U.S. patent application number 10/688115 was filed with the patent office on 2004-07-01 for intranasal immunization with detoxified lipooligosaccharide from nontypeable haemophilus influenzae or moraxella.
Invention is credited to Gu, Xin-Xing.
Application Number | 20040126381 10/688115 |
Document ID | / |
Family ID | 46300158 |
Filed Date | 2004-07-01 |
United States Patent
Application |
20040126381 |
Kind Code |
A1 |
Gu, Xin-Xing |
July 1, 2004 |
Intranasal immunization with detoxified lipooligosaccharide from
nontypeable haemophilus influenzae or moraxella
Abstract
The invention relates to intranasal immunization with detoxified
lipooligosaccharide from nontypeable Haemophilus influenzae or
Moraxella catarrhalis.
Inventors: |
Gu, Xin-Xing; (Potomac,
MD) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
46300158 |
Appl. No.: |
10/688115 |
Filed: |
October 17, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10688115 |
Oct 17, 2003 |
|
|
|
PCT/US01/32331 |
Oct 16, 2001 |
|
|
|
10688115 |
Oct 17, 2003 |
|
|
|
09789017 |
Feb 20, 2001 |
|
|
|
6607725 |
|
|
|
|
09789017 |
Feb 20, 2001 |
|
|
|
08842409 |
Apr 23, 1997 |
|
|
|
6207157 |
|
|
|
|
PCT/US01/32331 |
|
|
|
|
09610034 |
Jul 5, 2000 |
|
|
|
6685949 |
|
|
|
|
09610034 |
Jul 5, 2000 |
|
|
|
PCT/US99/00590 |
Jan 12, 1999 |
|
|
|
60288695 |
May 3, 2001 |
|
|
|
60016020 |
Apr 23, 1996 |
|
|
|
60071483 |
Jan 13, 1998 |
|
|
|
Current U.S.
Class: |
424/184.1 |
Current CPC
Class: |
A61K 2039/5555 20130101;
A61K 39/102 20130101; A61K 2039/55572 20130101; A61K 2039/543
20130101; C12P 19/04 20130101; A61K 2039/6037 20130101; A61K
39/1045 20130101 |
Class at
Publication: |
424/184.1 |
International
Class: |
C12Q 001/68; A61K
039/00; A61K 039/38 |
Claims
What is claimed is:
1. An aerosolizer for intranasal administration of an immunogenic
composition comprising an immunizing amount of Nontypeable
Haemophilus influenzae (NTHi) or Moraxella catarrhalis
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 wherein
said dLOS and said immunogenic carrier are covalently linked by a
linker, and a mucosal adjuvant or delivery system.
2. A method for inducing an immunological response comprising
intranasal administration of an immunogenic composition comprising
an immunizing amount of Nontypeable Haemophilus influenzae (NTHi)
or Moraxella catarrhalis 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 wherein said dLOS and said immunogenic carrier
are covalently linked by a linker, and a mucosal adjuvant or
delivery system, whereby colonization by NTHi or M catarrhalis is
inhibited or otitis media or other respiratory disease caused by
NTHi or M catarrhalis infection is prevented.
3. The aerosolizer or method of claims 1 or 2, wherein said mucosal
adjuvant or delivery system comprises aluminum salts.
4. The aerosolizer or method of claims 1 or 2, wherein said mucosal
adjuvant or delivery system comprises chitosan.
5. The aerosolizer or method of claims 1 or 2, wherein said mucosal
adjuvant or delivery system comprises cytokines.
6. The aerosolizer or method of claims 1 or 2, wherein said mucosal
adjuvant or delivery system comprises saponins.
7. The aerosolizer or method of claims 1 or 2, wherein said mucosal
adjuvant or delivery system comprises muramyl dipeptide (MDP)
derivatives.
8. The aerosolizer or method of claims 1 or 2, wherein said mucosal
adjuvant or delivery system comprises CpG oligos.
9. The aerosolizer or method of claims 1 or 2, wherein said mucosal
adjuvant or delivery system comprises lipopolysaccharide (LPS) of
gram-negative bacteria.
10. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises monophosphoryl lipid
A (MPL)
11. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises polyphosphazenes.
12. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises emulsions.
13. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises virosomes.
14. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises Iscoms.
15. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises cochleates.
16. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises
poly(lactide-co-glycolides) (PLG) microparticles.
17. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises poloxamer
particles.
18. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises virus-like
particles.
19. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises heat-labile
enterotoxin (LT) B subunit.
20. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises cholera toxin (CT) B
subunit.
21. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises mutant toxins.
22. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises microparticles.
23. The aerosolizer or method of claims 1 or 2, wherein said
mucosal adjuvant or delivery system comprises liposomes.
Description
RELATED APPLICATIONS
[0001] This application is a continuation and claims the benefit of
priority of International Application No. PCT/US01/32331 filed Oct.
16, 2001, designating the United States of America and published in
English, which claims the benefit of priority of U.S. Provisional
Application No. 60/288,695 filed May 3, 2001, and for U.S. purposes
only, PCT/US01/32331 is a continuation-in-part of U.S. patent
application Ser. No. 09/789,017 filed Feb. 20, 2001, issued as U.S.
Pat. No. 6,607,725, which is a divisional of U.S. patent
application Ser. No. 08/842,409 filed Apr. 23, 1997, issued as U.S.
Pat. No. 6,207,157, which claims the benefit of priority of U.S.
pat. Appl. No. 60/016,020 filed Apr. 23, 1996, and PCT/US01/32331
is also a continuation-in-part of U.S. patent application Ser. No.
09/610,034 filed Jul. 5, 2000, pending, which is a continuation of
Intl. pat. Appl. No. PCT/US99/00590 filed Jan. 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 Jan. 13, 1998; the disclosures of such related
applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to intranasal immunization with
detoxified lipooligosaccharide from nontypeable Haemophilus
influenzae or Moraxella catarrhalis.
BACKGROUND OF THE INVENTION
[0003] 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 Ann Intern Med 99:344-350). Moraxella
(Branhamella) catarrhalis (Catlin, B. W. 1990 Clin Microbiol Rev
3:293-320; Doem, G. V. 1986 Diagn Microbiol 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 J Infect 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 Am J Med
88:9s.sup.-14S), especially those with chronic obstructive
pulmonary diseases (Nicotra, B. et al. 1986 Arch Intern Med
146:890-893) or compromised immune systems (Alaeus, A. and G.
Stiernstedt Scand J Infect Dis 23:115-116; Enright, M. C and H.
McKenzie. 1997 J Med Microbiol 46:360-371).
[0004] 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 Immun 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 Ser 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-lipooligosaccharid- e (dLOS)-protein conjugates from
NTHi protected against otitis media in chinchillas.
[0005] Moraxella (Branhamella) catarrhalis (M. catarrhalis) is an
important 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-molecular-weight proteins (HMP) from nontypeable Haemophilus
influenzae 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 catarrhalis diseases.
[0006] 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 catarrhalis-caused otitis
media in children and other NTHi- and M catarrhalis-caused diseases
in children and adults.
SUMMARY OF THE INVENTION
[0007] The invention relates to intranasal immunization with
detoxified lipooligosaccharide from nontypeable Haemophilus
influenzae or Moraxella catarrhalis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows the proposed chemical structure of lipid A from
nontypeable Haemophilus influenzae 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).
[0009] FIG. 2 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).
[0010] FIG. 3. Immunohistochemistry 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 immunized mice, and
(C) anti-IgG staining in dLOS-TT immunized mice (magnification
400.times.). 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 immunized with
dLOS-TT. The nasal tissue of the control mice was not stained with
anti-IgA (or anti-IgG).
[0011] FIG. 4. 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).
[0012] FIG. 5. Binding reactivity of nasal wash (IgA) 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.
[0013] FIG. 6. Binding reactivity of serum (IgG) to homologous
strain and five heterologous strains in whole-cell ELISA. The
observed binding reactivity was similar to the one observed in
nasal wash from mice immunized with the dLOS-TT (FIG. 5).
[0014] FIG. 7. 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 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.
[0015] FIGS. 8A-C. Specific antibody-forming cells induced by
dLOS--CRM conjugate measured by ELISPOT assay. See Table 4,
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.
[0016] FIGS. 9A-C. Specific antibody-forming cells initiated by
different dLOS-protein conjugates. See Table 6, footnote a. NALT:
nasal-associated lymphoid tissue, NP: nasal passage, CLN: cervical
lymph node, PP: Peyer's patch.
[0017] FIGS. 10A-B. Comparison of protective effect induced by
different dLOS-protein conjugates in bacterial clearance from mouse
nasopharynx and lungs. See Table 6, footnote a. One week after the
last immunization, mice were challenged with 2.times.10.sup.8 CFU
of M catarrhalis strain 25238 per ml 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.
[0018] FIG. 11. Comparison of protective effect from different
immunization regimens in bacterial clearance from mouse
nasopharynx. See Table 7, footnote a. One week after the last
immunization, mice were challenged with 2.times.10.sup.8 CFU of M
catarrhalis strain 25238 per ml in a nebulizer, and nasal washes
were collected at 6 h postchallenge. Left two bars: intranasal
immunization, right two bars: subcutaneous injection.
[0019] FIG. 12. Comparison of protective effect from different
immunization regimens in bacterial clearance from mouse lungs. See
Table 7, footnote a. One week after the last immunization, mice
were challenged with 2.times.10.sup.8 CFU of M catarrhalis strain
25238 per ml in a nebulizer, and lungs were collected at 6 h
postchallenge. Left two bars: intranasal immunization, right two
bars: subcutaneous injection.
[0020] FIG. 13. Kinetics of bacterial recovery from mouse
nasopharynx challenged with M catarrhalis strain 25238. Mice were
intranasally administered 4 times at 1-week intervals with 10 .mu.l
of PBS containing a mixture of 5 .mu.g of dLOS--CRM and 1 .mu.g of
CT, or 10 .mu.l of PBS. One week after the last immunization, mice
were challenged with 5.times.10.sup.8 CFU of M. catarrhalis 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.
[0021] FIG. 14. Kinetics of bacterial recovery from mouse lungs
challenged with M catarrhalis strain 25238. See description of FIG.
13.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The invention relates to an immunogenic composition
comprising an immunizing amount of Nontypeable Haemophilus
influenzae (NTHi) or Moraxella catarrhalis 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.
[0023] In accordance with the present invention, it has now been
surprisingly found that mucosal administration, preferably
intranasally, of NTHi or M catarrhalis 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, 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.
[0024] 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 catarrhalis or
prevent otitis media and other respiratory diseases caused by NTHi
or M catarrhalis infection by mucosal administration, preferably
intranasal administration, to the host of an effective amount of
NTHi or M catarrhalis 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.
[0025] Moreover, in another aspect, the present invention provides
use of an effective amount of NTHi or M catarrhalis
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 catarrhalis or prevent otitis
media and other respiratory diseases caused by NTHi or M
catarrhalis infection.
[0026] The present invention relates to a conjugate vaccine
comprising nontypeable Haemophilus influenzae (NTHi) or Moraxella
catarrhalis lipooligosaccharide (LOS) from 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 linked by a linker. LOS may be extracted from NTHi or M
catarrhalis and purified according to conventional processes. NTHi
and M catarrhalis lipooligosaccharides may be of any 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 Immun 55:2902-2911), but the LOS
used for the conjugates herein was highly cross-reactive to the
majority of NTHi clinical isolates. For M catarrhalis, three major
LOS serotypes: A, B and C are cited (Vaneechoutte, M. G. et al.
1990 J Clin Microbiol 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.
[0027] A proposed chemical structure of lipid A from nontypeable
Haemophilus influenzae lipooligosaccharide (LOS) is shown in FIG.
1. A proposed chemical structure of lipid A from Moraxella
catarrhalis lipooligosaccharide (LOS) is shown in FIG. 2. 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).
[0028] 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.
[0029] After removal of one or more primary O-linked fatty acids,
dLOS is optionally conjugated to a linker, 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 linker capable of stably and efficiently
conjugating dLOS to an immunogenic carrier protein is contemplated.
The use of linkers is well known in the conjugate vaccine field
(see Dick et al. Conjugate Vaccines, J. M. Cruse and R. E. Lewis,
Jr., eds. Karger, New York, pp. 48-114, 1989).
[0030] dLOS may be directly covalently bonded to the carrier. 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 linkers include, for example,
heterodifunctional linkers such as .epsilon.-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 Vaccines, J. M. Cruse and R. E. Lewis, Jr.,
eds. Karger, New York, pp. 48-114, 1989).
[0031] The presence of a carrier increases the immunogenicity 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.
[0032] 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 contemplated
for use in the present invention include tetanus toxin/toxoid,
diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera
toxin/toxoid, pertussis toxin/toxoid and Clostridium perfringens
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.
[0033] CRMs include CRM197, antigenically equivalent to diphtheria
toxin (Pappenheimer et al. 1972 Immunochem 9:891-906) and CRM3201,
a genetically manipulated variant of pertussis toxin (Black 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.
[0034] Outer membrane proteins include high molecular weight
proteins (HMPs), P4 and P6 from nontypeable Haemophilus influenzae
and CD and USPA from Moraxella catarrhalis. For a list of other
outer membrane proteins, see PCT WO98/53851.
[0035] 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-octul- osonic acid (KDO) 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, U.S. Pat. Nos. 5,153,312 and
5,204,098; and EP 0 497 525; and EP 0 245 045.
[0036] 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 molar ratio of dLOS to carrier
is between about 15 and about 75, preferably between about 25 and
about 50.
[0037] 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 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 administration; 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).
[0038] 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.
[0039] For use in the present invention, the lipooligosaccharide is
combined with a mucosal adjuvant or delivery system. See Singh, M.
& O'Hagan, D., November 1999 Nature Biotechnology 17:1075-1081;
and Ryan, E. J. et al. August 2001 Trends in Biotechnology
19:293-304. Suitable mucosal adjuvants and delivery systems are
listed in the table below.
1TABLE Mucosal Adjuvants and Delivery Systems Aluminum salts
Chitosan Cytokines (e.g., IL-1, IL-2, IL-12, IFN-.gamma., 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, MF59) 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
[0040] 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
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.
[0041] 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 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.
[0042] Liquid sprays and drops are normally easier to prepare than
gels and other viscous compositions. Additionally, they are
somewhat more convenient to administer, 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.
[0043] 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 Remington 's Pharmaceutical Sciences, a
reference book in the field. Obviously, the choice of suitable
carriers 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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 humans or
other mammals can be determined without undue experimentation by
the skilled artisan from experiments involving mice, rabbits,
chinchillas, etc.
[0051] 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.
[0052] 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 carrier 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.
[0053] 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 prime/boost 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 .mu.g, preferably
from 1 to 50 .mu.g, more preferably from 1 to 10 .mu.g of
lipooligosaccharide. A dose is advantageously under a volume of
from 0.1 to 2 ml.
[0054] 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
dose(s) is (are) parenterally administered or vice versa.
Intranasal Immunization with Lipooligosaccharide-Based Conjugate
Vaccine from Nontypeable Haemophilus influenzae Inhibits Bacterial
Colonization in Mouse Nasopharynx.
[0055] Previous studies reported as Gu, X. X. et al. 1996 Infect
Immun 64:4047-4053 and Gu, X. X. et al. 1997 Infect Immun
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 (CT) significantly 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 10.sup.6 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.sup.7 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 serum 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.
[0056] Animals. Female BALB/c mice (6 weeks) were purchased from
Taconic farms Inc. (Germantown, N.Y.). The mice were in an animal
facility in accordance with National Institutes of Health
guidelines under animal study protocol 1009-01.
[0057] 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 previously (Gu, X. X. et al. 1995 Infect
Immun 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 .mu.g of dLOS and
901 .mu.g of TT per ml with a molar ratio of dLOS to TT at
35:1.
[0058] Bacterial growth and LOS puriflcation. 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.degree. C. under 5% CO.sub.2 for 8 h and
transferred to 200 ml of 3% brain heart infusion medium (Difco
Laboratories, Detroit, Mich.) containing NAD (5 .mu.g/ml) and hemin
(2 .mu.g/ml) (Sigma Chemical Co., St. Louis, Mo.) in a 500-ml
bottle. The bottle was incubated at 150 rpm in an incubator shaker
(model G-25; New Brunswick Scientific, Co. Edison, N.J.) at
37.degree. C. overnight. The culture was transferred to five
2.8-liter baffled Fembach flasks, each of which contained 1.4
liters of the same medium. The flasks were shaken at 140 rpm and
maintained at 37.degree. C. for 24 h. The culture was centrifuged
at 15,000.times.g at 4.degree. 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 150:76-85; Warburg,
O. and W. Christian 1942 Biochem Z 310:385-421).
[0059] 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 Immun
60:3201-3208). LOS (160 mg), each lot, was dried over
P.sub.2O.sub.5 for 3 days, suspended in 16 ml of anhydrous
hydrazine (Sigma), and incubated at 37.degree. C. for 2 h with
mixing every 15 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.times.g at
5.degree. 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 mixture was
ultracentrifuged at 150,000.times.g at 5.degree. C. for 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% by weight. For all
material and reagent preparations, glassware was baked and
pyrogen-free water was used.
[0060] 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. X. and C. M. Tsai 1993
Infect Immun 61:1873-1880; Staros, J. V. et al. 1986 Anal Biochem
156:220-222). dLOS (70 mg) was dissolved in 7 ml of 345 mM ADH (the
molar ratio of ADH to LOS is .about.100:1 based on an estimated
3,000 M.sub.r for dLOS) (Gibson, B. W. et al. 1993 J Bacteriol
175:2702-2712; Helander, J. M. et al. 1988 Eur J Biochem
177:483-492). N-Hydroxysulfosuccinimide was added to a
concentration of 8 mM, the pH was adjusted to 4.8 with 1 M HCl, 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 HCl for 3 h at room
temperature. It was adjusted to pH 7.0 with NaOH and passed through
the G-50 column 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 J Immunol Methods
94:65-72) by measuring the A.sub.490 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 Biochem 28:250-256; Kemp, A. H. and M.
R. A. Morgan 1986 J Immunol Methods 94:65-72).
[0061] 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-.sup.125I).
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 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 (M.sub.r 150,000) and HMP (M.sub.r
120,000) was .about.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 HCl. The reaction mixture was
adjusted to pH 7.0, centrifuged at 1,000.times.g for 10 min, and
passed through a column (1.6 by 90 cm) of Sephacryl S-300 in 0.9%
NaCl. 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 Biochem 28:250-256; Smith, P. K. et al. 1985 Anal Biochem
150:76-85).
[0062] Immunization and sample collection. Mice were immunized
nasally with 10 .mu.l of phosphate-buffered saline (PBS) containing
a mixture of 5 .mu.g of dLOS-TT and 1 .mu.g of cholera toxin (List
Biological Laboratories, Campbell, Calif.) as an adjuvant. Control
mice intranasally received 10 .mu.l of PBS containing 5 .mu.g of
dLOS and/or 1 .mu.g of CT. Each dose was pipetted into the mouse
nostril (5 .mu.l 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 while 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 .mu.l 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.
[0063] Preparation of single cell suspension. On day 35, nasal
passages, nasal-associated lymphoid tissues (NALTs), spleens,
cervical lymph nodes (CLNs), lungs, small intestines 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 Immunol 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 .mu.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.
[0064] 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 .mu.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.
[0065] 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 .mu.l of
strain 9274 LOS (10 .mu.g/ml) and incubated overnight at 4.degree.
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.degree. C. with 5% CO.sub.2 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.degree. C., the plates were washed five times with PBS-T, and
incubated with 5 .mu.g/ml of avidin-peroxidase conjugates (Sigma)
in PBS-T for 1 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.
[0066] 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% formalin 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 .mu.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, Calif.) for 1 h and developed in
0.05% 3,3'-diaminobenzidine-0.01% H.sub.2O.sub.2 substrate medium
in 0.1M PBS for 8 min.
[0067] 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.degree. C. under 5% CO.sub.2 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.about.6.times.10.sup.6 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
.mu.l of the bacterial suspension on day 35. Six hours
postchallenge, nasal washes were collected and diluted serially in
PBS, and 50 .mu.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 based on the same procedure except
only one control group (CT) was included since no significant
difference was found between control groups.
[0068] 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 .mu.l of the suspension and evaporated
at 37.degree. 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.
[0069] 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.degree. 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 J Clin Microbiol 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 2 h.
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.
[0070] 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: H.sub.1:
P<0, significantly).
RESULTS
[0071]
2TABLE 1 Murine antibody responses to NTHi 9274 LOS elicited by
dLOS-TT conjugate GM antibody ELISA titers (.+-.SD range).sup.b
Immunogen.sup.a Isotype Saliva Nasal wash BALF.sup.c Fecal Extract
Serum DLOS-TT + CT IgA 63 (28-140)** 452 (205-990)** 128 (24-692)**
16 (6-42)** 125 (61-257)** IgG 13 (4-38)** 16 (8-31)** 25 (8-27)**
6 (4-8) 320 (131-780)** IgM 5 (5) 5 (4-7) 6 (4-11) 5 (4-7) 10 (10)
DLOS + CT IgA 6 (4-9) 7 (4-12) 6 (4-9) 5 (5) 12 (8-20) IgG 5 (5) 6
(4-8) 6 (4-9) 5 (5) 10 (10) IgM 5 (5) 5 (5) 5 (5) 5 (5) 10 (10) CT
IgA 5 (5) 5 (5) 5 (5) 5 (5) 10 (10) IgG 5 (5) 5 (5) 5 (5) 5 (5) 10
(10) IgM 5 (5) 5 (5) 5 (5) 5 (5) 10 (10) .sup.aTen mice from each
group were given an intranasal immunization on days 0, 7, 14, 21
and 28 with dLOS-TT + CT, dLOS + CT or CT alone. External and serum
samples were collected at 1 week after the last immunization.
.sup.bLOS antibodies were measured by ELISA using strain 9274 LOS
as a coating antigen. Symbols: dLOS-TT + CT group versus either
dLOS + CT or CT group: p < 0.01. .sup.cBALF: Bronchoalveolar
lavage fluid.
[0072]
3TABLE 2 LOS-specific AFCs in mucosal and lymphoid tissues elicited
by dLOS-TT conjugate LOS-specific AFCs/10.sup.6 MNCs (mean .+-.
SD).sup.b Nasal Immunogen.sup.a Isotype NALT.sup.c Passage
SMG.sup.c Lung Intestine CLN.sup.c Spleen dLOS-TT + CT IgA 27 .+-.
8 528 .+-. 40 9 .+-. 1 12 .+-. 2 6 .+-. 1 9 .+-. 1 4 .+-. 1 IgG 0 9
.+-. 6 0 0 0 10 .+-. 8 3 .+-. 2 IgM 0 0 0 0 0 0 0 DLOS + CT IgA 0 2
.+-. 2 0 0 0 0 0 IgG 0 0 0 0 0 0 0 IgM 0 0 0 0 0 0 0 CT IgA 0 0 0 0
0 0 0 IgG 0 0 0 0 0 0 0 IgM 0 0 0 0 0 0 0 .sup.aSee Table 1,
footnote a. .sup.bLOS-specific AFCs were detected by ELISPOT assay
using strain 9274 LOS as a coating antigen. The numbers of AFCs
were obtained from each of six mice per group. .sup.cNALT: nasal
associated lymphoid tissue; SMG: submandibular gland; and CLN:
cervical lymph node.
[0073]
4TABLE 3 Effect of intranasal immunization with dLOS-TT conjugate
on bacterial clearance of heterologous NTHi strains from mouse
nasopharynx Strain Bacterial recovery GM Bacterial
concentration.sup.a (.+-.SD range) reduction (cfu/ml)
Immunogen.sup.b (cfu/ml) (%).sup.c 1479 dLOS-TT + CT 1324 (36-5676)
50% (5 .times. 10.sup.9) CT 2643 (841-8303) 2019 dLOS-TT + CT 2870
(935-8807) 29% (6 .times. 10.sup.9) CT 4054 (1369-12006) 3198
dLOS-TT + CT 3347 (1259-8902) 65%* (4 .times. 10.sup.9) CT 9727
(3336-28365) 5657 DLOS-TT + CT 780 (340-1792) 63%* (5 .times.
10.sup.9) CT 2041 (531-7998) 7502 DLOS-TT + CT 2050 (726-5793) 57%*
(6 .times. 10.sup.9) CT 4788 (1779-12835) .sup.aFive LOS prototype
strains were used for nasopharyngeal challenge and mouse nasal
washes were collected at 6 h postchallenge from 10 mice of each
group. .sup.bSee Table 1, footnote a. .sup.cdLOS-TT + CT group
versus CT group, symbols: p < 0.05.
[0074] 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).
[0075] 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 (528 per 10.sup.6 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.
[0076] Immunohistochemical staining of the nose.
Immunohistochemical staining of noses with anti-IgA (FIG. 3)
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.
[0077] 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
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. 4. 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).
[0078] 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).
[0079] 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)
(FIGS. 5 and 6). 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 low
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. 7). 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.
[0080] Conclusions. Intranasal 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 (NALT, CLN and spleen). It also generated
significant 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 pneumoniae,
group B Streptococci or Haemophilus influenzae type b (Bergquist,
C., T. Lagergard, and J. Holmgren 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 catarrhalis Conjugated to a Protein Elicits Protection in
a Mouse Model of Colonization.
[0081] Moraxella catarrhalis is a significant cause of otitis media
in children. Lipooligosaccharide (LOS) is a major surface antigen
of M catarrhalis 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
catarrhalis 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.
catarrhalis 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 (NALT), 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 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 catarrhalis through intranasal immunization, and provides
effective bacterial clearance in the mouse nasopharynx and lungs.
Therefore, it is envisioned as being an efficient route for
vaccines to prevent otitis media and lower respiratory tract
infections caused by M catarrhalis.
[0082] Animals. Female BALB/c mice (6-8 weeks old) were purchased
from Taconic farms Inc. (Germantown, N.Y.).
[0083] Conjugate vaccine. Purification of LOS from M catarrhalis
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).
[0084] LOS purification. Type A strain ATCC 25238 was grown on
chocolate agar at 37.degree. C. in 5% CO.sub.2 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.degree. C. overnight. The culture was
transferred to six 2.8-liter baffled Fembach flasks, each of which
contained 1.4 liters of tryptic soy broth. The flasks were shaken
at 110 rpm and maintained at 37.degree. C. for 24 h. The culture
was centrifuged at 15,000.times.g and 4.degree. 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 Immun 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 Biochem
Z 310:384-421).
[0085] 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
hydrazine (Sigma Chemical Co., St. Louis, Mo.) and incubated at
37.degree. 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.times.g and 5.degree.
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.times.g and
5.degree. 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.
[0086] 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 Immun 61:1873-1880). dLOS (70 mg) was dissolved
in 7 ml of 345 mM ADH (molar ratio of ADH to LOS is .about.100 to
1, based on an estimated M.sub.r of 3,000 for dLOS) (Edebrink, P.
1994 Carbohydr 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 (Kemp, A. H.,
and M. R. A. Morgan 1986 J Immunol Methods 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 Biochem
28:250-256; Kemp, A. H., and M. R. A. Morgan 1986 J Immunol Methods
94:65-72).
[0087] 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-.sup.125I). 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 15 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 (M.sub.r, 150,000)
and HMP (M.sub.r, 120,000) was .about.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%
NaCl. 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 Anal Biochem 28:250-256; Smith, P. K. et al. 1985 Anal Biochem
150:76-85).
[0088] 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 .mu.g of dLOS-protein at 1.about.2-week
intervals, respectively. The total volume of administration is 10
.mu.l for i.n. inoculation, or 0.2 ml for s.c. injection with or
without Ribi 700 (25 .mu.g/mouse) or cholera toxin (CT, 1
.mu.g/mouse) adjuvant. One week after the last immunization, nasal
washes, saliva, lung lavage, fecal extracts, and sera were
collected.
[0089] 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 catarrhalis
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.
[0090] 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.
[0091] Bacterial aerosol challenge. The bacterial aerosol
challenges were carried out one week after the last immunization in
an inhalation exposure system (Glas-col, Terre Haute, Ind.) (Hu, W.
G. et al. 1999 Vaccine 18:799-804). Conditions were as follows:
challenge dose of bacteria, 108 to 5.times.10.sup.8 CFU/ml in the
nebulizer; nebulizing time, 40 min; vacuum flowmeter, 60 standard
ft.sup.3/h; and compressed air flowmeter, 10 ft.sup.3/h.
[0092] 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 .mu.l 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. In addition, sera, nasal washes and
lung homogenates were collected for antibody quantification.
[0093] 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.
RESULTS
[0094]
5TABLE 4 Murine antibody responses against LOS of M. catarrhalis
strain 25238 elicited by dLOS-CRM conjugate vaccine Immunization
Antibody GM antibody ELISA titers.sup.b Group.sup.a class Nasal
wash Lung lavage Saliva Fecal Extract Serum {circle over
(1)}dLOS-CRM IgA 169 (36-782)**.sup.c 144 (41-501)** 30 (7-124)**
21 (3-159)* 48 (14-167)** IgG 14 (6-31)** 26 (8-83)** 3 3 56
(8-412)** IgM 3 3 3 3 48 (14-167)** {circle over (2)} dLOS IgA 26
(13-53)** 5 (2-13) 5 (3-9) 3 10 IgG 3 3 3 3 10 IgM 3 3 3 3 26
(18-39)** {circle over (3)} PBS IgA 3 3 3 3 10 IgG 3 3 3 3 10 IgM 3
3 3 3 10 .sup.aMice were intranasally immunized 4 times at 1-week
intervals with 10 .mu.l of PBS containing a mixture of 5 .mu.g of
dLOS-CRM and 1 .mu.g of CT, or 10 .mu.l of PBS containing a mixture
of 5 .mu.g of dLOS and 1.mu. of CT, or 10 .mu.l of PBS containing 1
.mu.g of CT, respectively. .sup.bGeometric mean (.+-.SD range) of
six to eight mice. .sup.cGroup 1, or group 2 versus group 3: *, P
< 0.05, **, P < 0.01.
[0095]
6TABLE 5 Effect of intranasal immunization with dLOS-CRM on
bacterial recovery of homologous strain 25238 in mouse nasopharynx
and lungs.sup.a Nasopharynx Lung Bacterial Bacterial Bacterial
Bacterial recovery.sup.b reduction.sup.c recovery.sup.b
reduction.sup.c Immunogen (CPU/lung) (%) (CFU/mouse) (%) {circle
over (1)}dLOS-CRM 91 75 1290 87 (41-201).sup.d (555-2998).sup.d
{circle over (2)} dLOS 354 2 8872 8 (188-669) (6468-12169) {circle
over (3)} PBS 362 0 9656 0 (250-516) (8130-11472) .sup.aSee Table
4, footnote a. One week after the last immunization, mice were
challenged with 2 .times. 10.sup.8 CFU of M. catarrhalis strain
25238 per ml in a nebulizer, and nasal washes and lungs were
collected at 6 h postchallenge, respectively. .sup.bGeometric mean
(.+-.SD range) of six to eight mice. .sup.cCompared with group 3.
.sup.dP < 0.01 compared with group 2 or group 3.
[0096]
7TABLE 6 Murine antibody responses against LOS of M. cat strain
25238 elicited by different dLOS-protein conjugates Immunization
Antibody GM antibody ELISA titers.sup.b Group.sup.a class Nasal
wash Lung lavage Saliva Fecal Extract Serum {circle over
(1)}dLOS-CRM IgA 231 (61-877)**.sup.c 105 (24-462)** 12 (4-32)** 5
(2-12) 56 (24-133)** IgG 16 (9-29)** 26 (7-97)** 4 (2-6) 5 (3-10)
123 (36-418)** IgM 3 13 (6-32)** 3 3 30 (12-74)** {circle over (2)}
dLOS IgA 103 (35-307)** 52 (10-274)** 5 (2-13) 5 (2-11) 34
(14-86)** IgG 11 (4-31)** 10 (3-32)* 4 (2-9) 4 (2-9) 45 (20-103)**
IgM 3 7 (4-13)** 3 3 20 (9-45)* {circle over (3)} dLOS-UspA IgA 26
(10-65)** 17 (10-31)** 4 (2-7) 5 (2-11) 17 (10-31)* IgG 6 (3-15)**
6 (3-12)** 3 6 (2-24) 20 (11-35)** IgM 3 5 (3-10)* 3 3 17 (10-31)*
{circle over (4)} PBS IgA 3 3 3 3 10 IgG 3 3 3 3 10 IgM 3 3 3 3 10
.sup.aMice were intranasally immunized 4 times at 1-week intervals
with 10 .mu.l of PBS containing a mixture of 5 .mu.g of dLOS-CRM,
dLOS-TT or dLOS-UspA and 1 .mu.g of CT, or 10 .mu.l of PBS
containing 1 .mu.g of CT, respectively. .sup.bGeometric mean
(.+-.SD range) of eight mice. .sup.cGroup 1, 2, or 3 versus group
4: *, P < 0.05; **, P < 0.01.
[0097]
8TABLE 7 Murine antibody responses against LOS of M. catarrhalis
strain 25238 elicited by dLOS-CRM conjugate through different
immunization regimens Immunization Antibody GM antibody ELISA
titers.sup.b Immunogen.sup.a route class Nasal wash Lung homogenate
Serum {circle over (1)}dLOS-CRM intranasal IgA 118 (26-545) 111
(23-530) 78 (18-348) IgG 20 (5-73) 73 (13-396) 156 (20-1192) IgM 3
31 (11-87) 26 (13-53) {circle over (2)} PBS intranasal IgA
3**.sup.c 3** 10** IgG 3** 3** 10** IgM 3 3** 10** {circle over
(3)} dLOS-CRM subcutaneous IgA 13 (8-22)** 15 (7-31)** 17 (8-40)*
IgG 15 (9-27) 161 (22-1185) 536 (60-4805) IgM 3 31 (11-87) 17
(8-40) {circle over (4)} PBS subcutaneous IgA 3** 3** 10** IgG 3**
3** 10** IgM 3 3** 10** .sup.aMice were intranasally administered 4
times at 1-week intervals with 10 .mu.l of PBS containing a mixture
of 5 .mu.g of dLOS-CRM and 1 .mu.g of CT, or 10 .mu.l of PBS
containing 1 .mu.g of CT, or subcutaneously injected 3 times at
2-week intervals with 0.2 ml of a mixture of 5 .mu.g of dLOS-CRM
and 1 .mu.g of CT, or 0.2 ml of PBS containing 1 .mu.g of CT,
respectively. .sup.bGeometric mean (.+-.SD range) of eight mice.
.sup.cGroup 2, 3, or 4 versus group 1: *, P < 0.05; **, P <
0.01.
[0098] Conclusions. Intranasal immunization with dLOS--CRM induced
both mucosal and systemic immunity (Table 4, FIGS. 8A-C).
Intranasal immunization with dLOS--CRM significantly enhanced M
catarrhalis clearance from mouse nasopharynx and lungs (Table 5).
Different conjugate vaccines elicited similar protection against M
catarrhalis (Table 6, FIGS. 9A-C and 10A-B). Compared to
subcutaneous injection, intranasal immunization with dLOS--CRM
showed a higher level of bacterial clearance from mouse nasopharynx
and lungs (Table 7, FIGS. 11 and 12). At each time point, immunized
mice significantly reduced bacterial recovery from nasopharynx and
lungs, and bacterial recovery became undetectable within 24 h
postchallenge (FIGS. 13 and 14).
[0099] 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.
* * * * *