Note: Descriptions are shown in the official language in which they were submitted.
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INTRANASAL IMMUNIZATION WITH DETOXIFIED
LIPOOLIGOSACCHARIDE FROM NONTYPEASLE HAEMOPHILUS
INFLUENZAEORMORAd~ELLA CATARRHALIS
This application claims the benefit of priority of U.S. Pat Appl No 60/288,695
filed
May 3, 2001, and for U.S. purposes only, is a continuation-in-part of U.S. Pat
Appl No
09/789,017 filed February 20, 2001, which is a divisional of U.S. Pat Appl No
08/842,409
filed April 23, 1997, which claims the benefit of priority of U.S. Pat Appl No
60/016,020
filed April 23, 1996, and is also a continuation-in-part of U.S. Pat Appl No
09/610,034
filed July 5, 2000, which is a continuation of Intl Pat Appl No
PCT/LTS99/00590 filed
January 12, 1999, designating the United States of America and published in
English,
which claims the benefit of priority of U.S. Pat Appl No 60/071,483 filed
January 13, 1998;
the disclosures of such related applications are incorporated herein by
reference.
Field of the Invention
The invention relates to intranasal immunization with detoxified
lipooligosaccharide from nontypeable Haemophilus influenzae or Mo~axella
cataf°f°halis.
Back~,round of the Invention
Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis
media
(OM) in children and respiratory tract diseases in adults (Klein, J.O. et al.
1992 Adv Pediatr
39:127-156; Murphy, T.F. et al. 1987 Rev Infect Dis 9:1-15; Musher, D.M. et
al. 1983 Anya
Intern Med 99:344-350). Mo~axella (By~anhamella) catarrhalis (Catlin, B.W.
1990 Clin
Mice~biol Rev 3:293-320; Doern, G.V. 1986 Diagn Micr~obiol Infect Dis 4:191-
201;
Enright, M.C., and H. McKenzie 1997 J Med Microbiol 46:360-371) is recognized
as the
third-most-common pathogen causing otitis media and sinusitis in children,
after
Streptococcus pneumoniae and nontypeable Haemophilus influenzae (Bluestone,
C.D. 1986
Drugs 31(Suppl. 3):132-141; Faden, H. et al. 1994 Jlnfect Dis 169:1312-1317).
This gram-
negative diplococcus is also a cause of respiratory tract infections in adults
(Boyle, F.M. et
al. 1991 Med J Aust 154:592-596; Sarubbi, F.A. et al. 1990 Ana J Med 88:95-
14S),
especially those with chronic obstructive pulmonary diseases (Nicotra, B. et
al. 1986 Arch
InteYn Med 146:890-893) or compromised immune systems (Alaeus, A. and G.
Stiernstedt
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Scand J Infect Dis 23:115-116; Enright, M.C. and H. McKenzie. 1997 J Med
Microbiol
46:360-371).
Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis
media
in children and of pneumonitis in adults with depressed resistance.
Lipooligosaccharide
(LOS) is a major surface antigen of NTHi and elicits bactericidal and opsonic
antibodies.
Gu, X.X. et al. 1996 Infect Imnaun 64:4047-4053 prepared detoxified LOS (dLOS)
protein
conjugates from NTHi for use as experimental vaccines. LOS from NTHi 9274 was
treated
with anhydrous hydrazine and had its toxicity reduced to clinically acceptable
levels.
Hydrazine treatment of NTHi LOS resulted in a 10,000-fold reduction in the
level of
"endotoxin", which is at clinically acceptable levels (W.H.O. Expert Committee
on
Biological Standardization 1991 W.H.O. Tech Rep Sen 814:15-37). dLOS was bound
to
tetanus toxoid (TT) or high-molecular-weight proteins (HMPs) from NTHi through
a linker
of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratio of the
dLOS
to protein carriers ranged from 26:1 to 50:1. The antigenicity of the
conjugates was similar
to that of the LOS alone as determined by double immunodiffusion. Subcutaneous
or
intramuscular injection of the conjugates elicited a 28- to 486-fold rise in
the level of
immunoglobulin G antibodies in mice to the homologous LOS after two or three
injections
and a 169- to 243-fold rise in the level of immunoglobulin G antibodies in
rabbits after two
injections. The immunogenicity of the conjugates in mice and rabbits was
enhanced by
formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits,
conjugate-
induced LOS antibodies induced complement-mediated bactericidal activity
against the
homologous strain 9274 and prototype strain 3189. These results indicate that
a detoxified
LOS-protein conjugate is a candidate vaccine for otitis media and pneumonitis
caused by
NTHi. Gu, X.X. et al. 1997 Infect Immun 65:4488-4493 determined that
subcutaneous or
intramuscular injections of detoxified-lipooligosaccharide (dLOS)-protein
conjugates from
NTHi protected against otitis media in chinchillas.
MoYaxella (Bs°anharnella) cataf°n7aalis (M. catan~halis) is an
importmt cause of otitis
media and sinusitis in children and of lower respiratory tract infections in
adults.
Lipooligosaccharide (LOS) is a major surface antigen of the bacterium and
elicits
bactericidal antibodies. Treatment of the LOS from strain ATCC 25238 with
anhydrous
hydrazine reduced its toxicity 20,000-fold, as assayed in the Limulus
amebocyte lysate
(LAL) test. The detoxified LOS (dLOS) was coupled to tetanus toxoid (TT) or
high-
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molecular-weight proteins (HMP) from nontypeable Haemophilus influerazae
through a
linker of adipic acid dihydrazide to form dLOS-TT or dLOS~HMP. The molar
ratios of
dLOS to TT and HMP conjugates were 19:1 and 31:1, respectively. The
antigenicity of the
two conjugates was similar to that of the LOS, as determined by double
immunodiffusion.
Subcutaneous or intramuscular injection of both conjugates elicited a 50- to
100-fold rise in
the geometric mean of immunoglobulin G (IgG) to the homologous LOS in mice
after three
injections and a 350- to 700-fold rise of anti-LOS IgG in rabbits after two
injections. The
immunogenicity of the conjugate was enhanced by formulation with
monophosphoryl lipid
A plus trehalose dimycolate. In rabbits, conjugate-induced antisera had
complement-
mediated bactericidal activity against the homologous strain and heterologous
strains of M.
catarrhalis. These results indicate that a detoxified LOS-protein conjugate is
a candidate
for immunization against M. catarf~halis diseases.
Current pediatric immunization programs include too many injections in the
first
months of life. Oral or nasal vaccine delivery eliminates the requirement for
needles.
There is a need for mucosal vaccines against NTHi- and M. catarYhalis- caused
otitis media
in children and other NTHi- and M. catar~halis- caused diseases in children
and adults.
Summary of the Invention
The invention relates to intranasal immunization with detoxified
lipooligosaccharide from nontypeable Haemophilus influenzae or
Mof°axella catarYlZalis.
Brief Description of the Drawings
Figure A shows the proposed chemical structure of lipid A from nontypeable
Haemophilus ifafluehzae lipooligosaccharide (LOS). R=site of attachment of the
oligosaccharide chain. Hydrazine treatment of LOS removes primary O-linked
fatty acids
from 3-hydroxy groups of diglucosamine (*) and secondary O-linked fatty acids
from
hydroxy groups of 3-hydroxy fatty acids of lipid A (arrow).
Figure B shows the proposed chemical structure of lipid A from Moraxella
catarrhalis lipooligosaccharide (LOS). R=site of attachment of the
oligosaccharide chain.
Hydrazine treatment of LOS removes primary O-linked fatty acids from 3-hydroxy
groups
of diglucosamine (*) and secondary O-linked fatty acids from hydroxy groups of
3-hydroxy
fatty acids of lipid A (arrow).
Figure 1. Tmmunohistochemistry with anti-IgA and anti-IgG staining in the
nose.
(A) Anti-IgA (or anti-IgG) staining in control mice, (B) anti-IgA staining in
dLOS-TT
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immunized mice, and (C) anti-IgG staining in dLOS-TT immunized mice
(magnification
400x). Intranasal immunization with dLOS-TT dramatically increased the
staining with
IgA of the mucous blanket, and glandular cells in the nose as compared with
the staining in
the control mice. However, staining with anti-IgG was strongly shown only at
the vessels
of the nasal tissue in mice irrnnunized with dLOS-TT. The nasal tissue of the
control mice
was not stained with anti-IgA (or anti-IgG).
Figure 2. Bacterial clearance of NTHi strain 9274 from mouse nasopharynx.
Immunization schedules and mouse grouping were shown in Table 1, footnote a.
Mice
were challenged with strain 9274 into the nose 1 wk after the last
immunization and nasal
washes were collected at 6 h post-challenge. Mice immunized with dLOS-TT and
CT
showed a significant reduction of bacterial recovery by 74% or 76% when
compared to
those of the mice immunized with CT alone or dLOS and CT (*, p<0.05).
Figure 3. Binding reactivity of nasal wash (IgA) or serum (IgG) to homologous
strain and five heterologous strains in whole-cell ELISA. The nasal wash from
mice
immunized with dLOS-TT bound strongly to the homologous strain 9274 and the
heterologous strains 3198, 5657 and 7502 but weakly to strains 1479 and 2019
(A). Similar
binding reactivity was observed in serum from mice immunized with the dLOS-TT
(B).
Figure 4. Silver-stained SDS-PAGE patterns (A) and Western blot analysis (B
and
C) of homologous strain and five heterologous strains. Lanes 1 through 6
contain strains
20. 1479, 2019, 3198, 5657, 7502 and 9274. Nasal wash (IgA) from mice
immunized with
dLOS-TT was reactive strongly to LOSS from strains 9274, 3198, 5657, and 7502,
weakly
to 1479 but not to 2019 (B). However, sera (IgG) from mice immunized with the
dLOS-TT
were reactive to all LOSS with strong binding in strains 9274, 3198, 5657 and
7502 (C).
Arrows show each LPS of Ra (upper arrow) and Rc (lower arrow) mutants as
markers from
Salmonella minnesota.
Figure I. Specific antibody-forming cells induced by dLOS-CRM conjugate
measured by ELISPOT assay. See Table I, footnote a. (A) IgA-forming cells per
million of
lymphoid cells; (B) IgG-forming cells per million of lymphoid cells; (C) IgM-
forming cells
per million of lymphoid cells. NALT: nasal-associated lymphoid tissue, NP:
nasal passage,
CLN: cervical Lymph node, PP: Peyer's patch.
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Figure II. Specific antibody-forming cells initiated by different dLOS-protein
conjugates. See Table III, footnote a. NALT: nasal-associated lymphoid tissue,
NP: nasal
passage, CLN: cervical lymph node, PP: Peyer's patch.
Figure III. Comparison of protective effect induced by different dLOS-protein
conjugates in bacterial clearance from mouse nasopharynx and Lungs. See Table
III,
footnote a. One week after the last immunization, mice were challenged with 2
x 10$ CFU
of M. catarnhalis strain 25238 per mI in a nebulizer, and nasal washes and
Lungs were
collected at 6 h postchallenge. The CFU of bacterial recovery from CT group
compared to
that of other group: P < 0.01.
Figure IV. Comparison of protective effect from different immunization
regimens
in bacterial clearance from mouse nasopharynx. See Table IV, footnote a. One
week after
the last immmuzation, mice were challenged with 2 x 108 CFU of M. catanf~halis
strain
25238 per ml in a nebulizer, and nasal washes were collected at 6 h
postchallenge. Green
bars: intranasal immunization, red bars: subcutaneous injection.
Figure V. Comparison of protective effect from different immunization regimens
in bacterial clearance from mouse lungs. See Table IV, footnote a. One week
after the last
immunization, mice were challenged with 2 x 10$ CFU of M. catarnhalis strain
25238 per
ml in a nebulizer, and lungs were collected at 6 h postchallenge. Green bars:
intranasal
immunization, red bars: subcutaneous injection.
Figure VI. Kinetics of bacterial recovery from mouse nasopharynx challenged
with
M. cataf°nhalis strain 25238. Mice were intranasally administered 4
times at 1-week
intervals with 10 ~I of PBS containing a mixture of 5 ~.g of dLOS-CItM and 1
~,g of CT, or
10 ~1 of PBS. One week after the last ixmnunization, mice were challenged with
5 x 108
CFU of M. catay~rhalis strain 25238 per ml in a nebulizer, and nasal washes or
lungs were
collected at 0, 3, 6, 12, 24 h postchallenge, respectively. At each time
point, immunized
mice significantly reduced bacterial recovery from nasopharynx and lungs, and
bacterial
recovery became undetectable within 24 h, postchallenge.
Figure VII. Kinetics of bacterial recovery from mouse lungs challenged with M.
catarr-halis strain 25238. See description of Fig. VI.
Detailed Description of the Invention
The invention relates to an immunogenic composition comprising an immunizing
amount of Nontypeable Haefnophilus influenzae (NTHi) or Moraxella
cataf°r~halis
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lipooligosaccharide (LOS) from which at least one primary O-linked fatty acid
has been
removed to fonn detoxified LOS (dLOS) and an immunogenic carrier covalently
linked
thereto, optionally where the dLOS and the immunogenic carrier are covalently
linked by a
linker, and a mucosal adjuvant or delivery system.
In accordance with the present invention, it has now been ~ surprisingly found
that
mucosal administration, preferably intranasally, of NTHi or M. catar~halis
lipooligosaccharide (LOS) from which at least one primary O-linked fatty acid
has been
removed to form detoxified LOS (dLOS) and an imrnunogenic Garner covalently
linked
thereto, optionally where the dLOS and the immunogenic Garner are covalently
linked by a
I O linker, elicits an immunological response and can even inhibit
colonization by NTHi or M.
catarrhalis and prevent otitis media and other respiratory diseases caused by
NTHi or M.
catarrhalis infection.
Accordingly, in one aspect, the present invention provides a method for
inducing an
immunological response in a host, preferably a human host, to inhibit
colonization by NTHi
or M. cata~~halis or prevent otitis media and other respiratory diseases
caused by NTHi or
M. cata~~halis infection by mucosal administration, preferably intranasal
adminstration, to
the host of an effective amount of NTHi or M. cata~Yhalis lipooligosaccharide
(LOS) from
which at least one primary O-linked fatty acid has been removed to form
detoxified LOS
(dLOS) and an immunogenic carrier covalently linked thereto, optionally where
the dLOS
and the immunogenic carrier are covalently linked by a linker, and a mucosal
adjuvant or
delivery system.
Moreover, in another aspect, the present invention provides use of an
effective
amount of NTHi or M. cata~rhalis lipooligosaccharide (LOS) from which at least
one
primary O-linked fatty acid has been removed to form detoxified LOS (dLOS) and
an
immunogenic carrier covalently linked thereto, optionally where the dLOS and
the
immunogenic carrier are covalently linked by a linker, and a mucosal adjuvant
or delivery
system, for mucosal administration, preferably intranasal administration, to a
host,
preferably a human host, for inducing an immunological response to inhibit
colonization by
NTHi or M. catarrl2alis or prevent otitis media and other respiratory diseases
caused by
NTHi or M. cataYrlaalis infection.
The present invention relates to a conjugate vaccine comprising nontypeable
Haemophilus influenzae (NTHi) or Moraxella catar-~laalis lipooligosaccharide
(LOS) from
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which at least one primary O-linked esterified fatty acid has been removed to
form
detoxified LOS (dLOS), and an immunogenic carrier covalently linked thereto,
optionally
where the dLOS and immunogenic carrier are covalently linlced by a linker. LOS
may be
extracted from NTHi or M. catar~rhalis and purified according to conventional
processes.
NTHi and M. catarrhalis lipooligosaccharides may be of airy serotype. As a
matter of
example, serotypes I, II, III, IV and V for NTHi are cited (Campagnari, A.A.
et al. 1987
Infect Immun 55:882-887; Partick, C.C. et al. 1987 Infect Immu>z 55:2902-
2911), but the
LOS used for the conjugates herein was highly cross-reactive to the majority
of NTHi
clinical isolates. For M. catar~halis, three major LOS serotypes: A, B and C
are cited
(Vaneechoutte, M.G. et al. 1990 J Clirz Mic~obiol 28:182-187). One or several
lipooligosaccharides may be concomitantly administered by the mucosal route.
In
particular, the medicament, i.e., the vaccine, for mucosal administration may
contain
several lipooligosaccharides, each of a particular serotype.
A proposed chemical structure of lipid A from nontypeable Haezzzophilus
influerzzae
lipooligosaccharide (LOS) is shown in Fig. A. A proposed chemical structure of
lipid A
from Mo>"axella cata~rhalis lipooligosaccharide (LOS) is shown in Fig. B. The
O-linked
esterified fatty acids shown by the asterisks are defined as primary O-linked
fatty acids and
those shown by the arrows are defined as secondary O-linked fatty acids. The
conjugate
vaccine may also comprise LOS from which both primary O-linked fatty acids
have been
removed. In addition to the removal of at least one primary O-linked fatty
acid from LOS,
one or both of the secondary O-linked fatty acids may also be removed. The
number of
primary and secondary O-linked fatty acids removed by hydrazine treatment, or
by
treatment with any other reagent capable of hydrolyzing these linkages, will
depend on the
time and temperature of the hydrolysis reaction. The determination of the
number of fatty
acid chains which have been removed during the reaction can be determined by
standard
analytical methods including mass spectrometry and nuclear magnetic resonance
(NMR).
Although the use of hydrazine for detoxification of LOS from NTHi or M.
catarrhalis is described herein, the use of any reagent or enzyme capable of
removing at
least one primary O-linked fatty acid from LOS is within the scope of the
present invention.
For example, other bases such as sodium hydroxide, potassium hydroxide, and
the like may
be used.
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After removal of one or more primary O-linked fatty acids, dLOS is optionally
conjugated to a linlcer, such as adipic acid dihydrazide (ADH), prior to
conjugation to an
immunogenic carrier protein, such as tetanus toxoid (TT). Although ADH is the
preferred
linker, the use of any linlcer capable of stably and efficiently conjugating
dLOS to an
immunogenic earner protein is contemplated. The use of linkers is well known
in the
conjugate vaccine field (see Dick et al. Cofajugate hacciraes, J.M. Cruse and
R.E. Lewis,
Jr., eds. Karger, New Yorlc, pp. 48-1 I4, 1989).
dLOS may be directly covalently bonded to the earner. This may be
accomplished,
for example, by using the cross-linking reagent glutaraldehyde. However, in a
preferred
embodiment, dLOS and the carrier are separated by a linker. The presence of a
linker
promotes optimum immunogenicity of the conjugate and more efficient coupling
of the
dLOS with the carrier. Linkers separate the two antigenic components by chains
whose
length and flexibility can be adjusted as desired. Between the bifunctional
sites, the chains
can contain a variety of structural features, including heteroatoms and
cleavage sites.
Linkers also permit corresponding increases in translational and rotational
characteristics of
the antigens, increasing access of the binding sites to soluble antibodies.
Besides ADH,
suitable linleers include, for example, heterodifunctional linkers such as s-
aminohexanoic
acid, chlorohexanol dimethyl acetal, D-glucuronolactone and p-nitrophenyl
amine.
Coupling reagents contemplated for use in the present invention include
hydroxysuccinimides and carbodiimides. Many other linkers and coupling
reagents known
to those of ordinary skill in the art are also suitable for use in the
invention (e.g. cystamine).
Such compounds are discussed in detail by Dick et al. (Dick et al. Conjugate
Iraccines,
J.M. Cruse and R.E. Lewis, Jr., eds. Karger, New York, pp. 48-114, 1989).
The presence of a earner increases the irmnunogenicity of the dLOS. Polymeric
immunogenic carriers can be a natural or synthetic material containing a
primary and/or
secondary amino group, an azido group or a carboxyl group. The carrier may be
water
soluble or insoluble.
Any one of a variety of immunogenic carrier proteins may be used in the
conjugate
vaccine of the present invention. Such classes of proteins include pili, outer
membrane
proteins and excreted toxins of pathogenic bacteria, nontoxic or "toxoid"
forms of such
excreted toxins, nontoxic proteins antigenically similar to bacterial toxins
(cross-reacting
materials or CRMs) and other proteins. Nonlimiting examples of bacterial
toxoids
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contemplated for use in the present invention include tetanus toxin/toxoid,
diphtheria
toxin/toxoid, detoxified P. aerugifaosa toxin A, cholera toxin/toxoid,
pertussis toxin/toxoid
and CZostridiufrZ perfringeras exotoxins/toxoid. The toxoid forms of these
bacterial toxins
are preferred. The use of viral proteins (i.e. hepatitis B surface/core
antigens; rotavirus VP
7 protein and respiratory syncytial virus F and G proteins) is also
contemplated.
CRMs include CRM197, antigenically equivalent to diphtheria toxin
(Pappenheimer et al. 1972 Immuhoclaem 9:891-906) and CRM3201, a genetically
manipulated variant of pertussis toxin (Blaclc et al. 1988 Science 240:656-
659). The use of
immunogenic carrier proteins from non-mammalian sources including keyhole
limpet
hemocyanin, horseshoe crab hemocyanin and plant edestin is also within the
scope of the
invention.
Outer membrane proteins include high molecular weight proteins (HMPs), P4 and
P6 from nontypeable Haemophilus ihfluenzae and CD and USPA from Mo~axella
cata~rhalis. For a list of other outer membrane proteins, see PCT W098/53851.
There are many coupling methods which can be envisioned for dLOS-protein
conjugates. In the disclosure set forth below, dLOS is selectively activated
by 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC)-mediated ADH derivatization of the
terminal 3-
deoxy-D-manno-2-octulosonic acid (I~DO) group of dLOS, followed by EDC-
mediated
coupling to TT. Alternatively, another method for producing the instant
conjugates
involves cystamine derivatization of dLOS, by, for example, EDC-mediated
derivatization,
followed by disulfide conjugation to N-succimidyl-3-(2-pyridyldithio)
propionate
derivatized protein. Other methods well known in the art for effecting
conjugation of
oligosaccharides to immunogenic carrier proteins are also within the scope of
the invention.
Such methods are described in, for example, LT.S. Patent Nos. 5,153,312 and
5,204,098;
and EP 0 497 525; and EP 0 245 045.
The molar ratio of ADH to dLOS in the reaction mixture is typically between
about
10:1 and about 250:1. A molar excess of ADH is used to ensure more efficient
coupling and
to limit dLOS-dLOS coupling. In a preferred embodiment, the molar ratio is
between about
50:1 and about 150:1; in a most preferred embodiment, the molar ratio is:
about 100:1.
Similar ratios of AH-dLOS to both TT and HMP in the reaction mixture are also
contemplated. In a preferred embodiment, one ADH per dLOS is present in the AH-
dLOS
conjugate. In another preferred embodiment, in the final dLOS-carrier protein
conjugate, the
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molar ratio of dLOS to carrier is between about 1 S and about 7S, preferably
between about 2S
and about S0.
Immunogenic compositions including vaccines may be prepared as inhalables,
sprays and the like (e.g., nasal spray, aerosol spray or pump spray and the
like), e.g., as
S liquid solutions or emulsions, etc. Aerosol spray preparations can be in a
pressurized
container with a suitable propellant such as a hydrocarbon propellant. Pump
spray
dispensers can dispense a metered dose or, a dose having a particular particle
or droplet
size. Pump spray dispensers are commercially available, e.g., from Valois of
America, Inc.,
Connecticut. Nasal spray dispensers are commonly fabricated from a flexible
material such
as plastic and cause a spray to dispense in response to being squeezed. Anti-
inflammatories,
such as "Vanceril" are commercially available in oral and nasal aerosol form
for mucosal
administration; the anti-inflammatory "Vancerase" is commercially available in
a pump
spray dispenser for nasal achninistration; cold remedies such as "Dristan" are
commercially
available in nasal spray (squeeze) dispensers (so that the reader is aware
that aerosol, pump
and squeeze dispensers are known and available).
The lipooligosaccharide may be mixed with pharmaceutically acceptable
excipients
which are ~ compatible therewith. Such excipients may include water, saline,
dextrose,
glycerol, ethanol, and combinations thereof. The immunogenic compositions and
vaccines
may further contain auxiliary substances, such as wetting or emulsifying
agents, pH
buffering agents, or mucosal adjuvants or delivery systems to enhance the
effectiveness
thereof.
For use in the present invention, the lipooligosaccharide is combined with a
mucosal adjuvant or delivery system. See Singh, M. & O'Hagan, D., Nov 1999
Natuf~e
Bioteclznology 17:1075-1081; and Ryan, E.J. et al. Aug 2001 T~efzds in
Biotechnology
2S 19:293-304. Suitable mucosal adjuvants and delivery systems are listed in
the table below.
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Table: Mucosal Adjuvants and Delivery Systems
Aluminum salts
Chitosan
Cytokines (e.g., IL-1, IL-2, IL-12, IFN-y, GM-CSF)
Saponins (e.g., QS21)
Muramyl dipeptide (MDP) derivatives
CpG oligos
Lipopolysaccharide (LPS) of gram-negative bacteria
Monophosphoryl Lipid A (MPL)
Polyphosphazenes
Emulsions (e.g., Freund's, SAF, MFS9)
Virosomes
Iscoms
Cochleates
Poly(lactide-co-glycolides) (PLG) microparticles
Poloxamer particles
Virus-like particles
Heat-labile enterotoxin (LT), LT B subunit
Cholera toxin (CT), CT B subunit
Mutant toxins (e.g., LTK63 and LTR72)
Microparticles
Liposomes
The mucosal administration preferably is effected intranasally, e.g., to the
olfactory
mucosa, to provide protection to the host against both bacterial colonization
and systemic
S infection. The intranasal administration also may provide protection to the
host against
pulmonary infection as well as protection to the host against an infection
starting as a
pulmonary infection. However, the mucosal administration can also involve
respiratory
mucosa, gingival mucosa or alveolar mucosa. Thus, the administration can be
perlingual or
sublingual or into the mouth or respiratory tract; but intranasal
administration is preferred.
Compositions of the invention, especially for nasal administration, are
conveniently
provided as isotonic aqueous solutions, suspensions or viscous compositions
which may be
buffered to a selected pH. The viscous compositions may be in the form of
gels, lotions,
ointments, creams and the like and will typically contain a sufficient amount
of a
thickening agent so that the viscosity is from about 2500 to 6500 cps,
although more
1S viscous compositions, even up to 10,000 cps may be employed. Viscous
compositions have
a viscosity preferably of 2500 to 5000 cps, since above that range they become
more
difficult to administer.
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Liquid sprays and drops are normally easier to prepare than gels and other
viscous
compositions. Additionally, they are somewhat more convenient to achninister,
especially
in multi-dose situations. Viscous compositions, on the other hand can be
formulated within
the appropriate viscosity range to provide longer contact periods with mucosa,
such as the
nasal mucosa.
Suitable nontoxic pharmaceutically acceptable carriers, and especially nasal
carriers, will be apparent to those skilled in the art of pharmaceutical and
especially nasal
pharmaceutical formulations. For those not skilled in the art, reference is
made to the text
entitled Remihgton's Phaf~maceutical Scieyaces, a reference book in the field.
Obviously, the
choice of suitable Garners will depend on the exact nature of the particular
mucosal dosage
form, e.g., nasal dosage form, required [e.g., whether the composition is to
be formulated
into a solution such as a nasal solution (for use as drops or as a spray), a
nasal suspension, a
nasal ointment, a nasal gel or another nasal form]. Preferred mucosal and
especially nasal
dosage forms are solutions, suspensions and gels, which normally contain a
major amount
of water (preferably purified water) in addition to the antigen (PspA). Minor
amounts of
other ingredients such as pH adjusters (e.g., a base such as NaOH),
emulsifiers or
dispersing agents, buffering agents, preservatives, wetting agents and jelling
agents (e.g.,
methylcellulose) may also be present. The mucosal (especially nasal)
compositions can be
isotonic, i.e., it can have the same osmotic pressure as blood and lacrimal
fluid.
The desired isotonicity of the compositions of this invention may be
accomplished
using sodium chloride, or other pharmaceutically acceptable agents such as
dextrose, boric
acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
Sodium
chloride is preferred particularly for buffers containing sodium ions.
Viscosity of the compositions may be maintained at the selected level using a
pharmaceutically acceptable thickening agent. Methylcellulose is preferred
because it is
readily and economically available and is easy to work with. Other suitable
thickening
agents include, for example, xanthan gum, carboxymethyl cellulose,
hydroxypropl
cellulose, carbomer, and the like. The preferred concentration of the
thickener will depend
upon the agent selected. The important point is to use an amount which will
achieve the
selected viscosity. Viscous compositions are normally prepared from solutions
by the
addition of such thickening agents.
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Compositions within the scope of this invention can contain a humectant to
inhibit
drying of the mucous membrane and to prevent irritation. Any of a variety of
pharmaceutically acceptable humectants can be employed including, for example
sorbitol,
propylene glycol or glycerol. As with the thickeners, the concentration will
vary with the
selected agent, although the presence or absence of these agents, or their
concentration, is
not an essential feature of the invention.
Enhanced absorption across the mucosal and especially nasal membrane can be
accomplished employing a pharmaceutically acceptable surfactant. Typically
useful
surfactants for compositions include polyoxyethylene derivatives of fatty acid
partial esters
of sorbitol anhydrides such as Tween 80, Polyoxyl 40 Stearate, Polyoxyethylene
50
Stearate and Octoxynol. The usual concentration is form 1% to 10% based on the
total
weight.
A pharmaceutically acceptable preservative can be employed to increase the
shelf
life of the compositions. Benzyl alcohol may be suitable, although a variety
of
preservatives including, for example, Parabens, thimerosal, chlorobutanol, or
bezalkonium
chloride may also be employed. A suitable concentration of the preservative
will be from
0.02% to 2% based on the total weight although there may be appreciable
variation
depending upon the agent selected.
Those skilled in the art will recognize that the components of the
compositions must
be selected to be chemically inert with respect to the lipooliogosaccharide.
This will present
no problem to those skilled in chemical and pharmaceutical principles, or
problems can be
readily avoided by reference to standard texts or by simple experiments (not
involving
undue experimentation), from this disclosure.
The therapeutically effective compositions of this invention are prepared by
mixing
the ingredients following generally accepted procedures. For example the
selected
components may be simply mixed in a blender, or other standard device to
produce a
concentrated mixture which may then be adjusted to the final concentration and
viscosity
by the addition of water or thickening agent and possibly a buffer to control
pH or an
additional solute to control tonicity. Generally the pH may be from about 3 to
7.5.
Compositions can be administered in dosages and by techniques well known to
those
skilled in the medical arts taking into consideration such factors as the age,
sex, weight, and
condition of the particular patient, and the mucosal route of administration.
Dosages for
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humans or other mammals can be determined without undue experimentation by the
skilled
artisan from experiments involving mice, rabbits, chinchillas, etc.
The vaccine composition which is administered intranasally as provided herein
may
be formulated in any convenient manner and in a dosage formulation consistent
with the
mode of administration and the elicitation of a protective response. The
quantity of antigen
to be administered depends on the subject to be immunized and the form of the
antigen.
Precise amounts and form of the antigen to be administered depend on the
judgement of the
practitioner. However, suitable dosage ranges are readily determinable by
those skilled in
the art and may be of the order of micrograms to milligrams. Suitable regimes
for initial
administration and booster doses also are variable, but may include an initial
administration
followed by subsequent administrations.
In summary, the lipooligosaccharides may conventionally be used in the
preparation
of the medicament e.g., vaccine. In particular, the lipooligosaccharides may
be formulated
with a diluent or a pharmaceutically acceptable Garner e.g., a buffer or a
saline. The
vaccine may additionally contain usual ingredients such as a stabilizer or as
already
mentioned above, a mucosal adjuvant or delivery system. In a general manner,
these
products are selected according to standard pharmaceutical practices as
described in
Remington's Pharmaceutical Sciences, a reference book in the field.
In a vaccination protocol, the vaccine may be administered by the mucosal
route, as
a unique dose or preferably, several times e.g., twice, three or four times at
week or month
intervals, according to a primelboost mode. The appropriate dosage depends
upon various
parameters, including the number of valencies contained in the vaccine, the
serotypes of the
lipooligosaccharides and the age of the recipient. It is indicated that a
vaccine dose suitably
contain per valency, from 0.5 to 100 ~,g, preferably from 1 to 50 ~,g, more
preferably from
1 to 10 ~,g of lipooligosaccharide. A dose is advantageously under a volume of
from 0.1 to
2 ml.
The vaccination protocol may be a strict mucosal protocol or a mix protocol in
which the priming dose of the vaccine is administered by the mucosal e.g.,
intranasal route
and the boosting doses) is (are) parenterally administered or vice versa.
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Intranasal Immunization with Lipooligosaccharide-based Conjugate Vaccine
from Nontypeable Haerraophilus irafluenzae Inhibits Bacterial Colonization in
Mouse
Nasopharynx.
Previous studies reported as Gu, X.X. et al. 1996 Infect ImmurZ 64:4047-4053
and
Gu, X.X, et al. 1997 Infect Iryarnun 65:4488-4493 demonstrated that systemic
immunization
with detoxified lipooligosaccharide (LOS) conjugate vaccines from nontypeable
Haemophilus influenzae (NTHi) elicited LOS-specific antibodies in mice and
rabbits and
resulted in protection against experimental otitis media in chinchillas. In
this disclosure,
we investigated if intranasal immunization with such a detoxified LOS-tetanus
toxoid
(dLOS-TT) vaccine would generate protective immunity against NTHi in a mouse
model of
nasopharyngeal colonization. The results demonstrated that intranasal
immunization with
dLOS-TT plus adjuvant cholera toxin (GT) siguficantly induced LOS-specific IgA
antibodies in mouse external secretions, especially in nasal wash (90-fold)-
followed by
bronchoalveolar lavage fluid (25-fold), saliva (13-fold) and fecal extract (3-
fold). LOS-
specific IgA antibody forming cells were also found in mucosal and lymphoid
tissues with
the highest number in nasal passage (528 per 106 cells). In addition, the
intranasal
immunization elicited a significant rise of LOS-specific IgG (32-fold) and IgA
(13-fold) in
serum. When these immunized mice were challenged through the nose with 10'
live
bacteria of strain 9274, the vaccine group showed a significant reduction of
NTHi by 74%
and 76%, compared to that of control groups with CT alone or dLOS plus CT
(p<0.05).
Negative correlations were found between bacterial counts and the levels of
nasal wash IgA
or IgG, saliva IgA or senun IgG. The clearance of five heterologous strains
were
investigated and revealed a significant clearance in strains 3198, 5657 and
7502 but not in
strains 1479 and 2019. These data indicate that intranasal immunization with
dLOS-TT
vaccine elicits both mucosal and systemic immunity against NTHi colonization
in a mouse
model of nasopharyngeal colonization. Therefore, it is envisioned as a useful
strategy in
humans to inhibit NTHi colonization and prevent otitis media and other
respiratory diseases
caused by NTHi infection.
Animals. Female BALB/c mice (6 weeks) were purchased from Taconic farms Inc.
(Germantown, NY). The mice were in an animal facility in accordance with
National
Institutes of Health guidelines under animal study protocol 1009-01.
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NTHi LOS and conjugate vaccine. NTHi strain 9274 and five prototype strains
1479, 2019, 3198, 5657 and 7502 were obtained from M.A. Apicella, University
of Iowa
(Campagnari, A.A. et al. 1987 Infect Immun 55:882-887). LOS of NTHi strain
9274 was
extracted from cells by hot phenol water, and then purified by gel filtration
as described
S previously (Gu, X.X. et al. 1995 Infect Imrnun 63:4115-4120). Protein
content was about
1% and nucleic acid content was less than 1 %. Detoxification of the LOS,
conjugation of
dLOS to TT, and characterization of dLOS-TT from strain 9274 were described
previously
(Gu, X.X. et al. 1996 Infect Immun 64:4047-4053). The composition of dLOS-TT
was 638
~,g of dLOS and 901 ~.g of TT per ml with a molar ratio of dLOS to TT at 35:1.
Bacterial growth and LOS purification. NTHi 9274, isolated from middle ear
fluid removed from a patient with OM, was provided by M. A. Apicella,
University of
Iowa. The strain was grown on chocolate agar at 37°C under S% COZ for
8 h and
transferred to 200 ml of 3% brain heart infusion medium (Difco Laboratories,
Detroit,
Mich.) containing NAD (S p,g/ml) and heroin (2 ~.g/ml) (Sigma Chemical Co.,
St. Louis,
1S Mo.) in a S00-ml bottle. The bottle was incubated at 1S0 rpm in an
incubator shaker
(model G-2S; New Brunswick Scientific, Co. Edison, N.J.) at 37°C
overnight. The culture
was transferred to five 2.8-liter baffled Fernbach flasks, each of which
contained 1.4 liters
of the same medium. The flasks were shaken at 140 rpm and maintained at
37°C for 24 h.
The culture was centrifuged at 15,000 x g at 4°C for 30 min to separate
the cells and the
supernatant. LOS was purified from cells by a modified phenol-water extraction
(Gu, X.X.
et al. 1995 Infect Immun 63:4115-4120) and from the culture supernatant by gel
filtration
(Gu, X.X. and Tsai, C.M. 1993 Anal Biochem 196:311-318). The protein and
nucleic acid
contents of both purified LOSS were less than 1 % (Smith, P.K. et al. 1985
Anal Biochem
150:76-8S; Warburg, O. and W. Christian 1942 Biochem Z 310:385-421).
2S Detoxification of LOS. Anhydrous hydrazine treatment of lipopolysaccharides
(LPSs) under mild condition removes esterified fatty acids from lipid A
(Gupta, R.K. et al.
1992 Infect Imnl.un 60:3201-3208). LOS (160 mg), each lot, was dried over PZOS
for 3
days, suspended in 16 ml of anhydrous hydrazine (Sigma), and incubated at
37°C for 2 h
with mixing every 1 S min. This suspension was cooled on ice and added
dropwise to cold
acetone in an ice bath until a precipitate formed (>_90% acetone). The mixture
was
centrifuged at 5,000 x g at S°C for 30 min. The pellet was washed twice
with cold acetone
and dissolved in pyrogen-free water at a final concentration of 20 mg/ml. The
reaction
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mixture was ultracentrifuged at 150,000 x g at 5°C fox 3 h. The
supernatant was freeze-
dried and passed through a column (1.6 by 90 cm) of Sephadex G-50 (Pharmacia
LKB
Biotechnology, Uppsala, Sweden), eluted with 25 mM ammonium acetate, and
monitored
with a differential refractometer (R-400; Waters, Milford, Mass.). The eluate
was assayed
for carbohydrate by the phenol-sulfuric acid method (Dubois, M. et al. 1956
Anal Biochem
28:250-256). The carbohydrate-containing fractions were pooled, freeze-dried
three times
to remove the salt, and designated dLOS. The yields of the dLOS from three
lots ranged
from 48 to 55°I° by weight. For all material and reagent
preparations, glassware was baked
and pyrogen-free water was used.
Derivatization or dLOS. Adipic acid dihydrazide (ADH) (Aldrich Chemical Co.,
Milwaukee, Wis.) was bound to the carboxyl group of the KDO moiety of the dLOS
to
form adipic hydrazide (AH)-dLOS derivatives with 1-ethy-3-(3-
dimethylaminopropyl)carbodiimide HCl (EDC) and N hydroxysulfosuccinimide
(Pierce)
(Gu, X.K. and C.M. Tsai 1993 Infect In2mun 61:1873-1880; Status, J.V. et al.
1986 Afaal
Biocheyn 156:220-222). dLOS (70 mg) was dissolved in 7 ml of 345 mM ADH (the
molar
ratio of ADH to LOS is 100:1 based on an estimated 3,000 Mr for dLOS) (Gibson,
B.W. et
al. 1993 J Bacte~iol 175:2702-2712; Helander, J.M. et al. 1988 Eu~ J Biochena
177:483-
492). N Hydroxysulfosuccinimide was added to a concentration of 8 mM, the pH
was
adjusted to 4.8 with 1 M HC1, and EDC was added to a concentration of 0.1 M.
The
reaction mixture was stirred and maintained at pH 4.8 ~ 0.2 with 1 M HC1 for 3
h at room
temperature. It was adjusted to pH 7.0 with NaOH and passed through the G-50
colunul as
described above. The eluate was assayed for carbohydrate and for AH by a
modification of
a previously described method (Kemp, A.H. and M.R.A. Morgan 1986 JImnZUnol
Methods
94:65-72) by measuring the A49° of AH groups. The peaks containing both
carbohydrate
and AH were pooled, freeze-dried three times to remove the salt, and
designated AH-dLOS.
AH-dLOS was measured for its composition with dLOS and ADH as standards
(Dubois,
M. et al. 1956 Anal Biochetn 28:250-256; Kemp, A.H. and M.R.A. Morgan 1986 J
Immunol Methods 94:65-72).
Conjugation of AH-dLOS to proteins. TT was obtained from Connaught
Laboratories, Inc., Swiftwater, Pa. HMP was purified from NTHi 12 (Barenkamp,
S.J. 1996
Infect Immun 64:1246-1251). AH-dLOS was coupled to carboxyl groups on TT or
HMP at
pH 5.6 with EDC. AH-dLOS (20 mg) was dissolved in 2 ml of water and mixed with
10
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WO 02/089839 PCT/USO1/32331
mg of TT (5.9 mg/ml) or with 8 mg of HMP (4 mg/ml). The molar ratio of AH-dLOS
to
both TT (Mr 150,000) and HMP (Mr 120,000) was 100:1. The pH was adjusted to
5.6
with 0.1 M HCl, and EDC was added to a concentration of 0.1 M. The reaction
mixture
was stirred for 1 to 3 h at room temperature; the pH was maintained at 5.6 ~
0.2 with 0.1 M
HC1. The reaction mixture was adjusted to pH 7.0, centrifuged at 1,000 x g for
10 min,
and passed through a column (1.6 by 90 cm) of Sephacryl S-300 in 0.9% NaCI.
Peaks that
contained both protein and carbohydrate were pooled and designated dLOS-TT or
dLOS
HMP. Both conjugates were analyzed for their composition of carbohydrate and
protein
with dLOS and bovine serum albumin (BSA) as standards (Dubois, M. et al. 1956
Anal
1Q Biochem 28:250-256; Smith, P.K. et al. 1985 Anal Biochem 150:76-85).
Immunization and sample collection. Mice were immunized nasally with 10 ~,l of
phosphate-buffered saline (PBS) containing a mixture of 5 ~g of dLOS-TT and 1
pg of
cholera toxin (List Biological Laboratories, Campbell, CA) as an adjuvant.
Control mice
intranasally received 10 ~.1 of PBS containing 5 ~,g of dLOS and/or 1 ~,g of
CT. Each dose
was pipetted into the mouse nostril (5 ~,1 each side) under anesthesia with
intraperitoneal
injection of 0.1 ml of 2% ketamine and 0.2 % xylazine. Immunizations were
given 5 times
on days 0, 7, 14, 21 and 28. On day 35, one set of mice was used for bacterial
challenge
wlule another set was used for sample collections only described as follows.
Nasal washes,
saliva, bronchoalveolar lavage fluids (BALFs), fecal extracts, and sera were
collected from
mice of each group under anesthesia as described before (Kurono, Y. et al.
1999 J Infect
Dis 180:122-132). Briefly, salivary samples were obtained following
intraperitoneal
injection with 0.1 ml of 0.1% pilocarpine (Sigma, St. Louis, MO) in PBS to
induce salivary
secretion. Blood samples were collected from axillary artery. After removal of
the
mandible, the nasal cavity was gently flushed from posterior opening of the
nose with 200
p,1 of PBS and nasal washes were collected from the anterior openings of the
nose. BALF
was obtained by irrigation with 1 ml of PBS through a blunted needle inserted
into the
trachea after incision. Fecal extract samples were obtained by adding weighed
pellets to
PBS containing 0.01% sodium azide (100 mg of fecal samples/ml) according to
the method
of deVos and Dick (Gu, X.X. et al. 1996 Infect Immun 64:4047-4053). Blood and
fecal
samples were centrifuged, and the supernatants were collected.
Preparation of single cell suspension. On day 35, nasal passages, nasal-
associated
lymphoid tissues (NALTs), spleens, cervical lymph nodes (CLNs), lungs, small
intestines
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WO 02/089839 PCT/USO1/32331
and submandibular glands (SMGs) were collected from mice. Single cell
suspensions were
prepared from nasal passages, NALTs, spleens, CLNs, lungs and SMGs by a gentle
teasing
through stainless steel mesh (Asanuma, H. et al. 1997 J ImmurZOl Methods
202:123-131).
Small intestines were dissociated with 0.5 mg/ml collagenase Type IV (Sigma)
to obtain
single-cell suspensions after removal of Peyer's patches. Each single-cell
suspension
sample except for NALTs, spleens and CLNs was centrifuged over a discontinuous
Percoll
gradient (Pharmacia, Uppsala, Sweden), and mononuclear cells (MNCs) at the
interface of
the 40% and 75% layers were collected. Then, MNCs were suspended in complete
medium
(1 liter of RPMI1640 supplemented with 1% of nonessential amino acid solution,
1 mM
HEPES, 100,000 U of penicillin, 100 ~,g of streptomycin, 40 mg of gentamicin,
and 10%
fetal calf serum). The number and viability of MNCs were examined by trypan
blue dye
exclusion.
Detection of LOS-specific antibodies by ELISA. Specific anti-LOS antibodies in
nasal wash, saliva and serum were determined by ELISA with strain 9274 LOS as
coating
antigen (10 ~.g/ml) (Gu, X.X. et al. 1996 Infect Immun 64:4047-4053). Samples
of naive
mice were served as negative controls. The negative controls gave optical
density readings
of less than 0.1 for IgA, IgG and IgM in serum, and 0.01 in external
secretions. The
antibody endpoint titer was defined as the highest dilution of samples giving
an optical
density two-fold greater than that of the negative controls at 30 min.
Detection of LOS-specific antibody-forming cells (AFCs) by enzyme-linked
immunospot (ELISPOT) assay. For the enumeration of LOS-specific immunoglobulin-
producing cells, the numbers of LOS-specific IgA-, IgG-, and IgM-producing
cells in
NALT, NP, SMG, spleen, CLN, lung, and small intestine were determined with
ELISPOT
assay (Kodama, S. et al. 2000 Infect Immun 68:2294-2300.). Briefly, 96-well
filtration
plates with a nitrocellulose base (Millititer HA; Millipore Corp., Bedford,
Mass.) were
coated with 100 ~,1 of strain 9274 LOS (10 ~,g/ml) and incubated overnight at
4°C. The
plates were washed three times with PBS and then blocked with complete medium
for 1 h.
After removing the blocking medium, test cells in complete medium were added
at various
concentrations and cultured at 37°C with 5% C02 for 6 h. After the
incubation, the plates
were washed thoroughly with PBS and then with PBS containing 0.05 % Tween 20
(PBS-
T). For capture of secreting antibodies, biotinylated goat anti-mouse IgA,
IgG, or IgM
(Sigma) was added in PBS-T at 1:1,000. After overnight incubation at
4°C, the plates were
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WO 02/089839 PCT/USO1/32331
washed five times with PBS-T, and incubated with 5 ~,g/ml of avidin-peroxidase
conjugates
(Sigma) in PBS-T for I h at room temperature. After washing with PBS-T and PBS
three
times for each, spots were developed in 4-chloro-1-naphthol solution for 10
min. The
' reaction was stopped by washing with water. The plate were dried and dark
blue-purple
colored spots were counted as LOS-specific AFCs under a stereo microscope.
Immunohistochemistry for IgA-, IgG-, IgM-positive cells in the nose. For
histological observation, the mice were euthanized on day 35 and then perfused
transcardially with PBS, followed by perfusion with 10% neutral buffered
formalin. Mouse
heads were removed and fixed in 10% fonnalin for 6. hr and decalcified with
0.12 M
ethylenediamine tetraacetic acid (EDTA, pH 7.0) for 2 weeks. After
dehydration, the
tissues were embedded in paraffin. For detection of IgA, IgG, IgM-positive
cells in the
nose, vertical-serial section (6 ~,m thickness) were prepared. Specimens were
dehydrated
through a graded series of ethanol and treated with 3% hydrogen peroxide in
absolute
methanol for 30 min. Sections were exposed to 5% normal goat serum in PBS for
30 min
and then incubated overnight with biotinylated goat anti-mouse IgA, IgG, or
IgM in 1
bovine serum albumin (BSA)-PBS. After rinsing with PBS, sections were
incubated with
avidin-biotin complex (Vector Laboratories, Burlingame, CA) for 1 h and
developed in
' 0.05% 3,3'-diaminobenzidine-0.01% H20z substrate medium in O.1M PBS for 8
min.
Bacterial challenge in nasopharynx. To examine the effect of the dLOS-TT
vaccine on NTHi clearance in nasopharynx, the mice immunized with different
antigens
were challenged with the homologous strain 9274. The strain was grown on
chocolate agar
at 37°C under 5 % COZ for 16 h, and then 3 - 5 clones were transferred
to another plate and
incubated for 4 h. A bacterial suspension was prepared to the concentration of
4 ~ 6 x 106
CFU/ml in PBS and stored on ice until use. The bacterial concentration was
determined by
a 65% transmission at wavelength 540 nm, and confirmed by counting the
colonies after
overnight incubation. The mice were intranasally inoculated with 10 ~1 of the
bacterial
suspension on day 35. Six hours postchallenge, nasal washes were collected and
diluted
serially in PBS, and 50 ~l of the diluted samples were plated on chocolate
agar. Bacterial
colonies were counted after overnight incubation. To investigate correlation
between
antibody levels and bacterial clearance of strain 9274, saliva, BALF, fecal
extract and
serum samples were collected from each mouse simultaneously. To examine the
effect of
the vaccine on heterologous NTHi, strains 1479, 2019, 3198, 5657 and 7502 were
used
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CA 02446355 2003-10-30
WO 02/089839 PCT/USO1/32331
based on the same procedure except only one control group (CT) was included
since no
significant difference was found between control groups.
Whole cell ELISA. To examine the cross-reactivity of antibodies in nasal wash
(IgA) and sera (IgG) elicited by the vaccine against heterologous NTHi
strains, the
homologous strain 9274 and strains 1479, 2019, 3198, 5657 and 7502 were
suspended in
PBS to an optical density of 65% transmission at 540 nm. Microtiter plates
were coated
with 100 ~,l of the suspension and evaporated at 37°C. Other steps were
the same as
described for the LOS ELISA except 3% of BSA-PBS was used for blocking and
1:15
dilution used for nasal wash or serum samples.
Western blot analysis. For characterization of antibodies in external
secretions and
sera, Western blot analysis was performed with the homologous strain 9274 and
five
heterologous strains. Each bacterial suspension was adjusted to a protein
concentration of 2
mg/ml. The suspensions were boiled at 100°C for 10 min in digestion
buffer, subjected to
SDS-PAGE in a 15% polyacrylamide gel and then transferred onto nitrocellulose
membranes at 250 mA for 6 h (Gu, X.X. et al. 1992 .l Clin Mic~obiol 30:2047-
2053). After
blocking with 3% BSA-Tris buffered saline (TBS) for 1 h, the membranes were
incubated
with nasal wash or serum sample (1:10) for 3 h, followed by biotinylated goat
anti-mouse
IgA or IgG for 2h. The membranes were washed with TBS-T, and incubated with
avidin-
peroxidase conjugate for 1 h. After washing with TBS, the membranes were
developed
with 4-chloro-1-naphthol solution. A duplicate gel was silver-stained after
SDS-PAGE.
Statistical analysis. Antibody levels were expressed as the geometric mean
(GM)
ELISA titers (reciprocal) of n independent observations (~ SD range). AFCs
were
expressed as a mean of n independent observations (~ SD). Bacterial
concentration was
expressed as GM CFU of n independent observations (~ SD). Differences between
vaccine
and control groups were determined using Student's t-test and P values smaller
than 0.05
were considered significant. Correlation between bacterial concentration and
IgA or IgG
titer was analyzed by Pearson's product moment method (null hypothesis: Ho: P
= 0;
alternative hypothesis: Hl: P < 0, significantly).
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CA 02446355 2003-10-30
WO 02/089839 PCT/USO1/32331
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CA 02446355 2003-10-30
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CA 02446355 2003-10-30
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24
CA 02446355 2003-10-30
WO 02/089839 PCT/USO1/32331
LOS-specific immune responses in external secretions and serum samples.
LOS-specific immune responses were elicited significantly by intranasal
immunization
with dLOS-TT and CT but not controls (Table 1). LOS-specific IgA titers in
external
secretions and in serum were increased by dLOS-TT and CT, especially in nasal
wash (90-
fold), BALF (26-fold), saliva (13-fold) and serum (13-fold), whereas slight
increase of
LOS-specific IgA in fecal extract was found (3-fold) when compared to that of
CT controls.
LOS-specific IgG titers in serum were increased significantly with dLOS-TT and
CT by
32-fold, while LOS-specific IgG antibodies in external secretions except for
fecal extract
were also elevated by 3 to 5-fold when compared to that of CT controls. No LOS-
specific
IgM was detected and no difference of antibody titers found between two
control groups:
dLOS plus CT and CT alone (p > 0.05).
LOS-specific antibody-forming cells (AFCs) in mucosal effector tissues.
Intranasal immunization with dLOS-TT and CT resulted in detection of LOS-
specific IgA
AFCs in all tissues tested, including distant organs such as intestine and
spleen (Table 2).
The majority of LOS-specific IgA AFCs were located in nasal passage (52~ per
106 cells),
followed by a small amount in other tested tissues. The dominant isotype of
LOS-specific
AFCs was IgA, followed by small numbers of IgG but not IgM. LOS-specific IgG
AFCs
were only detected in nasal passage, CLN and spleen. Intranasal immunization
with dLOS
and CT elicited 2 LOS-specific IgA AFCs in nasal passage but not in other
tested tissues.
No AFC was found in any tissues from mice immunized with CT.
Immunohistochemical staining' of the nose. Immunohistochemical staining of
noses with anti-IgA (Fig. 1) revealed that the mouse immunized with the dLOS-
TT vaccine
showed positive staining in the mucous blanket and glandular tissues (B) as
compared with
the control mouse (A). A large number . of IgA-positive cells were found in
nasal
subepithelial layer and nasal glands. In contrast, staining with anti-IgG in
the mouse
immunized with the dLOS-TT vaccine was only seen in the area of the vessels
but not the
glandular tissue (C). The nasal mucosa of the control mice was not stained
with anti-IgG,
and both vaccine-immunized and control mice showed no staining with anti-IgM
in the
nose.
Bacterial clearance from nasopharynx. Since intranasal immunization with
dLOS-TT vaccine induced high levels of LOS-specific IgA antibodies in nasal
wash and
IgG antibodies in serum, it was important to examine whether the NTHi LOS
specific
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CA 02446355 2003-10-30
WO 02/089839 PCT/USO1/32331
immune responses contributed to the clearance of NTHi colonization in the
nasal tract.
Bacterial colonization of the homologous strain inoculated into the mouse
nasopharynx is
shown in Fig. 2. The mice immunized with dLOS-TT and CT showed a significant
reduction of bacterial recovery by 74% or 76% when compared to those of the
mice
immunized with CT alone or dLOS and CT (p<0.05). Relationship between LOS-
specific
antibody titers and bacterial counts from nasopharynx was further analyzed in
nasal wash,
saliva, BALF, fecal extract and serum from dLOS-TT and CT immunized and CT
immunized mice. Negative correlation with bacteria was found in nasal wash IgA
(r =
0.56, p = 0.0085) or IgG (r = -0.63, p = 0.0025), saliva IgA (r = -0.45, p =
0.0447), or
serum IgG (r = -0.65, p = 0.014).
Heterologous bacterial clearance from nasopharynx. Since strain 9274 LOS
contains common LOS epitopes, bacterial clearance of heterologous strains was
performed
in mice immunized with or without dLOS-TT in CT (Table 3). Significant
inhibition in
bacterial colonization was seen in 3 out of 5 strains (3198, 5657 and 7502)
with a reduction
of 57 to 65%, when compared to the mice immunized with CT alone (p<0.05).
Cross-reactivity of LOS antibodies with heterologous strains. The cross-
reactivity of antibodies elicited by NTHi 9274 dLOS-TT and CT against
heterologous
strains was analyzed by whole cell ELISA with both nasal wash (mainly IgA) and
sera
(mainly IgG) (Fig. 3). Nasal wash IgA bound strongly to not only the
homologous strain
but also the heterologous strains 3198, 5657, and 7502 when compared with the
controls.
Binding reactivity of serum IgG also showed the same tendency as the nasal
wash IgA.
However, bindings to the heterologous strains 1479 and 2019 were weak in both
nasal wash
and serum antibodies. The control mice showed love background binding in nasal
wash and
medium background in serum to all strains. Both nasal wash and serum samples
were
further tested in Western blot with all above strains (Fig. 4). Nasal wash IgA
from mice
immunized with dLOS-TT and CT was reactive strongly to LOSS of strains 9274
and 3198,
moderately to 5657 and 7502, and weakly to 1479 LOS but not 2019 LOS. However,
the
serum IgG was reactive to all with a strong binding to LOSS of strains 9274
and 3198.
Conclusions. Tntranasal immunization with a NTHi dLOS-TT conjugate vaccine
elicited LOS-specific IgA antibodies in local and distant external secretions
as well as
LOS-specific IgA AFCs in mucosal effector tissues (nasal passage, SMG, lung
and
intestine) and lymphoid tissues (HALT, CLN and spleen). It also generated
significant
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CA 02446355 2003-10-30
WO 02/089839 PCT/USO1/32331
LOS-specific IgG antibodies in serum. This is the first demonstration at
intranasal
administration of a LOS-based conjugate eliciting antigen-specific mucosal and
systemic
immune responses although several recent studies have shown similar results by
capsular
polysaccharide conjugates from Streptococcal pneunaoniae, group B Streptococci
or
Haemophilus infl'uerazae type b (Bergquist, C., T. Lagergard, and J. Hohngren
1998 Apmis
106:800-806; Jakobsen, H. et al. 1999 Infect Immun 67:4128-4133; Jakobsen, H.
et al.
1999 Infect Immun 67:5892-5897; Shen, X. et al. 2000 Infect Immun 68:5749-
5755). In
summary, intranasal immunization with a LOS-based conjugate vaccine elicited
LOS-
specific mucosal and systemic immunity, which inhibited not only the
homologous but also
the heterologous bacterial adherence in a mouse model of nasopharyngeal
colonization.
Therefore, it is envisioned as being effective in humans with an appropriate
mucosal
adjuvant or delivery system to inhibit NTHi colonization and prevent otitis
media and other
respiratory diseases caused by NTHi infection.
Intranasal Immunization with Detoxified Lipooligosaccharides from Moraxella
catarrlzalis Conjugated to a Protein Elicits Protection in a Mouse Model of
Colonization.
Monaxella catay~~halis is a significant cause of otitis media in children.
Lipooligosaccharide (LOS) is a major surface antigen of M. catarnhalis and a
potential
vaccine candidate. In order to address the mucosal immunity of detoxified LOS
(dLOS)-
protein conjugate vaccines and their potential roles on mucosal surfaces,
BALB/c mice
were immunized intranasally with a mixture of dLOS-CRM (the diphtheria toxin
cross-
reactive mutant protein) and cholera toxin (CT) as an adjuvant, dLOS plus CT,
or CT only.
After immunization, the animals were aerosolly challenged with M. catar~halis
strain
25238. Immunization with dLOS-CRM generated a significant increase in
secreting IgA
and IgG in nasal washes, lung lavage and saliva, and serum IgG, IgM and IgA
against LOS
of M. cataYrhalis as detected by an indirect enzyme-linked immunosorbent assay
(ELISA).
The dLOS-CRM also elicited LOS-specific IgA, IgG, and IgM antibody-forming
cells
(AFCs) in different lymphoid tissues as measured by an enzyme-linked
immunospot
(ELISPOT) assay. LOS-specific IgA AFCs were found in the nasal passages,
spleens,
nasal-associated lymphoid tissues (HALT), cervical lymph nodes (CLN), lungs,
and small
intestines. LOS-specific IgG and IgM AFCs were only detected in the spleens,
CLN and
nasal passages. Furthermore, the dLOS-CRM vaccine generated a significant
bacterial
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CA 02446355 2003-10-30
WO 02/089839 PCT/USO1/32331
clearance in the nasopharynx and lungs when compared to the controls (P< 0.01)
following
an aerosol challenge with the homologous strain 25238. A comparison of dLOS-
CRM,
dLOS-TT and dLOS-UspA through intranasal immunization resulted in similar
protection
against M. catarrhalis. Intriguingly, intranasal immunization with dLOS-CRM
containing
CT showed a higher level of bacterial clearance in both sites when compared to
subcutaneous injections with dLOS-CRM plus CT adjuvant. These data indicate
that
dLOS-CRM induces specific mucosal and systemic immunity against M. cata~~halis
through intranasal immunization, and provides effective bacterial clearance in
the mouse
nasopharynx alld lungs. Therefore, it is envisioned as being an efficient
route for vaccines
to prevent otitis media and lower respiratory tract infections caused by M.
cata~rhalis.
Animals. Female BALB/c mice (6-8 weeks old) were purchased from Taconic
farms Inc. (Germantown, NY).
Conjugate vaccine. Purification of LOS from M. catar~halis strain 25238,
detoxification of the LOS, and conjugation of dLOS to carrier protein
including CRM, TT,
UspA were performed as described previously (Gu, X.X. et al. 1998 Infect Immun
66:1891
1897).
LOS purification. Type A strain ATCC 25238 was grown on chocolate agar at
37°C in 5% COZ for 8 h and transferred to 250 ml of 3% tryptic soy
broth (Difco
Laboratories, Detroit, Mich.) in a 500-ml bottle. The bottle was incubated at
110 rpm in an
incubator shaker (model G-25; New Brunswick Scientific Co., Edison, N.J.) at
37°C
overnight. The culture was transferred to six 2.8-liter baffled Fernbach
flasks, each of
which contained 1.4 liters of tryptic soy broth. The flasks were shaken at 110
rpm and
maintained at 37°C for 24 h. The culture was centrifuged at 15,000 ~ g
and 4°C for 10 min
to collect the cells. The cell pellets were washed once with 95% ethanol,
twice with
acetone, and twice with petroleum ether (Masoud, H. et al. 1994 Can J Chem
72:1466-
1477) and dried to a powder. The LOS was extracted from cells (Gu, X.X. et al.
1995 Infect
Imnaun 63:4115-4120), and the protein and nucleic acid contents of the LOS
were less than
1% (Smith, P. K. et al. 1985 Anal Biochem 150:76-85; Warburg, O., and W.
Christian.
1942 Biochefn Z 310:384-421).
Detoxification of LOS. Anhydrous hydrazine treatment of LOS removes esterified
fatty acids from lipid A (Gu, X.X. et al. 1996 Infect Immun 64:4047-4053;
Gupta, R. K. et
al. 1992 Infect Immun 60:3201-3208). LOS (160 mg) was suspended in 16 ml of
anhydrous
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CA 02446355 2003-10-30
WO 02/089839 PCT/USO1/32331
hydrazine (Sigma Chemical Co., St. Louis, Mo.) and incubated at 37°C
for 3 h with
mixing. This suspension was cooled on ice and added dropwise with cold acetone
until a
precipitate formed. The mixture was centrifuged at 5,000 X g and 5°C
for 30 min. The
pellet was washed twice with cold acetone, dissolved in pyrogen-free water at
a final
concentration of 10 to 20 mg/ml, and then ultracentrifuged at 150,000 X g and
5°C for 3 h.
The supernatant was passed through a column (1.6 by 90 cm) of Sephadex G-50
(Pharmacia LKB Biotechnology, Uppsala, Sweden) eluted with 25 mM ammonium
acetate
and monitored with a differential refractometer (R-400; Waters, Milford,
Mass.). The eluate
was assayed for carbohydrate by a phenol-sulfuric acid method (Dubois, M. et
al. 1956
Anal Biochem 28:250-256). The carbohydrate-containing fractions were pooled,
freeze-
dried, and designated dLOS.
Derivatization of dLOS. Adipic acid dihydrazide (ADH; Aldrich Chemical Co.,
Milwaukee, Wis.) was bound to dLOS to form adipic hydrazide (AH)-dLOS
derivatives,
using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) and N-
hydroxysulfosuccinimide (sulfo-NHS) (Pierce) (Gu, X.X., and C.M. Tsai 1993
Infect
Irnnaura 61:1873-1880). dLOS (70 mg) was dissolved in 7 ml of 345 mM ADH
(molar ratio
of ADH to LOS is 100 to 1, based on an estimated Mr of 3,000 for dLOS)
(Edebrink, P.
1994 CaYbohydn Res 257:269-284). Sulfo-NHS was added to a concentration of 8
mM, the
pH was adjusted to 4.8, and EDC was added to a concentration of 0.1 M. The
reaction
mixture was stirred and maintained at pH 4.8 for 3 h. The reaction mixture was
adjusted to
pH 7.0 and passed through the G-50 column as described above. The eluate was
assayed for
carbohydrate and for AH (I~emp, A. H., and M. R. A. Morgan 1986 J Immunol
Meth~ds
94:65-72). The peaks containing both carbohydrate and AH were pooled, freeze-
dried, and
designated AH-dLOS. AH-dLOS was measured for its composition, using dLOS and
ADH
as standards (Dubois, M. et al. 1956 Anal Biochern 28:250-256; Kemp, A. H.,
and M. R. A.
Morgan 1986 Jlrnnzunol Methods 94:65-72).
Conjugation of AH-dLOS to proteins. TT was obtained from Connaught
Laboratories Inc., Swiftwater, Pa., and HMP was purified from NTHi strain 12
(Barenkamp, S. J. 1996 Infect Immun 64:1246-1251). AH-dLOS was coupled to TT
or
HMP to form conjugates (Gu, X.X., and C.M. Tsai 1993 Infect Immun 61:1873-
1880).
Briefly, AH-dLOS (30 mg) was dissolved with 3 ml of water and mixed with I S
mg of TT
(5.9 mg/ml) or with 12 mg of HMP (4 mg/ml). The molar ratio of AH-dLOS to both
TT
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CA 02446355 2003-10-30
WO 02/089839 PCT/USO1/32331
(Mr, 150,000) and HMP (Mr, 120,000) was 100 to 1. The pH was adjusted to 5.4,
and
EDC was added to a concentration of 0.05 to 0.1 M. The reaction mixture was
stirred, and
the pH was maintained at 5.4 for 3 h. The reaction mixture was adjusted to pH
7.0,
centrifuged, and passed through a column (1.6 by 90 cm) of Sephacryl S-300 in
0.9% NaCI.
Peaks that contained both protein and carbohydrate were pooled and designated
dLOS-TT
or dLOS-HMP. Both conjugates were analyzed for their composition of
carbohydrate and
protein, using dLOS and bovine serum albumin (BSA) as standards (Dubois, M. et
al. 1956
Ahal Biochem 28:250-256; Smith, P. I~. et al. 1985 Ahal Biochem 150:76-85).
Immunization and sample collection. Mice, 6-8 for each group, were immunized
intranasally (i.n.) 4 times, or subcutaneously (s.c.) 3 times, with PBS or 5
~,g of dLOS-
protein at 1~2-week intervals, respectively. The total volume of
administration is 10 ~,1 for
i.n. inoculation, or 0.2 ml for s.c. injection with or without Ribi 700 (25
~,g/mouse) or
cholera toxin (CT, 1 ~,g/mouse) adjuvant. One week after the last
immunization, nasal
washes, saliva, lung lavage, fecal extracts, and sera were collected.
Detection of LOS-specific antibodies by ELISA. The titers of LOS specific
antibodies in nasal washes, saliva, lung lavage, fecal extracts and sera were
determined by
ELISA using M. catarrlzalis strain 25238 LOS as a coating antigen. The
antibody endpoint
titer was defined as the highest dilution of sample giving an A405 twofold
greater than that
of negative controls.
Detection of LOS-specific antibody-forming cells (AFCs). Mononuclear cells
were taken from the nasal passage, spleen, nasal-associated lymphoid tissue,
cervical lymph
node, Peyer's patch and lung. Numbers of LOS-specific IgA-, IgG-, and IgM-
producing
cells in each tissue were determined by an enzyme-linked immunospot (ELISPOT)
assay.
Bacterial aerosol challenge. The bacterial aerosol challenges were carned out
one
week after the last immunization in an inhalation exposure system (Glas-col,
Terre Haute,
Ind.) (Hu, W.G. et al. 1999 haccihe 18:799-804). Conditions were as follows:
challenge
dose of bacteria, 10$ to 5x108 CFUIml in the nebulizer; nebulizing time, 40
min; vacuum
flowmeter, 60 standard ft3/h; and compressed air flowmeter, 10 ft3/h.
Measurement of bacterial clearance from mouse nasopharynx and lungs. At 6
h postchallenge, mice lungs were removed, and homogenated in 5 ml of PBS for 1
min at
low speed in a tissue homogenizer (Stomacher Lab System Model 80, Seward,
London,
UK). At the same time, nasal washes were obtained by flushing the nasal cavity
with 200
-3 0-
CA 02446355 2003-10-30
WO 02/089839 - PCT/USO1/32331
p.1 of PBS. The appropriately diluted or undiluted lung homogenates, and nasal
washes
were plated on chocolate agar plates, and the bacterial colonies were counted
after
overnight incubation. W addition, sera, nasal washes and lung homogenates were
collected
for antibody quantification.
Statistical analysis. The viable bacteria were expressed as the geometric mean
CFU of n independent observations + standard deviation. Geometric means of
reciprocal
antibody titers were determined. Significance was determined by Student's t
test.
-31-
CA 02446355 2003-10-30
WO 02/089839 PCT/USO1/32331
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CA 02446355 2003-10-30
WO 02/089839 PCT/USO1/32331
Conclusions. Intranasal immunization with dLOS-CRM induced both mucosal and
systemic immunity (Table I, Fig. I). Intranasal immunization with dLOS-CRM
significantly enhanced M. catar~rhalis clearance from mouse nasopharynx and
lungs (Table
II). Different conjugate vaccines elicited similar protection against M.
catar~rhalis (Table
III, Fig. II and III). Compared to subcutaneous injection, intranasal
immunization with
dLOS-CRM showed a higher level of bacterial clearance from mouse nasopharynx
and
lungs (Table IV, Fig. IV and V). At each time point, immunized mice
significantly reduced
bacterial recovery from nasopharynx and lungs, and bacterial recovery became
undetectable
within 24 h postchallenge (Fig. VI and VII).
*******
While the present invention has been described in some detail for purposes of
clarity and understanding, one skilled in the art will appreciate that various
changes in form
and detail can be made without departing from the true scope of the invention.
All
references referred to above are hereby incorporated by reference.
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