an .alpha. chain of L6
type, in which the heptose II residue of the inner core bears in
position O-3 and in position O-6 or O-7 a phosphoethanolamine (PEA)
substituent; and [0177] (ii) an LOS obtained according to
preparation process (ii) or an N. meningitidis LOS in particular
constituted by a lipid A, an inner core, an .alpha. chain of L6
type, in which the heptose II residue of the inner core bears in
position O-3 a phosphoethanolamine (PEA) substituent and does not
bear a PEA substituent in positions O-6 and O-7, [0178] 2) A
vaccine composition according to the invention, which comprises:
[0179] (i) an LOS obtained according to preparation process (i) or
an N. meningitidis LOS in particular constituted by a lipid A, an
inner core, an .alpha. chain of L6 type, in which the heptose II
residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7; and [0180] (ii) an N.
meningitidis LOS in particular constituted by a lipid A, an inner
core, an .alpha. chain of L6 type, in which the heptose II residue
of the inner core bears in position O-6 or O-7 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in position O-3, [0181] 3) A vaccine composition
according to the invention which comprises: [0182] (i) an LOS
obtained according to preparation process (i) or an N. meningitidis
LOS in particular constituted by a lipid A, an inner core, an
.alpha. chain of L6 type, in which the heptose II residue of the
inner core bears in position O-3 a phosphoethanolamine (PEA)
substituent and does not bear a PEA substituent in positions O-6
and O-7; and [0183] (ii) an N. meningitidis LOS in particular
constituted by a lipid A, an inner core, an .alpha. chain of L8
type, in which the heptose II residue of the inner core bears in
position O-3 a phosphoethanolamine (PEA) substituent and does not
bear a PEA substituent in positions O-6 and O-7, [0184] 4) A
vaccine composition according to the invention which comprises:
[0185] (i) an LOS obtained according to preparation process (i) or
an N. meningitidis LOS in particular constituted by a lipid A, an
inner core, an .alpha. chain of L6 type, in which the heptose II
residue of the inner core bears in position O-3 and in position O-6
or O-7 a phosphoethanolamine (PEA) substituent; and [0186] (ii) an
N. meningitidis LOS in particular constituted by a lipid A, an
inner core, an .alpha. chain of L8 type, in which the heptose II
residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7, [0187] 5) A vaccine
composition according to the invention, which comprises: [0188] (i)
an LOS obtained according to preparation process (iii) or an N.
meningitidis LOS in particular constituted by a lipid A, an inner
core, an .alpha. chain of L8 type, in which the heptose II residue
of the inner core bears in position O-3 and in position O-6 or O-7
a phosphoethanolamine (PEA) substituent; and [0189] (ii) an LOS
obtained according to preparation process (ii) or an N.
meningitidis LOS in particular constituted by a lipid A, an inner
core, an .alpha. chain of L6 type, in which the heptose II residue
of the inner core bears in position O-3 a phosphoethanolamine (PEA)
substituent and does not bear a PEA substituent in positions O-6
and O-7, [0190] 6) A vaccine composition according to the
invention, which comprises: [0191] (i) an LOS obtained according to
preparation process (iii) or an N. meningitidis LOS in particular
constituted by a lipid A, an inner core, an .alpha. chain of L8
type, in which the heptose II residue of the inner core bears in
position O-3 and in position O-6 or O-7 a phosphoethanolamine (PEA)
substituent; and [0192] (ii) an LOS obtained according to
preparation process (ii) or an N. meningitidis LOS in particular
constituted by a lipid A, an inner core, an .alpha. chain of L6
type, in which the heptose II residue of the inner core bears in
position O-3 and in position O-6 or O-7 a phosphoethanolamine (PEA)
substituent.
[0193] A vaccine/pharmaceutical composition according to the
invention is especially useful for treating or preventing an
infection caused by N. meningitidis, such as meningitis caused by
N. meningitidis, meningococcemias and complications that may derive
therefrom such as purpura fulminans and septic shock; and also
arthritis and pericarditis caused by N. meningitidis.
[0194] It may be manufactured in a conventional manner. In
particular, a therapeutically or prophylactically effective amount
of the essential constituent of the vaccine, which is the LOS, is
combined with a pharmaceutically acceptable support or diluent.
Advantageously, it may also comprise a pharmaceutically acceptable
adjuvant.
[0195] For use in a composition according to the invention, the
LOS(s) (are) advantageously formulated as liposomes.
[0196] Additionally, a composition according to the invention may
comprise one or more additional vaccine antigens of N.
meningitidis; for example one or more N. meningitidis polypeptides.
In a particularly preferred form, a composition according to the
invention may comprise (i) the subunit B (TbpB) of the human
transferrin receptor, which is an outer membrane lipoprotein of a
certain number of non-enteric Gram-negative bacteria such as
Neisseriae, e.g. N. meningitidis; or (ii) a lipidated N-terminal
fragment thereof. In the latter case the lipidated TbpB or a
lipidated fragment thereof may act both as a vaccine antigen and as
an LOS adjuvant.
[0197] In N. meningitidis, the strains are divided into two
isotypes: isotypes I and II, which differ according to the length
of the TbpB amino acid chain (EP 560 969 and EP 586 266). With
regard to isotype I, the reference strain is strain B16B6. With
regard to isotype II, the reference strain is strain M982.
[0198] According to one advantageous embodiment, a composition
according to the invention additionally comprises N. meningitidis
isotype I or II lipidated TbpB or lipidated TbpB of each of isotype
I and II strains. To this end, the lipidated TbpB of N.
meningitidis isotype I strain may be that of strain B16B6; and the
lipidated TbpB of N. meningitidis isotype II strain may be that of
strain M982.
[0199] Therefore, it is cited as a matter of example: [0200] 1) A
vaccine composition of the invention, which comprises: [0201] (i) a
LOS obtained according to the preparation process (ii) or a N.
meningitidis LOS in particular in particular constituted by a lipid
A, an inner core, an .alpha. chain of L6 type, in which the heptose
II residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7; and [0202] (ii) the lipidated
TbpB of a N. meningitidis strain of isotype II or a lipidated
N-terminal fragment thereof. [0203] 2) A vaccine composition of the
invention, which comprises: [0204] (i) a LOS obtained according to
the preparation process (ii) or a N. meningitidis LOS in particular
in particular constituted by a lipid A, an inner core, an .alpha.
chain of L6 type, in which the heptose II residue of the inner core
bears in position O-3 a phosphoethanolamine (PEA) substituent and
does not bear a PEA substituent in positions O-6 and O-7; and
[0205] (ii) the lipidated TbpB of a N. meningitidis strain of
isotype II or a lipidated N-terminal fragment thereof; and [0206]
(iii) the lipidated TbpB of a N. meningitidis strain of isotype I
or a lipidated N-terminal fragment thereof. [0207] 3) A vaccine
composition of the invention, which comprises: [0208] (i) a LOS
obtained according to the preparation process (ii) or a N.
meningitidis LOS in particular in particular constituted by a lipid
A, an inner core, an .alpha. chain of L6 type, in which the heptose
II residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7; and [0209] (ii) a N.
meningitidis LOS in particular in particular constituted by a lipid
A, an inner core, an .alpha. chain of L8 type, in which the heptose
II residue of the inner core bears in position O-3 a
phosphoethanolamine (PEA) substituent and does not bear a PEA
substituent in positions O-6 and O-7; or A LOS obtained according
to the preparation process (iii); or a N. meningitidis LOS in
particular in particular constituted by a lipid A, an inner core,
an .alpha. chain of L8 type, in which the heptose II residue of the
inner core bears in position O-3 and in position O-6 or O-7, a
phosphoethanolamine (PEA) substituent; and [0210] (iii) the
lipidated TbpB of a N. meningitidis strain of isotype II or a
lipidated N-terminal fragment thereof; and optionally [0211] (iv)
the lipidated TbpB of a N. meningitidis strain of isotype I or a
lipidated N-terminal fragment thereof.
[0212] When a vaccine composition of the invention comprises one or
more TbpB(s) this (these) latter can be formulated with the LOS in
liposomes or be simply mixed with liposomes LOS (LOS formulated in
liposomes).
[0213] The amounts of LOS per vaccine dose which are sufficient to
achieve the abovementioned aims, and which are effective from an
immunogenic, prophylactic or therapeutic point of view, depend on
certain parameters that include the individual treated (adult,
adolescent, child or infant), the route of administration and the
administration frequency.
[0214] The amount of LOS per dose which is sufficient to achieve
the abovementioned aims is in particular between 5 and 500 .mu.g,
advantageously between 10 and 200 .mu.g, preferably between 20 and
100 .mu.g, entirely preferably between 20 and 80 .mu.g or between
20 and 60 .mu.g, limits included.
[0215] The term "dose" employed above should be understood to
denote a volume of vaccine administered to an individual in one
go--i.e. at a time T. Conventional doses are of the order of a
milliliter, for example 0.5, 1 or 1.5 ml; the definitive choice
depending on certain parameters, and in particular on the age and
the status of the recipient. An individual can receive a dose
divided up into injections at several injection sites on the same
day. The dose may be a single dose or, if necessary, the individual
may also receive several doses a certain time apart--it being
possible for this time apart to be determined by those skilled in
the art.
[0216] It may be administered by any conventional route in use in
the prior art, e.g. in the vaccines field, in particular enterally
or parenterally. The administration may be carried out as a single
dose or as repeated doses a certain time apart. The route of
administration varies as a function of various parameters, for
example of the individual treated (condition, age, etc.).
[0217] Finally, the invention also relates to: [0218] a method for
inducing in a mammal, for example a human, an immune response
against N. meningitidis, according to which an immunogenically
effective amount of a composition according to the invention is
administered to the mammal so as to induce an immune response, in
particular a protective immune response against N. meningitidis;
and [0219] a method for prevention and/or treatment of an infection
caused by N. meningitidis, according to which a prophylactically or
therapeutically effective amount of a composition according to the
invention is administered to an individual in need of such a
treatment.
Experimental Data
[0220] A Experimental Data Relating to the Strains Derived from N.
meningitidis C708
1. Materials & Methods
1.1 Transformation of Strain C708
[0221] Strain C708 is cultured in BHI (Brain Heart Infusion) agar
medium at 37.degree. C. under an atmosphere containing 10%
CO.sub.2. The bacterial lawn is harvested in BHI liquid medium
complemented with 5 mM MgCl.sub.2 to obtain a bacterial suspension
at 10.sup.9 cfu/ml (cfu: colony-forming unit).
[0222] To 900 .mu.l of the BHI liquid medium+5 mM MgCl.sub.2 are
added 10 .mu.g of DNA necessary for the transformation (linearized
plasmid); followed by 100 .mu.l of the bacterial suspension
(10.sup.8 microorganisms). The transformation medium is incubated
for 30 minutes at 37.degree. C., 10% CO.sub.2. 500 .mu.l of this
preparation (i.e. about 5.10.sup.7 cfu) serve to inoculate 4.5 ml
of BHI+5 mM MgCl2. The bacteria are left to regenerate for 2 hours
at 37.degree. C., 10% CO.sub.2. Next, starting with this
suspension, dilutions of BHI+5 mM MgCl.sub.2 are made. 300 .mu.l of
a dilution containing about 30 000 cfu are plated out on a 140 mm
agar BHI dish. The dishes are placed at 37.degree. C., 10% CO.sub.2
for at least 20 hours.
1.2. Blotting of the Transformant Colonies
[0223] The colonies are transferred onto 137 mm Hybond-XL membrane
(GE Healthcare; # RPN 137S). The microorganisms deposited on the
membrane are lysed in denaturing buffer (0.5 M NaOH, 1.5 M NaCl).
The membranes are washed with neutralizing buffer (0.5 M Tris, 1.5
M NaCl, pH 7.5); transferred into SSC 2.times. medium; and then
dried. The DNA is bound by incubation for 2 hours at 80.degree.
C.
1.3. Detection of the Transformants by Hybridization with a Probe
Labeled with .sup.33P dCTP
[0224] Labeling of the probe is obtained by PCR amplification using
the Ready-to-Go PCR Beads kit (GE Healthcare); the labeled probe is
then purified on a ProbeQuant G50 Microcolumn column (GE
Healthcare).
[0225] The membranes to be hybridized are placed in threes in 50 ml
of Rapid Hyb buffer (GE Healthcare) for 15 minutes at 65.degree.
C., with slow stirring, for prehybridization. The probe labeled and
denatured beforehand for 2 minutes at 95.degree. C. is added to the
membranes. The final probe concentration is 5 ng/ml. Hybridization
is allowed to continue for 2 hours at 65.degree. C., with slow
stirring.
[0226] The membranes are then subjected to successive washes by
working as follows: [0227] in low stringency buffer (2.times.SSC,
0.1% SDS (weight/vol)) 15 minutes at ambient temperature with slow
stirring; [0228] in medium stringency buffer (1.times.SSC, 0.1% SDS
(weight/vol)) 20 minutes at 65.degree. C. with slow stirring; and
[0229] low stringency buffer (0.1.times.SSC, 0.1% SDS (weight/vol))
45 minutes at 65.degree. C. with slow stirring.
[0230] Once dried, the membranes are revealed by autoradiography
(Biomax MR film).
1.4. Detection of the Transformants by Hybridization with an
Oligonucleotide Labeled with [.gamma..sup.32P] ATP
[0231] Labeling of the 5' end of the oligonucleotide is performed
in the following reaction medium (the amounts indicated are those
corresponding to the hybridization of an amount of oligonucleotide
necessary for the hybridization of 3 membranes in a dish):
TABLE-US-00005 free 5'-OH oligonucleotide 3 .mu.l max i.e. 10 pmol
10 X phosphorylation buffer 1 .mu.l i.e. 1 X [.gamma.-.sup.32P] ATP
10 mCi/ml 5 .mu.l i.e. 50 .mu.Ci T4 kinase (10 U/.mu.l) 1 .mu.l
i.e. 10 U H.sub.2O qs 10 .mu.l
[0232] The reaction medium is incubated at 37.degree. C. for 30
minutes. Next, the T4 kinase is inactivated by heating for 10
minutes at 70.degree. C.
[0233] The membranes to be hybridized are placed in threes in 60 ml
of Rapid Hyb buffer (GE Healthcare) for 15 minutes at 48.degree.
C., with slow stirring, for prehybridization. The prehybridization
buffer is removed and replaced with 50 ml of hybridization buffer
as follows: 5.times.SSC, 5.times. Denhardt's solution, 0.5% SDS
(weight/vol.) and 100 .mu.g/ml of salmon sperm DNA at 10 mg/ml
sonicated and denatured for 5 minutes at 100.degree. C.
[0234] The labeled oligonucleotide (10 .mu.l) is added to the
membranes. The hybridization is allowed to continue overnight at a
temperature 5.degree. C. below the Tm of the oligonucleotide, with
gentle stirring.
[0235] The membranes are then subjected to successive washes by
working in the order as follows: [0236] in low stringency buffer
(2.times.SSC, 0.1% SDS (weight/vol) 5 minutes at the Tm of the
oligonucleotide -5.degree. C., with slow stirring; [0237] in medium
stringency buffer (1.times.SSC, 0.1% SDS (weight/vol) 15 minutes at
the Tm of the oligonucleotide -5.degree. C., with slow stirring;
and [0238] in low stringency buffer (0.1.times.SSC, 0.1% SDS
(weight/vol) 10 minutes at the Tm of the oligonucleotide -5.degree.
C., with slow stirring.
[0239] Once dried, the membranes are revealed by autoradiography
(Biomax MR film).
2. Construction of A Strain of N. meningitidis Expressing An LOS
Having An .alpha. Chain Is That of An LOS of Immunotype L6 and
Comprising in Each of the Positions O3 and O6 of the Heptose II
(hep II) Residue of the Inner Core A Phosphoethanolamine (PEA)
Substituent
[0240] The starting strain used is N. meningitidis strain C708 of
serogroup A and immunotype L6 having, inter alia, the following
characteristics: [0241] an active lgtA gene (gene switched "ON");
[0242] an lgtB gene--(non-functional gene); [0243] an inactive lgtG
gene (switched "OFF"); [0244] a truncated lpt3 gene; [0245] an
active lpt6 gene; and [0246] an active lot3 gene.
[0247] Strain C708 was filed on 11 Mar. 2008 at the Collection
Nationale de Culture de Microorganisme, 25 rue du Dr Roux 75015
Paris, according to the terms of the treaty of Budapest. This
strain bears the order number CNCMI-3942.
[0248] Strain C708 comprises a truncated lpt3 gene. To modify it
such that the LOS bears a PEA substituent in position O3, it is
chosen to replace by homologous recombination the truncated lpt3
gene with the complete (full-length) lpt3 gene of the strain of N.
meningitidis FAM18 serogroup C (strain made available worldwide to
research laboratories). The strain resulting therefrom will be
referred to for greater convenience as C708 lpt3 FL.
2.1. PCR (Polymerase Chain Reaction) Amplification of the
Full-Length (FL) lpt3 Gene of N. meningitidis Strain FAM18
[0249] 100 ng of genomic DNA of the strain FAM18 were used for
amplification with Platinum.RTM. Taq DNA polymerase High Fidelity
(Invitrogen, #11304-011).
[0250] The pair of primers is as follows (pair No. 1):
TABLE-US-00006 (SEQ ID NO: 8) CG GAATTC GCC GTC TCA A ATG AAA AAA
TCC CTT TTC GTT CTC (Tm = 55.9.degree. C.); and (SEQ ID NO: 9) AA
CTGCAG TCA TTG CGG ATA AAC ATA TTC CG (Tm = 57.1.degree. C.); (the
EcoRI and PstI sites are respectively underlined).
[0251] The following mixture was used for amplification:
TABLE-US-00007 Components Volume Final concentration 10X High
Fidelity PCR buffer 5 .mu.l 1X 10 mM dNTP mixture 1 .mu.l 0.2 mM of
each 50 mM MgSO.sub.4 2 .mu.l 2 mM Mixture of primers (10 .mu.M
each) 1 .mu.l 0.2 .mu.M of each Genomic DNA x .mu.l 100 ng Platinum
.RTM. Taq High Fidelity 0.2 .mu.l 1.0 unit Nuclease-free water qs
50 .mu.l final Does not apply
[0252] The thermocycler program is as follows:
TABLE-US-00008 Initial denaturing: 94.degree. C. for 30 seconds 30
cycles of: denaturing: 94.degree. C. for 30 seconds hybridization:
55.degree. C. for 30 seconds extension: 68.degree. C. for 1
minute/kb of PCR product.
[0253] After the reaction, 1/10 of the PCR product was deposited on
agarose gel for verification.
2.2. Construction of a Transformation Vector
[0254] The PCR product, on the one hand, and the plasmid pUC19, on
the other hand, were subjected to double digestion with EcoRI and
PstI for 2 hours at 37.degree. C. 10 units of each enzyme per .mu.g
of DNA in REact2 buffer (Invitrogen) were used.
[0255] The PCR fragment was then inserted into the linearized pUC19
vector. The ligations were performed in a final volume of 20 .mu.l
with 50 ng of vector, 0.5 U of T4 DNA ligase (Invitrogen) and 1
.mu.l of 10 mM ATP (Invitrogen) for 16 hours at 16.degree. C. The
ligase was then inactivated by heating for 10 minutes at 65.degree.
C.
[0256] The vector thus obtained was transferred via the
electroporation technique into a strain of E. coli XL1 blue MRF
resistant to kanamycin and made electrocompetent. The parameters
adopted for the electroporation are as follows: capacitance: 500
.mu.FD; resistance: 200 ohms; voltage: 1700 volts.
[0257] Selection of the transformed clones was performed by plating
out on 100 .mu.g/ml ampicillin LB dishes. Authentification of 10 of
the 50 positive clones was performed by PCR amplification. 100% of
the clones had the expected profile. Finally, the lpt3 gene in a
plasmid of one of these clones (plasmid pM1222) was verified by
sequencing.
2.3. Transformation of Strain C708 and Detection of the Homologous
Recombination Event
2.3.1. Transformation
[0258] 40 .mu.g of pM1222 were digested with 400 U of EcoRI and 10
.mu.g were used to transform the strain C708 according to the
method described in section A.1.1.
[0259] After transformation, the bacteria were plated out onto 17
140 mm Petri dishes at a theoretical concentration of 30 000 cfu
per dish; i.e. 510 000 cfu (colony-forming units) and were then
placed overnight at 37.degree. C. The dishes were then placed at
+4.degree. C. for 30 minutes.
[0260] After 24 hours at 37.degree. C., counting of the control
dishes made it possible to estimate the number of cfu as 27 000 per
dish for the mutant.
2.3.2. Clone Selection
[0261] The recombination event, i.e. the replacement of the lpt3 TR
(truncated) gene with the lpt3 FL (full-length) gene, was detected
after transferring the clones onto hybridization membrane and
hybridizing with a probe labeled with .sup.33P dCTP according to
the methods described in sections A.1.2. and A.1.3.
[0262] Selection of the positive clones was made by hybridization
of the DNA bound to the membranes with a DNA probe labeled with
.sup.33P dCTP corresponding to the truncated part of the lpt3 gene,
which is thus present only in the recombinant clones.
Preparation of the Probe
[0263] In order to obtain the 270-bp lpt3 probe, 10 ng of the
plasmid pUClpt3 were used for PCR amplification with Platinum.RTM.
Taq DNA polymerase High Fidelity (Invitrogen, #11304-011).
[0264] The pair of primers is as follows (pair No. 2):
TABLE-US-00009 (SEQ ID NO: 10) CGC CGA ATA CTT TAT CTT GAG GC (Tm =
60.6.degree. C.); and (SEQ ID NO: 11) CTC GCC AAA GAG CAG GGC (Tm =
60.5.degree. C.).
[0265] For amplification, the following mixture was used:
TABLE-US-00010 Components Volume Final concentration 10X High
Fidelity PCR buffer 5 .mu.l 1X 10 mM dNTP mixture 1 .mu.l 0.2 mM of
each 50 mM MgSO.sub.4 2 .mu.l 2 mM Mixture of primers (10 .mu.M
each) 1 .mu.l 0.2 .mu.M of each Plasmid pUC x .mu.l 100 ng Platinum
.RTM. Taq High Fidelity 0.2 .mu.l 1.0 unit Nuclease-free water qs
50 .mu.l final Does not apply
[0266] The thermocycler program is as follows:
TABLE-US-00011 Initial denaturing: 94.degree. C. for 30 seconds 30
cycles of: Denaturing: 94.degree. C. for 30 seconds Hybridization:
55.degree. C. for 30 seconds Extension: 68.degree. C. for 45
seconds
[0267] After the reaction, 1/10 of the PCR products was deposited
on agarose gel to ensure the specificity of the amplicon, and the
PCR fragment was then purified using the QIAquick PCR Purification
Kit (Qiagen, #28104).
Hybridization and Revelation
[0268] The steps of labeling of the probe, hybridization, washing
and revelation were performed as described in section A.1.3.
[0269] About 460 000 cfu were tested. After exposure with the
BioMax MR films, the autoradiographs revealed 5 positive spots
(C708 containing an lpt3 FL gene) each on a different membrane.
Screening and Authentification of the Positive Clones
[0270] After locating on the Petri dish, part of the zone taken up
around the positive clones was stored in freezing medium (M199
medium, 20% fetal calf serum, 10% glycerol) and the other part was
used for the PCR authentification.
[0271] To do this, each of the samples collected was first taken up
in 80 .mu.l of BHI broth, so as to plate out 30 .mu.l of this
suspension, as a mini-lawn on a BHI dish.
[0272] The remaining volume was centrifuged for 5 minutes at 6000
rpm and the pellet was then taken up in 50 .mu.l of nuclease-free
water. The microorganisms were lysed for 5 minutes at 95.degree. C.
and the supernatant, which serves as the matrix for the PCR
reaction, was collected after centrifugation.
[0273] For each collected sample corresponding to a positive spot
and for the controls, PCR amplification with the pair of primers
that served for the amplification of the 270 bp C708 lpt3 probe
(pair No. 2) was performed with the Expand Long Template PCR kit
(Roche) as described below.
TABLE-US-00012 Components Volume Final concentration 10X ELT PCR
buffer 5 .mu.l 1X dNTP mixture (10 mM of each) 2 .mu.l 0.4 mM of
each mixture of primers (10 .mu.M of each) 1.5 .mu.l 0.3 .mu.M of
each DNA matrix 40 .mu.l Does not apply Polymerase ELT 0.75 .mu.l
3.75 units Nuclease-free water qs 50 .mu.l Does not apply
[0274] The thermocycler program is as follows:
TABLE-US-00013 Initial denaturing: 94.degree. C. for 2 min 10
cycles of: denaturing: 94.degree. C. for 10 seconds hybridization:
54.degree. C. for 30 seconds extension: 68.degree. C. for 45
seconds 20 cycles of: denaturing: 94.degree. C. for 15 seconds
hybridization: 54.degree. C. for 30 seconds extension: 68.degree.
C. for 45 seconds + 20 sec/cycle Final elongation: 68.degree. C.
for 7 min
[0275] After the reaction, 1/10 of the PCR products was deposited
on agarose gel for verification. Four of the 5 clones had the
expected profile. The frequency of production of a true positive
clone was 1/115 000 cfu tested.
[0276] The following step consisted in isolating a pure clone. To
do this, one of the heterogeneous positive clones was plated out as
isolated cfus and several of these cfus (40) were analyzed by PCR,
with the pairs of primers 1 or 2.
[0277] Each cfu was resuspended in 100 .mu.l of nuclease-free
water, 30 .mu.l were deposited on a BHI dish and the remaining 70
.mu.l were lysed for 5 minutes at 95.degree. C., and the
supernatant, which serves as the matrix for the PCR reaction, was
collected after centrifugation.
[0278] The PCRs were performed with Platinum.RTM. Taq High Fidelity
(Invitrogen) as already described for the amplification of the lpt3
probe. The hybridization temperature was 54.degree. C.
[0279] After the reaction, 1/10 of the PCR products was deposited
on agarose gel for verification. Five of the 40 clones proved to be
pure clones.
[0280] The mini-lawn of pure clones was taken up in freezing
medium, divided into 100 .mu.l aliquots and stored at -70.degree.
C. The purity and the identity of this freezing material were
validated.
3. Construction of A Strain of N. meningitidis Expressing An LOS
Having An .alpha. Chain is That of An LOS of Immunotype L6 And
Comprising Only in Position O3 of the heptose II (hep II) residue
of the inner core a phosphoethanolamine (PEA) substituent
[0281] The starting strain used is N. meningitidis strain C708 lpt3
FL obtained as described previously. The objective is to inactivate
the lpt6 gene of this strain by deletion of a central part of the
gene.
3.1. PCR (Polymerase Chain Reaction) Amplification of the
Full-Length lpt6 Gene of N. meningitidis Strain Z2491 of Serogroup
A (Gene NMA 0408)
[0282] 100 ng of genomic DNA of strain Z2491 (strain made available
worldwise to research laboratories) were used for amplification
with Platinum.RTM. Taq DNA polymerase High Fidelity (Invitrogen,
#11304-011).
[0283] The pair of primers is as follows (pair No. 3):
TABLE-US-00014 (SEQ ID NO: 12) CG GAATTC GCC GTC TCA A GGT TGC CTA
TGT TTT CCT GTT TTT G (Tm = 59.7.degree. C.); and (SEQ ID NO: 13)
AA CTGCAG CTA ACG GGC AAT TTT CAA AAC GTC (Tm = 59.3.degree. C.);
(the EcoRI and PstI sites are respectively underlined).
[0284] For amplification the following mixture was used:
TABLE-US-00015 Components Volume Final concentration 10X High
Fidelity PCR buffer 5 .mu.l 1X 10 mM dNTP mixture 1 .mu.l 0.2 mM of
each 50 mM MgSO.sub.4 2 .mu.l 2 mM Mixture of primers (10 .mu.M
each) 1 .mu.l 0.2 .mu.M of each Genomic DNA x .mu.l 100 ng Platinum
.RTM. Taq High Fidelity 0.2 .mu.l 1.0 unit Nuclease-free water qs
50 .mu.l final Does not apply
[0285] The thermocycler program is as follows:
TABLE-US-00016 Initial denaturing: 94.degree. C. for 30 seconds 30
cycles of: denaturing: 94.degree. C. for 30 seconds hybridization:
55.degree. C. for 30 seconds extension: 68.degree. C. for 1
minute/kb of PCR product.
[0286] After the reaction, 1/10 of the PCR product was deposited on
agarose gel for verification.
3.2. Construction of Vector pM1223 (pUC19 lpt6 FL)
[0287] The PCR product, on the one hand, and plasmid pUC19, on the
other hand, were subjected to double digestion with EcoRI and PstI
for 2 hours at 37.degree. C. 10 units of each enzyme per .mu.g of
DNA were used in the buffer REact2 (Invitrogen).
[0288] The PCR fragment was then inserted into the linearized pUC19
vector. Ligations were performed on a final volume of 20 .mu.l with
50 ng of vector, 0.5 U of T4 DNA ligase (Invitrogen) and 1 .mu.l of
10 mM ATP (Invitrogen) for 16 hours at 16.degree. C. The ligase was
then inactivated by heating for 10 minutes at 65.degree. C.
[0289] The vector thus obtained was transferred via the
electroporation technique into a strain of E. coli XL1 blue MRF
resistant to kanamycin and made electrocompetent. The parameters
adopted for the electroporation are as follows: capacitance: 500
.mu.FD; resistance: 200 ohms; voltage: 1700 volts.
[0290] Selection of the transformed clones was performed by plating
out onto 100 .mu.g/ml ampicillin LB dishes. Authentification of the
positive clones (presence of an lpt6 FL gene) was performed by NdeI
enzymatic digestion after extraction of the DNA by miniprep. Out of
20 clones analyzed, 6 had the expected profile. The recombinant
plasmid of the selected clone was named pM1223.
3.3. Deletion of the Central Part of the lpt6 Gene Originating from
Strain Z2491 Construction of a Transformation Vector
[0291] With the Expand Long Template PCR kit (Roche), a reverse PCR
was performed using the plasmid pM1223 with the aid of the
following pair of primers (pair No. 4):
TABLE-US-00017 (SEQ ID NO: 14) CG GGATCC CAT CGA CAC GAA CGC CGC
(Tm = 60.5.degree. C.); and (SEQ ID NO: 15) CG GGATCC CCG CGC TTA
ACG ACT ACA TC (Tm = 59.4.degree. C.); (the BamHI sites are
underlined).
[0292] This makes it possible to amplify again the plasmid while
deleting the part that it is desired to remove (808 bp).
[0293] The following mixture was used for amplification:
TABLE-US-00018 Components Volume Final concentration 10X ELT PCR
buffer 5 .mu.l 1X dNTP mixture (10 mM of each) 2 .mu.l 0.4 mM of
each Mixture of primers (10 .mu.M of each) 1.5 .mu.l 0.3 .mu.M of
each DNA matrix (pM1223) 40 .mu.l Does not apply Polymerase ELT
0.75 .mu.l 3.75 units Nuclease-free water qs 50 .mu.l Does not
apply
[0294] The thermocycler program is as follows:
TABLE-US-00019 Initial denaturing: 94.degree. C. for 2 minutes 10
cycles of: denaturing: 94.degree. C. for 10 seconds hybridization:
54.degree. C. for 30 seconds extension: 68.degree. C. for 3 minutes
20 cycles of: denaturing: 94.degree. C. for 15 seconds
hybridization: 54.degree. C. for 30 seconds extension: 68.degree.
C. for 3 minutes + 20 sec/cycle Final elongation: 68.degree. C. for
7 minutes
[0295] After the reaction, 1/10 of the PCR products was deposited
on agarose gel.
[0296] After purification on a QiaQuick column, the PCR product was
digested with BamHI at a rate of 10 U of enzyme per .mu.g of DNA.
Once digested, it was purified by electroelution and then extracted
with phenol-chloroform.
[0297] Self-ligation of the vector was performed in a final volume
of 20 .mu.l with 0.5 U of T4 DNA ligase (Invitrogen) and 1 .mu.l of
10 mM ATP (Invitrogen) for 16 hours at 16.degree. C. The ligase was
then inactivated by heating for 10 minutes at 65.degree. C.
[0298] The final step consisted in transferring the vector thus
ligated into E. coli as described for pM1222. Authentification of
the positive clones was performed by NdeI-PstI enzymatic digestion
after extraction of the DNA by miniprep. Out of the 4 clones
analyzed, 100% had the expected profile.
[0299] The recombinant plasmid of the selected positive clone was
named pM1224, and this clone was stored in glycerol at -70.degree.
C. The presence in the plasmid pM1224 of an lgt6 gene with its
central part deleted was confirmed by sequencing.
3.4. Transformation of Strain C708 lpt3 FL And Detection of the
Homologous Recombination Event
Transformation
[0300] 10 .mu.g of plasmid pM1224 were linearized with EcoRI at a
rate of 10 units of enzyme per .mu.g of plasmid to be digested in
the appropriate buffer for 2 hours at 37.degree. C.
[0301] The transformation of strain C708 with plasmid pM1224 was
performed according to the technique described in section
A.1.1.
[0302] After transformation, the bacteria were plated out on 16 140
mm Petri dishes at a theoretical concentration of 50 000 cfu per
dish; i.e. 800 000 cfu. The dishes were placed overnight at
37.degree. C. and then placed for 30 minutes at +4.degree. C.
Clone Selection: Preparation of the Probe, Hybridization And
Revelation
[0303] The recombination event was detected after colony blotting
and hybridization with an oligonucleotide labeled with
.gamma..sup.32P dATP.
[0304] The recombination event, i.e. the replacement of the lpt6 FL
gene with the lpt6 TR gene, was detected after transferring the
clones onto membrane and hybridization with a labeled probe
according to the methods described in sections A.1.2. and
A.1.4.
[0305] The clones transferred onto membranes were subjected to
lysis and washing steps. The DNA is bound to the membranes by
placing them for 2 hours at 80.degree. C.
[0306] Selection of the positive clones was performed by
hybridization of the DNA bound to Hybond N+ membranes with a
radioactive oligonucleotide whose sequence overlaps the two
recombigenic ends. This is the following oligonucleotide: GTC GAT
GGG ATC CCC GCG CTT AAC G (SEQ ID NO: 16) (Tm=69.5.degree. C.).
[0307] About 840 000 cfu were tested. After exposure with BioMax MR
films, the autoradiographs revealed 16 positive spots (C708
containing an lpt6 TR gene) divided among 9 different
membranes.
Screening And Authentification of the Positive Clones
[0308] For each of the 16 positive spots, after detection on the
Petri dish, part of the zone collected around the positive clones
was stored in freezing medium (M199, 20% FCS, 10% glycerol) and the
other part was used for the PCR authentification.
[0309] To do this, the collected samples were first taken up in 80
.mu.l of BHI broth, so as to plate out, as a mini-lawn on a BHI
dish, 30 .mu.l of each suspension.
[0310] The remaining volume was centrifuged for 5 minutes at 6000
rpm, and the pellet was then taken up in 50 .mu.l of nuclease-free
water. The microorganisms were lysed for 5 minutes at 95.degree. C.
and the supernatant, which serves as the PCR reaction matrix, was
collected after centrifugation.
[0311] For each collected sample corresponding to a positive spot,
a PCR amplification PCR was performed with the Platinum.RTM. Taq
High Fidelity kit (Invitrogen) and the following pair of primers
(pair No. 5)
TABLE-US-00020 (SEQ ID NO: 17) CCG ACT GGC GGA ATT GGG (TM =
60.5.degree. C.); and (SEQ ID NO: 18) CCC ATT TCT TCC TGA CGG AC
(Tm = 59.4.degree. C.).
[0312] The following mixture was used for amplification:
TABLE-US-00021 Components Volume Final concentration 10X High
Fidelity PCR buffer 5 .mu.l 1X 10 mM dNTP mixture 2 .mu.l 0.2 mM of
each 50 mM MgSO.sub.4 2 .mu.l 2 mM Mixture of primers (10 .mu.M
each) 1 .mu.l 0.2 .mu.M of each DNA matrix x .mu.l 100 ng Platinum
.RTM. Taq High Fidelity 0.2 .mu.l 1.0 unit Nuclease-free water qs
50 .mu.l final Does not apply
[0313] The thermocycler program is as follows:
TABLE-US-00022 Initial denaturing: 94.degree. C. for 1 minute 30
cycles of: denaturing: 94.degree. C. for 30 seconds hybridization:
55.degree. C. for 30 seconds extension: 68.degree. C. for 50
seconds.
[0314] After the reaction, 1/10 of the PCR product was deposited on
agarose gel, for verification. Two candidates out of 16 proved to
be true positives: i.e. a frequency of production of one true
positive clone per 425 000 cfu tested.
[0315] The following step consisted in isolating a pure clone. To
do this, one of the 2 hetero-geneous positive clones was plated out
as isolated cfus and several of these cfus (24) were analyzed by
PCR, with the pair of primers No. 5.
[0316] Each cfu was resuspended in 50 .mu.l of nuclease-free water,
20 .mu.l were deposited on a BHI dish and the remaining 30 .mu.l
were lysed for 5 minutes at 95.degree. C. and the supernatant,
which serves as PCR reaction matrix, was collected after
centrifugation.
[0317] The PCRs were performed with the Platinum.RTM. Taq High
Fidelity kit (Invitrogen) as already described for the screening of
the collected samples of the positive spots.
[0318] After the reaction, 1/5 of the PCR products was deposited on
agarose gel for verification.
[0319] Only one clone out of 24 proved to be a true positive.
[0320] The mini-lawn of the pure positive clone was taken up in
freezing medium, divided into 100 .mu.l aliquots and stored at
-70.degree. C. The purity and identity of this frozen material were
confirmed.
4. Construction of a N. meningitidis Strain Expressing An LOS
Having An .alpha. Chain Which is That of An LOS of Immunotype L8
and Comprising A Single Phosphoethanolamine (PEA) Substituent in
Position O3 of the Heptose II (hep II) Residue of the Inner
Core
[0321] N. meningitidis strain C708 lpt3 FL obtained as described
previously in section A.3. is used as starting strain. The goal is
to inactivate the lgtA gene of this strain by deletion of a central
part of the gene.
4.1. PCR (Polymerase Chain Reaction) Amplification of the
Full-Length lgtA Gene of N. meningitidis Strain MC58 of Serogroup B
(Gene NMB 1929)
[0322] 100 ng of genomic DNA of strain MC58 (strain made available
worldwise to research laboratories) were used for amplification
with Platinum.RTM. Taq DNA polymerase High Fidelity (Invitrogen,
#11304-011).
[0323] The pair of primers is as follows:
TABLE-US-00023 (SEQ ID NO: 19) CG GAATTC GCC GTC TCA A ATG CCG TCT
GAA GCC TTC AG (Tm = 59.4.degree. C.); and (SEQ ID NO: 20) AA
CTGCAG AAC GGT TTT TCA GCA ATC GGT (Tm = 60.6.degree. C.); (the
EcoRI and PstI sites are respectively underlined).
[0324] For amplification the following mixture was used:
TABLE-US-00024 Components Volume Final concentration 10X High
Fidelity PCR buffer 5 .mu.l 1X 10 mM dNTP mixture 1 .mu.l 0.2 mM of
each 50 mM MgSO.sub.4 2 .mu.l 2 mM Mixture of primers (10 .mu.M
each) 1 .mu.l 0.2 .mu.M of each Genomic DNA x .mu.l 100 ng Platinum
.RTM. Taq High Fidelity 0.2 .mu.l 1.0 unit Nuclease-free water qs
50 .mu.l final Does not apply
[0325] The thermocycler program is as follows:
TABLE-US-00025 Initial denaturing: 94.degree. C. for 30 seconds 30
cycles of: denaturing: 94.degree. C. for 30 seconds hybridization:
55.degree. C. for 30 seconds extension: 68.degree. C. for 1
minute/kb of PCR product.
[0326] After the reaction, 1/10 of the PCR product was deposited on
agarose gel for verification.
4.2. Construction of Vector pUC19 lgtA FL)
[0327] The PCR product obtained in A.4.1., on the one hand, and
plasmid pUC19, on the other hand, were submitted to a double
digestion with EcoRI and PstI for 2 hours at 37.degree. C. 10 units
of each enzyme per .mu.g of DNA were used in the buffer REact2
(Invitrogen).
[0328] The PCR fragment was then inserted into the linearized pUC19
vector. Ligations were performed under a final volume of 20 .mu.l
with 50 ng of vector, 0.5 U of T4 DNA ligase (Invitrogen) and 1
.mu.l of 10 mM ATP (Invitrogen) for 16 hours at 16.degree. C. The
ligase was then inactivated by heating for 10 minutes at 65.degree.
C.
[0329] The vector thus obtained was transferred by the
electroporation technique into E. coli strain XL1 blue MRF
kanamycin-resistant and made electro competent. The parameters
adopted for the electroporation are as follows: capacitance: 500
.mu.FD; resistance: 200 ohms; voltage: 1700 volts.
[0330] Selection of the transformed clones was performed by plating
out onto 100 .mu.g/ml ampicilline LB dishes. Authentification of
the positive clones (presence of an lgtA FL gene) was performed by
NdeI enzymatic digestion after extraction of the DNA by miniprep.
1/5 of the analyzed clones had the expected profile.
4.3. PCR Amplification of the Erythromycine (erm) Gene
[0331] PCR amplification of the erythromycine cassette (erm) was
achieved using plasmid pMCG10 as template with primers allowing the
introduction of restriction sites BamHI and XbaI.
[0332] The primer pair is as follows:
TABLE-US-00026 (SEQ ID NO: 21) CG GGATCC GGA AGG CCC GAG CGC AGA
AGT (Tm: 65.7.degree. C.); and (SEQ ID NO: 22) GC TCT AGA CAA CTT
ACT TCT GAC AAC GAT CGG (Tm: 61.degree. C.)
[0333] For amplification the following mixture was used:
TABLE-US-00027 Components Volume Final concentration 10X Pfu turbo
buffer 5 .mu.l 1X 10 mM dNTP mixture 0.4 .mu.l 0.2 mM of each 50 mM
MgSO.sub.4 2 .mu.l 2 mM Mixture of primers (10 .mu.M each) 1 .mu.l
0.2 .mu.M of each pMGC10 x .mu.l 10 ng Pfu turbo (Stratagene) 0.2
.mu.l 2.5 units Nuclease-free water qs 50 .mu.l final Does not
apply
[0334] The thermocycler program is as follows:
TABLE-US-00028 Initial denaturing: 95.degree. C. for 30 seconds 30
cycles of: denaturing: 95.degree. C. for 30 seconds hybridization:
55.degree. C. for 30 seconds extension: 72.degree. C. for 10
min.
4.4. Construction of Plasmid pUC19 lgtA TR (Deletion of the Central
Part of the lgtA Gene by Reverse PCR)
[0335] With the Expand Long Template PCR kit (Roche), a reverse PCR
was achieved using plasmid pUC19 with the double objective of
removing the central part of lgtA and of creating two restriction
sites (BamHI and XbaI). The following pair of primers was used:
TABLE-US-00029 (SEQ ID NO: 23) CG GGATCC GCC AAT TCA TCC AGC CCG
ATG (Tm = 61.8.degree. C.); and (SEQ ID NO: 24) CG TCTAGA CCC GGT
TCG ACA GCC TTG (Tm = 60.5.degree. C.); (the BamHI and XbaI sites
are underlined).
[0336] This makes it possible to amplify AGAIN the plasmid while
deleting the part that it is desired to remove.
[0337] The following mixture was used for amplification:
TABLE-US-00030 Components Volume Final concentration 10X ELT PCR
buffer 5 .mu.l 1X dNTP mixture (10 mM of each) 2 .mu.l 0.4 mM of
each Mixture of primers (10 .mu.M of each) 1.5 .mu.l 0.3 .mu.M of
each 10 ng of pUC19 lgtA FL Does not apply Polymerase ELT 0.75
.mu.l 3.75 units Nuclease-free water qs 50 .mu.l Does not apply
[0338] The thermocycler program is as follows:
TABLE-US-00031 Initial denaturing: 94.degree. C. for 2 minutes 10
cycles of: denaturing: 94.degree. C. for 10 seconds hybridization:
55.degree. C. for 30 seconds extension: 68.degree. C. for 1 minute
per kbs 20 cycles of: denaturing: 94.degree. C. for 15 seconds
hybridization: 55.degree. C. for 30 seconds extension: 68.degree.
C. for 1 minute per kbs + 20 sec/cycle Final elongation: 68.degree.
C. for 7 minutes
[0339] After the reaction, 1/10 of the PCR products was deposited
on agarose gel for verifying the size (3.2 kbs). The PCR product
was purified on a QiaQuick column.
[0340] The final step has consisted in transferring the plasmid
into E. coli strain XL1 blue MRF kanamycin-resistant and made
electrocompetent as described above e.g. for pM1222.
Authentification of the positive clones (lgtA with a deleted
central region) was performed by enzymatic digestion after
extraction of the DNA by miniprep.
4.5. Construction of Plasmid pUC19 lgtA::erm
[0341] The PCR product erm, on the one hand, and the plasmid pUC19
lgtA TR obtained by reverse PCR, on the other hand, were submitted
to a double digestion with BamHI and XbaI under the following
conditions:
[0342] 2 .mu.g of DNA were mixed with 20 units of XbaI in 60 .mu.L
of buffer 2 (InVitrogen) for 2 hours 37.degree. C. Then XbaI was
inactivated by heating. 7 .mu.L of NaCl 1 M, 20 BamHI units and 1
.mu.L of buffer 2 were added. The reaction was performed for 2
hours at 37.degree. C.
[0343] The digestion products were then deposited on a 0.8% agarose
gel and after migration, bands were cut out for further
electroelution (plasmid band migrates at 3.2. kbs).
[0344] Upon purification, the linearized plasmid and the digested
PCR product erm were ligated together under ligation conditions as
described previously. The ligation product has been used to
transform as previously described, E. coli strain XL1 blue MRF
kanamycin-resistant and made electro-competent. The recombinant
clones were analyzed by enzymatic digestion. 4/11 of the analyzed
clones had the expected digestion profile.
4.6. Transformation of Strain C708 lpt3 FL lpt6 TR and Detection of
the Homologous Recombination Event
[0345] 10 .mu.g of plasmid pUC19 lgtA::erm were linearized with
EcoRI at a rate of 10 units of enzyme per .mu.g of plasmid to be
digested in the appropriate buffer for 2 hours at 37.degree. C.
[0346] The transformation of strain C708 was performed according to
the technique described in section A.1.1.
[0347] After transformation, 1.24 10.sup.8 bacteria were plated out
on BHI+2 .mu.g /mL erythromycine and incubated overnight at
37.degree. C. The transformation rate was 1/2.5 10.sup.6.
B. Experimental Data Relating to the Vaccine Compositions
[0348] 1. Preparation of Lipidated rTbpB M982 and B16B6
[0349] In the interest of simplifying the language and reading, the
term "rTbpB" or "TbpB" will subsequently be simply indicated.
1.1. Production
[0350] Strains Expressing rTbpB M982 and B16B6
[0351] The expression strains are E. coli BL21 strains respectively
containing plasmids TG9219 and TG9216. These plasmids contain in
particular a kanamycin-selectable marker and the polynucleotide
encoding the rTbpB from N. meningitidis strain M982 (pTG9219) or
B16B6 (pTG9216) (the sequences of which are as described in patent
EP 586 266) fused to the E. coli RlpB (real lipoprotein B) signal
sequence and placed under the control of the arabinose promoter
(araB).
Culture
[0352] Three frozen samples of E. coli BL21/pTG9219 or E. coli
BL21/pTG9216 strain (each 1 ml) are used to inoculate 3 liters of
LB (Luria Broth) medium divided up in Erlenmeyer flasks. The
incubation is continued for 15 to 18 h at 37.degree. C.
[0353] This preculture is used to inoculate a fermenter containing
TGM16 medium (9 g/L yeast extract, 0.795 g/L K.sub.2SO.sub.4, 3.15
g/L K.sub.2HPO.sub.4, 0.75 g/L NaCl, 0.005 g/L
CaCl.sub.2.2H.sub.2O, 0.021 g/L FeCl.sub.3.6H.sub.2O, 0.69 g/L
MgSO.sub.4.7H.sub.2O, 37.5 g/L salt-free casein acid hydrolysate)
supplemented with 20 g/L glycerol, in a proportion of 10%
(vol./vol).
[0354] The culturing is continued at 37.degree. C. with shaking, at
a pressure of 100 mbar and with an air feed of 1 L/min/L of
culture, while readjusting, over time, the glycerol concentration
to 20 g/L (e.g. at OD.sub.600 of 15.+-.2). When the OD.sub.600 is
between 21 and 27, the rTbpB expression is induced by adding
arabinose so as to obtain a final concentration of 10 g/L. After
one hour of induction, the culture is stopped by cooling to around
10.degree. C.
[0355] The bacterial pellets are recovered by centrifugation and
stored in the cold.
1.2. Purification
[0356] Extraction of Membranes Containing the rTbpB
LOS Extraction
[0357] A bacterial pellet equivalent to one liter of culture
(approximately 72 g of microorganisms, wet weight) is thawed at a
temperature of 20.degree. C. +/-5.degree. C. The thawed (or
partially thawed) microorganisms are resuspended with 800 ml of a
solution, at ambient temperature, of 50 mM Tris HCl, 5 mM EDTA, pH
8.0. 9 protease inhibitor tablets (7 Complete Mini, EDTA free
tablets; ROCHE ref 11836170001+two Complete, EDTA free tablets;
ROCHE ref 11836170001) are immediately added. Since some of the
microorganisms lyze spontaneously, 4 .mu.l of benzonase (1 IU of
DNAse activity/ml final concentration; Merck ref. K32475095) are
also added. The incubation is continued at +4.degree. C. for 45
minutes with magnetic stirring after homogenization with a Turrax
(15 sec.).
[0358] 4 ml of 1M MgCl.sub.2 are then added so as to be at a final
concentration of 5 mM. The magnetic stirring is continued for 10
minutes. Centrifugation at 15 000 g for 45 minutes makes it
possible to harvest the pellet (pellet P1; versus supernatant 51)
containing the rTbpB protein.
[0359] A second extraction is carried out: homogenization with a
Turrax in 800 ml of the 50 mM Tris HCl buffer containing 5 mM EDTA,
pH 8.0, and stirring for 30 min. MgCl.sub.2 (8 ml of a molar
solution) is added. The incubation is continued for 10 minutes. The
suspension is centrifuged at 15 000 g for 1 hour 30.
Bacterial Lysis
[0360] The pellet is resuspended with 1400 ml of 50 mM Tris HCl
supplemented with 4 protease inhibitor tablets with 8 .mu.l of
benzonase. The solution is homogenized with a Turrax for 15
seconds. The lysis is carried out at +4.degree. C. for 30 minutes
through the addition of 14 ml (10 mg/ml final concentration) of
lysozyme at 100 mg/ml in 25 mM Na acetate, 50% glycerol.
[0361] The suspension is centrifuged at 30 000 g for 30 minutes
(pellet P2 containing the protein; versus supernatant S2 containing
the contaminants of rTbpB). The pellet containing the membranes can
be frozen at this stage.
Washing of Membrane Fragments
[0362] The lysis pellet P2 is taken up in 50 mM Tris HCl (1100 ml).
After homogenization, (Turrax 15 seconds), it is washed for one
hour at +4.degree. C. A centrifugation is carried out as previously
at 30 000 g for 30 minutes. The pellet (P3; versus supernatant S3)
is frozen at -45.degree. C. 50 mM Tris HCl buffer makes it possible
to remove a small amount of protein (supernatant S3) and
solubilizes only very little rTbpB.
[0363] The pellet P3 is taken up in 50 mM Tris HCl buffer
containing 8M urea, pH 8.0 (800 ml). This buffer makes it possible
to remove a part of the contaminating proteins without solubilizing
the membranes containing the rTbpB. After homogenization (without
using a Turrax), the solution is then stirred for one hour at
+4.degree. C. A centrifugation is carried out as previously at 30
000 g for 30 minutes, which makes it possible to obtain a membrane
pellet which can be frozen.
Membrane Solubilization
[0364] The thawed membrane pellet is solubilized with 780 ml of 50
mM Tris HCl buffer containing 6 mM EDTA, 2M urea and 4% elugent, at
pH 7.5. The presence of the detergent at 4% and of the 2M urea
makes it possible to solubilize the pellet. The solution is stirred
at +4.degree. C. overnight (minimum 16 h). Centrifugation of the
solution at 30 000 g (1 hour at +4.degree. C.) leaves only a small
pellet (P4) containing a few impurities. The supernatant S4
containing the rTbpB protein is recovered for loading on a first
cation exchange column (QS I).
Purification by Anion Exchange Chromatography on Q Sepharose at pH
7.5
[0365] Two successive chromatographies are carried out. The product
of the first chromatography is collected and then subsequently
loaded, after a dialysis step, on a second chromatography column
which uses different conditions (absence of EDTA).
1.sup.st Chromatography, in the Presence of EDTA (Chromatography QS
I)
[0366] A column of 600 ml (K50, diameter 20 cm.sup.2) of Q
Sepharose Fast Flow gel (ref 17-0510-01 GE Healthcare) is mounted,
tamped in equilibration buffer, 50 mM Tris HCl containing 6 mM
EDTA, 2M urea and 1% elugent, at pH 7.5, at the flow rate of 8
ml/minute.
[0367] The supernatant S4 (approximately 845 ml) is loaded at the
flow rate of 6 ml/minute. The direct eluate (part which does not
attach to the column during loading of the sample) contains the
protein of interest, rTbpB. This eluate (1150 ml) is taken and then
dialyzed at +4.degree. C. (for 6 days) against 6 liters of 50 mM
Tris HCl buffer containing 2 M urea and 1% elugent, pH 7.5, in
order to reduce the EDTA concentration to 1 mM and to remove
NaCl.
2.sup.nd Chromatography (QS II), without EDTA
[0368] A K50 column of 490 ml of new Q Sepharose Fast Flow gel is
equilibrated in 50 mM Tris HCl buffer containing 2 M urea and 1%
elugent, pH 7.5.
[0369] The dialyzed solution (1080 ml) is loaded on the column
(flow rate 6 ml/minute); then 5 saline elution steps in this same
buffer are carried out: 20 mM, 50 mM, 100 mM, 250 mM and 1 M NaCl
(working flow rate 6 ml/minute). The rTbpB protein is eluted from
the column at two salt concentrations (50 mM and 100 mM). The 50 mM
elution fraction is the fraction of interest, since the rTbpB
protein therein is the purest and is present in a greater amount
(2.6 times more protein than in the 100 mM NaCl fraction).
[0370] The pH of the fraction corresponding to the 50 mM NaCl
elution peak is decreased, with magnetic stirring, to pH 5.5 by
adding 1.7N acetic acid. The solution (860 ml) is dialyzed against
5 liters of 10 mM sodium acetate buffer containing 1M urea and 0.2%
elugent, pH 5.5 (24 hours at +4.degree. C.) and then against 4
liters of 10 mM sodium acetate buffer containing 1M urea and 0.2%
elugent, pH 5.5 (17 hours at +4.degree. C.).
Purification by Cation Exchange Chromatography on SP Sepharose
(SPI) at pH 5.5
[0371] A K50 column or 100 ml of new SP Sepharose Fast Flow gel (Ge
Healthcare, ref 17-0729-01) is equilibrated in 10 mM sodium acetate
buffer containing 1M urea and 0.2% elugent, pH 5.5.
[0372] The dialyzed protein solution (850 ml) is loaded on the
column (flow rate 6 ml/minute). Then, five saline elution steps are
carried out: 50 mM, 100 mM, 250 mM, 500 mM and 1 M NaCl, in the
buffer mentioned above.
[0373] The rTbpB protein is eluted exclusively in the 250 mM NaCl
fraction and the low-molecular-weight contaminants are eliminated
essentially in the direct eluate (40%). About 35 mg of purified
rTbpB M982 are thus obtained and slightly less for rTbpB B16B6.
Dialysis and Concentration of the SPI Product (250 mM
Fractions)
[0374] The fractions corresponding to the 250 mM elution peak of
the SPI column are combined (volume 274 ml). The pH of the solution
is brought back up to pH 7.3 by adding, with stirring,
approximately 800 .mu.l of 0.5N NaOH. The solution is dialyzed at
+4.degree. C. (Spectra Por 1: cutoff threshold 6-8000 D) against
two 10 liter baths of PBS containing 0.2% elugent, pH 7.1 (66 hours
and 22 hours).
[0375] The dialysate is concentrated to a volume of 21.1 ml by
frontal diafiltration concentration on a 30 kD Amicon membrane in
PBS (ref. PBTK06510).
[0376] The concentrate obtained is then again dialyzed against 2
liters of PBS containing 0.2% elugent, pH 7.1 (Slide A Lyser ref
66810: cutoff threshold 10 kD).
[0377] The solution is then filtered aseptically through a 0.22
.mu.m Millex filter with Durapore membrane (Millipore ref. SLGV
033RS). The purified rTbpB protein batch obtained is frozen at
-80.degree. C. The protein concentration is 1642 .mu.g/ml.
1.3. Preparation of rTbpB for Injection
[0378] The rTbpB solution obtained in section B.1.2. is treated by
adsorption on Bio-Beads.TM. SM-2 in order to remove the excess
Elugent.TM. detergent (surfactant in particular constituted of
alkyl glucosides) which could destabilize the LOS liposomes.
Activation of Bio-Beads.TM.
[0379] About 2.5 ml of methanol are added to 500 mg of
Bio-Beads.TM. and the mixture is homogenized intermittently for 15
min at ambient temperature. After a settling-out period, the
supernatant is removed. This washing operation is repeated
twice.
[0380] About 5 ml of ultra-filtered sterile water are then added
and the mixture is homogenized intermittently for 15 min at ambient
temperature. After a settling-out period, the supernatant is
removed. This washing operation is repeated twice.
[0381] About 5 ml of PBS are then added and the mixture is
homogenized intermittently for 15 min at ambient temperature. It is
stored at 5.degree. C. and used the same day.
[0382] At the end, the weight of the Bio-Beads.TM. has increased by
a factor R (equal to approximately 1.2).
Removal of the Detergent by Adsorption on Bio-Beads.TM.
[0383] The rTbpB solution obtained in section 1.2. contains 2 mg/ml
of Elugent.TM.. The amount of Bio-.TM. that has to be used is
determined according to the amount of Elugent.TM. to be
removed.
[0384] For one ml of the rTbpB solution obtained in section B.1.2.,
29.times.R mg of activated Bio-Beads.TM. are added. The mixture is
vigorously stirred for one hour at ambient temperature. The maximum
amount of liquid is then recovered and a final concentration of
0.001% of merthiolate is added thereto. The whole process is
carried out under sterile conditions.
2. Preparation of the Purified LOS
Culture
[0385] Eight ml of frozen sample of any one of N. meningitidis C708
serogroup A strains described hereinabove or N. meningitidis strain
A1 serogroup A that exclusively expresses immunotype L8 and exhibit
a LOS bearing two PEAs, one in position 3, one in position 6 of
heptose II, are used to inoculate 800 ml of Mueller-Hinton medium
(Merck) supplemented with 4 ml of a solution of glucose at 500 g/l
and divided up in Erlenmeyer flasks. The culture is continued with
shaking at 36.+-.1.degree. C. for approximately 10 hours.
[0386] 400 ml of a solution of glucose at 500 g/l and 800 ml of a
solution of amino acids are added to the preculture. This
preparation is used to inoculate a fermentor containing
Mueller-Hinton medium, at an OD.sub.600 nm close to 0.05. The
fermentation is continued at 36.degree. C., at pH 6.8, 100 rpm,
pO.sub.2 30% under an initial airstream of 0.75 l/min/l of
culture.
[0387] After approximately 7 hours (OD.sub.600 nm of approximately
3), Mueller-Hinton medium is added at a rate of 440 g/h. When the
glucose concentration is less than 5 g/l, the fermentation is
stopped. The final OD.sub.600 nm is commonly between 20 and 40. The
cells are harvested by centrifugation and the pellets are frozen at
-35.degree. C.
[0388] Purification (method adapted by Westphal & Jann, (1965)
Meth. Carbohydr. Chem. 5: 83)
[0389] The pellets are thawed and suspended with 3 volumes of 4.5%
(vol./vol.) phenol with vigorous stirring for 4 hours at
approximately 5.degree. C. The LOS is extracted by phenol
treatment.
[0390] The bacterial suspension is heated to 65.degree. C. and then
mixed vol./vol. with 90% phenol, with vigorous stirring for 50-70
min at 65.degree. C. The suspension is subsequently cooled to
ambient temperature and then centrifuged for 20 min at 11 000 g.
The aqueous phase is removed and stored, while the phenolic phase
and the interphase are harvested so as to be subjected to a second
extraction.
[0391] The phenolic phase and the interphase are heated to
65.degree. C. and then mixed with a volume of water equivalent to
that of the aqueous phase previously removed, with vigorous
stirring for 50-70 min at 65.degree. C. The suspension is
subsequently cooled to ambient temperature and then centrifuged for
20 min at 11 000 g. The aqueous phase is removed and stored, while
the phenolic phase and the interphase are harvested so as to be
subjected to a third extraction identical to the second.
[0392] The three aqueous phases are dialyzed separately, each
against 401 of water. The dialysates are then combined. One volume
of 20 mM Tris, 2 mM MgCl.sub.2 is added to 9 volumes of dialysate.
The pH is adjusted to 8.0.+-.0.2 with 4N sodium hydroxide.
[0393] Two hundred and fifty international units of DNAse are added
per gram of pellet. The pH is adjusted to 6.8.+-.0.2. The
preparation is placed at 37.degree. C. for approximately 2 hours
with magnetic stirring, and then subjected to filtration through a
0.22 .mu.m membrane. The filtrate is purified by passing it through
a Sephacryl S-300 column (5.0.times.90 cm; Pharmacia.TM.).
[0394] The fractions containing the LOS are combined and the
MgCl.sub.2 concentration is increased to 0.5M by adding powdered
MgCl.sub.2.6H.sub.2O, with stirring.
[0395] While continuing the stirring, dehydrated absolute alcohol
is added to give a final concentration of 55% (vol./vol.). The
stirring is continued overnight at 5.+-.2.degree. C., and then
centrifugation is carried out at 5000 g for 30 min at
5.+-.2.degree. C. The pellets are resuspended with at least 100 ml
of 0.5M MgCl.sub.2 and then subjected to a second alcoholic
precipitation identical to the preceding one. The pellets are
resuspended with at least 100 ml of 0.5M MgCl2.
[0396] The suspension is subjected to a gel filtration as
previously described. The fractions containing the LOS are combined
and filtration-sterilized (0.8-0.22 .mu.m) and stored at
5.+-.2.degree. C.
[0397] This purification method makes it possible to obtain
approximately 150 mg of LOS per liter of culture.
3. Preparation of [LOS] Liposomes by Detergent Dialysis
3.1. Preparation of Liposomes
[0398] The LOS liposomes are prepared by detergent dialysis.
Briefly, the lipids (EDOPC:DOPE) are made into the form of a lipid
film and taken up in 10 mM Tris buffer, and then dispersed in the
presence of 100 mM of octyl-.beta.-D-glucopyranoside (OG)
(Sigma-Aldrich ref O8001) and filtered sterilely. The LOS in 100 mM
OG is added sterilely. The lipids/LOS/OG mixture is then dialyzed
against 10 mM Tris buffer in order to remove the OG and to form the
liposomes.
Protocol
[0399] A lipid preparation in chloroform, of the lipids that will
be used to produce the liposomes, is prepared. A dry film is
obtained by complete evaporation of the chloroform.
[0400] A dry film of 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine
(EDOPC or ethyl-DOPC) and of
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in an
EDOPC:DOPE mole ratio of 3 to 2 is obtained by mixing 12.633 ml of
a solution of EDOPC (Avanti Polar Lipids ref 890704) at 20 mg/ml in
chloroform and 7.367 ml of a solution of DOPE (Avanti Polar Lipids
ref 850725) at 20 mg/ml in chloroform, and evaporating off the
chloroform until it has completely disappeared.
[0401] The dry film is taken up with 30 ml of 10 mM Tris buffer, pH
7.0, so as to obtain a suspension containing 13.333 mg of lipids/ml
(8.42 mg/ml of EDOPC and 4.91 mg/ml of DOPE). The suspension is
stirred for 1 hour at ambient temperature and then sonicated for 5
min in a bath.
[0402] 3.333 ml of a sterile 1M solution of
octyl-.beta.-D-glucopyranoside (OG) (Sigma-Aldrich ref O8001) in 10
mM Tris buffer, pH 7.0, are then added, still with stirring, so as
to obtain a clear suspension of lipids at 12 mg/ml, 100 mM OG and
10 mM Tris buffer. The stirring is continued for 1 h at ambient
temperature on a platform shaker. Filtration is then carried out
sterilely through a Millex HV 0.45 um filter.
[0403] A composition is prepared, under sterile conditions, by
bringing together LOS and lipids in a lipids:LOS mole ratio of 250
(0.160 mg/ml of LOS, 9.412 mg/ml of lipids and 100 mM of OG). 40 ml
of such a composition are obtained from mixing the following
preparations:
[0404] 2.005 ml of 10 mM Tris buffer, pH 7.0; 0.223 ml of 100 mM OG
in 10 mM Tris; 31.373 ml of the EDOPC:DOPE suspension having a mole
ratio of 3:2, at 12 mg/ml in 100 mM OG, 10 mM Tris; and 6.4 ml of a
sterile suspension of LOS at 1 mg/ml in 100 mM OG, 10 mM Tris.
[0405] After stirring for one hour at ambient temperature, the
suspension is transferred sterilely into 4 sterile 10 ml dialysis
cassettes. Each cassette is dialyzed 3 times (24 hrs-24 hrs-72 hrs)
against 200 volumes of 10 mM Tris, pH 7.0, i.e. 2 l.
[0406] The liposomes are recovered under sterile conditions. The
increase in volume after dialysis is approximately 30%.
[0407] Merthiolate and NaCl are added to this preparation so as to
obtain a preparation of liposomes in 10 mM Tris, 150 mM NaCl, pH
7.0, 0.001% merthiolate, which ultimately contains approximately
110 .mu.g/ml of LOS and 7 mg/ml of lipids, of which there are
approximately 4.5 mg/ml of EDOPC and approximately 2.5 mg/ml of
DOPE (theoretical concentrations).
[0408] The LOS liposomes are stored at +5.degree. C.
3.2. Preparation of the Injectable Materials
[0409] The liposomes are adjusted to the required LOS concentration
(in particular required for the immunogenicity tests) in 10 Mm
Tris, 150 Mm NaCl, pH 7.4. The merthiolate concentration is
maintained at 0.001%.
4. Preparation of An [LOS] Liposomes +rTbpB Mixture
[0410] rTbpB in PBS (section B.1.3.) is mixed with [LOS] liposomes
(section B.3.) in an rTbpB:LOS weight:weight ratio equal to 1. The
volume is then adjusted with 10 mM Tris buffer containing 150 mM
NaCl, pH 7.4, so as to obtain a preparation in which each of the
components (rTbpB and LOS) is at a concentration of 80 .mu.g/ml.
The merthiolate concentration is maintained at 0.001%.
5. Production of An Endotoxoid (LOS Detoxified by Complexation with
a Peptide Analog of Polymyxin B)
[0411] This endotoxoid is prepared as described in patent
application WO 06/108586. Briefly, one volume of a solution of
purified LOS at 1 mg/ml, sterilized by filtration through a 0.22
.mu.m membrane, is mixed with one volume of a solution of SAEP2-L2
peptide at 1 mg/ml, sterilized by filtration through a 0.22 .mu.m
membrane.
[0412] The SAEP2-L2 peptide is a peptide with an antiparallel
dimeric structure, of formula:
##STR00007##
[0413] A precipitate forms immediately. Mixing is carried out for 5
min at ambient temperature, and then the mixture is left to stand
overnight at 4.degree. C. The precipitate is harvested by
centrifugation at 3000 rpm for 10 min. The pellet is washed 5 times
with one volume of pyrogen-free sterile water, pH 7.2. Finally, the
pellet is resuspended in 10 mM Tris buffer containing 150 mM NaCl
and Tween 80, pH 7.4, so as to obtain a suspension at 1 mg/ml,
calculated based on the wet weight of the precipitate. The
suspension is stored at 4.degree. C.
6. Preparation of An Endotoxoid+rTbpB Mixture
[0414] rTbpB in PBS (obtained as described in section B.1.3.) is
mixed with endotoxoid (section B.5.) in a weight:weight ratio equal
to 1. The volume is then adjusted with 10 mM Tris buffer containing
150 mM NaCl and 0.05% Tween 80 so as to obtain a preparation in
which each of the components is at a concentration of 80
.mu.g/ml.
7. Immunogenicity Study No. 1 in Rabbits
[0415] The various formulations tested were produced as described
in one of the preceding sections.
7.1. Immunization of Rabbits
[0416] Twenty-four 7-week-old female NZ-KBL rabbits (Charles River
Lab.) are divided up into 5 test groups of four and 2 control
groups of two.
[0417] The female rabbits of each group receive, in a volume of 0.5
ml, divided up into 2 concomitant intramuscular injections in the
legs, at DO, D21 and D42: [0418] Group A: 40 .mu.g of liposomes
[LOS .alpha. chain L6, PEA-3]in 10 mM Tris, 150 mM NaCl, pH 7.4
buffer; [0419] Group B: 40 .mu.g of liposomes [LOS .alpha. chain
L6, PEA-3] and 40 .mu.g M982 rTbpB in 10 mM Tris, 150 mM NaCl, pH
7.4 buffer; [0420] Group C: 40 .mu.g of liposomes [LOS .alpha.
chain L6, PEA-3, PEA-6]in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer;
[0421] Group D: 40 .mu.g of liposomes [LOS .alpha. chain L6, PEA-3,
PEA-6] and 40 .mu.g M982 rTbpB [0422] in 10 mM Tris, 150 mM NaCl,
pH 7.4 buffer; [0423] Group E: 40 .mu.g of LOS .alpha. chain L6,
PEA-3 in endotoxoid form and 40 .mu.g M982 rTbpB [0424] in 10 mM
Tris, 150 mM NaCl, 0.5% Tween, pH 7.0 buffer; [0425] Group F
(control): 40 .mu.g rTbpB and empty liposomes in 10 mM Tris, 150 mM
NaCl, pH 7.4 buffer; and [0426] Group G (control): empty liposomes
in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer.
[0427] A blood sample is taken from the animals for analysis at DO,
D42 (before the third injection) and at D56.
7.2. Assaying of Anti-LOS Antibodies by ELISA
[0428] This assay is automated (Staccato automation system,
Caliper) according to the following protocol:
[0429] The wells of Dynex.TM. 96-well plates are impregnated with,
for each of the groups, 1 .mu.g of LOS homolog in 1.times. PBS
(phosphate buffered saline) buffer, pH 7.1, 10 mM MgCl.sub.2, and
the plates are incubated for 2 hours at 37.degree. C. and then
overnight at 4.degree. C. The plates are blocked by adding, to the
wells, 150 .mu.l of PBS containing 0.05% of Tween 20 and 1%
(weight/vol) of skimmed milk powder (PBS-Tween-milk). The plates
are incubated for 1 hour at 37.degree. C.
[0430] Serial doubling dilutions of the test samples are prepared
in PBS--0.05% Tween--1% milk. The plates are incubated for 90 min
at 37.degree. C. and then washed 3 times with PBS+Tween 20 at
0.05%.
[0431] A peroxidase-anti-mouse IgG or peroxidase-anti-rabbit IgG
conjugate in PBS-Tween-milk is added to the wells and the plates
are incubated for 90 min at 37.degree. C. The plates are washed
three times. 100 .mu.l of a ready-to-use solution of TMB
(3,3',5,5'-tetramethylbenzidine, substrate for peroxidase) are
distributed per well. The plates are incubated in the dark for 20
min at ambient temperature. The reaction is stopped by adding 100
.mu.l of 1M HCl per well.
[0432] The optical density is measured at 450-650 nm with an
automatic reader (Multiskan Ascent). In the absence of standard,
the antibody titers are determined as being the reciprocal dilution
giving an optical density of 1.0 on a tendency curve (CodUnit
software). The antibody detection threshold is 1.3 log.sub.10 ELISA
unit. For each titer below this threshold, an arbitrary value of
1.3 log.sub.10 is assigned.
7.3. Measurement of the Bactericidal Activity of IgGs Purified
Against N. meningitidis Strains Heterologous to the Strain C708
(SBA Test)
[0433] IgGs were purified from pooled sera by affinity
chromatography using HiTrap rProtein A FF column (GE
Healthcare/Amersham Biosciences) according to the supplier's
recommendations.
[0434] On the basis of the purified IgGs, serial twofold dilutions
are carried out in gelatin-containing Dulbecco's PBS with calcium
and magnesium ions. The dilutions are carried out in a 96-well
plate for a final volume of 50 .mu.l per well.
[0435] The bactericidal activity of the purified IgGs has been
tested against the strains mentioned in the table which
follows:
[0436] Twenty-five .mu.l of a culture of N. meningitidis in the
exponential phase (4.times.10.sup.3 CFU/ml) in BHI medium in the
absence of an agent which chelates iron in free form (so as to
avoid expressing TbpB), and also 25 .mu.l of baby rabbit complement
at 1/1.5, are added to each well. The plate is incubated for one
hour at 37.degree. C., with shaking.
[0437] Fifty .mu.l of the mixture of each well are then deposited
on bioMerieux Mueller-Hinton agar plates and incubated overnight at
37.degree. C. under 10% CO.sub.2. The number of clones is
counted.
[0438] There are three controls: [0439] bacteria+baby rabbit
complement, without test serum ("complement" control); [0440]
bacteria+inactivated baby rabbit complement, without test serum
("microorganism" control); and [0441] bacteria+inactivated baby
rabbit complement+test serum (serum control).
[0442] The bactericidal titer is expressed as the inverse of the
dilution giving 50% bacterial death by comparison with the
"complement" control.
7.4. Results and Discussion
ELISAs
[0443] FIG. 3 gives the ELISA titers expressed as log.sub.10 of the
anti-LOS IgGs of the rabbit sera of groups A, B, C, D, E and F. In
white, the titers of the sera before immunization; shaded, those of
the sera of which samples were taken at D42 after the second
immunization; and darkly shaded, those of the sera of which samples
were taken at D56.
[0444] The ELISA titers at times D42 and D56 show that the LOS
obtained from each of the strains C708 constructed as described
previously is immunogenic. This immunogenicity is increased by the
presence of lipidated rTbpB for each period under consideration
(which confirms the adjuvant power of this lipoprotein).
SBA Test
[0445] The bactericidal activity has been established in "fold
increase" as being the purified IgG bactericidal titer of a
considered group: negative control group titer ratio. As such, the
seroconversion rate in "fold increase" expresses the bactericidal
titer increase. It is considered that the bactericidal activity is
significant when a "fold increase" equal or above x 8 is
obtained.
[0446] Purified IgGs from control group G exhibit against all
tested strains a fold increase inferior to x 4.
[0447] The fold increase values obtained with purified IgGs from
groups B and D against 18 strains are reported in table IV
hereinafter:
TABLE-US-00032 TABLE IV Strains included in the cross SBA study,
cultured in the absence of chelating agent LOS in liposomes :
Epidemio- L6 PEA-3 + L6 PEA-3, -6 + TbpB logical Lipidated
Lipidated IT Isotype complex Name TbpB M982 TbpB M982 L3 II ST-32
BZ83 <.times.4 <.times.4 II ST-41/44 LNP23015 .times.8
.times.8 II ST-41/44 LNP22979 <.times.4 <.times.4 II ST-41/44
BZ138 .times.8 .times.4 II ST-41/44 95/46 .times.8 .times.4 II
S3032 <.times.4 <.times.4 II LNP22763 <.times.4
<.times.4 II M982 .times.256 .times.168 II NG144/82 .times.16
.times.16 II NGF26 <.times.4 <.times.4 L4- II ST-8 BZ163
.times.8 .times.4 like ST-8 NGH41 .times.8 <.times.4 L8 II 8680
<.times.4 <.times.4 II ST-41/44 RH873 <.times.4
<.times.4 L1 II ST-41/44 92/123 <.times.4 <.times.4 I
ST-269 No28 LO .times.4 <.times.4 05-2606 II ST-269 No 60 AA
.times.16 .times.16 07-1734 N.d. II EG327 <x4 <x4 * LOS in
liposomes + adjuvant (lipidated rTbpB)
[0448] Purified IgGs obtained after immunisation with the group B
composition (40 .mu.g liposomes [LOS cha ne .alpha. L6, PEA-3] and
40 .mu.g rTbpB M982) have then been tested against a larger number
of strains. The results are reported in tables V and VI
hereinafter.
8. Immunogenicity Study No. 2 in Rabbits
[0449] The various test formulations were manufactured as described
in one of the preceding sections.
8.1. Immunization of Rabbits
[0450] Twenty-four 7-week-old NZ KBL female rabbits (Charles River
Lab.) are dispatched into 4 test groups of four (groups A to D) and
4 groups of 2 (groups E and H).
[0451] The female rabbits of each group receive in a volume of 0.5
ml divided among 2 concomitant intramuscular injections into the
legs, on D0, D21 and D42: [0452] Group A: 40 .mu.g of liposomes
[LOS .alpha. chain L8, PEA-3, PEA-6] et 40 .mu.g rTbpB M982, [0453]
in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer; [0454] Group B: 40 .mu.g
of liposomes [LOS .alpha. chain L8, PEA-3, PEA-6] and 40 .mu.g
rTbpB B16B6, in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer; [0455]
Group C: 40 .mu.g of liposomes [LOS .alpha. chain L8, PEA-3] and 40
.mu.g rTbpB M982, in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer; [0456]
Group D: 40 .mu.g of liposomes [LOS .alpha. chain L8, PEA-3,
PEA-6]in 10 mM Tris, 150 mM NaCl, pH 7.4 buffer; [0457] Group E: 40
.mu.g rTbpB M982 and 40 .mu.g of LOS-free liposomes in 10 mM Tris,
150 mM NaCl, 0.5% Tween, pH 7.0 buffer; [0458] Group F: 40 .mu.g
rTbpB B16B6 and 40 .mu.g of LOS-free liposomes in 10 mM Tris, 150
mM NaCl, 0.5% Tween, pH 7.0 buffer; [0459] Group G: 40 .mu.g of
liposomes [LOS .alpha. chain L8, PEA-3]in 10 mM Tris, 150 mM NaCl,
pH 7.4 buffer; [0460] Group H: 10 mM Tris, 150 mM NaCl, pH 7.4
buffer
[0461] Blood is collected from the animals for analysis at D0, D42
(before the third injection) and at D56.
8.2. Measurement of the Bactericidal Activity of Purified IgGs
against Strains of N. meningitidis Heterologous to the Strain
C708
[0462] IgGs were purified from pooled sera by affinity
chromatography using the HiTrap rProtein A FF column (GE
Healthcare/Amersham Biosciences) according to the manufacturer's
recommendations.
[0463] Using the purified IgGs, twofold serial dilutions are
performed in Dulbecco's gelatinized PBS containing calcium and
magnesium ions. The dilutions are performed in a 96-well plate for
a final volume of 50 .mu.l per well.
[0464] The bactericidal activity of the purified IgGs was tested
against the strains mentioned in Table V below:
[0465] 25 .mu.l of an N. meningitidis culture in the exponential
phase (4.times.10.sup.3 CFU/ml) in BHI medium+50 .mu.M of Desferal
(agent for chelating iron in free form, to allow the expression of
TbpB) and 25 .mu.l of baby rabbit complement at 1/1.5 are added to
each well. The plate is incubated for one hour at 37.degree. C.
with stirring.
[0466] 50 .mu.l of the mixture in each well are then deposited on
bioMerieux Mueller-Hinton agar dishes and incubated overnight at
37.degree. C. under 10% CO.sub.2. The number of clones is
counted.
[0467] There are three controls: [0468] bacteria+baby rabbit
complement, without test serum ("complement" control); [0469]
bacteria+inactivated baby rabbit complement, without test serum
("microorganism" control); and [0470] bacteria+inactivated baby
rabbit complement+test serum (serum control).
[0471] The bactericidal titer is expressed as being the inverse of
the dilution giving 50% bacterial death by comparison with the
"complement" control.
8.3. Results and Discussion
[0472] SBA Test 34 N. meningitidis strains have been tested for
cross bactericidal activity. Their names are to be seen in table V
hereinafter. The results are expressed in fold increase according
to the calculation methodology described in section B.7.4.
hereinabove.
[0473] As expected, purified IgGs from negative control
immunisation group do not show any bactericidal activity against
any of the strains.
[0474] Purified IgGs from immunisation groups D and G (LOS not
adjuvanted with TbpB) exhibit a bactericidal activity of less
interest. For simplicity'sake, table V hereinafter only shows the
SBA results expressed in fold increase , obtained with purified
IgGs from groups A, B, C, E and F of the second study together with
the results obtained with the purified IgGs of group B of the first
study.
[0475] Table VI shows the SBA results expressed in fold increase ,
of purified IgGs from immunisation group B of the first study and
of group A of the second study against 22 strains cultured in
presence/absence of Desferal.
[0476] Table VII shows for a variety of vaccine compositions, the
percentage of protection deduced from the cross SBA studies
including 34 strains cultured in presence of Desferal.
TABLE-US-00033 TABLE V LOS in liposomes: Empty liposomes+ 34
strains included in the cross L6 PEA-3 + L8 PEA-3, -6 + L8 PEA-3 +
L8 PEA-3, -6 + Lipidated Lipidated SBA study, cultured in the
Lipidated Lipidated Lipidated Lipidated TbpB TbpB presence of
Desferal TbpB M982 TbpB M982 TbpB M982 TbpB B16B6 M982 B16B6 TbpB
Study no1 Study no 2 Study no 2 Study no 2 Study no 2 Study no 2 IT
Isotype Name Group B Group A Group C Group B Group E Group F L3 II
BZ83 .times.32 .times.32 .times.16 <.times.4 .times.16
<.times.4 II LNP23015 .times.16 .times.16 .times.16 <.times.4
.times.16 <.times.4 II LNP20443 <.times.4 .gtoreq..times.4
<.times.4 <.times.4 <.times.4 <.times.4 II LNP22979
.times.8 .times.32 .times.8 <.times.4 <.times.4 <.times.4
II BZ138 .times.512 .times.256 .times.256 <.times.4 .times.256
<.times.4 II 95/46 .times.64 .times.16 .times.8 <.times.4
.times.64 <.times.4 II S3032 .times.4 .times.4 .times.4
<.times.4 <.times.4 <.times.4 II LNP22763 .times.4
.times.4 <.times.4 <.times.4 <.times.4 <.times.4 II
M982 >.times.1024 .times.512 .times.512 <.times.4 .times.256
<.times.4 II NG144/82 .times.64 .times.138 .times.64
<.times.4 .times.128 <.times.4 II H44/76 .times.8 .times.4
.times.4 <.times.4 <.times.4 <.times.4 II MC58 .times.32
.times.8 .times.4 <.times.4 .times.16 <.times.4 II NGPB24
.times.8 .times.4 .times.8 <.times.4 .times.16 <.times.4 II
NGF26 .times.4 .times.4 <.times.4 <.times.4 <.times.4
<.times.4 L4- II BZ163 .times.16 .times.16 .times.8 .times.4
<.times.4 <.times.4 like I NGP20 <.times.4 <.times.4
<.times.4 .times.1024 <.times.4 .times.1024 I M986
<.times.4 <.times.4 <.times.4 .times.1024 <.times.4
.times.512 L4 II M2 .times.16 <.times.4 <.times.4
<.times.4 <.times.4 <.times.4 L8 II 8680 .times.4
.times.256 .times.256 .times.16 <.times.4 <.times.4 II RH873
.times.16 .times.256 .times.256 .times.64 .times.8 <.times.4 L1
II 92/123 .times.8 .times.32 .times.32 .times.16 .times.4
<.times.4 II M101/93 .times.16 .times.16 .times.16 <.times.4
.times.4 <.times.4 II 1000 <.times.4 .times.4 .times.4
.times.8 <.times.4 <.times.4 I No 28 LO 05- .times.16
.times.8 .times.16 .times.128 .times.8 .times.16 2606 II No 60 AA
07- .times.256 .times.128 .times.256 .times.64 .times.32
<.times.4 1734 L2 II BZ157 .times.8 <.times.4 <.times.4
<.times.4 <.times.4 <.times.4 II BZ232 .times.16 .times.32
.times.16 <.times.4 .times.16 <.times.4 I B16B6 <.times.4
<.times.4 <.times.4 .times.1024 <.times.4 .times.512 N.d.
II 30 .times.128 .times.128 .times.64 <.times.4 .times.16
<.times.4 II 62 .times.4 .times.8 .times.4 <.times.4
<.times.4 <.times.4 I FAM18 <.times.4 <.times.4
<.times.4 .times.1024 <.times.4 .times.512 II 90/94 .times.16
<.times.4 .times.4 <.times.4 .times.4 <.times.4 II 22
<.times.4 .times.16 .times.32 .times.16 <.times.4
<.times.4 II EG327 <.times.4 <.times.4 <.times.4
<.times.4 <.times.4 <.times.4 In Table V hereinabove: IT
means immunotype L6 means a LOS bearing an .alpha. chain of type L6
L8 means a LOS bearing an .alpha. chain of type L8 PEA-3 means that
the LOS bears a unique PEA substituent in position 3 of heptose II
PEA-3, -6 means that the LOS bears a PEA substituent in position 3
and a PEA substituent in position 6 of heptose II
TABLE-US-00034 TABLE VI LOS in liposomes: L6 PEA-3 + L8 PEA-3, -6 +
Lipidated TbpB M982 Lipidated TbpB M982 Strains included in the
cross SBA study, cultured Study no 1 Group B Study no 2 Group A in
presence (Desferal)/absence of chelating Without With Without With
TbpB Epidemiological chelating chelating chelating chelating IT
Isotype complex Name agent agent agent agent L3 II ST-32 BZ83
<.times.4 .times.32 <.times.4 .times.32 II ST-41/44 LNP23015
.times.8 .times.16 <.times.4 .times.16 II ST-41/44 LNP22979
<.times.4 .times.8 <.times.4 .times.32 II ST-41/44 BZ138
.times.8 .times.512 .times.32 .times.256 II ST-41/44 95/46 .times.8
.times.64 <.times.4 .times.16 II S3032 <.times.4 .times.4
<.times.4 .times.4 II LNP22763 <.times.4 .times.4
<.times.4 .times.4 II M982 .times.256 >.times.1024 .times.64
.times.512 II NG144/82 .times.16 .times.64 .times.4 .times.128 II
H44/76 <.times.4 .times.8 <.times.4 .times.4 II NGF26
<.times.4 .times.4 <.times.4 .times.4 II 62 <.times.4
.times.4 <.times.4 .times.8 L4- II ST-8 BZ163 .times.8 .times.8
.times.8 .times.16 like L8 II 8680 <.times.4 .times.4 .times.128
.times.256 II ST-41/44 RH873 <.times.4 .times.16 .times.64
.times.256 L1 II ST-41/44 92/123 <.times.4 .times.8 .times.16
.times.32 I ST-269 No28 LO .times.4 .times.16 <.times.4 .times.8
05-2606 II ST-269 No 60 AA .times.16 .times.256 .times.64
.times.128 07-1734 N.d. II EG327 <.times.4 <.times.4
<.times.4 <.times.4 L2 II BZ157 <.times.4 .times.8
<.times.4 <.times.4 II BZ232 <.times.4 .times.16
<.times.4 .times.32 I B16B6 <.times.4 <.times.4
<.times.4 <.times.4
TABLE-US-00035 TABLE VII % of protection deduced from cross SBA
studies including 34 strains cultured in the presence Vaccine
composition of Desferal L6 PEA O3 + TbpB M982 61.8% L8 PEA O3, O6 +
TbpB M982 55.9% L6 PEA O3 + L8 PEA O3, O6 + TbpB M982 70.6% L8 PEA
O3 + TbpB M982 52.9% L6 PEA O3 + L8 PEA O3 + TbpB M982 67.6% L8 PEA
O3, O6 + TbpB B16B6 .sup. 32% L6 PEA O3 + TbpB M982 + TbpB B16B6
73.5% L8 PEA O3, O6 + TbpB M982 + TbpB B16B6 58.8-67.6% .sup. L6
PEA O3 + L8 PEA O3, O6 + TbpB 82.4% M982 + TbpB B16B6 L8 PEA O3 +
TbpB M982 + TbpB B16B6 64.7%
Sequence CWU 1
1
2415PRTArtificial SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is a
peptide of 2 to 5 and preferably 3 or 4 amino acid residues, in
which at least 2 amino acid residues are independently chosen from
Lys, Hyl (hydroxylysine), Arg and His.MISC_FEATURE(3)..(3)Xaa is a
peptide of 3 to 7 and preferably 4 or 5 amino acid residues, which
comprises at least two and preferably three amino acid residues
chosen from Val, Leu, Ile, Phe, Tyr and Trp.MISC_FEATURE(5)..(5)Xaa
is optional (this position may or may not be empty) and is an amino
acid residue or a peptide formed from 2 to 3 amino acid residues.
1Xaa Cys Xaa Cys Xaa1 5210PRTArtificial SequenceSynthetic 2Lys Thr
Lys Cys Lys Phe Leu Lys Lys Cys1 5 10310PRTArtificial
SequenceSynthetic 3Lys Thr Lys Cys Lys Phe Leu Leu Leu Cys1 5
10411PRTArtificial SequenceSyntheticMISC_FEATURE(4)..(4)Xaa is
hydroxylysine 4Lys Arg His Xaa Cys Lys Arg Ile Val Leu Cys1 5
10511PRTArtificial SequenceSynthetic 5Lys Arg His Cys Val Leu Ile
Trp Tyr Phe Cys1 5 10610PRTArtificial SequenceSynthetic 6Lys Thr
Lys Cys Lys Phe Leu Leu Leu Cys1 5 10712PRTArtificial
SequenceSyntheticMISC_FEATURE(1)..(1)Xaa is hydroxylysine 7Xaa Arg
His Lys Cys Phe Tyr Trp Val Ile Leu Cys1 5 10842DNAArtificial
SequenceSynthetic 8cggaattcgc cgtctcaaat gaaaaaatcc cttttcgttc tc
42931DNAArtificial SequenceSynthetic 9aactgcagtc attgcggata
aacatattcc g 311023DNAArtificial SequenceSynthetic 10cgccgaatac
tttatcttga ggc 231118DNAArtificial SequenceSynthetic 11ctcgccaaag
agcagggc 181243DNAArtificial SequenceSynthetic 12cggaattcgc
cgtctcaagg ttgcctatgt tttcctgttt ttg 431332DNAArtificial
SequenceSynthetic 13aactgcagct aacgggcaat tttcaaaacg tc
321426DNAArtificial SequenceSynthetic 14cgggatccca tcgacacgaa
cgccgc 261528DNAArtificial SequenceSynthetic 15cgggatcccc
gcgcttaacg actacatc 281625DNAArtificial SequenceSynthetic
16gtcgatggga tccccgcgct taacg 251718DNAArtificial SequenceSynthetic
17ccgactggcg gaattggg 181820DNAArtificial SequenceSynthetic
18cccatttctt cctgacggac 201938DNAArtificial SequenceSynthetic
19cggaattcgc cgtctcaaat gccgtctgaa gccttcag 382029DNAArtificial
SequenceSynthetic 20aactgcagaa cggtttttca gcaatcggt
292129DNAArtificial SequenceSynthetic 21cgggatccgg aaggcccgag
cgcagaagt 292232DNAArtificial SequenceSynthetic 22gctctagaca
acttacttct gacaacgatc gg 322329DNAArtificial SequenceSynthetic
23cgggatccgc caattcatcc agcccgatg 292426DNAArtificial
SequenceSynthetic 24cgtctagacc cggttcgaca gccttg 26
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