Note: Descriptions are shown in the official language in which they were submitted.
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COMBINED THERAPY AND PROPHYLAXIS FOR GENITAL TRACT
INFECTIONS
Cross-Reference to Related Applications
[0001] This application claims priority to U.S. Patent Application
No. 16/138,526,
filed on September 21, 2018, which is a continuation-in-part of U.S. Patent
Application No.
15/824,700, filed on November 28, 2017, the disclosures of each of which are
incorporated
herein by reference.
Statement Regarding Federally Sponsored Research
[0002] This invention was made with government support under grant
AI074791
awarded by the National Institutes of Health. The government has certain
rights in the
invention.
Field of the Invention
[0003] The invention relates to compositions comprising IL-12 and
outer membrane
vesicles from Neisseria gonorrhoeae, and methods for using such compositions
for treatment
of genital tract infections.
Background of the Invention
[0004] Genital tract infection by Neisseria gonorrhoeae gives rise to
gonorrhea,
which is the second most frequent reportable infectious disease in the US
affecting >300,000
individuals per annum, although the real incidence is believed to be at least
double that
number. The worldwide incidence of gonorrhea is estimated to be >100 million
cases per
year. Women bear the brunt of the infection, because untreated gonorrhea can
ascend into the
upper reproductive tract and give rise to pelvic inflammatory disease and
tubal scarring,
leading to infertility and risk for ectopic pregnancy which can be life-
threatening. Yet a large
proportion of infected women, variously given as up to 50% or even more, can
be
asymptomatically infected, thereby increasing the risk of spreading the
infection among their
sexual contacts. Men by contrast usually become aware of their infection
within a few days
and are therefore impelled to seek treatment. New-born infants can become
infected in the
eyes as a result of delivery through an infected birth canal, and this can
lead to blindness if
left untreated. Untreated gonorrhea is also known to increase the risk for
acquiring and
transmitting HIV up to 5-fold. Treatment depends upon antibiotics, but N.
gonorrhoeae has
quickly become resistant to each class of antibiotics used against it,
including most recently
the fluorquinolones (ciprofloxacin), and the currently recommended antibiotics
are
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cephalosporins. However, resistance to these has begun to emerge, making N.
gonorrhoeae
multiple-drug-resistant. Despite various efforts, no vaccine against N.
gonorrhoeae is
currently available. Vaccine efforts are complicated by the extensive
antigenic variability of
N. gonorrhoeae, in which most major surface antigens, including
lipooligosaccharide (LOS),
porin, pilin, and the opacity proteins (Opa) are subject to phase-variable
expression (LOS,
Opa, pilus), allelic variation (porin, Opa), or recombinatorial expression
(pilin). Thus options
for treatment and control of the disease are becoming limited. A puzzling but
well-known
feature of gonorrhea is that recovery from infection does not lead to
protective immunity
against re-infection, and repeated infections are common.
.. Summary of the Invention
[0005] This disclosure provides a method of reducing the risk of
developing N.
gonorrhoeae infections by administering to an individual N. gonorrhoeae
antigens and IL-12
incorporated in polymeric microspheres. The N. gonorrhoeae antigens may be in
the form of
outer membrane vesicles or microvesicles. Other forms of antigens (such as
purified or semi-
purified) may also be used. The N. gonorrhoeae antigen preparation (such as
OMVs) and the
IL-12 microspheres may be delivered in a single composition or different
compositions, by
the same route or different routes, at the same time or different times, over
a same time
period and delivery regimen or different time period and delivery regimens.
For example, the
N gonorrhoeae OMVs and IL-12 microspheres can be delivered intravaginally, or
may be
delivered intranasally.
[0006] In one aspect, this disclosure provides a method for treatment
of cervico-
vaginal infections by local application of IL-12 incorporated in polymeric
microspheres.
While not intending to be bound by any particular theory, it is considered
that application of
IL-12 incorporated in polymeric microspheres locally to mucosal surfaces
enhances the
body's own immune response against an existing infection resulting in
reduction or
elimination of that infection and/or generation of immunity against repeat
infection. In one
embodiment, the amount is sufficient to promote Thl- driven response against
the
microorganisms causing the infection. The amount of IL-12 may be sufficient to
provide a
therapeutic effect, a prophylactic effect, or both against the causative
microorganisms.
Infections that can be treated by the present method include, but are not
limited to, those that
are caused by N gonorrhoeae, C. trachomatis or both. An example of a polymer
that can be
used for microencapsulation of IL-12 is polylactic acid.
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[0007] In one aspect, this disclosure provides a composition
comprising OMVs
prepared from N. gonorrhoeae and IL-12 containing microspheres (ms) suitable
for
intravaginal delivery.
[0008] In one aspect, this disclosure provides a kit for intravaginal
delivery of OMVs
prepared from N. gonorrhoeae and IL-12 containing microspheres. The OMVs and
IL-12 ms
may be present as separate compositions or the same composition. The kit may
comprise
multiple doses of the OMV composition and the IL-12 composition and
instructions for
administration, which may include instruction on frequency, length of
administration
regimen, mode of administration and the like.
Brief Description of the Drawings
[0009] For a fuller understanding of the nature and objects of the
invention, reference
should be made to the following detailed description taken in conjunction with
the
accompanying drawings, in which:
[0010] Fig. 1 is a graph showing the effects of intravaginal
treatment with 1 g of IL-
12 encapsulated in polylactic acid (PLA) microspheres on the course of vaginal
infection
with N. gonorrhoeae in mice;
[0011] Fig. 2 is a graph showing the effects of intravaginal
treatment with IL-12
microspheres during primary infection with N. gonorrhoeae (Fig. 1) on the
course of
secondary vaginal infection with N. gonorrhoeae in mice.
[0012] Fig. 3 is a graph showing the effects of intravaginal treatment with
1 g of
soluble vs. microencapsulated IL-12 on the course of vaginal infection with N.
gonorrhoeae
in mice.
[0013] Fig. 4 is a graph showing the effect of intravaginal IL-12
microsphere (ms)
treatment on primary gonococcal infection in BALB/c mice. (A) IL-12 ms dose
optimization
experiment. Microspheres containing the stated doses of IL-12 were given on
days 0, 2, 4, 6,
8; n = 8 mice per group. N. gonorrhoeae (Ngo) burden was monitored daily by
vaginal swab
culture. Significant differences in infection burdens were found between mice
treated with
2.0 g (p < 0.01), 1.0 g (p < 0.01), or 0.5 g (p < 0.05) of microencapsulated
IL-12 and
controls (ANOVA). (B) Time course of infection in mice treated with IL-12 ms,
soluble IL-
12, IL-17 ms, or control ms, or in untreated mice; cytokine dose = 1.0 [tg
given on days ¨1, 1,
3, 5, 7; n = 8 mice per group. Significant differences in infection burdens
were found between
mice treated with IL-12 ms (p < 0.01) or IL-17 ms (p ¨0.01) and controls
(ANOVA). (C)
Data from the experiment shown in B plotted as percentage of mice remaining
infected under
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the indicated cytokine treatments. Infection was cleared significantly faster
in mice treated
with IL-12 ms (p < 0.0001) or IL-17 ms (p < 0.001) than in controls (Kaplan-
Meier). (D)
Cytokine expression in isolated ILN cells from sham-infected or infected mice
with IL-12
ms, IL-17 ms, or control ms treatment; n = 7 mice per group. Expression of IFN-
y, IL-4, and
IL-17 in CD4+ T cells isolated at day 5 after infection was analyzed by flow
cytometry. (E)
RT-PCR analysis of IFN-y, IL-4, and IL-17 mRNA levels in vaginal tissue
harvested at day 3
from sham-infected or infected mice with IL-12 ms, IL-17 ms, or control ms
treatment; n = 7
mice per group. Cytokine gene expression levels detected by RT-PCR were
normalized
relative to expression of I3-actin and set at 1.0 for sham-infected group. (F)
Phenotypic profile
of vaginal cells isolated on day 5 from sham-infected or infected mice treated
with IL-17 ms
or control ms; n = 7 mice per group. (G) Vaginal and (H) serum anti-gonococcal
IgA and
IgG antibody responses in sham-infected or infected mice with IL-12 ms, IL-17
ms, or
control ms treatment; n = 7 mice per group. Vaginal washes and sera were
collected 15 days
after inoculation, and gonococcus-specific and total IgA and IgG were measured
by ELISA.
Results from one representative out of three independent experiments are
shown. In D ¨ H, #
p < 0.05; * p < 0.01 (unpaired t test);
[0014] Fig. 5 is a graph showing the effect of intravaginal IL-12
microsphere (ms)
treatment during primary infection on secondary gonococcal infection. (A) Time
course of
secondary infection in mice treated with IL-12 ms, soluble IL-12, IL-17 ms, or
control ms
during primary infection, or in previously sham-infected mice with or without
IL-12 ms
treatment; n = 8 mice per group. Significant differences in infection burdens
were found
between mice previously treated with IL-12 ms (p ¨0 .01) and controls (ANOVA).
(B) Data
from the experiment shown in A plotted as percentage of mice remaining
infected after
reinfection under the indicated treatments during primary infection. Infection
was cleared
significantly faster in mice previously treated with IL-12 ms (p < 0.0001)
than in controls
(Kaplan-Meier). (C) Flow cytometric analysis of cytokine expression in ILN
CD4+ T cells
isolated at day 5 from reinfected mice treated with IL-12 ms, IL-17 ms, or
control ms during
primary infection, or from mice that were sham-infected in both primary and
secondary
phases ("sham-reinfected"); n = 7 mice per group. (D) RT-PCR analysis of IFN-
y, IL-4, and
IL-17 mRNA levels in vaginas harvested at day 3 from sham-reinfected or
reinfected mice
treated with IL-12 ms, IL-17 ms, or blank ms during primary infection; n = 7
mice per group.
Cytokine gene expression levels detected by RT-PCR were normalized relative to
expression
of I3-actin and set at 1.0 for sham-reinfected group. (E) Vaginal and (F)
serum anti-
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gonococcal IgA and IgG antibody responses to secondary infection in sham-
reinfected or
reinfected mice treated with IL-12 ms, IL-17 ms, or blank ms during primary
infection; n = 7
mice per group. Vaginal washes and sera were collected 15 days after
inoculation, and
gonococcus-specific and total IgA and IgG were measured by ELISA. Results from
one
representative out of three independent experiments are shown. In C ¨ F, #p <
0.05; *p <
0.01 (unpaired t test).
[0015] Fig. 6 shows intravaginal (I.vag) immunization with gonococcal
OMV plus
IL-12/ms induced resistance to genital infection with N. gonorrhoeae, and
generated an
immune response. a: Mice were immunized 3 times at 7-day intervals with OMV
(4011g
protein) from strain FA1090 plus control (blank) ms or IL-12/ms (111g IL-12);
control mice
were sham-immunized with either blank ms, or with IL-12/ms alone. Two weeks
after the last
immunization, all mice were challenged by i.vag. inoculation with N.
gonorrhoeae strain
FA1090 (5 x 106 CFU), and infection was monitored by vaginal swabbing and
plating. Left
panel: recovery (CFU) of N. gonorrhoeae (mean SEM, N=8 mice), * P <0.01
(ANOVA);
right panel: % of animals remaining infected at each time point, P <0.01
(Kaplan-Meier
analysis, log-rank test, OMV plus IL-12/ms vs. OMV plus blank ms). b: Vaginal
wash (left)
and serum (right) antibodies against strain FA1090 in samples collected after
termination
(day 15), shown as mean SEM, N=5 samples; #P <0.05, * P <0.01, Student's t.
c:
Intracellular cytokine staining in CD4+ cells recovered from ILN at
termination (day 15),
shown as mean SEM, N=3 samples, % of CD4+ staining for each cytokine; * P
<0.01
Student's t. d: Mice were immunized twice at a 14-day interval with gonococcal
(Ngo) OMV
(4011g protein) plus blank ms or IL-12/ms (111g IL-12); control mice were sham-
immunized
with blank ms alone or with NTHI OMV (4011g protein) plus IL-12/ms (111g IL-
12). Two
weeks later, all mice were challenged with N. gonorrhoeae FA1090 (5 x 106
CFU). Left
panel: recovery (CFU) of N. gonorrhoeae (mean SEM, N=8 mice), * P <0.01
(ANOVA,
gonococcal OMV plus IL-12/ms vs. gonococcal OMV plus blank ms); right panel: %
of
animals remaining infected at each time point, P <0.0001 (Kaplan-Meier
analysis, log-rank
test, gonococcal OMV plus IL-12/ms vs. gonococcal OMV plus blank ms).
[0016] Fig. 7 shows antibody responses generated by immunization with
gonococcal
OMV plus IL-12/ms, prior to gonococcal challenge. a: Vaginal wash (left panel)
and serum
(right panel) antibodies assayed by ELISA 2 weeks after the last immunization
with 1, 2, or 3
doses of gonococcal OMV (4011g protein) plus IL-12/ms (111g IL-12). Control
samples were
obtained from mice sham-immunized with blank ms (3 doses); additional mice
were
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immunized 3x with gonococcal OMV plus blank ms. Data shown as mean SEM, N=5
samples, # P <0.05, * P <0.01 relative to control samples (ANOVA). Duration of
vaginal
wash (b) and serum (c) antibodies in mice immunized with 2 doses of FA1090 OMV
plus
blank ms or IL-12/ms; data shown as mean SEM, N=5 samples; C, control samples
from
unimmunized mice.
[0017] Fig. 8 shows: a: T cell cytokine responses in ILN cells
induced by
immunization with gonococcal OMV plus IL-12/ms 2 weeks after the last
immunization with
1, 2, or 3 immunizations with gonococcal OMV (4011g protein) plus IL-12/ms
(111g IL-12).
Control ILN were obtained from mice sham-immunized with blank ms (3 doses) and
additional mice were immunized 3x with gonococcal OMV plus blank ms. Data
shown as
mean SEM, N=3 samples, % of CD4+ or CD8+ cells staining for each cytokine. *
P < 0.01
(Student's t test) comparing immunization with IL-12/ms vs. blank ms. b:
Duration of IFNy
responses in CD4+ ILN cells 1-6 months after two immunizations with gonococcal
OMV plus
IL-12/ms or with OMV plus blank ms. Data shown as mean SEM, N=3 samples, % of
CD4+
cells staining for IFNy; C, control ILN from unimmunized mice.
[0018] Fig. 9 shows resistance to gonococcal (FA1090) challenge
persisted for at
least 6 months after immunization with two doses of gonococcal (FA1090) OMV
plus IL-
12/ms. Left panel: recovery (CFU) of N. gonorrhoeae (mean SEM, N=8 mice), * P
<0.01
(ANOVA, gonococcal OMV plus IL-12/ms vs. gonococcal OMV plus blank ms); right
panel:
% of animals remaining infected at each time point, P <0.001 (Kaplan-Meier
analysis, log-
rank test, gonococcal OMV plus IL-12/ms vs. gonococcal OMV plus blank ms).
[0019] Fig. 10 shows resistance to heterologous gonococcal challenge.
a: One month
after immunization with FA1090 OMV plus IL-12/ms or blank ms, mice were
challenged
with N. gonorrhoeae strain FA1090 (homologous challenge) or strain MS11
(heterologous
challenge). Left panel: recovery (CFU) of N. gonorrhoeae (mean SEM, N=8
mice), * P
<0.001 (ANOVA, for comparisons shown); right panel: % of animals remaining
infected at
each time point, P <0.02 for FA1090 challenge, IL-12/ms vs. blank ms; P <0.001
for MS11
challenge, IL-12/ms vs. blank ms (Kaplan-Meier analysis, log-rank test). b:
Mice immunized
with MS11 OMV were resistant to challenge with N. gonorrhoeae FA1090. Left
panel:
recovery (CFU) of N. gonorrhoeae (mean SEM, N=8 mice), P <0.01 (ANOVA); right
panel: % of animals remaining infected at each time point), P <0.01 (Kaplan-
Meier analysis,
log-rank test). c: Mice immunized with FA1090 OMV were resistant to challenge
with N.
gonorrhoeae FA19. Left panel: recovery (CFU) of N. gonorrhoeae (mean SEM, N=8
mice),
* P <0.01 (ANOVA, for comparisons shown); right panel: % of animals remaining
infected
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at each time point, P <0.01, IL-12/ms vs. blank ms for FA1090 challenge; P
<0.0001, IL-
12/ms vs. blank ms for FA19 challenge (Kaplan-Meier analysis, log-rank test),
N=8 mice. d:
Mice immunized with FA19 OMV were resistant to challenge with N. gonorrhoeae
FA1090.
Left panel: recovery (CFU) of N. gonorrhoeae (mean SEM, N=8 mice), P <0.01
(ANOVA);
right panel: % of animals remaining infected at each time point), P <0.01
(Kaplan-Meier
analysis, log-rank test). e: Mice immunized with FA1090 OMV were resistant to
challenge
with clinical isolate GC68. Left panel: recovery (CFU) of N. gonorrhoeae (mean
SEM, N=8
mice), P <0.01 (ANOVA); right panel: % of animals remaining infected at each
time point),
P <0.01 (Kaplan-Meier analysis, log-rank test).
[0020] Fig. 11 shows immunoproteomics of gonococal OMV. a.: SDS-PAGE of
OMV preparations from N. gonorrhoeae strains FA1090, MS11, and FA19, stained
with
Coomassie blue. b.: Western blot analysis of mouse sera tested on gonococcal
OMV
preparations separated by SDS-PAGE. Lane 1, control serum from a mouse
immunized with
FA1090 OMV plus blank ms, tested against FA1090 OMV; lanes 2-4, serum #1 from
a
mouse immunized with FA1090 OMV plus IL-12/ms, tested against OMV from FA1090
(lane 2), MS11 (lane 3), or FA19 (lane 4); lane 5, serum #2 from a mouse
immunized with
FA1090 OMV plus IL-12/ms, tested against OMV from FA1090; lane 6, antibody H5
(anti-
porin PI133) tested against FA1090 OMV. c.-e.: proteome maps of gonococcal OMV
derived
from FA1090 (c), MS11 (d), and FA19 (e) revealed by 2D electrophoresis and
Flamingo
fluorescent staining (left panels) and their corresponding immunoblots (right
panels) obtained
by probing with mouse serum #2. Immunoreactive spots subjected to MS/MS
analysis are
labeled as spots 1 and 2 (arrows). Molecular mass marker (kDa) indicated on
the left.
[0021] Fig. 12 shows resistance to challenge induced by immunization
with
gonococcal OMV plus IL-12/ms depended on IFNy and B cells. a: Course of
infection
(FA1090) in IFNy-ko vs wild-type mice immunized with FA1090 OMV; left panel,
recovery
(CFU) of N. gonorrhoeae (mean SEM, N=8 mice), = P <0.01 (ANOVA); right panel,
% of
animals remaining infected at each time point, P <0.0001 for wild-type mice,
IL-12/ms vs.
blank ms (Kaplan-Meier analysis, log-rank test). b: Course of infection
(FA1090) in MT vs.
wild-type mice immunized with FA1090 OMV; left panel, recovery (CFU) of N.
gonorrhoeae (mean SEM, N=8 mice), = P <0.01 (ANOVA); right panel, % of
animals
remaining infected at each time point, P <0.0001 for wild-type mice, IL-12/ms
vs. blank ms
(Kaplan-Meier analysis, log-rank test). Vaginal wash (c) and serum (d)
antibody responses in
IFNy-ko vs wild-type (mean SEM, N=5 samples) assayed at termination (day 13).
IgA and
IgG responses in vaginal wash and serum were significant (P <0.05, Student's
t, OMV plus
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blank ms vs. OMV plus IL-12/ms) for wild-type mice, but not for IFNy-ko mice.
e: T cell
cytokine responses in MT vs. wild-type mice (mean SEM, N=3 samples) assayed
at
termination (day 13). IFNy response to immunization with OMV plus blank ms vs.
OMV
plus IL-12/ms was significant (P <0.01) for both wild-type and MT mice
(ANOVA). f:
Course of infection (FA1090) in CD4-ko vs wild-type mice immunized with FA1090
OMV;
left panel, recovery (CFU) of N. gonorrhoeae (mean SEM, N=8 mice), # P <0.05,
= P <0.01
(ANOVA) for comparisons shown; right panel, % of animals remaining infected at
each time
point, P <0.001 for wild-type mice IL-12/ms vs. blank ms, P <0.01 for CD4-ko
mice IL-
12/ms vs. blank ms (Kaplan-Meier analysis, log-rank test). g: Course of
infection (FA1090)
.. in CD8-ko vs wild-type mice immunized with FA1090 OMV; left panel, recovery
(CFU) of
N. gonorrhoeae (mean SEM, N=8 mice), # P <0.05, = P <0.01 (ANOVA) for
comparisons
shown; right panel, % of animals remaining infected at each time point, P
<0.001 for wild-
type mice IL-12/ms vs. blank ms, P <0.02 for CD8-ko mice IL-12/ms vs. blank ms
(Kaplan-
Meier analysis, log-rank test).
[0022] Fig. 13 is a replicate of Fig. 1: I.vag. immunization with
gonococcal OMV
plus IL-12/ms induced resistance to genital infection with N. gonorrhoeae and
generated an
immune response. A: Mice were immunized 3 times at 7-day intervals with OMV
(4011g
protein) from strain FA1090 plus control (blank) ms or IL-12/ms (111g IL-12);
control mice
were sham-immunized with either blank ms, or with IL-12/ms alone. Two weeks
after the last
immunization, all mice were challenged by i.vag. inoculation with N.
gonorrhoeae strain
FA1090 (5 x 106 CFU), and infection was monitored by vaginal swabbing and
plating. Left
panel: recovery (CFU) of N. gonorrhoeae (mean SEM, N=8 mice), P <0.01 (ANOVA,
OMV plus IL-12/ms vs. OMV plus blank ms); right panel: % of animals remaining
infected
at each time point, P <0.001 (Kaplan-Meier analysis, log-rank test, OMV plus
IL-12/ms vs.
.. OMV plus blank ms). B: Vaginal wash (left) and serum (right) antibodies
against strain
FA1090 in samples collected after termination (day 15), shown as mean SEM,
N=5
samples.
C: Intracellular cytokine staining in CD4+ cells recovered from ILN at
termination (day 15),
shown as mean SEM, N=3 samples, % of CD4+ staining for each cytokine. D: Mice
were
immunized twice at a 14-day interval with gonococcal (Ngo) OMV (4011g protein)
plus blank
ms or IL-12/ms (111g IL-12); control mice were sham-immunized with blank ms
alone or
with NTHI OMV (4011g protein) plus IL-12/ms (111g IL-12). Two weeks later, all
mice were
challenged with N. gonorrhoeae FA1090. Left panel: recovery (CFU) of N.
gonorrhoeae
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(mean SEM, N=8 mice), * P <0.001 (ANOVA, gonococcal OMV plus IL-12/ms vs.
gonococcal OMV plus blank ms); right panel: % of animals remaining infected at
each time
point, P <0.001 (Kaplan-Meier analysis, log-rank test, gonococcal OMV plus IL-
12/ms vs.
gonococcal OMV plus blank ms).
[0023] Fig. 14 shows examples of flow cytometry plots for data shown in
Fig. 6C.
Intracellular cytokine staining of ILN cells after clearance of infection (day
15) in mice
immunized as shown. X-axes (FL4H): cytokine fluorescence; Y-axes (PE2): CD4
fluorescence (A-L); CD8 fluorescence (M-P).
[0024] Fig. 15 shows responses induced by immunization with OMV
prepared from
NTHI; samples collected 2 weeks after immunization as shown. A: Vaginal wash
antibodies
(mean SEM, N=5 samples); B: serum antibodies (mean SEM, N=5 samples); C: T
cell
cytokines in ILN cells (mean SEM, N=3 samples). * P <0.01 vs. control samples
(Student's
t test).
[0025] Fig. 16 shows IgG subclass of anti-gonococcal antibody
responses (mean
SEM, N=5 samples) in vaginal wash (A) and serum (B) induced by immunization
with
gonococcal OMV plus IL-12/ms. # P <0.05, * P <0.01 vs control samples
(Student's t test).
[0026] Fig. 17 shows: A: Specificity of T cell responses in ILN for
gonococcal
antigen. ILN CD4+ T cells from mice immunized with FA1090 OMV plus blank ms or
IL-
12/ms were preloaded with CFSE and cultured in vitro in the presence of
antigen-presenting
cells for 3 days with (stim) or without FA1090 cells. Proliferation was
assayed by flow
cytometry after surface staining for CD4, and cytokine secretion was measured
by ELISA.
Data shown as mean SEM, N=7 samples. * P <0.01 vs control samples (Student's
t test). B:
RT-PCR analysis of RNA extracted from vaginal tissue 3 days after the last
immunization (1,
2, or 3 doses) with OMV plus blank ms or IL-12/ms. Data shown as mean SEM,
N=7
samples. * P < 0.01 vs samples from mice immunized with OMV plus blank ms
(Student's t
test). C: Persistence of IFNy response in CD4 + ILN cells harvested 1-6 months
after
immunization with OMV plus IL-12/ms. Data shown as mean SEM, N=7 samples.
[0027] Fig. 18 shows responses in mice challenged with N. gonorrhoeae
FA1090 6
months after immunization with FA1090 OMV plus IL-12/ms. A: Vaginal wash
antibodies
(mean SEM, N=5 samples), B: serum antibodies (mean SEM, N=5 samples), C:
cytokine
production by CD4 + ILN cells (mean SEM, N=3 samples). # P <0.05, * P <0.01
vs control
samples from sham-infected mice (Student's t test).
[0028] Fig. 19 shows responses in mice immunized with gonococcal OMV
plus balnk
ms or IL-12/ms after heterologous gonococcal challenge. A, B, C: Mice
immunized with
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FA1090 OMV and challenged with MS11; A: Vaginal wash antibodies, B: serum
antibodies
to MS11 (mean SEM, N=5 samples); C: cytokine responses in CD4+ ILN cells
(mean
SEM, N=3 samples). D, E, F: Mice immunized with FA1090 OMV and challenged with
FA19; D: Vaginal wash antibodies, E: serum antibodies to FA19 (mean SEM,
N=5); F:
-- cytokine responses in CD4+ ILN cells (mean SEM, N=3 samples). # P <0.05, *
P <0.01 vs
control samples from sham-infected mice (Student's t test).
[0029] Fig. 20 is a replicate of Fig. 7A,B,F,G: I.vag. immunization
with gonococcal
OMV plus IL-12/ms in immunodeficient mice. A: Course of infection (FA1090) in
IFNy-ko
vs wild-type mice immunized with FA1090 OMV; left panel, recovery (CFU) of N.
-- gonorrhoeae (mean SEM, N=8 mice), = P <0.01 (ANOVA); right panel, % of
animal
remaining infected at each time point, P <0.001 for wild-type mice, IL-12/ms
vs. blank ms
(Kaplan-Meier analysis, log-rank test). B: Course of infection (FA1090) in MT
mice
immunized with FA1090 OMV; left panel, recovery (CFU) of N. gonorrhoeae (mean
SEM,
N=8 mice). C: Course of infection (FA1090) in CD4-ko mice immunized with
FA1090
-- OMV; left panel, recovery (CFU) of N. gonorrhoeae (mean SEM, N=8 mice);
right panel,
% of animal remaining infected at each time point, P <0.01 (Kaplan-Meier
analysis, log-rank
test). D: Course of infection (FA1090) in CD8-ko vs wild-type mice immunized
with
FA1090 OMV; left panel, recovery (CFU) of N. gonorrhoeae (mean SEM, N=8
mice); right
panel, % of animal remaining infected at each time point, P <0.01 (Kaplan-
Meier analysis,
-- log-rank test).
[0030] Fig. 21 shows the effect of i.n. immunization with gonococcal
OMV (strain
FA19) plus IL-12/ms on gonococcal challenge infection in BALB/c mice. A:
recovery
(CFUs) of N. gonorrhoeae (mean s.e.m., N=8 mice), P < 0.01 (ANOVA, gonococcal
OMV
plus IL-12/ms vs. gonococcal OMV plus blank ms); B: percentage of animals
remaining
-- infected at each time point.
[0031] Fig. 22 shows the effect of immunization with different doses
of gonococcal
OMV (strain FA1090) plus IL-12/ms on heterologous gonococcal challenge
infection with
strain FA19 in BALB/c mice. Low dose: 6011g, mid dose: 3011g, low dose: 1511g
of OMV
protein per dose, plus 1tg IL-12/ms. Left panel: recovery (CFUs) of N.
gonorrhoeae (means
-- s.e.m., N=8 mice); right panel: percentage of animals remaining infected
at each time point.
[0032] Fig. 23 shows A: comparison of the effects of intranasal or
intravaginal
immunization with gonococcal OMV (3011g protein; strain FA1090) plus 1tg IL-
12/ms on
heterologous gonococcal challenge infection with strain FA19 in BALB/c mice.
Left panel:
recovery (CFUs) of N. gonorrhoeae (means s.e.m., N=8-9 mice); right panel:
percentage of
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animals remaining infected at each time point. B: serum, C: vaginal wash, D:
salivary
antibodies against N. gonorrhoeae in samples collected after clearance of the
infection at
termination (2 weeks after challenge); * P<0.01 compared with controls
(Student t test).
[0033] Fig. 24 shows a comparison of the effects of intranasal or
intravaginal
immunization with gonococcal OMV (3011g protein; strain FA1090) plus 1tg IL-
12/ms on
homologous gonococcal challenge infection with strain FA1090 in BALB/c mice.
Left
panel: recovery (CFUs) of N. gonorrhoeae (means s.e.m., N=8-9 mice); right
panel:
percentage of animals remaining infected at each time point.
[0034] Fig. 25 shows the effect of intranasal immunization with
gonococcal OMV
.. (3011g protein, strain FA1090) plus 1tg IL-12/ms on heterologous gonococcal
challenge
infection with strain MS11 in BALB/c mice. Mice were given either one dose of
OMV plus
IL-12/ms, or two doses with a 2 week interval; control mice were given 2 doses
of blank ms.
Left panel: recovery (CFUs) of N. gonorrhoeae (means s.e.m., N=9 mice); right
panel:
percentage of animals remaining infected at each time point.
[0035] Fig. 26 shows the effect of intranasal immunization with gonococcal
OMV (30
tg protein, strain MS11) plus 1 tg IL-12/ms on heterologous gonococcal
challenge infection
with strain FA1090 in BALB/c mice. Left panel: recovery (CFUs) of N.
gonorrhoeae (means
s.e.m., N=8-9 mice); right panel: percentage of animals remaining infected at
each time
point.
[0036] Fig. 27 shows the effect of intranasal immunization with gonococcal
OMV
(3011g protein, strain MS11) plus 1tg IL-12/ms on heterologous gonococcal
challenge
infection with strain FA19 in BALB/c mice. Left panel: recovery (CFUs) of N.
gonorrhoeae
(means s.e.m., N=9 mice); right panel: percentage of animals remaining
infected at each
time point.
Description of the Invention
[0037] The present invention is based on our studies which have
helped to unfold the
ways in which N. gonorrhoeae prevents the immune system from mounting
effective immune
responses against it. We provide here a novel approach to overcome the ability
of N.
gonorrhoeae to suppress immune response against it.
[0038] In an aspect, the method comprise reducing the likelihood of an
individual
contracting a N. gonorrhoeae infection by administration of an antigenic
preparation from N.
gonorrhoeae and IL-12 present in microspheres (ms). An example of an antigenic
preparation
from N. gonorrhoeae is OMVs. The OMVs and the IL-12 ms may be delivered
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intravaginally. In one embodiment, the OMVs and the IL-12 ms may be delivered
intranasally. The OMVs and the IL-12 ms may be delivered by separate routes of
administration. For example, one may be delivered intravaginally and the other
intranasally.
In one embodiment, the OMVs and the IL-12 ms are delivered by the same route
of
administration, e.g, intranasally.
[0039] In an embodiment, the OMVs and the IL-12 ms are delivered in
the same
composition. Amounts of OMVs and IL-12 ms per dose can be such that an immune
response
is elicited. The OMVs can be in the range of 0.01 to 1 mg of protein per dose.
In one
embodiment, the OMVs can be in the range of 0.01 to 2 mg of protein per dose.
In
embodiments, OMVs can be 10, 50, 100, 250, 500, 750, 1,000, 1250, 1,500, 1,750
or 2,000
[tg protein per dose. In an embodiment, OMVs can be in the range of 15-300 [tg
protein per
dose. In one embodiment, the OMV dose can be 50 to 1,000 [tg protein. For a 70
kg human,
that corresponds to a OMV dose of about 0.7 to 28 microgram protein per kg
body weight. In
one embodiment, the OMVs can be from 0.5 to 30 [tg protein/kg body weight. The
IL-12 (in
microspheres) can be 1 to 500 g/dose. That corresponds to about 14 ng to 7
g/kg body
weight (assuming a 70 kg body weight). In an embodiment, IL-12 can be 10 ng to
10 g/kg
body weight. In one embodiment, the IL-12 can be 10, 50, 100, 200, 300, 400 or
500 g/dose.
In an embodiment, the IL-12 can be 10 to 300 ng or 5 to 300 ng per kg body
weight. In an
embodiment, IL-12 can be 0.5 to 20 micrograms in microspheres per dose.
Determining the
amount of microspheres can be done based on the loading of IL-12. The
compositions may be
provided in carriers, buffers and the like or may be lyophilized. The two
components may be
provided separately, and can be combined just before administration or may be
administered
separately. In one embodiment, the composition does not have any added free
soluble IL-12.
Any leaked IL-12 from the microspheres prior to administration is considered
to be
insignificant. Even if free soluble IL-12 is present, it is not considered to
contribute to the
present effects and method. Rather, encapsulated IL-12 in microspheres was
required in the
composition to see the protective effects.
[0040] Outer membrane vesicles can be prepared from the outer
membranes of N.
gonorrhoeae. For example, OMVs can be prepared from the outer membranes of a
cultured
strain of Neisseria gonorrhoeae spp. OMVs can be obtained from a N.
gonorrhoeae grown in
broth or solid medium culture. The preparation may comprise separating the
bacterial cells
from the culture medium (e.g. by filtration or by a low-speed centrifugation
and the like),
lysing the cells (e.g. by addition of detergent, osmotic shock, sonication,
cavitation,
homogenization and the like) and separating an outer membrane fraction from
cytoplasmic
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molecules (e.g. by filtration; or by differential precipitation or aggregation
of outer
membranes and/or outer membrane vesicles, or by affinity separation methods;
or by a high-
speed centrifugation).
[0041] The compositions can be administered preferably as multiple
doses with an
interval in between. For example, at least two doses can be administered with
an interval of at
least one week in between. The interval may be from one to three weeks. In one
embodiment,
two doses are used with an interval of about 2 weeks in between.
[0042] In one embodiment, the IL-12 formulations of the present
invention are
sustained release formulations. In one embodiment, IL-12 is delivered as
incorporated (also
referred to herein as encapsulated or microencapsulated) in polymeric
microparticles (also
referred to herein as microspheres). In one embodiment, the microparticles are
biodegradable
and biocompatible. Preparation techniques for such microspheres are known in
the art. See
for example, U.S. Patent numbers 6,143,211; 6,235,244; 6,616,869; and
7,029,700, the
disclosures of which pertaining to methods and compositions for preparation of
microspheres
are incorporated herein by reference. In one embodiment, a phase inversion
technique is used
to prepare microencapsulated IL-12. In general, a biodegradable polymer is
dissolved in a
solvent (such as dichloromethane or other organic solvent) and then a mixture
is formed by
adding micronized IL-12 (i.e. lyophilized mixtures of IL-12 and excipient such
as polyvinyl
pyrrolidone) to the polymer dissolved in the solvent. A non-solvent (such as
alcohol or
hexane) is then introduced causing spontaneous formation of microencapsulated
IL-12.
Examples of biodegradable polymers include polymers of lactic acid and
glycolic acid,
polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid),
poly(valeric acid),
poly(caprolactone), poly(hydroxybutyrate), poly(lactide-co-glycolide) and
poly(lactide-co-
caprolactone), and natural polymers. In one embodiment, the microspheres are
composed of a
polymer of lactic acid (polylactic acid (PLA)).
[0043] In one embodiment, the IL-12 containing microspheres degrade
by hydrolysis
slowly over time, releasing the encapsulated IL-12. The microspheres are
suspended before
use and can also be delivered in an acceptable buffered physiological saline
solution. In one
embodiment, slow release of IL-12 over a period of time such as approximately
4 days allows
for continuous stimulation of locally present immune cells without elevating
the
concentration of IL-12 in the local tissues or the circulation to potentially
harmful levels. The
microspheres are made of biodegradable materials. In one embodiment, the
hydrolytic
product of the microspheres is lactic acid, a harmless product of normal
metabolism. PLA is a
component of absorbable sutures and has been in use for that purpose for many
decades, and
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is therefore considered safe. Microencapsulated IL-12 has been shown to be
stable in storage
at ambient temperatures and to have a long shelf-life.
[0044] The microspheres are in the range of 10 nm to 10 microns. The
microspheres
may be suspended in pharmaceutically acceptable medium such as a physiological
buffer. In
one embodiment, the loading of IL-12 is from 0.1 to 10 [tg per mg of the
particles. In one
embodiment, the loading is from 1 to 5 [tg IL-12 per mg of the particles. In
one embodiment,
the loading is from about 2.5 [tg IL-12 per mg of the particles.
[0045] The IL-12 formulations can be used in amounts that will result
in therapeutic
and/or prophylactic effects. An effective dose in mice was observed to be 1
[tg of IL-12.
Determining the effective dosage for humans is within the purview of
clinicians and other
individuals involved in the treatment of such infections. Generally, the
amount administered
depends upon various factors including the severity of the infection, the
weight, health and
age of the individual. Such factors can be readily determined by a clinician.
In one
embodiment, the dose may be from 1 [tg to 200 [tg of IL-12 per day. In some
embodiments,
the dose is 1, 5, 10, 15, 20, 50, 75, 100, 125, 150, 175 and 200 [tg of IL-12
per dose and all
integers between 1 and 200 [tg and all ranges therebetween. The dosage
required may be less
if used in conjunction with an antimicrobial agent.
[0046] The dosage may be repeated as necessary. For example, the
administration
may be repeated daily, multiple times in a day, or at longer intervals, such
as at intervals of 2-
4 days, weekly or monthly. In one embodiment, the administration is repeated
at intervals
from 1 day to 1 month (28, 29, 30 or 31 days) or beyond that and all intervals
therebetween.
The treatment regimen may be repeated as necessary. In some embodiments, the
dosage is
administered every 2, 3, 4, 5, 6, 7, 10, or 14 days, or longer.
[0047] In one embodiment, the present invention provides a method of
treating
genital tract infections in a female subject by intravaginal application of IL-
12 incorporated
in polymeric microspheres. The infections that can be treated by this method
include
bacterial, fungal, parasitic, viral and the like. In one embodiment, the
amount is sufficient to
promote Thl- driven response against the microorganisms causing the infection.
In one
embodiment, the amount is sufficient to provide a therapeutic effect, a
prophylactic effect, or
both against the causative microorganisms. The term "treated" or "treatment"
as used herein
means to reduce or eliminate an infection. An infection is considered to be
reduced when the
underlying cause of the infection is reduced.
[0048] In one embodiment, the method of the present invention is
useful for treating
genital tract infections, such as cervico-vaginal infections, caused by
bacteria, such as N.
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gonorrhoeae. The method comprises the steps of providing local (intravaginal)
application of
the cytokine interleukin-12 (IL-12) incorporated in biodegradable,
biocompatible
microspheres. In one embodiment, the dose is sufficient to promote Thl-driven
immune
responses against infection with N. gonorrhoeae. In one embodiment, the
invention provides
a method for therapy or prophylaxis or both for cervico-vaginal gonococcal
infection (i.e.,
gonorrhea) by means of local administration of IL-12 microspheres. While not
intending to
be bound by any particular theory, it is considered that this method works, at
least in part, by
reversing the ability of N. gonorrhoeae to interfere with the host's immune
responses.
[0049] In one embodiment, the IL-12 formulation is delivered locally
to the mucosal
surface of the genital tract of an individual. In one embodiment, the
individual is not already
receiving IL-12, or has not been administered IL-12 prior to the initiation of
the present
method. In one embodiment, the individual is not receiving IL-12 via any other
administration mode. In one embodiment, the formulation contains no other
therapeutic
agent, no other prophylactic agent, or no other agent that is both therapeutic
and prophylactic.
In one embodiment, the formulation does not contain the infection causing
microorganism
(such as in an inactivated form) or an antigen therefrom, and the individual
has not been
and/or is not being administered the inactivated microorganism or an antigen
therefrom. In
another embodiment, the formulation may be delivered to an individual who is
already
receiving treatment (other than IL-12) for genital tract infection (such as
gonococcal
infection).
[0050] In one embodiment, the invention further comprises the step of
administering
an antimicrobial agent to the individual. For example, in one embodiment, the
method of this
invention comprises the steps of identifying an individual who is suffering
from or has been
diagnosed with an infection of the genital tract, delivering to the genital
tract locally (such as
intravaginally) a composition comprising a therapeutically effective, a
prophylactically
effective, or both therapeutically and prophylactically effective amount of a
composition
comprising IL-12 in biodegradable polymeric microspheres, and optionally
administering to
the individual one or more antimicrobial agents (such as antibiotics,
antifungal or antiviral
agents). The antimicrobial agents may be administered prior to, during or
after the
administration of the IL-12 formulation. An example of such a treatment is the
administration
of antibacterial agents, such as antibiotics. Examples of suitable antibiotics
used for genital
tract infections include fluorquinolones, cephalosporins, azithromycin,
Ceftriaxone,
doxycycline, and Cefixime.
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[0051] In one embodiment, the administration of the microencapsulated
IL-12 as
described here reduces the N. gonorrhoeae infection. In one embodiment, the
infection is
eliminated. The presence or absence of infection or the level of infection may
be tested by
routine microbiological methods (such as culture and testing). In one
embodiment, the
infection may be tested by obtaining vaginal swab and testing for the presence
of bacteria
(such as by the ability to form colonies), or by nucleic acid amplification
methods.
[0052] In another embodiment, the administration of the
microencapsulated IL-12 as
described here reduces the N. gonorrhoeae infection and reduces the risk of
repeat infection
of N. gonorrhoeae after the treatment with microencapsulated IL-12 has been
stopped. While
.. not intending to be bound by any particular theory, it is considered that
the prophylactic
effect of IL-12 is achieved by stimulation of the immune system. In one
embodiment, the
administration of IL-12 does not significantly increase the level of IL-12 in
the systemic
circulation. In one embodiment, the serum level of IL-12 does not increase to
greater than 50
picograms/ml.
[0053] For intravaginal applications, the formulations of the present
invention can be
delivered as applied to an article of manufacture acting as a carrier. For
example, the
formulations may be incorporated into or onto and then delivered via an
insert, an applicator,
tablet, suppository, vaginal ring, vaginal sponge, tampon and the like. The
formulation may
also be delivered in the form of a liquid, cream, gel, lotion, ointment,
paste, spray and the
like.
[0054] The pharmaceutical formulations may optionally include
pharmaceutically
acceptable carriers, buffers, diluents, solubilizing or emulsifying agents,
and various salts.
Such additives are well known in the art. See, e.g., Remington's
Pharmaceutical Sciences,
18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042).
[0055] An advantage of local application of microencapsulated IL-12 as
described
herein is that it can provide a sustained effect while avoiding problems of
potential systemic
toxicity.
[0056] In one embodiment, the present invention is used for treating
genital
Chlamydia trachomatis infection (chlamydia). Chlamydia is another sexually
transmitted
disease (STD) of even more frequent occurrence than gonorrhea, and is the most
frequently
reported infectious disease in the US, thought to affect up to 3 million
individuals per annum
(>92 million worldwide). It is also a major cause of pelvic inflammatory
disease in women
and its sequelae (infertility and risk for ectopic pregnancy). Therefore in
one embodiment,
local (intravaginal) application of IL-12 incorporated in polymeric
microspheres is used to
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promote local Thl- immune responses for therapy and prophylaxis against C.
trachomatis. In
one embodiment, the method of the present invention is used to treat
urinogenital infections
due to N. gonorrhoeae and C. trachomatis. This may be advantageous in the STD
clinic
setting because gonorrhea and chlamydia present with similar signs and
symptoms, and the
differential diagnosis may depend on identifying the causative organism.
Furthermore, mixed
infections with both are common. In other embodiments, other genital tract
infections could
also be treated with (intravaginal) application of microencapsulated IL-12 to
enhance local
immunity against them.
[0057] In other embodiments, local application of microencapsulated
IL-12 is used in
the treatment of other local mucosal infections where the normal immune
response is
insufficient to eliminate them. Examples include: bronchitis and chronic
obstructive
pulmonary disease (respiratory tract), otitis media (middle ear infection,
which is the most
frequent reason for pediatric office visits in the US), Helicobacter pylori
infection (which
causes gastric ulcer and can lead to gastric cancer), and possibly periodontal
disease (which
afflicts most adults from age 35 onwards and is the main cause of tooth loss
in adults).
[0058] In one embodiment, the IL-12 may be administered with a
gonococcal antigen
containing vaccine. For example, IL-12 can be administered with a locally
administered non-
living gonococcal vaccine. As described in Example 3, we have immunized mice
i.vag. with
a gonococcal outer membrane vesicle (OMV) preparation administered with or
without IL-
12/ms. OMV contain most of the surface antigens in native conformation, not
denatured by
heat or chemical inactivation. The results demonstrate the generation of a Thl-
driven,
antibody-dependent, protective immune response that persists for at least
several months and
is effective against antigenically diverse strains of N. gonorrhoeae. As
described in Example
4, immunization can also be carried out via intranasal route.
[0059] The OMV and the IL-12 microspheres may be administered to female or
male
subjects. When administered to a male subject, the compositions may be
delivered
intranasally, and when administered to a female subject, the compositions may
be delivered
intravaginally and/or intranasally.
[0060] The following examples are provided to further illustrate the
disclosure. These
examples are not intended to be restrictive.
EXAMPLE 1
[0061] The invention has been demonstrated in the mouse model of
vaginal
gonococcal infection. Details of the mouse model can be found in Jerse,
Infect. Immun. 67:
5699-5708; 1999.
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[0062] Intravaginal treatment of mice with IL-12 microspheres (111g)
on days 0, 2,
and 4 of primary vaginal infection (on day 0) with N. gonorrhoeae resulted in
accelerated
clearance of the infection, compared to control mice given blank microspheres
(See Fig. 1).
[0063] Fig. 1 illustrates the effect of intravaginal treatment with
1tg of IL-12
encapsulated in PLA microspheres (on days 0, 2, and 4) on the course of
vaginal infection
with N. gonorrhoeae in mice. Data shown as mean SEM cfu of N. gonorrhoeae
recovered
from vaginal swabs taken daily; N = 8 mice per group. Control mice were given
blank
microspheres. Mice were treated with antibiotic on day 14 and then rested for
secondary
infection (See Fig. 2).
[0064] When mice that were treated with IL-12 microspheres during primary
vaginal
gonococcal infection were allowed to recover, treated with antibiotic
(ceftriaxone) on day 14,
rested and then reinfected one month later with N. gonorrhoeae, the secondary
infection was
cleared faster than in control mice given blank microspheres during primary
infection (See
Fig. 2). Normally, secondary infection is considered to clear with the same
kinetics as
primary infection, and there is little or no antibody response developed.
[0065] Fig. 2 illustrates the effect of intravaginal treatment with
IL-12 microspheres
during primary infection with N. gonorrhoeae (See Fig. 1) on the course of
secondary vaginal
infection with N. gonorrhoeae in mice. Control mice were given blank
microspheres. Data
shown as mean SEM cfu of N. gonorrhoeae recovered from vaginal swabs taken
daily; N =
8 mice per group.
[0066] Further, the effect of intravaginal treatment with
microencapsulated IL-12 (IL-
12 microspheres) was compared with soluble IL-12 on the course of mouse
genital tract
infection with Neisseria gonorrhoeae. For this purpose, 1tg of IL-12 was
instilled
intravaginally in a group of 8 mice in free soluble form (dissolved in sterile
phosphate-
buffered physiological saline) on days 0, 2, 4, 6, 8, and 10 after infection
with N.
gonorrhoeae (i.e., every other day until the infection was cleared), in direct
comparison with
mice treated with IL-12 microspheres and a control group treated with vehicle
only. Mice
treated with IL-12 microspheres cleared the infection within 7 days, much
faster than the
control group, whereas mice treated with soluble IL-12 cleared the infection
at the same rate
as the control group (See Fig. 3). Data shown as mean SEM cfu of N.
gonorrhoeae
recovered from vaginal swabs taken daily; N = 8 mice per group.
[0067] The results show that local intravaginal treatment with
soluble IL-12 had no
effect on the course of infection, whereas IL-12 microspheres accelerated
clearance, as
described previously.
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EXAMPLE 2
[0068] This example describes another set of experiments that
illustrate the
effectiveness of intravaginal application of IL-12 microspheres on N.
gonorrhoeae vaginal
infection.
MATERIALS AND METHODS
[0069] Mice: BALB/c mice were purchased from Jackson Laboratories
(Bar Harbor,
ME), and were maintained under standard conditions in the Laboratory Animal
Facility at the
University at Buffalo. All animal use protocols were approved by the
Institutional Animal
Care and Use Committee of the University at Buffalo.
[0070] Bacteria: N. gonorrhoeae FA1090 were cultured on GC agar
supplemented
with hemoglobin and ISOVITALEX , an enrichment medium (BD Diagnostic Systems,
Franklin Lakes, NJ). Growth was checked for colony morphology consistent with
Opa
protein and pilus expression, and gonococci were harvested from plates and the
cell density
was determined. Opa expression as was: Opa A, B/D/G, E/K.
[0071] Microspheres: Cytokines were encapsulated into poly-lactic acid
(PLA)
microspheres using the Phase Inversion Nanoencapsulation (PIN) technology as
follows.
Briefly, recombinant IL-12 (mouse or human) is mixed with excipients including
sucrose
(0.1%, w/w) and polyvinylpyrrolidone in water and then is lyophilized. The
lyophilized
material is dissolved in tertyl butyl alcohol (TBA) and is mixed with
polylactic acid (PLA)
resomer dissolved in TBA (1 to 3 ratio, vol:vol for micronized IL-12 and PLA
solution). This
solution is then poured into 100x volume of heptane to induce formation of the
particles. The
particles are then filtered and lyophilized. Three formulations were produced:
(a) control
microspheres containing no cytokine or antibody; (b) murine IL-12 (0.2511g/mg
particles);
and (c) murine IL-17 (0.2511g/mg particles).
[0072] Mouse Vaginal Infection Model: Female mice between 7 and 9 weeks old
were infected vaginally on day 0 with live N. gonorrhoeae FA1090 as previously
described.
Vaginal mucus was quantitatively cultured daily on GC agar supplemented with
selective
antibiotics to determine the bacterial colonization loads. The limit of
detection was 100 CFU
recovered per mouse. Intravaginal treatments with microsphere preparations
were given
.. every second day from day 0 to day 8, by instillation of 40 1 suspensions
in PBS of
microspheres containing IL-12 or IL-17, or control microspheres.
[0073] Cell Isolation and Flow Cytometry: Mice were sacrificed and
the iliac
lymph nodes (ILN) and genital tracts were excised aseptically. ILN were teased
in Hanks'
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buffered salt solution to release cells. Vaginal single-cell suspensions were
prepared by
enzymatic digestion. Isolated cells were washed with staining buffer twice,
then incubated
with the indicated antibodies for 30 min on ice, washed twice, and analyzed on
a
FACSCalibur cytometer. For determination of intracellular cytokine expression,
cells were
restimulated with phorbol myristate acetate-ionomycin-GOLGISTOP , a protein
transport
inhibitor (eBioscience, San Diego, CA) for 5 h, and then fixed with
CYTOFIX/CYTOPERM , a fixation/permeabilization solution, (eBioscience).
Antibodies to
mouse CD4, CD8, CD19, CD11b, CD11 c, NKG2D, Gr-1, IFN-y, IL-4, and IL-17A
conjugated with fluorescein isothiocyanate, phycoerythrin, or allophycocyanin
were
purchased from eBioscience.
[0074] Cytokine ELISA: IL-12p70, IFN-y, IL-4, IL-5, and IL-17A levels
in serum or
vaginal wash samples were measured in triplicate using ELISA kits purchased
from
eBioscience.
[0075] Real-time RT-PCR: Total cellular RNA of whole vaginas
harvested from the
mice was isolated with RNEASY , RNA purification Mini Kits (Qiagen, Valencia,
CA), and
was transcribed to cDNA using the ISCRIPTTm cDNA synthesis kit (Bio-Rad,
Hercules, CA).
Real-time RT-PCR was performed on an ICYCLER IQ real-time PCR detection
system
(Bio-Rad) using SYBR Green dye, a fluorescent dye, (Bio-Rad) for real-time
monitoring of
the PCR. Relative quantification of target genes was analyzed based on the
threshold cycle
(Ct) determined by Bio-Rad IQ Tm 5 optical system software.
[0076] Assay of Serum and Mucosal Antibodies: Samples of saliva,
vaginal wash,
and serum were collected from individual mice on day 15 post-inoculation.
Gonococcus-
specific IgA, IgG, and IgM in saliva, sera, and vaginal washes and total IgA,
IgG, and IgM
concentrations in secretions were assayed by ELISA.
[0077] Statistical Analysis: Data are expressed as the means standard
errors of the
means (SEM). Data on the effects of IL-12-, IL-17-, anti-TGF-0-, anti-IL-10-
loaded versus
blank microsphere treatments on vaginal N. gonorrhoeae infection were analyzed
using
repeated-measures analysis of variance (ANOVA) with Bonferroni corrected post-
hoc testing
of pair-wise comparisons. Kaplan-Meier analysis with log-rank testing was also
used to
compare infection clearance. Data from in vitro experiments were analyzed by
unpaired two-
tailed t tests to compare the mean values between two selected groups. P <
0.05 was
considered statistically significant.
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RESULTS
[0078] Intravaginal Administration of IL-12 Microspheres Protects
Mice against
Genital Tract N gonorrhoeae Infection. To examine the therapeutic effect of IL-
12-loaded
microspheres, groups of female BALB/c mice were infected with N. gonorrhoeae
and the
bacterial burden was monitored daily by vaginal swab culture. Preliminary dose-
ranging
experiments showed that intravaginal instillation of microspheres containing
1.0 [tg of IL-12
every second day was sufficient to accelerate clearance of the infection
relative to treatment
with blank microspheres; no further enhancement of clearance was obtained with
2.0 [tg of
microencapsulated IL-12, and lower doses were progressively less effective
(Fig. 4A).
[0079] Untreated or blank microsphere-treated mice cleared the infection in
¨15 days
(Fig. 4B, C). Intravaginal instillation of microencapsulated IL-12 at the
optimal 1.0 [tg dose
significantly reduced the recoverable N. gonorrhoeae load starting from day 4,
and these
mice cleared the infection by day 7, 8 days earlier than blank microsphere-
treated or
untreated mice (Fig. 4B, C). The infection did not relapse after treatment
ceased on day 7. In
contrast, intravaginal administration of free, soluble IL-12 was completely
ineffective in
enhancing clearance of N. gonorrhoeae (Fig. 4B, C).
[0080] Intravaginal administration of IL-17-loaded microspheres at
the optimal dose
(1.0 g) also accelerated clearance of N. gonorrhoeae infection, but to a
lesser extent than IL-
12 microspheres given on the same schedule (Fig. 4B, C).
[0081] Treatment With IL-12 Microspheres Enhances Thl and Antibody
Responses to Vaginal Gonococcal Infection. To elucidate the mechanisms
underlying the
therapeutic effects of IL-12, we characterized the local immune responses to
genital
gonococcal infection in mice treated with IL-12-loaded or blank microspheres.
Single-cell
suspensions were prepared from ILN and vaginas of 7 mice per group at 3, 5, 7,
and 14 days
after inoculation with N. gonorrhoeae or vehicle only for evaluation by flow
cytometry to
detect intracellular IFN-y, IL-4, and IL-17. Starting on day 3 after
inoculation, IL-17+/CD4+ T
cells were observed in the local draining ILN, with production peaking at day
5 and
continuing for the duration of infection. At day 5, approximately 22% of CD4+
T cells
present in the ILN of control-treated infected mice were IL-17k, whereas only
¨3.5% were
IFN-y+ and few IL-4+/CD4+ T cells were detected (Fig. 4D). IL-12 microsphere
treatment
markedly enhanced Thl immune responses to N. gonorrhoeae, indicated by
significantly
increased numbers of IFN-y+/CD4+ T cells (Fig. 4D). In contrast, IL-12
microspheres did not
change Th2 or Th17 responses as the numbers of IL-4+/CD4+ and IL-17+/CD4+ T
cells in ILN
were similar between the treated groups (Fig. 4D). RT-PCR analyses showed that
IFN-y, but
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not IL-4 or IL-17 mRNA expression was elevated in the vaginas of infected mice
following
IL-12 microsphere treatment (Fig. 4E). Although IL-17 microspheres ameliorated
gonococcal
infection, this treatment was not associated with enhanced Thl or Th2
responses (Fig. 4D, E),
but there was increased influx of Gr-1+ neutrophils into the genital tract
(Fig. 4F).
[0082] We also measured by ELISA IL-12p70, IFN-y, IL-4, and IL-17
concentrations
in vaginal wash and serum collected 7 days after inoculation. IL-12 (176.5
48.6 pg/ml) was
detected in vaginal wash from infected mice treated with IL-12 microspheres.
Low levels of
IL-12 (41.7 10.7 pg/ml) were found in the serum of these mice, suggesting
that the effects
of IL-12 microsphere treatment on gonococcal infection did not result
primarily from the
passage of the cytokine into the circulation. Consistent with the flow
cytometric studies, IFN-
y was present in the vaginal wash (32.6 9.8 pg/ml) and serum (43.3 11.5
pg/ml) of
infected mice treated with IL-12 microspheres, but IL-4 and IL-17 were not
detected. None of
these cytokines was detected in control-treated infected mice.
[0083] IL-12 can stimulate humoral immune responses in an IFN-y-
dependent
manner or directly. We therefore determined whether IL-12 microsphere
treatment during N.
gonorrhoeae infection led to the production of anti-gonococcal antibodies in
vaginal wash,
saliva, and serum collected 15 days after inoculation. IgM antibodies were at
low levels with
little difference between experimental groups (data not shown). No salivary
gonococcus-
specific antibody was detected in any group of mice (data not shown). N.
gonorrhoeae
infection of control-treated mice did not significantly elevate gonococcus-
specific IgA or IgG
antibodies in either vaginal washes or sera. However, IL-12 microsphere
treatment increased
vaginal and serum specific IgG antibody (Fig. 4G, H), as well as vaginal
specific IgA
antibody production (Fig. 4G).
[0084] Treatment with IL-12 Microspheres Induces Protective
Anamnestic
Immunity against Secondary N gonorrhoeae Infection. We further assessed
whether IL-
12 microsphere treatment resulted in the generation of immune memory and
protective
immunity against reinfection. Groups of mice infected with N. gonorrhoeae were
treated with
IL-12-loaded or blank microspheres, and after the infection had run its
course, the mice were
treated with ceftriaxone (300 i.p.) on day 15 to ensure complete
elimination of the
gonococci. An additional group of sham-infected mice treated with IL-12
microspheres was
used to evaluate the possible persistent effect of IL-12 in the absence of
infection. Five to six
weeks later, all mice were inoculated with N. gonorrhoeae of the same strain
without any
further treatment. As observed previously, primary infection of control-
treated mice did not
protect them against subsequent secondary challenge: the duration and
bacterial burden of
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secondary gonococcal infection in previously blank microsphere-treated mice
were the same
as for the primary infection of age-matched naïve mice (Fig. 5A, B). In
contrast, intravaginal
treatment with IL-12-loaded microspheres during primary infection protected
the mice
against secondary infection: reinfected mice that had been treated with IL-12
microspheres
during the primary infection resisted the challenge more effectively than
controls (Fig. 5A,
B). However, previous IL-12 microsphere treatment of sham-infected mice did
not induce
protection against subsequent infection (Fig. 5A, B). This result also
excluded the possibility
that any persisting microspheres still affected the secondary N. gonorrhoeae
infection.
[0085] Flow cytometric and RT-PCR analyses of ILN cells and vaginas
taken on day
5 and day 3 of secondary infection, respectively, indicated that the
protective effect of
previous IL-12 microsphere treatment on secondary gonococcal infection was
also associated
with significantly enhanced Thl (IFN-y) responses (Fig. 5C, D). There was also
a robust
specific secondary antibody response in IL-12 microsphere-treated mice after
they were
rechallenged with N. gonorrhoeae. Gonococcus-specific IgA and IgG antibodies
in vaginal
washes and IgG antibodies in sera of reinfected mice previously treated with
IL-12
microspheres were significantly higher than those of control groups (Fig. 5E,
F).
[0086] In contrast to the effects of IL-12 microsphere treatment,
treatment with IL-17
microspheres during primary gonococcal infection did not lead to any
protective immunity to
secondary gonococcal infection, or induce any anamnestic T cell or antibody
responses (Fig.
5A-F).
EXAMPLE 3
[0087] This example describes that the present experimental vaccine
induces Thl-
driven immune responses and resistance to Neisseria gonorrhoeae infection in a
murine
model.
[0088] RESULTS
[0089] Intravaginal immunization of mice with gonococcal OMV plus IL-
12/ms
accelerates clearance of challenge infection with N gonorrhoeae. Groups of 8
female
BALB/c mice were immunized i.vag. with gonococcal OMV (strain FA1090; 4011g
protein)
plus IL-12/ms (111g IL-12), or with OMV plus control (blank) ms; two
additional control
groups were sham-immunized with IL-12/ms or with blank ms alone. Immunizations
were
repeated 1 week and 2 weeks later, and all mice were challenged after a
further 2 weeks by
i.vag. instillation of N. gonorrhoeae FA1090 (5 x 106 CFU). Control (sham-
immunized)
mice, or mice immunized with OMV plus blank ms cleared the infection
commencing at day
7 post-challenge and were all cleared by day 15; median days of clearance were
10-13. There
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was no significant difference in the clearance rates between these three
control groups
(Fig.6a). However, mice immunized with OMV plus IL-12/ms cleared the infection
beginning at day 6 and were all cleared by day 9; median clearance =7.5 days
compared to 12
days in mice immunized with OMV plus blank ms (P <0.01, Kaplan-Meier; Table 1
and Fig.
6a). This experiment was repeated twice more with similar results (see Table 1
and Fig.13).
Further examples of replication of this finding can be seen in subsequent
experiments
reported below, e.g., Figs. 6d, 9, 10a, and c (and Figs. 10b, d, and e for
other gonococcal
strains), and Figs. 12a, b, f, and g (with C57BL/6 mice).
[0090]
Table 1: Summary data from immunization experiments using homologous
challenge (BALB/c mice).
Expt Group Vaccine Challenge Median P Notes
OMV Adjuvant strain clearance (Kaplan-
strain day Meier)
1 a - Blank ms FA1090 11 a ¨
b ¨ c Data
b - IL-12 ms FA1090 12 NS from
c FA1090 Blank ms FA1090 12 Fig.6a
d FA1090 IL-12 ms FA1090 7.5 <0.01
2 a - Blank ms FA1090 9 a ¨
b ¨ c Data
b - IL-12 ms FA1090 10 NS from
Fig.
c FA1090 Blank ms FA1090 9.5 13a
d FA1090 IL-12 ms FA1090 6 <0.001
3 a - Blank ms FA1090 23.5
a ¨ b ¨ c Replicate
b - IL-12 ms FA1090 24 NS of
Fig.6a
c FA1090 Blank ms FA1090 22.5
<0.0001
d FA1090 IL-12 ms FA1090 10.5
4 a - Blank ms FA1090 14 a
vs. b Data
b FA1090 Blank ms FA1090 14 NS from
c FA1090 IL-12 ms FA1090 8 b vs. c --
Fig. 6d
<0.0001
d NTI-II IL-12 ms FA1090 13 c vs. d
<0.0001
5 a - Blank ms FA1090 11 a vs. b
Data
b FA1090 Blank ms FA1090 10 NS from
Fig.
c FA1090 IL-12 ms FA1090 7 b vs. c
13d
<0.001
d NTI-II IL-12 ms FA1090 9.5 c vs. d
<0.001
6 a - Blank ms FA1090 9.5 a vs. b
Data
NS from Fig.
b FA1090 Blank ms FA1090 10 9
(tested
c FA1090 IL-12 ms FA1090 6.5
<0.001at 6 mos)
7 a - Blank ms FA1090 11.5 a vs. b
Replicate
NS of Fig. 9
b FA1090 Blank ms FA1090 11 <0001
(tested at
.
c FA1090 IL-12 ms FA1090 7 4m05)
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[0091] Serum and vaginal wash samples collected after clearance (at
termination, day
15 post-inoculation) were assayed for antibodies against intact gonococci
(FA1090) by
ELISA. This showed that mice immunized with OMV plus IL-12/ms had developed
the
highest levels of vaginal and serum IgG and IgA antibodies, whereas those mice
immunized
with OMV plus blank ms developed much lower levels of these antibodies (Fig.
6b). Mice
that were unimmunized and sham-infected showed no antibodies detectable above
assay
background at the starting dilutions, and mice immunized with blank ms alone
and infected
also did not develop detectable antibodies (Fig. 6b). Iliac lymph node (ILN)
mononuclear
cells collected at the same time were stained for surface CD4 expression and
for intracellular
cytokines, and analyzed by flow cytometry. This revealed that only mice
immunized with
OMV plus IL-12/ms generated CD4+/IFNy+ (and CD8+/IFNy+) T cells, whereas no
mice
developed significant numbers of CD4+/IL-4+ T cells (Fig. 6c; see also Fig.
14). However, all
mice that were infected with N. gonorrhoeae regardless of immunization
developed CD4+/IL-
17+ T cells (Fig. 6c and Fig. 14).
[0092] Further experiments were performed to determine the minimum number
of
immunizations required to induce immune resistance to infection. A single dose
of OMV plus
IL-12/ms given i.vag. did not consistently generate resistance to challenge,
but two doses of
OMV (4011g protein) plus IL-12/ms (111g IL-12) given at an interval of 2 weeks
were
sufficient to induce similar resistance to infection; median clearance =8 days
(Fig. 6d; Table
1). In addition, control immunization with OMV prepared from non-typable
Haemophilus
influenzae (NTHI) plus IL-12/ms failed to induce resistance to N. gonorrhoeae;
median
clearance =13 days (Fig. 6d; Table 1). This induced antibodies to NTHI but not
to N.
gonorrhoeae (Figs. 15a and b) and generated IFNy-producing CD4+ and CD8+ cells
in the
ILN (Fig. 15c).
[0093] Intravaginal immunization with gonococcal OMV plus IL-12/ms induces
persistent gonococcus-specific antibody responses and Thl cellular responses.
To
characterize the local and systemic immune responses after immunization with
1, 2, or 3
doses of gonococcal OMV plus IL-12/ms and before challenge, serum, vaginal
washes, and
ILN were collected from immunized and control mice 2 weeks after the last
immunization.
Serum anti-gonococcal IgM antibodies were at low levels with little difference
between
experimental groups. IgA and IgG antibodies were not detectable above
background in
vaginal wash or serum samples of mice given blank ms alone. Intravaginal
immunization
with 3 doses of gonococcal OMV plus blank ms elevated vaginal and serum anti-
gonococcal
IgA and IgG antibodies but to a lesser degree than immunization with OMV plus
IL-12/ms
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(Fig.7a). In contrast, immunization with one dose of OMV plus IL-12/ms induced
low levels
of anti-gonococcal antibodies in both serum and vaginal wash; a second dose
elevated
antibody production, but no further elevation was seen after 3 doses (Fig.7a).
Antibodies
appeared to be specific for N. gonorrhoeae, as they were not detected above
control levels
against E. coil or NTHI. Assay of IgG subclass antibodies in both vaginal wash
and serum
showed a predominance of IgG2a, with lesser amounts of IgG1 and IgG2b and low
levels of
IgG3 (Fig. 16). The production of anti-gonococcal IgA and IgG antibodies in
vaginal wash
and serum peaked at 3 months after immunization with 2 doses of OMV plus IL-
12/ms, and
were still detectable at 6 months after immunization (Figs. 7b and c).
[0094] Flow cytometric analysis of ILN cells revealed increased numbers of
IFNy+/CD4+ and IFNy+/CD8+ T cells from mice immunized with OMV plus IL-12/ms
compared with those from control mice (Fig. 8a). As observed with the antibody
responses,
one immunization was sufficient to induce IFNy production, and it was further
elevated by 2
immunizations; 3 doses did not further increase it. In contrast, immunization
with OMV plus
IL-12/ms did not significantly increase the numbers of IL-4+/CD4+ and IL-
17+/CD4+ T cells
relative to controls (Fig. 8a). To determine whether the induced IFNy+/CD4+
(and
IFNy+/CD8+) T cells were specific for gonococcal antigens, CD4+ cells isolated
from ILN
were preloaded with CF SE, cultured in vitro for 3 days in the presence of
antigen-presenting
cells with gonococcal OMV or without stimulation as controls, and their
proliferation was
assessed by flow cytometry. CD4+ cells from the ILN of immunized mice
proliferated
significantly more, and produced significantly more IFNy, in response to
stimulation in vitro,
than the cells from control mice (Fig. 17a). No production of IL-4 was seen,
but IL-17 was
generated by CD4+ T cells cultured with gonococcal OMV (Fig. 17a). IFNy
production by
ILN CD4+ T cells remained elevated, albeit at slowly declining levels, for 4
months after
immunization (Fig. 8b).
[0095] In addition, vaginas were excised from euthanized mice 3 days
after the last
immunization and RNA was extracted from the whole tissue. RT-PCR analysis
showed that,
in comparison to controls, immunization with gonococcal OMV plus IL-12/ms
significantly
enhanced the expression of mRNA for IFNy but not for IL-4 or IL-17 (Fig. 17b).
IFNy
mRNA expression in vaginal tissue, and production of IFNy by ILN CD4+ cells
remained
elevated for up to 2 months following i.vag. immunization with gonococcal OMV
plus IL-
12/ms (Fig. 17c). These findings support the cytokine expression results
obtained with cells
from the draining ILN.
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[0096] Duration of vaccine-induced resistance to infection. To
evaluate the
duration of immune resistance, groups of 8 mice were immunized with gonococcal
OMV
plus IL-12/ms, and were challenged with the same strain (FA1090) of N.
gonorrhoeae at 2, 4,
or 6 months after immunization. Compared to age-matched controls that were
either sham-
immunized or immunized with OMV plus blank ms, mice immunized with OMV plus IL-
12/ms were resistant to N. gonorrhoeae infection when challenged at 2 or 4
months after
immunization; median clearance in controls =11-11.5 days vs. 7 days in
immunized mice
(Table 1 = Suppl. Table 1). Similar results were obtained in a replicate
experiment when mice
were challenged 6 months after immunization; median clearance in controls =9.5-
10 days vs.
6.5 days in mice immunized with OMV plus IL-12/ms (Fig. 9; Table 1). After
termination
anti-gonococcal antibodies were detected in serum and vaginal washes (Figs.
18a and b).
IFNy- (but not IL-4-) secreting CD4+ T cells were present in ILN (Fig. 18c).
Notably, the
antibody and IFNy responses detected after challenge were higher than those
before challenge
(compare with Figs. 7b, c, and 8b) implying recall of memory. As observed
previously, IL-
17-secreting T cells were always found after challenge with N. gonorrhoeae,
regardless of
immunization (Fig. 18c). Longer time periods were not evaluated because mice
become
increasingly resistant to gonococcal infection as they age.
[0097] Immunization induces resistance to heterologous strains of N
gonorrhoeae. An important consideration for any vaccine is that it should be
effective against
different strains of the pathogen, as well as those antigenically homologous
to the
immunizing strain. N. gonorrhoeae is well known for its antigenic variability
involving most
of its surface antigens through multiple molecular mechanisms. We therefore
determined
whether i.vag. immunization with one strain of gonococcal OMV would be
effective against
challenge with other strains to a similar extent as challenge with the same
strain. At first,
mice (8 per group) were immunized i.vag. with OMV prepared from strain FA1090
plus IL-
12/ms or blank ms, and were challenged one month later with N. gonorrhoeae
strains
FA1090 or MS11 (5 x 106 CFU). Immunization with FA1090 OMV induced resistance
to
challenge with either FA1090 or MS11 to similar extents (Fig. 10a; Table 2).
After challenge
and clearance antibodies were elevated to similar levels against MS11 and the
Thl responses
indicated by IFNy+/CD4+ T cells in ILN were similarly enhanced (Figs. 19a, b,
and c). In a
reciprocal manner, immunization with MS11 OMV (plus IL-12/ms) induced
resistance to
challenge with strain FA1090 (Fig. 10b; Table 2).
[0098] Table 2 Summary data from immunization experiments using
heterologous
challenge (BALB/c mice).
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Expt Group Vaccine Challenge Median P
Notes
OMV Adjuvant strain clearance (Kaplan-
strain day Meier)
1 a FA1090 Blank ms FA1090 13
<0.002 Data from
b FA1090 IL-12 ms FA1090 8 Fig.
10a
c FA1090 Blank ms MS11 12
<0.0001
d FA1090 IL-12 ms MS11 7
2 a FA1090 Blank ms FA1090
10.5 Data from
b FA1090 IL-12 ms FA1090 7.5 <0.002
Fig. 10a
c FA1090 Blank ms MS11 11
<0.001
d FA1090 IL-12 ms MS11 8
3 a MS11 Blank ms FA1090
13.5 Data from
b MS11 IL-12 ms FA1090 9 <0.01
Fig. 10b
4 a MS11 Blank ms FA1090 11
Data from
b MS11 IL-12 ms FA1090 8 <0.01
Fig. 10b
a FA1090 Blank ms FA1090 10 <0.01
Data from
b FA1090 IL-12 ms FA1090 8 Fig.
10c
c FA1090 Blank ms FA19 10
d FA1090 IL-12 ms FA19 7 <0.0001
6 a FA1090 Blank ms FA1090 10
Data from
b FA1090 IL-12 ms FA1090 7 <0.01
Fig. 10c
c FA1090 Blank ms FA19 10
<0.0001
d FA1090 IL-12 ms FA19 6
7 a FA19 Blank ms FA1090 12
Data from
b FA19 IL-12 ms FA1090 8 <0.01
Fig. 10d
8 a FA19 Blank ms FA1090
9 Replicate of
b FA19 IL-12 ms FA1090 7 <0.01
Fig. 10d
9 a FA1090 Blank ms GC68 10
<0 01 Data from
.
b FA1090 IL-12 ms GC68 7 Fig.
10e
a FA1090 Blank ms GC68 12.5 <0 001
Replicate of
.
b FA1090 IL-12 ms GC68 8 Fig.
10e
11 a FA1090 Blank ms GC69
12.5 Additional
b FA1090 IL-12 ms GC69 8
<0.001 clinical
isolate
12 a FA19 Blank ms MS11 9
Additional
b FA19 IL-12 ms MS11 7
<0.01 heterologous
challenge
13 a MS11 Blank ms FA19 11
<001 Reciprocal
.
b MS11 IL-12 ms FA19 8
of expt. 12
[0099]
Gonococcal strains FA1090 and MS11 both possess porin of the same major
type (PorB.1B) although of different subtypes. Therefore, to determine whether
the major
porin type is integral to immune resistance, further experiments were
performed with strain
FA19 (PorB.1A). Immunization with FA1090 OMV (plus IL-12/ms) induced
resistance to
5 challenge with FA19 (Fig. 10c; Table 2). Antibody responses assayed
at termination showed
cross-reactivity against FA19, and IFNy+/CD4+ T cells in ILN were elevated
(Figs. 19d, e,
and g). Reciprocally, immunization with FA19 OMV induced resistance to
challenge with
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strain FA1090 (Fig. 10d; Table 2). Other immunization and challenge
combinations (i.e.,
MS11 against FA19, and vice versa) likewise showed similar cross-resistance
(Table 2).
[0100]
N. gonorrhoeae strains FA1090, MS11, and FA19 have been widely used in
many laboratories and extensively subcultured since their original isolation.
As a result, it is
possible that they have acquired mutations and become altered in some of their
characteristics. Therefore we also challenged immunized mice with novel
clinical strains that
have been minimally passaged in vitro since their isolation. Mice immunized
with FA1090
OMV plus IL-12/ms were also resistant to challenge with clinical isolates GC68
(a PorB.1B
strain; Fig. 10e; Table 2) and GC69 (PorB.1A; Table 2).
[0101] Antigens targeted by immunization-induced antibodies. When examined
by one-dimensional (1D) SDS-PAGE, the protein profiles of FA1090, MS11, and
FA19
OMV were similar, but with apparent quantitative as well as qualitative
variations (Fig. 11a).
Western blot analyses of serum from one mouse (#1) immunized with FA1090 OMV
plus IL-
12/ms against FA1090, MS11 or FA19 OMV separated by SDS-PAGE revealed IgG
antibodies reactive with bands migrating at approximately 35-80kDa, with
reactivity against
bands present in OMV from all three strains strains (Fig. 11b, lanes 2-4). One
of these bands
at approximately 35kDa may correspond to porin, as H5 antibody reacted with a
band of
similar mobility (Fig. 11b, lane 6). Another serum (#2) displayed strong
reactivity against
three bands migrating at approximately 45-65kDa (Fig. 11b, lane 5). In an
effort to identify
the ¨45-65-kDa antigens, we used immunoproteomic approaches consisting of two-
dimensional (2D) SDS-PAGE separation of OMV and parallel 2D SDS-PAGE followed
by
immunoblotting (2D-immunoblot) and mass spectrometry. The three 2D protein
maps of
OMV revealed by Flamingo staining showed numerous protein species and
significant
differences in the OMV proteome between FA1090, MS11, and FA19 strains (Figs.
11c, d,
and e). In contrast, the blotted protein maps showed two spots (Spot 1 and
Spot 2) of masses
corresponding to 45kDa and 43kDa, and pI 5.2 and 5.5, respectively (FA1090
OMV; Fig.
11c), or one spot (Spot 1) with an approximate mass 45kDa and pI 5.2 (MS11 and
FA19
OMV; Figs. lid and e). Mass spectrometry analysis of the tryptic peptides
obtained from
Spot 1 and Spot 2 (Figs. 11c, d, and e) revealed as top hits translation
elongation factor-Tu
(EF-Tu) and a putative periplasmic polyamine-binding protein, PotF3,
respectively (Table 3).
EF-Tu appeared as the most confident antigen as it was immunoreactive in all
three 2D-
immunoblots and was identified with the highest confidence (score ranging from
485.0 to
947.1) and coverage (64.2 to 90.6) in all OMV preparations (Table 3).
29
[0102]
Table 3 Protein identification of 2D-
SDS-PAGE gel spots from gonococcal OMV (Spot 1 and Spot 2; shown in Figs.
llc-e) recognized by sera from mice immunized with FA1090 OMVs plus IL-12/ms.
0
t..)
o
,-,
,o
Spot 1
o
Strain Accession Description Gene Score
Coverage MW [kDa] calc. pl Localization cee
u,
Q5F5Q8, tufl , NG01842
Cytoplasmidl t..)
oo
elongation factor Tu 583.1
71.8 42.91 5.30
YP_ 208891 tuf2, NG01858 Periplasmic
pyruvate
YP_207710 dehydrogenase subunit aceE, NG00565 159.8
27.1 99.53 5.80 Cytoplasmic
El
FA1090 molecular chaperone
YP- 209108 groL groEL, NG02095 101.9 38.1 57.31 5.03
Cytoplasmic
GroEL
carbamoyl phosphate
YP 207230 carA, NG00053 83.6
43.8 40.60 5.43 Cytoplasmic
- synthase small subunit
cell division protein
Inner P
YP 208577 ftsA, NG01529 77.6
54.4 44.03 5.52 .
-
FtsA membrane
.
.3
tufl , tuf2, NGEG 02139,
Cytoplasmicil
EEZ46817 elongation factor Tu 485.0
64.2 42.91 5.30 _.]
L..) NG01842, NGOT858
___________________________________________ Periplasmic ,
,,
molecular chaperone
r.,0
EEZ44759 NGEG 00029, NG02095 242.0
46.9 57.30 5.03 Cytoplasmic .
GroEL -
'
ATP synthase FOF1
E
EZ44814 NGEG 00084, NG01205 143.5
45.4 50.39 5.16 Unknown ,
FA19 subunit beta -
pyruvate
EEZ45398 aceE, NGEG 00668, NG00565 109.6 25.0 99.53 5.80
Cytoplasmic
dehydrogenase
FKBP-type peptidyl-
Outer
EEZ44683 prolyl cis-trans fkpA, NGEG_01946, NG01225
109.5 44.1 28.94 5.86
membrane
isomerase FkpA
tufl, tuf2, NGFG 02465,
AGU85180,
Cytoplasmidl
elongation factor Tu NGFG 02466, N-G01842, 947.1
90.6 42.91 5.30 1-d
AGU85181 Periplasmic n
NG0158
signal recognition
Inner cp
MS11 EEZ48906 particle-docking protein NGFG_01822, NG02060
94.5 44.4 44.30 5.22 w
membrane =
FtsY
1-
cio
polyamine ABC O-
potF3, NGFG_01435,
EEZ48268 transporter substrate- 89.4
45.5 41.29 5.96 Periplasmic w
NG01494
vi
yD
binding protein
o
carbamoyl-phosphate
EEZ47020 carA, NGFG_00187, NG00053 87.9 38.5 40.62 5.43
Cytoplasmic
synthase
pilus assembly protein
EEZ47069 NGFG 00236, NG00098 78.5
43.4 41.23 5.36 Unknown w
PilM -
o
1-
yD
1-
o
cio
vi
Spot 2
t,.)
cie
Strain Accession Description Gene Score
Coverage MW [kDa] calc. pl Localization
ABC transporter
periplasmic
YP 208544 potF3, NG01494 167.5
43.7 41.23 5.96 Periplasmic
- binding protein,
polyamine
ABC transporter
periplasmic
YP 207371 potF1 , NG00206 70.7
27.3 41.16 5.87 Periplasmic
- binding protein
polyamine
P
FA1090 carbamoyl
.
.
.3
phosphate
YP 207230 carA, NG00053 62.6
40.1 40.60 5.43 Cytoplasmic ,
(....) - synthase small
,
.-
subunit
.
Q5F5Q8, elongation factor
tufl , NG01842 .
,
55.5
35.3 42.91 5.30 Cytoplasmic 0
, YP_208891 Tu
tuf2, NG01858 ,,
,
succinyl-CoA
YP_208021 synthetase subunit sucC, NG00913 42.7
30.7 41.28 5.44 Cytoplasmic
beta
hypothetical
EEZ46094 potF3, NGEG_01364, NG01494 348.1 55.5 41.26 5.96
Periplasmic
protein
putrescine
transport system
EEZ45054 potF1 , NGEG_00324, NG00206
152.9 43.8 41.23 5.87 Periplasmic
substrate-binding
1-d
protein
n
FA19
pyruvate
EEZ45398 aceE, NGEG_00668, NG00565 146.0
32.0 99.53 5.80 Cytoplasmic cp
dehydrogenase
w
o
succinyl-CoA
1-
cio
ligase [ADP-
EEZ45783 sucC, NGEG_01053, NG00913 142.7
47.9 41.27 5.35 Cytoplasmic
forming] subunit
w
vi
beta
yD
o
EEZ44847 DNA polymerase
dnaN, NGEG_00117, NG00002 102.1
55.9 40.84 5.31 Cytoplasmic
III, beta subunit
polyamine ABC
0
transporter
potF3, NGFG_01435, NG01494 794.15
74.0 41.29 5.96 Periplasmic
EEZ48268
substrate-binding
protein
cio
putrescine ABC
transporter
potF1 , NGFG_00341, NG00206 293.78
50.4 41.20 5.87 Periplasmic
EEZ47174
substrate-binding
MS11 protein
pilus assembly
EEZ47069 NGFG00236, NG00098 131.0
48.3 41.23 5.36 Unknown
protein PilM _
tufl , tuf2, NGFG 02465,
AGU85180, elongation factor
NGFG 02466, N¨G01842, 110.6
58.6 42.91 5.30 Cytoplasmic/
AGU85181 Tu
Periplasmmicl
NG0158
EEZ48160 transaldolase NGFG 01327, NG01610 101.2
55.0 37.44 5.45 Cytoplasmic
1 Localization determined by Porcella et. al. 1996 (ref. 45).
1-d
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[0103] Immune resistance to N gonorrhoeae depends on IFNy and
antibody. To
determine whether the protective effect of immunization with OMV adjuvanted
with IL-
12/ms is dependent on IFNy or antibody responses, or on immunity governed by
CD4+ or
CD8+ T cells, we performed immunization experiments using mutant C57BL/6 mice
deficient
in IFNy (IFNy-ko) B cells ( MT), CD4+ T cells (CD4-ko), or CD8+ T cells (CD8-
ko). Groups
of 8 C57BL/6 wild-type (control) and immumnodeficient mice were immunized with
FA1090 OMV plus IL-12/ms or blank ms, and challenged with N. gonorrhoeae
FA1090 (5 x
106 CFU) one month later. The course of vaginal gonococcal infection was not
altered in
unimmunized immunodeficient mice relative to wild-type controls. All wild-type
and
immunodeficient mice started to reduce the recoverable gonococcal load from
day 7-11 and
had cleared the infection by day12-14 (median 9-13 days), similar to BALB/c
mice used in
experiments described in the previous sections (Figs. 12a, b, f, and g; Table
4).
[0104] Table 4 Summary data from immunization experiments using
immunodeficient mice (C57BL/6 background)
Expt Group Mouse Vaccine Challenge Median P Notes
strain OMV Adjuvant strain clearance (Kaplan
strain day -Meier)
1 a WT FA1090 Blank ms FA1090
11.5 <0.0001 Data from
b WT FA1090 IL-12 ms FA1090
7 Fig. 12a
c IFNy-ko FA1090 Blank ms
FA1090 11.5
NS
d IFNy-ko FA1090 IL-12 ms
FA1090 11.5
2 a WT FA1090 Blank ms FA1090
11 Data from
b WT FA1090 IL-12 ms FA1090
7 <0.0001 Fig. 12b
c MT FA1090 Blank ms FA1090
11
NS
d MT FA1090 IL-12 ms FA1090
10
3 a WT FA1090 Blank ms FA1090
9.5 Data from
b WT FA1090 IL-12 ms FA1090
7 <0.001 Fig. 12f
c CD4-ko FA1090 Blank ms
FA1090 11
<0.01
d CD4-ko FA1090 IL-12 ms
FA1090 9.5
4 a WT FA1090 Blank ms FA1090
12 Data from
b WT FA1090 IL-12 ms FA1090
7 <0.001 Fig. 12g
c CD8-ko FA1090 Blank ms
FA1090 12
<0.02
d CD8-ko FA1090 IL-12 ms
FA1090 9
5A a WT FA1090 Blank ms FA1090
10 <0.001 Data from
b WT FA1090 IL-12 ms FA1090
6.5 Fig. 20a
c IFNy-ko FA1090 Blank ms
FA1090 10
NS
d IFNy-ko FA1090 IL-12 ms
FA1090 10
6 c MT FA1090 Blank ms FA1090
13 NS Data from
d MT FA1090 IL-12 ms FA1090
13 Fig. 20b
7 c CD4-ko FA1090 Blank ms
FA1090 11 <0.01 Data from
d CD4-ko FA1090 IL-12 ms
FA1090 8 Fig. 20c
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8 c CD8-ko FA1090 Blank ms FA1090 13
Data from
CD8-ko FA1090 IL-12 ms FA1090 9
<0.01Fig. 20d
[0105] In contrast to wild-type mice, clearance of gonococcal
infection was not
accelerated in IFNy-ko or MT mice immunized with OMV plus IL-12/ms compared
to
immunization with OMV plus blank ms (Figs. 12a and b; Table 4; Figs. 20a and
b). Thus
deficiency of either IFNy or B cells abrogated the adjuvant effect of IL-12/ms
in generating
immune resistance to genital gonococcal infection. The production of
gonococcus-specific
vaginal and serum IgA and IgG antibodies induced by OMV plus IL-12/ms in wild-
type mice
was abrogated in IFNy-ko mice (Figs. 12c and d), and as expected there was no
generation of
IFNy by the ILN cells of immunized IFNy-ko mice (not shown). Likewise, in MT
mice
there was no detectable antibody response to immunization (not shown). In
contrast, the
numbers of IFNy+/CD4+ T cells in ILNs of MT mice immunized with gonococcal
OMV
plus IL-12/ms were not affected, and there was no IL-4 response, while IL-17
responses
remained unaltered (Fig. 10e = Fig. 5e). These findings indicate that
resistance induced by
immunization with gonococcal OMV plus IL-12/ms depended on both IFNy and B
cells, the
latter presumably to produce gonococcus-specific antibodies.
[0106] The protective effect of immunization with gonococcal OMV plus IL-
12/ms
was incompletely diminished in CD4-ko, and partially diminished also in CD8-ko
mice, in
comparison with wild-type controls (Figs. 12f and g; Table 4; Figs. 20c and d.
These findings
suggest that the requirement for CD4+ T cells to generate immune resistance
could be
partially compensated by other cells, including CD8+ or NK cells, which can
also produce
IFNy. However, CD8+ cells appeared to be less critical for protective
immunity.
DISCUSSION
[0107] We have demonstrated for the first time that a vaccine-induced
state of
immune resistance to genital gonococcal infection can be reliably generated by
an intact
mammalian immune system. This state of immunity appears to depend on antibody
production by B cells, and on the generation of IFNy mainly by CD4+ T cells.
I.vag.
vaccination of mice with gonococcal OMV plus IL-12/ms as an adjuvant induced
serum and
vaginal IgG and IgA antibodies against gonococcal antigens, and IFNy-secreting
CD4+ and
CD8+ T cells in the draining ILN. Both Thl cellular and antibody responses
persisted for
several months after immunization, and were capable of eliciting resistance to
challenge with
N. gonorrhoeae for at least 6 months, with the recall of immune memory. I.vag.
immunization with gonococcal OMV alone, either without adjuvant or with
control (blank)
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ms, induced only weak antibody responses with no detectable IFNy production,
and no
significant resistance to challenge infection. Control immunization with OMV
prepared from
NTHI plus IL-12/ms did not generate immune resistance or antibodies cross-
reactive with N.
gonorrhoeae, although an IFNy response was induced. Thus, while IFNy appears
to be
necessary for resistance to N. gonorrhoeae, without specific antibodies it is
not sufficient.
[0108] We report here that IL-12/ms, given i.vag as an adjuvant with
gonococcal
OMV vaccine, enhances Thl-driven protective immunity revealed by a
significantly
shortened course of genital gonococcal infection. It should be emphasized that
free soluble
IL-12 is ineffective and and that IL-12 encapsulated in ms were required for
the adjuvant
.. effect with the OMVs.
[0109] Given the well-known and extensive antigenic variation shown
by N.
gonorrhoeae, resistance extended to heterologous strains as well as against
the homologous
strain from which the OMV vaccine was prepared ws unexpected. Our results show
that
immunization with OMV derived from strain FA1090 enhances resistance equally
well
against strains MS11 and FA19, and vice versa, and that resistance extends to
clinical isolates
of N. gonorrhoeae in addition to these "laboratory strains". Among the major
gonococcal
surface antigens, we know that FA1090, MS11, and FA19 differ in their porin
(PorB)
molecules. FA1090 and MS11 possess different subtypes of PorB.1B, and FA19 has
PorB.1A
(Elkins et al., Mol. Microbiol. 14, 1059-1075 (1994)). Although not as well
characterized, the
Opa proteins encoded in their genomes differ (Hobbs et al., Front. Microbiol.
2, 123 (2011),
Cole et al., PLoS One 4, e8108 (2009), and their LOS are different (Erwin et
al., I Exp. Med.
184, 1233-1241 (1996)). Opa proteins and LOS glycan chains are also phase-
variable,
resulting in the expression of different antigenic epitopes (Apicella, M.A. et
at. Phenotypic
variation in epitope expression of the Neisseria gonorrhoeae
lipooligosaccharide. Infect.
.. Immun. 55, 1755-1761 (1987)).
[0110] Consistent with cross-protective immunity, ELISA analysis of
antibodies
induced by immunization revealed quantitatively similar levels of antibodies
detectable
against the different strains, with respect to both IgG and IgA in serum and
vaginal washes.
The antibodies appeared to be specific for N. gonorrhoeae as they were not
detected against
E. coil or NTHI, and they were not generated by immunization with OMV prepared
from
NTHI. Western blot analysis of serum IgG antibodies, however, revealed
evidence of
antigens shared between different strains of N. gonorrhoeae. Bands migrating
at 45-65kDa
migrated at higher molecular mass than major gonococcal outer membrane
proteins such as
porin and Opa which are in the range 30-40kDa.
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[0111] Our studies have identified two novel gonococcal vaccine
candidates, EF-Tu
in FA1090, MS11, and FA19 OMV, and PotF3 also in FA1090. Both proteins have
been
identified in quantitative proteomic profiling of cell envelopes and OMV
derived from four
common gonococcal isolates (Zielke et al., Molec. Cell. Proteomics 13, 1299-
1317 (2014)).
EF-Tu is of particular interest as it has been identified in both spots and in
all analyzed OMV.
EF-Tu is commonly perceived as a cytosolic GTP-binding protein and an
essential factor in
protein synthesis.
[0112] The present disclosure provides demonstration that individuals
can be
immunized against N. gonorrhoeae by the i.vag. administration of a non-living
vaccine
(OMV) with a Thl-driving adjuvant, IL-12/ms. These findings demonstrate the
feasibility of
a vaccine against N. gonorrhoeae despite previous setbacks and these findings
also shed light
on the type of immune responses that needs to be induced to generate
protective immunity.
METHODS
[0113] Mice. All mice, including wild-type BALB/c and C57BL/6 mice,
B6.129S7-
tinen-v.1 (iir-rivy -
deficient), B6.129S2-/ghmtmickna (B cell-deficient; also known as MT),
B6.129S2-Cd4tmlmaka (CD4-deficient), and B6.129S2-Cd8atml'IJ (CD8-deficient)
mice on
a C57BL/6 background, were purchased from Jackson Laboratories (Bar Harbor,
ME).
BALB/c mice were used for the experiments unless otherwise specified. Mice
were
maintained in a BSL2 facility in the Laboratory Animal Facility at the
University at Buffalo,
which is fully accredited by AAALAC. All animal use protocols were approved by
the
Institutional Animal Care and Use Committee of the University at Buffalo.
[0114] Bacteria. N. gonorrhoeae strain FA1090 was provided by Dr
Janne
Cannon (University of North Carolina at Chapel Hill); strain MS11 was provided
by Dr
Daniel Stein (University of Maryland); strain FA19, and clinical isolates were
obtained from
the collection of clinical strains maintained at the University of North
Carolina at Chapel
Hill. For use in the murine infection model, N. gonorrhoeae strains 9087 and
0336 were
transformed with the streptomycin-resistant rpsL gene from strain FA1090 to
generate strains
GC68 and GC69, respectively. E. colt K12 was provided by Dr Terry Connell
(University at
Buffalo). Non-typeable Haemophilus influenzae (NTHI) strain 6P24H1 was
provided by Dr
Timothy Murphy (University at Buffalo). N. gonorrhoeae was cultured on GC agar
supplemented with hemoglobin and ISOVITALEX , an enrichment medium (BD
Diagnostic
Systems, Franklin Lakes, NJ) and the resultant growth was checked for colony
morphology
consistent with Opa protein and pilus expression. NTHI was cultured on GC agar
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supplemented with hemoglobin only. E. coil was cultured on BHI agar. Bacteria
were
harvested from plates and the cell density was determined (Liu et al., Mucosal
Immunol. 5,
320-331 (2012)).
[0115] IL-12 microspheres. Murine IL-12 (Wyeth, Philadelphia, PA) was
encapsulated into poly-lactic acid microspheres using the Phase Inversion
Nanoencapsulation
technology as previously described except that bovine serum albumin was
replaced by
sucrose (0.1 %, w/w) (Egilmez et al., Methods Mol. Med. 75, 687-696 (2003)).
Blank
microspheres were prepared in the same way but without IL-12.
[0116] Gonococcal outer membrane vesicles (OMV). After 18-22 h
culture on
supplemented GC agar, N. gonorrhoeae was harvested from plates into ice-cold
lithium
acetate buffer (pH 5.8) and passed through a 25-gauge needle 10-12 times to
sheer the outer
membranes from the bacteria. The suspensions were spun in microfuge tubes at
13,000 RPM
for 1 min. The supernatants were collected and ultracentrifuged at 107,000xg
for 2 h. The
pellet was washed with 50 mM Tris-HC1 (pH 8.0) and resuspended in PBS. Protein
was
.. assayed with the Micro BCA protein kit (Thermo Scientific, Rockford, IL) or
RC DC Protein
Assay kit (Bio-Rad, Hercules, CA).
[0117] Immunization schedule and mouse vaginal infection model.
Groups of 8
female mice between 7 and 9 weeks old were immunized i.vag. with gonococcal
OMV (40 g
protein) of various strains as described, plus IL-12/ms (1 g IL-12) or blank
ms in a total
volume of 40 1 PBS; control groups were sham-immunized with IL-12/ms or with
blank ms
alone. Mice were immunized 1 to 3 times with a 7-14 day interval, as
indicated. After a
further 2 weeks to 6 months, immunized mice were infected with 5 x 106 CFU
live N.
gonorrhoeae as previously described (Jerse, Infect. Immun. 67, 5699-5708
(1999; Liu et al.,
Infect. Dis. 208, 1821-1829 (2013)), with the modification that 0.5 mg
Premarin (Pfizer,
Philadelphia, PA) was used as estradiol administered s.c. on days ¨2, 0, and
2. Vaginal swabs
collected daily were quantitatively cultured on GC agar supplemented with
hemoglobin,
ISOVITALEX (an enrichment medium) and selective antibiotics (vancomycin,
streptomycin, nisin, colistin, and trimethoprim) to determine the bacterial
colonization loads.
The limit of detection was 100 colony-forming units (CFU) recovered per mouse.
Gonococcal recovery was counted by an individual who was "blinded" to the
experimental
treatments, and all experiments were repeated 2 or 3 times for verification.
[0118] Assay of serum and mucosal antibodies. Samples of vaginal wash
and
serum were collected from the mice at the indicated time points (Liu et al.,
mBio 2: (2011).
Gonococcus-specific IgA, IgG, IgM, and IgG subclass antibodies IgGl, IgG2a,
IgG2b, and
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IgG3 in vaginal washes and sera were measured by ELISA on plates coated with
whole
gonococci, using undiluted vaginal wash and 10-fold diluted serum as starting
dilutions. 17,26
Total IgA, IgG, and IgM concentrations in secretions were assayed by ELISA on
plates
coated with anti-IgA, -IgG, or -IgM antibodies (Southern Biotech, Birmingham,
AL). H5
mouse monoclonal antibody (specific for N. gonorrhoeae porin serovar PIB3) or
affinity-
purified mouse IgA, IgG, and IgM (Southern Biotech) were used to establish
standard curves.
Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-
mouse IgA,
IgG, IgM, IgGl, IgG2a, IgG2b, or IgG3 antibody (Southern Biotech) and p-
nitrophenylphosphate substrate (Southern Biotech). Plates were read in a
VersaMax
microplate reader with SoftMax software (Molecular Devices, Sunnyvale, CA) or
an ELx800
Universal microplate reader with KC Junior software (Bio-Tek Instruments,
Winooski, VT).
Antibody data were expressed as relative (fold increase) to the antibody
levels detected in
control samples (from sham-immunized mice) assayed simultaneously.
[0119] Flow cytometry. Isolated cells were washed with staining
buffer twice, then
incubated with the indicated antibodies for 30 min on ice, washed, and
analyzed on a
FACSCalibur cytometer. For intracellular staining, cells were first fixed with
CYTOFIX/CYTOPERM (eBioscience). Antibodies to mouse CD4, CD8, IFNy, IL-4, and
IL-17A conjugated with FITC, PE, or allophycocyanin were purchased from
eBioscience.
[0120] Lymphocyte isolation and culture. Mononuclear cells were
isolated from
aseptically harvested ILN using Histopaque 1083 (Sigma-Aldrich, St Louis, MO)
density
gradient centrifugation and pooled from 2 or 3 mice to provide sufficient
numbers of cells for
culture. CD4 + T cells were purified through negative selection using a Dynal
CD4 cell
isolation kit (Invitrogen, Carlsbad, CA). Cells were cultured in 24-well
culture plates at a
density of 2 x 106 cells/ml in the presence of equal numbers of mitomycin C-
inactivated
.. spleen cells to serve as APC, either with no stimulus or with 2 x 107 N.
gonorrhoeae cells.
[0121] Proliferation assays. Cells were labeled with carboxymethyl
fluorescein
succinimide ester (CFSE; Sigma-Aldrich). CFSE-labeled cells were then washed
twice in
PBS, recounted, and stimulated as described above. Cultured cells were
harvested and then
stained with allophycocyanin-conjugated anti-mouse CD4 antibody. The data were
acquired
by gating on the CD4 + cell populations in a FACSCalibur cytometer. The
attenuation of
CF SE fluorescence was used to measure cell proliferation.
[0122] Cytokine ELISA. IFNy, IL-4, and IL-17A levels were measured in
triplicate
using ELISA kits purchased from eBioscience.
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[0123] Real-time RT-PCR. Total cellular RNA of whole vaginas
harvested from the
mice was isolated with RNEASY RNA purification Mini Kits (Qiagen, Valencia,
CA), and
was transcribed to cDNA using the 'SCRIPT' cDNA synthesis kit (Bio-Rad,
Hercules, CA).
Real-time RT-PCR was performed on an ICYCLER IQ detection system (Bio-Rad)
using
SYBR Green Dye (Bio-Rad) for real-time monitoring of the PCR. The primers
used were as
follows: IFNy, 5'-TACTGCCACGGCACAGTCATTGAA-3'(SEQ ID NO: 1), 5'-
GCAGCGACTCCTTTTCCGCTTCCT-3' (SEQ ID NO: 2); IL-4, 5'-
GAAGCCCTACAGACGAGCTCA-3'(SEQ ID NO: 3), 5'-
ACAGGAGAAGGGACGCCAT-3' (SEQ ID NO: 4); IL-17A, 5'-
TCAGGGTCGAGAAGATGCTG-3' (SEQ ID NO: 5), 5'-TTTTCATTGTGGAGGGCAGA-
3' (SEQ ID NO: 6); I3-actin, 5'-CCTAAGGCCAACCGTGAAAAG-3' (SEQ ID NO: 7), 5'-
GAGGCATACAGGGACAGCACA-3' (SEQ ID NO: 8). Relative quantification of target
genes was analyzed based on the threshold cycle (Ct) determined by Bio-Rad IQ
5 optical
system software.
[0124] Western Blot. N. gonorrhoeae OMV preparations were boiled for 5 min
in
sodium dodecyl sulfate (SDS) loading buffer containing 2-mercaptoethanol.
Protein
quantification was done with the RC DC Protein Assay kit. Ten micrograms of
protein from
each sample was separated on 10% polyacrylamide SDS electrophoresis gels.
Protein bands
were transferred onto nitrocellulose membranes using the electrophoresis
transfer system
(Bio-Rad, Hercules, CA, USA). The nitrocellulose membranes were blocked with
PBS
containing 3% skim milk overnight at 4 C before incubation for 2 h with serum
samples
diluted 1:200, or vaginal wash samples diluted 1:20 in PBS containing 3% skim
milk.
Specific antibodies bound to N. gonorrhoeae OMV preparations were detected
with
horseradish peroxidase-conjugated goat anti-mouse-IgG (Santa Cruz
Biotechnology, Paso
Robles, CA) at a dilution of 1:4000. The Pierce detection kit was used for
chemiluminescent
detection and images were collected with a ChemiDoc MP imaging system (Bio-
Rad).
[0125] Immunoproteomics. Protein concentration in OMV was measured
using DC
Protein Assay Kit (Bio-Rad). Samples of OMV [300 g and 50 g of protein, for
two
dimensional (2D) SDS-PAGE-MS/MS analysis and immunoblotting, respectively]
were
precipitated overnight in 90% acetone, washed twice with 100% ice-cold acetone
and air-
dried. Protein pellets were reconstituted in 2004, of rehydration buffer (7M
urea, 2M
thiourea, 2% CHAPS, 2% ASB-14, 1% DTT, 2mM TBP, 2% 3-10 IPG buffer, trace of
Orange G) and used to rehydrate pH 4-7 ReadyStrip IPG strips (Bio-Rad)
overnight at 25 C.
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Isoelectric focusing was carried out using the PROTEAN i12TM IEF System (Bio-
Rad) for a
total of 26,000Vh with the following settings: 50 A current limit, 8000V rapid
ramp for
26,000Vh, 750V hold. The second dimension (2D) SDS-PAGE was performed using
Criterion TGX Any kD gels (Bio-Rad). The proteins were stained overnight in
Flamingo
fluorescent stain (Bio-Rad) and the spots were visualized using the ChemiDoc
Imaging
System (Bio-Rad). For immunoblotting, separated proteins were transferred onto
PVDF
membranes using the TurboBlott transfer system (Bio-Rad). The membranes were
blocked
for 2h in 5% milk in PBS Tween, and probed by overnight incubation with sera
from
immunized mice, followed by incubation with anti-mouse HRP-conjugated
antibodies (Bio-
Rad). Spots were visualized using Clarity Western ECL Substrate and ChemiDoc
MP
Imaging System (Bio-Rad). Proteins on membranes were stained with Novex
Reversible
Membrane Protein Stain (InVitrogen) to overlay positions of selected "anchor"
spots with the
Flamingo-stained 2D gels. Matching spots were excised and the proteins were
trypsin
digested. Samples containing extracted peptides were desalted using ZipTip C18
(Millipore,
Billerica, MA) and eluted with 70% acetonitrile/0.1% TFA, and dried in a speed
vac.
Desalted peptides were brought up in 2% acetonitrile in 0.1% formic acid
(20pL) and
analyzed (20_,) by LC/ESI MS/MS with a Thermo Scientific Easy-nLC II (Thermo
Scientific, Waltham, MA) nano HPLC system coupled to a hybrid Orbitrap Elite
ETD
(Thermo Scientific) mass spectrometer. In-line de-salting was accomplished
using a reversed-
phase trap column (100pm x 20mm) packed with Magic C18AQ (5-pm 200A resin;
Michrom
Bioresources, Auburn, CA) followed by peptide separations on a reversed-phase
column
(75pm x 250mm) packed with Magic C18AQ (5-pm 100A resin; Michrom Bioresources)
directly mounted on the electrospray ion source. A 30-minute gradient from 7%
to 35%
acetonitrile in 0.1% formic acid at a flow rate of 400nL/min was used for
chromatographic
separations. The heated capillary temperature was set to 300 C and a spray
voltage of 2750V
was applied to the electrospray tip. The Orbitrap Elite instrument was
operated in the data-
dependent mode, switching automatically between MS survey scans in the
Orbitrap (AGC
target value 1,000,000, resolution 240,000, and injection time 250
milliseconds) with MS/MS
spectra acquisition in the linear ion trap (AGC target value of 10,000, and
injection time
100msec). The 20 most intense ions from the Fourier-transform (FT) full scan
were selected
for fragmentation in the linear trap by collision-induced dissociation with
normalized
collision energy of 35%. Selected ions were dynamically excluded for 15sec
with a list size
of 500 and exclusion mass by mass width 0.5. Data analysis was performed
using Proteome
Discoverer 1.4 (Thermo Scientific). All identified peptides were searched
against a N.
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gonorrhoeae database (FA1090, FA19, and MS11) combined with cRAP.fasta, a
database of
common contaminants (thegpm.org/crap/); this creates a list of proteins
commonly found in
proteomics experiments that are present by accident or unavoidable
contamination. Trypsin
was set as the enzyme with maximum missed cleavages set to 2. The precursor
ion tolerance
was set to 10 ppm and the fragment ion tolerance was set to 0.8 Da. Variable
modifications
included oxidation on methionine (+15.995 Da) and carbamidomethyl on cysteine
(+57.021
Da). Data were searched using Sequest HT. All search results were run through
Percolator for
scoring
[0126] Statistical analysis. Data are expressed as the mean
standard error of the
mean (SEM). Data on the effect of immunization on recovery of N. gonorrhoeae
after
inoculation were analyzed using two-way ANOVA for repeated measures with
Fisher's
protected least significant difference post-hoc tests. In addition, Kaplan-
Meier analysis with
log-rank tests was used to compare clearance of infection (defined as the
first of 3 successive
days of zero recovery) between treatment groups. For immune response data,
unpaired two-
tailed t tests were used to compare the mean values between two groups, or
ANOVA with
Bonferroni post-hoc tests was used for multiple comparisons. P <0.05 was
considered
statistically significant. Statistical analyses were performed using Microsoft
Excel or Prism 5
(GraphPad Software, San Diego, CA).
EXAMPLE 4
[0127] This examples describes the administration of OMVs and IL-12 ms
intranasally. Female mice (8 per group) were immunized intranasally with OMV
(4011g
protein, strain FA19) plus IL-12/ms (111g IL-12) or blank ms on days 0 and 14.
Two weeks
later all mice were challenged intravaginally with 5 x 106 CFU of N.
gonorrhoeae strain
FA1090. Vaginal swabs collected daily were diluted and cultured quantitatively
on GC agar
plates containing selective antibiotics. Results show that mice immunized with
OMVs plus
IL-12 ms cleared the infection significantly faster than the control groups (p
< 0.01, Fig. 21).
In addition, the bacterial colonization loads were significantly lower in the
mice immunized
with OMVs plus IL-12 ms.
EXAMPLE 5
[0128] Studies were carried out to further illustrate protective effect of
compositions
comprising OMV and IL-12 microspheres delivered via the intranasal route and
comparison
of the intranasal route with the intravaginal route.
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[0129] Mice were immunized intranasally (i.n.) with different doses
of OMV
prepared from N. gonorrhoeae strain FA1090: 1511g, 3011g, or 6011g of OMV
protein,
together with microencapsulated IL-12 (IL-12/ms; 1tg of IL-12), or with IL-
12/ms alone.
Immunizations were repeated 2 weeks later. Two weeks after the last dose, mice
were
challenged intravaginally with live N. gonorrhoeae strain FA19, and the course
of infection
was followed by daily vaginal swabbing and plating as described previously. As
shown in Fig
22, mice immunized with the low dose of 1511g OMV did not show accelerated
clearance of
the infection compared to the control group immunized with IL-12/ms alone
(median
clearance times: 10 days for both groups). Mice immunized with the usual (mid)
dose 3011g
or with an increased dose of 6011g OMV cleared the infection significantly
faster (median
clearance times 6 days and 7.5 days, respectively) than the control group
(P<0.001, Kaplan-
Meier, log-rank test).
[0130] Intranasal (i.n.) immunization was compared with intravaginal
(i.vag.)
immunization as follows. Mice were immunized with 3011g of gonococcal FA1090
OMV (as
protein) plus IL-12/ms (111g IL12) or blank microspheres (ms) either i.n. or
i.vag. and
immunizations were repeated after 2 weeks. Two weeks later, mice were
challenged i.vag.
with live N. gonorrhoeae strain FA19 (heterologous challenge), and the course
of infection
was followed by daily vaginal swabbing and plating as described previously. As
shown in Fig
23A-D, mice immunized with OMV plus IL-12/ms i.n. cleared the infection
comparably to
mice immunized i.vag. After clearance of the infections (2 weeks after
challenge) samples of
serum, vaginal wash, and saliva were collected for assay of IgG and IgA anti-
gonococcal
antibodies by ELISA. Mice immunized i.n. with IL-12/ms cleared the infection
comparably
to those immunized i.vag. with the same vaccine (Fig 23A). Mice immunized i.n.
with OMV
plus IL-12/ms developed IgG and IgA antibodies in serum (Fig 23B) and vaginal
wash (Fig
23C) at levels higher than those immunized i.vag. Mice immunized i.n. with OMV
plus IL-
12/ms also developed high levels of salivary IgA antibodies (Fig 23D).
[0131] When mice were challenged with the same strain as used for the
preparation of
the OMV, FA1090 (homologous challenge), mice immunized with OMV plus IL-12/ms
i.n.
(as described for Figure 3), cleared the infection comparably to those
immunized i.vag. (Fig
24).
[0132] Mice were immunized i.n. once or twice with gonococcal FA1090
OMV
(3011g protein) plus IL-12/ms (111g IL-12); control mice received blank ms.
Two weeks after
the second immunization, mice were challenged i.vag. with N. gonorrhoeae MS11
(heterologous challenge). As shown in Fig 25, mice immunized twice with OMV
plus IL-
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12/ms cleared the infection faster (median clearance time: 4 days) than those
immunized with
one dose (median clearance time 9 days) or than control mice (median clearance
time 7
days); P=0.0155 (Kaplan-Meier analysis, log-rank test).
[0133] Mice were immunized i.n. with OMV (3011g protein) prepared
from N.
gonorrhoeae MS11, plus either IL-12/ms (111g IL-12) or blank ms, and
challenged with
heterologous gonococcal strain FA1090. As shown in Fig 26, mice immunized with
MS11
OMV plus IL-12/ms cleared the infection faster (median clearance time: 7 days)
compared to
those immunized with MS11 OMV plus blank ms (median clearance times: 11 days),
or
compared to unimmunized control mice (median clearance time: 12 days);
P=0.0231
.. (Kaplan-Meier analysis, log-rank test).
[0134] Mice were immunized i.n. with OMV (3011g protein) prepared
from N
gonorrhoeae MS11, plus either IL-12/ms (111g IL-12) or blank ms, and
challenged with
heterologous gonococcal strain FA19. As shown in Fig 27, mice immunized with
MS11
OMV plus IL-12/ms cleared the infection faster (median clearance time: 5 days)
compared to
those immunized with MS11 OMV plus blank ms (median clearance time: 9 days),
or
compared to unimmunized control mice (median clearance time: 10 days).
P=0.0007
(Kaplan-Meier analysis, log-rank test)
[0135] It should be noted that the strains of N gonorrhoeae used in
these experiments
express different porin types. Gonococcal porin PorB is the major outer
membrane protein in
N gonorrhoeae, and is represented by two major types, PorB-1A and PorB-1B,
each having
many sub-types. Strain FA1090 has porin type PorB-1B, strain MS11 also has
porin type
PorB-1B although a different sub-type from FA1090, and strain FA19 has porin
type PorB-
1A. In addition, these strains express different Opa proteins and
lipooligosaccharide
structures, thus they represent widely different antigenic variants of N
gonorrhoeae.
[0136] Although the present invention has been described with respect
to one or more
particular embodiments, it will be understood that other embodiments of the
present
invention may be made without departing from the spirit and scope of the
present invention.
43
