U.S. patent application number 15/599556 was filed with the patent office on 2017-09-07 for ultrafiltration for preparing outer membrane vesicles.
This patent application is currently assigned to GLAXOSMITHKLINE BIOLOGICALS S.A.. The applicant listed for this patent is GLAXOSMITHKLINE BIOLOGICALS S.A.. Invention is credited to Ilio MARSILI, Roberto OLIVIERI, Fabio SABBATINI.
Application Number | 20170252699 15/599556 |
Document ID | / |
Family ID | 27763870 |
Filed Date | 2017-09-07 |
United States Patent
Application |
20170252699 |
Kind Code |
A1 |
OLIVIERI; Roberto ; et
al. |
September 7, 2017 |
ULTRAFILTRATION FOR PREPARING OUTER MEMBRANE VESICLES
Abstract
In place of a step of centrifugation during preparation of outer
membrane vesicles (OMVs) from bacteria, the invention utilises
ultrafiltration. This allows much larger amounts of OMV-containing
supernatant to be processed in a much shorter time. Thus the
invention provides a process for preparing bacterial OMVs,
comprising a step of ultrafiltration. The ultrafiltration step is
performed on an aqueous suspension of OMVs after they have been
prepared from bacteria and the OMVs remain in suspension after the
filtration step. The invention is particularly useful for preparing
OMVs from Neisseria meningitidis.
Inventors: |
OLIVIERI; Roberto; (Siena,
IT) ; SABBATINI; Fabio; (Siena, IT) ; MARSILI;
Ilio; (Siena, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE BIOLOGICALS S.A. |
Rixensart |
|
BE |
|
|
Assignee: |
GLAXOSMITHKLINE BIOLOGICALS
S.A.
Rixensart
BE
|
Family ID: |
27763870 |
Appl. No.: |
15/599556 |
Filed: |
May 19, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10564474 |
Nov 20, 2006 |
9687789 |
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PCT/IB04/02475 |
Jul 15, 2004 |
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15599556 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/095 20130101;
C12R 1/36 20130101; B01D 2311/04 20130101; C12N 1/005 20130101;
B01D 61/16 20130101; C12N 1/00 20130101; C07K 14/22 20130101; B01D
61/147 20130101; C12N 1/06 20130101; A61K 2039/6018 20130101; B01D
2311/04 20130101; B01D 2311/16 20130101; C12N 1/20 20130101; B01D
61/145 20130101; B01D 2311/2676 20130101; C12N 1/02 20130101; C12R
1/01 20130101 |
International
Class: |
B01D 61/14 20060101
B01D061/14; A61K 39/095 20060101 A61K039/095 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 13, 2003 |
GB |
0316560.2 |
Claims
1-16. (canceled)
17. Bacterial OMVs obtainable by a process comprising a step of
ultrafiltration.
18. A pharmaceutical composition comprising the OMVs of claim 17
and a pharmaceutically acceptable carrier.
19. The composition of claim 18, comprising an aluminium hydroxide
adjuvant and a histidine buffer.
20. The composition of claim 18, wherein the composition is
substantially free from whole bacteria.
21. A vial containing the composition of claim 18.
22. A syringe containing the composition of claim 18.
23. A method for raising an immune response in a patient,
comprising administering an immunogenic dose of the composition of
claim 18.
Description
[0001] All documents cited herein are incorporated by reference in
their entirety.
TECHNICAL FIELD
[0002] This invention is in the field of vesicle preparation for
immunisation purposes.
BACKGROUND ART
[0003] One of the various approaches to immunising against N.
meningitidis infection is to use outer membrane vesicles (OMVs). An
efficacious OMV vaccine against serogronp B has been produced by
the Norwegian National Institute of Public Health [e.g. ref. 1]
but, although this vaccine is safe and prevents NmB disease, its
efficacy is limited to the strain used to make the vaccine.
[0004] The `RIVM` vaccine is based on vesicles containing six
different PorA subtypes and has been shown to be immunogenic in
children in phase II clinical trials [2].
[0005] References 3 & 4 disclose a vaccine against different
pathogenic serotypes of serogroup meningococcus based on OMVs which
main a protein complex of 65-kDa. Reference 5 discloses a vaccine
comprising OMVs from genetically-engineered meningococcal strains,
with the OMVs comprising: at least one Class 1 outer-membrane
protein (OMP) but not comprising a Class 2/3 OMP. Reference 6
discloses OMVs comprising OMPs which have mutations in their
surface loops. Reference 7 discloses compositions comprising OMVs
supplemented with transferrin binding proteins (e.g. TbpA and TbpB)
and/or Cu,Zn-superoxide dismutase. Reference 8 discloses
compositions comprising OMVs supplemented by various proteins.
References 9 & 10 also describe OMV preparations from
meningococcus.
[0006] Reference 11 discloses a process for preparing OMV-based
vaccines, particularly for serogroup A meningococcus, comprising
the following 10 steps: (a) cultivating bacterial cells; (b)
concentrating the cultivated cells from step (a); (c) treating the
cells with a bile acid salt detergent at a pH sufficiently high not
to precipitate the detergent, for inactivating the bacteria,
disrupting the outer membrane of the bacteria and forming vesicles
of the outer membrane of the bacteria, said vesicles comprising
outer membrane components mainly presented in their native form;
(d) centrifuging the composition from step (c) at
10,000-20,000.times.g for about 1 to 2 hours to separate the outer
membrane vesicles from the treated cells and cell debris, and
collecting the supernatant; (e) performing a high speed
centrifugation of the supernatant from step (d) and collecting the
outer membrane vesicles in a pellet; (f) re-dispersing the pellet
from step (e) in a buffer by stirring at ambient temperature; (g)
performing a second high speed centrifugation in accordance with
step (e), collecting the outer membrane vesicles in a pellet; (h)
re-dispersing the pellet from step (g) in an aqueous medium
containing a stabilising agent by stirring at ambient temperature;
(i) performing a step-wise sterile filtration through at least two
filters of decreasing pore size of the re-dispersed composition
from step (h), ending with a filter of pore-size of about 0.2
.mu.m; and (j) optionally including the composition from step (i)
in a pharmaceutically acceptable carrier and/or adjuvant
composition.
[0007] It is an object of the present invention to provide an
improved process for preparing OMVs for use in vaccines, in
particular a process which can prepare a greater quantity of OMVs
in a shorter time, and particularly a process suitable for
industrial-scale use.
DISCLOSURE OF THE INVENTION
[0008] The invention is based on the finding that, compared to the
centrifugation used in step (e) of the process of reference 11,
ultrafiltration allows much larger amounts of OMV-containing
supernatant to be processed in a much shorter time (typically
>15 litres in 4 hours, compared to <1.5 litres in 10 hours).
As well allowing step (e) to be performed more quickly, the use of
ultrafiltration allows step (f) to be avoided because the OMVs
remains in suspension.
[0009] Thus the invention provides a process for preparing
bacterial OMVs, comprising a step of ultrafiltration. The
ultrafiltration step is performed on an aqueous suspension of OMVs
after they have been prepared from bacteria and the OMVs remain in
suspension alter the ultrafiltration step.
[0010] The invention also provides, in a process for preparing OMVs
from a bacterium, the improvement consisting of the use of
ultrafiltration of an OMV suspension in place of a step of
centrifugation.
[0011] The invention also provides a process for purifying
bacterial OMVs, wherein the process does not include a
centrifugation step in which the OMVs are pelleted, particularly a
centrifugation step performed on crude OMVs.
The Ultrafiltration Step
[0012] Ultrafiltration is a separation process whereby solvent is
removed from a solution (including a colloidal solution) or a
suspension by forcing it to flow through a membrane by the
application of a hydraulic pressure. Components in the solution
which are significantly larger than the solvent cannot pass through
the membrane. Ultrafiltration therefore separates components based
on size.
[0013] The ultrafiltration step preferably results in diafiltration
of the solution. In diafiltration, solvent and/or microsolutes
(e.g. salts) which are removed during ultrafiltration are replaced
by new solvent and microsolutes. In general, removal and
replacement occur at the same rate and the volume of the solution
is thus kept constant. The overall effect of the process is
therefore the replacement of original solvent/microsolutes with new
solvent/microsolutes. The process of the invention may thus include
a step of diafiltration.
[0014] The ultrafiltration is preferably cross-flow or tangential
flow ultrafiltration, in which the solution flows substantially
parallel to the membrane surface, rather than flowing perpendicular
to the surface as in ordinary filtration.
[0015] Preferred membranes for use in the ultrafiltration step have
a cut-off of about 300 kDa.
[0016] The ultrafiltration step preferably last less than 10 hours
e.g. between 2 and 6 hours, preferably between 3 and 5 hours e.g.
between 3.5 and 4.5 hours.
[0017] Membranes may be made from any suitable material e.g.
polyethersulphone.
Pre-Ultrafiltration Steps
[0018] Prior to the ultrafiltration step, the process of the
invention will typically comprise an initial step of cultivating
bacterial cells (e.g. in broth or in solid medium culture),
optionally followed by a step of collecting and/or concentrating
the cultivated cells by filtration or by a low-speed centrifugation
to pellet the cells). However, the invention may be performed on
bacteria which have already been cultured and/or harvested
separately. The bacterial culture preferably involves the use of
neither blood products nor material contaminated with a
transmissible spongiform encephalopathy agent.
[0019] The ultrafiltration step is performed on an aqueous
suspension of OMVs after they have been prepared from bacteria.
Prior to ultrafiltration, the process may therefore comprise a step
of OMV preparation in which cells are treated to disrupt their
outer membranes. The preparation of OMVs from meningococcus is
well-known in the art. Methods for obtaining suitable preparations
are disclosed in, for instance, references 1 to 25. Techniques for
forming OMVs include treating bacteria with a bile acid salt
detergent (e.g. salts of lithocholic acid, chenodeoxycholic acid,
ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic
acid, etc., with sodium deoxycholate [26 & 27] being preferred
for treating Neisseria) at a pH sufficiently high not to
precipitate the detergent [11]. Other techniques may be performed
substantially in the absence of detergent [28] using techniques
such as sonication, homogenisation, microfluidisation, cavitation,
osmotic shock, grinding, French press, blending, etc.
[0020] After OMV formation and prior to ultrafiltration, the OMVs
are preferably separated from bacterial cells and cell debris.
Separation can conveniently be achieved by centrifugation at
10,000-20,000.times.g for about 1 to 2 hours). OMVs remain in the
supernatant and can then be subjected to ultrafiltration according
to the invention, rather than to ultracentrifugation as in the
prior art. Other methods for separating outer membrane fractions
from cytoplasmic molecules may involve filtration (e.g. cross flow
filtration), differential precipitation or aggregation of outer
membranes and/or OMVs, affinity separation methods using ligands
that specifically recognize outer membrane molecules, etc. Use of a
closed filtration system may be preferred to avoid open handling of
infections bacteria.
[0021] In order to preserve the native conformation of proteins and
other labile outer membrane antigens, mild conditions will
generally be selected for preparation of OMVs. Heat inactivation of
bacteria (e.g. at 56.degree. C. or higher) is thus preferentially
avoided, as is solvent denaturation.
Post-Ultrafiltration Steps
[0022] After the ultrafiltration step, the OMVs may be further
treated.
[0023] For example, the OMVs may be sterilised. Sterilisation is
preferably a final step before packaging as a pharmaceutical, and
can conveniently be achieved by filter sterilisation. Although OMVs
will pass through a standard 0.22 .mu.m filters, these can rapidly
become clogged by other material, and so it is preferred to perform
sequential steps of filter sterilisation through a series of
filters of decreasing pore size, finishing with a standard
sterilisation filter (e.g. a 0.22 .mu.m filter). Examples of
preceding filters would be those with pore size of 0.8 .mu.m, 0.45
.mu.m, etc. Filter sterilisation advantageously occurs at ambient
temperature or above, rather than at refrigeration temperatures.
Vesicle flexibility is higher at ambient temperature and larger
vesicles (.about.0.2 .mu.m) can thus pass through a 0.22 .mu.m
filter more easily, giving less clogging of filters.
[0024] The OMVs may also be centrifuged (e.g. ultracentrifuged)
after ultrafiltration takes place. Thus, in some embodiments, the
invention does not completely replace the use of
ultracentrifugation during OMV preparation, but removes al least
one step of ultracentrifugation relative to ref. 11. A normal
ultracentrifugation requires about 13 hours for 1.3 litres of OMV
suspension, and so a large volume of OMVs requires a large
ultracentrifugation resource. Ultrafiltration according to the
invention can be used to reduce the volume which has to be
ultracentrifuged (by around 3-fold) and so can improve throughput
even though ultracentrifugation is not wholly avoided.
[0025] The OMVs may be combined with pharmaceutical carriers and/or
adjuvants and/or stabilisers. For example, pellet(s) from
ultracentrifugation can be re-suspended (e.g. in a sucrose
solution, preferably about 3% sucrose) and then subjected to filter
sterilisation as described above.
[0026] OMVs may be sonicated. Sonication is particularly useful
between re-suspension of centrifugation pellets and
sterilisation.
[0027] After re-suspension, OMV preparations preferably contain
between 500 and 2000 mg of protein per millilitre e.g. between 900
and 1800 mg/ml, or 1000.+-.100 mg/ml.
Overall Process for Preparing Sterile OMVs
[0028] In general, therefore,, the process of the invention will
include the following steps: (1) cultivating bacterial cells; (2)
collecting the cultivated cells; (3) OMV formation; (4) separation
of OMV from cell debris, to give an aqueous suspension of OMV; (5)
ultrafiltration; (6) centrifugation and re-suspension to collect
purified OMV; and (7) sterilisation. pH may be adjusted at any
stage as required. Similarly, dilution as appropriate can be
used.
[0029] Step (5) in this process replaces steps (e) and (f) from
reference 11.
The Bacterium
[0030] The bacterium from which OMVs are prepared may be
Gram-positive, but it is preferably Gram-negative. The bacterium
may be from any suitable genus, including Moraxella (e.g. M.
catarrhalis [29, 30]), Shigella (e.g. S. flexneri [31, 32]),
Pseudomonas (e.g. P. aeruginosa [31, 32]), Treponema (e.g. T.
pallidum [33]), Haemophilus (e.g. H. influenza [9, 10]),
Porphyromonas (e.g. P. gingivalis [34]) or Helicobacter (e.g. H.
pyori [35]), but it is preferably from the Neisseria genus.
Preferred Neisseria species are N. meningitidis, N. lactimica [36]
and N. gonorrhoeae [37 & 38]. Within N. meningitidis, any of
serogroups A, C, W135 and Y may be used, but it is preferred to
prepare vesicles from serogroup B.
[0031] Preferred strains within serogroup B are MC58, 2996, H4476,
394/98 and New Zealand strain 98/254. The best serotypes and
strains to use, however, will depend on the strains prevalent in a
particular geographical location. For example, the meningococcus
can be of any serotype (e.g. 1, 2a, 2b, 4, 14, 15, 16, etc.), of
any serosubtype (P1.2; P1.4; P1.5, 2; P1.7, 16; P1.7, 16b; P1.9;
P1.9, 15; P1.2, 13; P1.13; P1.14; P1.15; P1.21, 16; P1.22, 14;
etc.) and of any immunotype (e.g. L1; L3, 3, 7; L10; etc.), and
preferred strains include: (a) B:4:P1.4; (b) B:4:P1.15; (c)
B:15:P1.7, 16; and (d) B:4:P1.7b, 4. The meningococcus may be from
any suitable lineage, including hyperinvasive and hypervirulent
lineages e.g. any of the following seven hypervirulent lineages:
subgroup I; subgroup III; subgroup IV-I; ET-5 complex; ET-37
complex; A4 cluster; lineage 3. These lineages have been defined by
multilocus enzyme electrophoresis (MLEE), but multilocus sequence
typing (MLST) has also been used to classify meningococci [ref. 39]
e.g. the ET-37 complex is the ST-11 complex by MLST, the ET-5
complex is ST-32 (ET-5), lineage 3 is ST-41/44, etc.
[0032] To reduce pyrogenic activity, it is preferred that the
bacterium should have low endotoxin (LPS) levels. Suitable mutant
bacteria are known e.g. mutant Neisseria [40] and mutant
Heliobacter [41]. Processes for preparing LPS-depleted outer
membranes from Gram-negative bacteria are disclosed in reference
42.
[0033] The bacterium may be a wild-type bacterium, or it may be a
recombinant bacterium. Preferred recombinant bacteria over-express
(relative to the corresponding wild-type strain) immunogens such as
NspA, protein 287 [8], protein 741 [8], TbpA, TbpB, superoxide
dismutase [7], etc. The bacterium may express more than one PorA
class I outer membrane protein e.g. 2, 3, 4, 5 or 6 of PorA
subtypes; P1.7, 16; P1.5, 2; P1.19, 15; P1.5c, 10; P1.12, 13; and
P1.7h,4 [e.g. refs. 12 & 14].
[0034] Other recombinant bacteria that can be used with the
invention have one or more mutations to decrease (or, preferably,
to knockout) expression of particular gene products. Preferred
genes for down-regulation and/or knockout include: (a) Cps, CtrA,
CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa,
Opc, PilC, PorA, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB
[9]; (b) CtrA, CtrB, CtrC, CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB,
LpxK, Opa, Opc, PhoP, PilC, PmrE, PmrF, PorA, SiaA, SiaB, SiaC,
SiaD, TbpA, and/or TbpB [10]; (c) lytic transglycosylase NMB0033
[43]: (d) ExbB, ExbD, rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB,
Opa, Opc, PilC, PorA, PorB, SiaA, SiaB, SiaC, SiaD, TbpA, and/or
TbpB [44]; and (e) CtrA, CtrB, CtrD, FrpB, OpA, OpC, PilC, PorA,
PorB, SiaD, SynA, SynB, and/or SynC [45].
Pharmaceutical Compositions
[0035] For human use, OMVs will generally be combined with a
pharmaceutically acceptable carrier.
[0036] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent, such as
antibodies or a polypeptide, genes, and other therapeutic agents.
The term refers to any pharmaceutical carrier that does not itself
induce the production of antibodies harmful to the individual
receiving the composition, and which can be administered without
undue toxicity. Suitable carriers can be large, slowly metabolized
macromolecules such as proteins, polysaccharides, polylactic acids,
polyglycolic acids, polymeric amino acids, amino acid copolymers,
and inactive virus particles. Such carriers are well known to those
of ordinary skill in the art. Pharmaceutically acceptable carriers
in therapeutic compositions can include liquids such as water,
saline, glycerol and ethanol. Ancillary substances, such as wetting
or emulsifying agents, pH buffering substances, and the like, can
also be present in such vehicles. Typically, the therapeutic
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid vehicles prior to injection can also be
prepared. Liposomes are included within the definition of a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
salts can also be present in the pharmaceutical composition, e.g.
mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and be salts of organic acids
such as acetates, propionates, malonates, benzonates, and the like.
A thorough discussion of pharmaceutically acceptable excipients is
available in reference 46. The composition will typically include
saline.
[0037] Once formulated, compositions can be administered directly
to a subject. Delivery will generally be accomplished by parenteral
injection (e.g. subcutaneously, intraperitoneally, intravenously or
intramuscularly, or to the interstitial space of a tissue) or by
mucosal administration (e.g. oral, pulmonary, rectal, vaginal,
intranasal [47, 48]) etc.). Transdermal applications, needles, and
gene guns or hyposprays may also be used. Intramuscular injection
is the preferred manner of delivery.
[0038] The dose and the means of administration of be inventive
pharmaceutical compositions are determined based on the specific
qualities of the therapeutic composition, the condition, age, and
weight of the patient, the progression of the disease, and other
relevant factors.
[0039] Neisseria infections affect various areas of the body and so
the compositions of the invention may be prepared in various forms.
For example, the compositions may be prepared as injectables,
either as liquid solutions or suspensions. Solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection
can also be prepared (e.g. a lyophilised composition). The
composition may be prepared for topical administration as an
ointment, cream or powder. The composition be prepared for oral
administration e.g. as a tablet or capsule, or as a syrup
(optionally flavoured). The composition may be prepared for
pulmonary administration e.g. as an inhaler, using a fine powder or
a spray. The composition may be prepared as a suppository or
pessary. The composition may be prepared for nasal, aural or ocular
administration e.g. as drops.
[0040] The OMVs of the invention may be combined with an adjuvant.
Preferred adjuvants to enhance effectiveness of the composition
include, but are not limited to: (A) MF59 (5% Squalene, 0.5% Tween
80, and 0.5% Span 85, formulated into submicron particles using a
microfluidizer) [see Chapter 10 of ref. 49; see also ref. 50]; (B)
microparticles (i.e. a particle of .about.100 nm to .about.150
.mu.m in diameter, more preferably .about.200 nm to -30 .mu.m in
diameter, and most, preferably .about.500 nm to .about.10 .mu.m in
diameter) formed from materials that are biodegradable and
non-toxic (e.g. a poly(.alpha.-hydroxy acid), a polyhydroxybutyric
acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.),
with poly(lactide-co-glycolide) being preferred, optionally treated
to have a negatively-charged surface (e.g. with SDS) or a
positively-charged surfaces (e.g. with a cationic detergent, such
as CTAB) [51 & 52]); (C) liposomes [see Chapters 13 and 14 of
ref. 49]; (D) ISCOMs [see Chapter 23 of ref. 49], which may be
devoid of additional detergent (53); (E) SAF, containing 10%
Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and
thr-MDP, either microfluidized into a submicron emulsion or
vortexed to generate a larger particle size emulsion [see Chapter
12 of ref. 49]; (F) Ribi.TM. adjuvant system (RAS), (Ribi
Immunochem) containing 2% Squalene, 0.2% Tween 80, and one or more
bacterial cell wall components from the group consisting of
monophosphorylipid A (MPL) trehalose dimycolate (TDM), and cell
wall skeleton (CWS), preferably MPL+CWS (Detox.TM.); (G) saponin
adjuvants, such as QuilA or QS21 [see Chapter 22 of ref. 49], also
known as Stimulon.TM.; (H) chitosan [e.g. 54]; (I) complete
Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (J)
cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-12, etc.), interferons (e.g. interferon-.gamma.),
macrophage colony stimulating factor, tumor necrosis factor, etc.
[see Chapters 27 & 21 of ref. 49]; (K) a saponin (e.g. QS21)+3
dMPL+IL-12 (optionally+a sterol) [55]; (L) monophosphoryl lipid A
(MPL) or 3-O-deacylated MPL (3dMPL) [e.g. chapter 21 of ref. 49];
(M) combinations of 3dMPL with, for example, QS21 and/or
oil-in-water emulsions [56]; (N) oligonucleotides comprising CpG
motifs [57] i.e. containing at least one CG dinucleotide; (O) a
polyoxyethylene ether or a polyoxyethylene ester [58]; (P) a
polyoxyethylene sorbitan ester surfactant in combination with an
octoxynol [59] or a polyoxyethylene alkyl ether or ester surfactant
in combination with at least one additional non-ionic surfactant
such as an octoxynol [60]; (Q) an immunostimulatory oligonucleotide
(e.g. a CpG oligonucleotide) and a saponin [61]; (R) an
immunostimulant and a particle of metal salt [62]; a saponin and an
oil-in-water emulsion [63]; (T) E. coli heat-labile enterotoxin
("LT")l , or detoxified mutants thereof, such as the K63 or R72
mutants [e.g. Chapter 5 of ref. 64]; (U) cholera toxin ("CT"), or
detoxified mutants thereof [e.g. Chapter 5 of ref. 64]; (V)
double-stranded RNA; (W) aluminium salts, such as aluminium
hydroxides (including oxyhydroxides), aluminium phosphates
(including hydroxyphosphates), aluminium sulfate, etc [Chapters 8
& 9 in ref. 49]; (X) monophosphoryl lipid A mimics, such as
aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [65];
(y) polyphosphazene (PCPP); (Z) a bioadhesive [66] such as
esterified hyaluronic acid microspheres [67] or a mucoadhesive
selected from the group consisting of cross-linked derivatives of
poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,
polysaccharides and carboxymethylcellulose. Other substances that
act as immunostimulating agents may also be used [e.g. see Chapter
7 of ref. 49].
[0041] Aluminium salts are preferred adjuvants for parenteral
immunisation. Mutant toxins are preferred mucosal adjuvants. The
use of an aluminium hydroxide adjuvant is most preferred,
particularly for intramuscular injection, and this adjuvant is
preferably used with a histidine buffer [68].
[0042] The invention provides a process for preparing a
pharmaceutical composition, comprising the steps of: (i) preparing
OMVs according to the invention; and (ii) formulating the OMVs as a
pharmaceutical Step (ii) may involve activities such as filtration,
addition of adjuvants, addition of buffer,, etc.
OMVs and OMV-Based Compositions
[0043] The invention provides OMVs obtained by a process of the
invention. The invention also provides components in essentially
their native form.
[0044] The invention also provides a composition comprising such
OMVs and a pharmaceutically acceptable carrier. The composition may
also comprise an adjuvant.
[0045] Compositions of the invention are preferably immunogenic
compositions, and are more preferably vaccine compositions. Such
compositions can be used to raise immune responses (e.g. antibody
responses) in a mammal (e.g. in a human, such as a child).
[0046] The pH of the composition is preferably between 6 and 8,
preferably about 7. The pH may be maintained by the use of a
buffer. The composition may be sterile and/or pyrogen-free. The
composition may be isotonic with respect to humans. The composition
may or may not include a preservative (e.g. thiomersa,
2-phenoxyethanol, etc.). Mercury-free compositions are
preferred.
[0047] The composition is preferably free from blood-derived
components. The composition is preferably free from transmissible
spongiform encephalopathy agents (e.g. prions). The composition is
preferably substantially free from whole bacteria, and in
particular from living bacteria.
[0048] The composition may include residual material from vesicle
preparation (e.g. detergent, preferably <0.4 .mu.g detergent per
.mu.g OMV protein). The composition may include soluble sugars e.g.
disaccaharides such as sucrose and/ trehalose. LPS content is
preferably <0.2 .mu.g per .mu.g OMV protein.
[0049] Compositions of the invention may be distributed in various
containers e.g. vials or pre-filled syringes. The use of glass
vials is preferred. These containers will generally be sterile and
hermetically-sealed. Each container preferably includes a single
dose e.g. 0.5 ml of liquid. Containers may be packaged singly or in
multiples e.g. a box of 10 vials. Once packaged, compositions of
the invention are preferably stored at between 2.degree. C. and
8.degree. C., but should not be frozen.
[0050] Vaccines of the invention may be prophylactic (i.e. to
prevent disease) or therapeutic to reduce or eliminate the symptoms
of a disease).
[0051] Compositions for administration to patients will comprise an
immunologically effective amount of the OMVs. An "immunologically
effective amount" is an amount sufficient to effect an immune
response in a patient, and more preferably a protective immune
response in a patient. The precise amount for a patient will depend
upon their size and health, the nature and extent of their
condition, and the therapeutics or combination of therapeutics
selected for administration. The effective amount for a given
situation is determined by routine experimentation and is within
the judgment of a physician. For purposes of the present invention,
an immunologically effective amount will generally be administered
at a dosage of from about 0.01 mg/kg to about 5 mg/kg, or about
0.01 mg/kg to about 50 mg/kg or about 0.05 mg/kg to about 10 mg/kg
of the composition of be invention in the individual to which it is
administered. A typical composition will include 50 .mu.g/ml of
protein.
[0052] In addition to OMV antigens, compositions of the invention
may include one or more of the following additional antigens:
[0053] a saccharide antigen from N. meningitidis serogroup A, C,
W135 and/or Y, such as the oligosaccharide disclosed in ref. 142
from serogroup C [see also ref. 69] or the oligosaccharides of ref.
146. [0054] antigens from Heliobacter pylori such as CagA [70 to
73], VacA [74, 75], NAP [76, 77, 78], HopX [e.g. 79], HopY [e.g.
79] and/or urease. [0055] a saccharide antigen from Streptococcus
pneumoniae [e.g. 80, 81, 82]. [0056] an antigen from hepatitis A
virus, such as inactivated virus [e.g. 83, 84]. [0057] an antigen
from hepatitis B virus, such as the surface and/or core antigens
[e.g. 84, 85]. [0058] an antigen from hepatitis C virus [e.g. 86].
[0059] a diphtheria antigen, such as a diphtheria toxoid [e.g.
chapter 3 of ref. 87]. [0060] a tetanus antigen, such as a tetanus
toxoid [e.g. chapter 4 of ref. 87]. [0061] an antigen from
Bordetella pertussis, such as pertussis holotoxin (PT) and
filamentous haemagglutinin (FHA) from B. pertussis, optionally also
in combination with pertactin and/or glutinogens 2 and 3 [e.g.
refs. 88 & 89]; whole-cell pertussis antigen may also be used.
[0062] a saccharide antigen from Hesemophilus influenzae B [e.g.
69]. [0063] polio antigen(s) [e.g. 90, 91] such as OPV or,
preferably, IPV. [0064] an antigen from N. gonorrhoeae [e.g. 92,
93, 94, 95]. [0065] an antigen from Chlamydia pneumoniae [e.g.
refs. 96 to 102]. [0066] an antigen from Porphyromonas gingivalis
[e.g. 103]. [0067] rabies antigen(s) [e.g. 104] such as lyophilised
inactivated virus [e.g. 105, RabAvert.TM.]. [0068] measles, mumps
and/or rubella antigens [e.g. chapters 9, 10 & 11 of ref.
87].
[0069] influenza antigen(s) [e.g. chapter 19 of ref. 87], such as
the heamagglutinin and/or neuraminidase surface proteins. [0070]
antigen(s) from a paramyxovirus such as respiratory syncytial virus
(RSV [106, 107]) and/or parainfluenza virus (PIV3 [108]). [0071] an
antigen from Moraxella catarrhalis [e.g. 109]. [0072] an antigen
from Streptococcus pyogenes (group A streptococcus) [e.g. 110, 111,
112]. [0073] an antigen from Staphylococcus aureus [e.g. 113].
[0074] an antigen from Bacillus anthracis [e.g. 114, 115, 116].
[0075] an antigen from a virus in the flaviviridae family (genus
Flavivirus), such as from yellow fever virus, Japanese encephalitis
virus, four serotypes of Dengue viruses, tick-borne encephalitis
virus, West Nile virus. [0076] a pestivirus antigen, such as from
classical porcine fever virus, bovine viral diarrhoea virus, and/or
border disease virus. [0077] a parvovirus antigen from parvovirus
B19. [0078] a prion protein (e.g. the CJD prion protein) [0079] an
amyloid protein, such as a beta peptide [117]. [0080] a cancer
antigen, such as those listed in Table 1 of ref. 118 or in tables 3
& 4 of ref. 119.
[0081] The inclusion of further N. meningitidis antigens is
preferred. In particular, the composition may include a saccharide
antigen from one or more (i.e. 1, 2, 3 or 4) of meningococcal
serogroups A, C, W135 and/or Y. Where fewer than 4 of these
additional serogroups are included, it is preferred to include at
least serogroup C e.g. C+A+W135, C+A+Y, C+W135+Y.
[0082] Where a saccharide or carbohydrate antigen is used, it is
preferably conjugated to a carrier protein in order to enhance
immanogenicity [e.g. refs. 120 to 129]. Preferred carrier proteins
are bacterial toxins or toxoids, such as diphtheria or tetanus
toxoids. The CRM.sub.197 diphtheria toxin mutant is particularly
preferred [130]. Other carrier polypeptides include the N.
meningitidis outer membrane protein [131], synthetic peptides [132,
133], heat shock proteins [134, 135], pertussis proteins [136,
137], protein D from H. influenzae [138], cytokines [139],
lymphokines [139], hormones [139], growth factors [139], toxin A or
B from C. difficile [140], iron-uptake proteins [141], etc.
Different saccharides can be conjugated to the same or different
type of carrier protein. Any suitable conjugation reaction can be
used, with any suitable linker where necessary. For meningococcal
conjugates [142-148], preferred carriers are diphtheria toxoid,
CRM197 and H. influenza protein D.
[0083] Toxic protein antigens may be detoxified where necessary
e.g. detoxification of pertussis toxin hy chemical and/or genetic
means [89].
[0084] Where a diphtheria antigen is included in the composition it
is preferred also to include tetanus antigen and pertussis
antigens. Similarly, where a tetanus antigen is included it is
preferred also to include diphtheria and pertussis antigens.
Similarly, where a pertussis antigen is included it is preferred
also to include diphtheria and tetanus antigens, DTP combinations
are thus preferred.
[0085] Antigens in the composition will typically be present at a
concentration of at least 1 .mu.g/ml each. In general, the
concentration of any given antigen will be sufficient to elicit an
immune response against that antigen.
[0086] As an alternative to using protein antigens in the
composition of the invention, nucleic acid encoding be antigen may
be used [e.g. refs. 149 to 157]. Protein components of the
compositions of the invention may thus be replaced by nucleic acid
(preferably DNA e.g. in the form of a plasmid) that encodes the
protein.
Methods of Treatment
[0087] The invention provides a method for raising an immune
response in a patient, comprising administering an immunogenic dose
of OMVs of the invention to the patient. The immune response is
preferably protective and preferably involves antibodies and/or
cell-mediated immunity. The method may raise a booster
response.
[0088] The patient is preferably a human. Where the vaccine is for
prophylactic use, the human is preferably a child (e.g. a toddler
or infant) or a teenager; where the vaccine is for therapeutic use,
the human is preferably a teenager or an adult. A vaccine intended
for children may also be administered to adults e.g. to assess
safety, dosage, immunogenictiy. The patient is preferably less than
20 years old e.g. 13-19 years old, 8-12 years old, 16-24 months
old, 6-8 months old, 6 weeks-5 months old.
[0089] Vaccines of the invention are preferably administered by
intramuscular injection. Typical sites for injection include the
upper thigh and the upper arm.
[0090] The invention also provides OMVs of the invention for use in
medicine.
[0091] The invention also provides the use of OMVs of the invention
in the manufacture of a medicament for treating and/or preventing
meningococcal infection and/or bacterial meningitis.
[0092] The invention may be used to elicit systemic and/or mucosal
immunity.
[0093] Dosage treatment can be a single dose schedule or a multiple
dose schedule. Multiple doses may be used in a primary immunisation
schedule and/or in a booster immunisation schedule e.g. a primary
immunisation schedule may involve three injections, with an
interval of about 6 weeks between each injection. A typical volume
for a single intramuscular liquld dose is 0.5 ml.
[0094] The term "OMV" as used herein includes any proteoliposomic
vesicle obtained by disrupting a bacterial outer membrane to form
vesicles of the outer membrane which include protein components of
the outer membrane. OMVs are prepared artificially from bacteria
(e.g. by detergent treatment) and are thus distinct from
microvesicles (MVs [158]) and `native OMVs` (`NOMs` [48]), both of
which are naturally-occurring membrane vesicles that form
spontaneously during bacterial growth and are released into culture
medium. MVs can be obtained by culturing in broth culture medium,
separating whole cells from the smaller blebs in the broth culture
medium, and then collecting the MVs from the cell-depleted medium.
Strains for use in production of MVs can generally be selected on
the basis of the amount of MVs produced in culture e.g. refs. 159
& 160 describe Neisseria with high MV production.
[0095] The term "comprising" can mean "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0096] The term "about" in relation to a numerical value x means,
for example, e.g. x.+-.10%.
[0097] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the dedinition of the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0098] FIG. 1 shows a process including two ultracentrifugation
steps without ultrafiltration.
[0099] FIG. 2 shows a process in which one ultracentrifugation step
has been replaced by an ultrafiltration step.
MODES FOR CARRYING OUT THE INVENTION
Example 1
OMVs from Meningococcal Serogroup B (Norwegian Strain)
[0100] N. meningitidis serogroup B (strain 44/76, from Norway) was
cultured on eight "selective medium for Meningococci" plates at
35.degree. C. in 5% CO.sub.2/air atmosphere for 24 hours. Cell were
harvested into 2 tubes with 12 ml Frantz' medium. Contents of tubes
were added to 2.times.500 ml flasks containing Frantz' medium (150
ml) and grown with shaking for 12 hours to obtain the correct
growth for transferring into 2.times.5000 ml flasks containing
Frantz' medium (1500 ml). The flasks were grown with shaking for a
further 12 hours to yield the inoculum. One flask was added to a
Chemap fermentor with 300 L capacity, containing 110 L of
pre-sterilised Frantz' medium and sterile-filtered dialysed yeast
extract. The pH after inoculation was 7.1, maintained at 7.0 with
3N NaOH. A surface aeration fermentation was performed, controlling
the amount of air O.sub.2 and stirrer applied, and cultivating for
10 hours at 35.degree. C. Growth was terminated at an OD.sub.590 mm
of 7.10, the fermentor was cooled under 15.degree. C., the air
supply was reduced and stirring continued at 100 rpm overnight.
[0101] Transfer of the bacterial suspension from the fermentor was
done by pressure to a Millipore CUF cross flow filtration unit
equipped with valves, pumps and a filter module with 4 Pellicon
P2B300VO5 polyethersulphone filters (300 kD cutoff). Initial
transfer of 30 L bacterial suspension was followed with a constant
volume concentration until the fermentor was emptied, and then a
further concentration was performed to give a volume of 5.5 L.
[0102] Concentrating the suspension was performed in the CFF unit
by circulating the suspension to be passing by the filters, with a
transmembrane pressure being continuously monitored and kept less
then 0.5 bar (observed: 0.5 bar at the end of concentration).
[0103] Adjustment of pH of the concentrated bacterial suspension
from pH 7.0 to 8.2 was done by adding, via a tubing system, 5 L of
0.1 M Tris-HCl buffer of pH 9 with 10 mM EDTA, followed by 15 min
stirring in the CFF unit to secure uniform conditions.
[0104] Inactivation/extraction of outer membrane (OM) material was
initiated by adding, via tubings 500 ml of an 0.1M Tris-HCl buffer
(pH 9) containing 10% deoxycholate (DOC), to give a final
concentration of 0.5%. Subsequently the suspension was circulated
in the CFF unit for 30 min, and the extracted suspension (9.5 L),
checked to be completely without living bacteria, was drained off
by pumping into a 25 L bottle.
[0105] In a first experiment (experiment A; FIG. 1), crude OMVs
were prepared by distributing the inactivated suspension to
centrifuge tubes of 500 ml, and centrifuging in a Beckman
centrifuge at 9000 rpm (16650.times.g) for 1 hour at 4.degree. C.,
cellecting of supernatant, 1.35 L of crude OMVs was purified by two
subsequent ultracentrifugations at 19000 rpm, 4.degree. C., for
13.6 hours and 6.8 hours respectively, collecting the pellet. The
pellet was suspended in 660 ml of 3% sucrose with magnetic stirring
at room temperature until homogeneous, obtaining a concentration of
the purified material of 1.52 g/L of total protein.
[0106] In a second experiment (experiment B; FIG. 2), 3 L of crude
OMVs were prepared from bacterial suspension using a CUF cross flow
filtration unit equipped with valves, pumps and a filter module
with 1 Pellicon P2B300V05 polyethersulphone filters (300 kD
cutoff), Initial transfer of 1 L crude OMVs was followed with a
constant volume concentration until the 3 L was finished, and then
diafiltered by adding 5 L of 0.05 M Tris-HCl buffer of pH 8.6 with
2 mM EDTA, 1% of DOC and 20% sucrose. The retentate obtained was
purged by ultracentrifugation at 19000 rpm. 4.degree. C., for 6.8
hours, cellecting the pellet. The pellet was suspended in 1 t of 3%
sucrose with magnetic stirring at room temperature until
homogeneous, obtaining a concentration of 0.85 g/L total protein in
the purified material.
[0107] Final purifications of OMV obtained by both experiment A and
B, after a dilution with 3% sucrose around 1.2 g/L of total
protein, were both performed at 20.degree. C. by filtering through
3 capsule filters (Gelman Science Suporlife DCF) in sequence, first
pre filters of 0.8 .mu.m and 0.45 .mu.m, respectively, then the
final sterile filtration (0.22 .mu.m), testing 836 ml of purified
material for experiment A, with an initial protein concentration of
1.1 mg/ml, and 1 L for experiment B. The OMV protein concentrations
after the filtration were 0.12 mg/ml and 0.59 mg/ml
respectively.
[0108] OMVs were characterised as follows:
TABLE-US-00001 Experiment A Experiment B Specification Deoxycholate
1.5 0.4 0.1-0.4 (.mu.g/g protein) DNA 0.004 0.004 <0.035
(.mu.g/g protein) Endotoxin 2.8 .times. 10.sup.3 2.6 .times.
10.sup.3 <20 .times. 10.sup.3 (UI/g protein) LPS 0.05 0.08
0.06-0.12 (.mu.g/g protein) SDS page 80 kDa 1.7 2.2 1-4 70 kDa 11.8
12.7 1-12 class I 24.6 25.1 22-32 class III 34.8 32.8 30-43 class
IV 12.0 12.2 9-18 class V 15.0 15.1 10-24
[0109] Thus the OMVs prepared using ultrefiltration have a similar
composition to those obtained by ultracentrifugation. In comparison
to the prior art method, however, the method of the invention is
much simpler and quicker.
Example 2
OMVs from Meningococcal Serogroup B (New Zealand Strain)
[0110] N. meningitidis serogroup B (strain NZ 98/254, from New
Zealand) was cultured as before, except that; (a) Catlin medium was
used in place of Frantz' medium; (b) the initial 150 ml cultures
were grown to a level ready for transferring into a Chemap
fermentor with 300 L capacity, containing 120 L of pre-sterilized
medium; (c) growth in the Chemap fermentor was for 12 hours; (d)
growth was terminated at OD.sub.690 mm of 5.90.
[0111] Transfer from the fermentor was as before, except that
concentration was performed until 5 L volume. Concentration was
performed as before.
[0112] pH was adjusted as before, except that: (a) the final pH was
8.6; (b) the amount of 0.1 M Tris-HCl buffer added was 6 L.
[0113] Inactivation/extraction was as before, except; (a) 600 ml of
the Tris-HCl buffer was added; (b) the volume of extracted
suspension was 19.5 L.
[0114] Preparation of crude OMVs was as before, except; (a)
centrifuge tubes were 1000 ml volume; (b) centrifugation was at
8000 rpm (16650.times.g), to give 17.5 L of supernatant.
[0115] Cross-flow filtration for purifying OMVs (in place of
centrifugation) was as in experiment B above, except: (a) using
17.5 L crude OMVs; bh) using two P2B300V05 polyethersulphone
filters (300 kD cutoff); (c) using an initial transfer of 4 L crude
OMVs; (d) diafiltration with 30 L Tris-HCl buffer; (e) pellet was
resuspended in 1.2 L of 3% sucrose; (f) the homogenous material was
further sonicated, and gave a final concentration of purified
material of 1.5 g/L total protein.
[0116] Final purification was as before, except: (a) filtration was
through two capsule filters (Sartoclean CA, Sartobran P) in
sequence, first pre-filters of 0.8+0.65 .mu.m, then a final sterile
filtration 0.45+0.22 .mu.m. The OMV protein concentration after the
filtration was 1.0 g/L.
[0117] OMVs were characterised as follows:
TABLE-US-00002 Example 2 Specification Deoxycholate 0.4 0.1-0.4
(.mu.g/g protein) DNA 0.0005 <0.035 (.mu.g/g protein) Endotoxin
5393 <20 .times. 10.sup.3 (UI/g protein) LPS 0.10 0.06-0.12
(.mu.g/g protein) SDS page 80 kDa 3.8 1-4 70 kDa 6.4 1-12 class I
18.7 22-32 class III + FbpA 31.3 30-43 class IV 10.7 9-18 class V
2.7 10-24 NspA 3.9 1-7
[0118] Thus the production method provided OMVs with a native
antigen mosaic and a strongly reduced level of LPS. In comparison
to the prior art method where two ultracentrifugation steps are
used, however, the invention is much simpler and quicker.
Example 3
OMVs from Meningococcal Serogroup B (New Zealand Strain)
[0119] Crude OMVs were prepared from the 98/254 strain as described
above. The pH was adjusted to between 7.5 and 9.0 (typically
between and 8.3 and 8.5) with buffer, and then concentrated up to
20 litres by ultrafiltration for between 3.5 and 4.5 hours using
Polysulphone Millipore Pellicon 2 cassettes with a surface area of
3 m..sup.2. The concentrate material was diafiltered against 7
volumes of a solution containing Tris-EDTA, 1% DOC and 20% sucrose
(`buffer B`), and then with 3 volumes of `buffer B1` (same as
`buffer B` but with only 0.5% DOC). The retentate was concentrated
again up to 4 litres and collected. The ultrafiltration system was
washed with buffer B1. The retentate was then washed, and OMVs
(retentate+washes) were stored at 2-8.degree. C. The bioburden in
the final material was aero, and endotoxin content was <0.05
UI/ml. The process showed excellent lot-to-lot consistency.
[0120] The stored material was centrifuged in a Beckman Coulter
Optima XL 100K ultracentrifuge using a type 19 rotor and 250 ml
Beckman bottles (220.+-.10 ml material per bottle), 19000 rpm for
408 minutes at 2-8.degree. C. Pellets were washed in 10 ml of a
sucrose solution, and were then re-suspended in 3% sucrose (60 ml
volume added) using a 700 rpm magnetic agitator (2.5 cm bar) in 250
ml Beckman bottles. Re-suspended material was sonicated for 300
minute at <20.degree. C. If necessary, the sonicated material
was diluted with 3% sucrose solution to give a final protein
concentration of 1.2 mg/ml. The bioburden in the final material was
zero, and the process showed excellent lot-to-lot consistency.
[0121] The OMVs were subjected to a final filtration step, first
through 0.8-0.65 .mu.m filters and then through 0.22 .mu.m filters.
The sonicated OMVs were passed into a sterile glass container with
Sartoclean CA 0.8-0.65 .mu.m 0.2 .mu.m pre-filters. This
pre-filtration was performed for 5-6 minutes with a peristaltic
pump using only one set of filters. The filtrate was then passed
into a second sterile glass container with Sartobran P 0.45-0.22
.mu.m 0.4 m.sup.4 filters. This filtration lasted 7-10 minutes,
again with peristaltic pumps. The pre-filters were first rinsed
with 500-600 ml of 3% sucrose, and the 0.22 .mu.m filters were
washed with 200 ml of 5% sucrose after filtration. Final OMV
material was stored at 2-8.degree. C., and contained <0.16 .mu.g
LPS per .mu.g of protein and <0.4 .mu.g DOC per .mu.g of
protein. Bioburden was zero. Protein content in the OMVs was
between .800 .mu.g/ml and .1000 .mu.g/ml.
[0122] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
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