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M A J O R A R T I C L E

Enhancement of Adaptive Immunity to


Neisseria gonorrhoeae by Local Intravaginal
Administration of Microencapsulated
Interleukin 12
Yingru Liu, Nejat K. Egilmez, and Michael W. Russell
Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, New York
Gonorrhea remains one of the most frequent infectious diseases, and Neisseria gonorrhoeae is emerging as re-
sistant to most available antibiotics, yet it does not induce a state of specic protective immunity against rein-
fection. Our recent studies have demonstrated that N. gonorrhoeae proactively suppresses host T-helper (Th) 1/
Th2-mediated adaptive immune responses, which can be manipulated to generate protective immunity. Here
we show that intravaginally administered interleukin 12 (IL-12) encapsulated in sustained-release polymer mi-
crospheres signicantly enhanced both Th1 and humoral immune responses in a mouse model of genital gono-
coccal infection. Treatment of mice with IL-12 microspheres during gonococcal challenge led to faster
clearance of infection and induced resistance to reinfection, with the generation of gonococcus-specic circulat-
ing immunoglobulin G and vaginal immunoglobulin A and G antibodies. These results suggest that local ad-
ministration of microencapsulated IL-12 can serve as a novel therapeutic and prophylactic strategy against
gonorrhea, with implications for the development of an effective vaccine.
Keywords. Neisseria gonorrhoeae; immunity; cytokine therapy; IL-12; microencapsulation.
Infection with gonorrhea does not induce a state of
specic protective immunity and as a result it can be
acquired repeatedly [1, 2]. The World Health Organiza-
tion estimates that >100 million new gonococcal infec-
tions occur worldwide every year [3]. No vaccine is
available against gonorrhea, and therapy depends upon
antibiotics [4]. However, antibiotic resistance of N. gon-
orrhoeae continues to emerge and is becoming prob-
lematic for effective treatment [5].
The mechanisms underlying the paucity of protec-
tive immune responses against Neisseria gonorrhoeae
infection remain elusive. It is generally believed that the
extensive and rapid antigenic variability of the gono-
coccus and its capacity to resist complement-mediated
bacteriolysis result inimmune evasion[1, 68]. However,
our recent studies using experimental murine infections
have demonstrated that N. gonorrhoeae proactively sup-
presses host T-helper (Th) 1 and Th2-mediated specic
immune responses [912]. Concomitantly, gonococcal
infection elicits Th17 responses, which recruit innate
defense mechanisms including the characteristic neu-
trophil inux, and interference with interleukin 17 (IL-
17) signaling prolongs the infection [13]. Induction of
transforming growth factor (TGF) and interleukin 10
(IL-10) is critically involved in the suppression of adap-
tive immunity by N. gonorrhoeae, and systemic treat-
ment with antiTGF- or/and antiIL-10 antibodies
reverses the natural capacity of N. gonorrhoeae to
inhibit Th1 and Th2 responses, allowing the develop-
ment of protective immune responses that not only ac-
celerate clearance of an existing infection but also
generate resistance to future infection [1012].
Received 29 January 2013; accepted 21 May 2013; electronically published 18
September 2013.
Presented in part: 18th International Pathogenic Neisseria Conference, Wrz-
burg, Germany, 914 September 2012.
Correspondence: Yingru Liu, MD, PhD, Department of Microbiology and Immu-
nology, Farber 138, University at Buffalo, 3435 Main St, Buffalo, NY 14214
(yingruli@buffalo.edu; yingru_ubc@yahoo.com).
The Journal of Infectious Diseases 2013;208:18219
The Author 2013. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
journals.permissions@oup.com.
DOI: 10.1093/infdis/jit354
Immunity to N. gonorrhoeae JID 2013:208 (1 December) 1821

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Interleukin 12 (IL-12) is a potent proinammatory cytokine
that strongly stimulates Th1-associated cellular immunity [14].
Some studies have shown that IL-12 also activates humoral im-
munity through both T-celldependent and T-cellindependent
mechanisms, and enhances immunoglobulin A (IgA) and im-
munoglobulin G (IgG) antibody responses to foreign proteins
and pathogens [1517]. It has therefore been exploited as an an-
titumor therapeutic as well as a vaccine adjuvant for protection
against bacterial and viral infectious diseases [1821]. However,
therapeutic use of soluble IL-12 requires repeated parenteral ad-
ministration in large doses resulting in systemic toxicity and
limited efcacy [22, 23]. To avoid this problem, we have used
biodegradable polymeric microparticles for the local and sus-
tained delivery of cytokines directly to the cancer microenviron-
ment [24]. Others have demonstrated that local administration
of similar formulations leads to effective particle uptake and
release of encapsulated therapeutics in the mucosa [25, 26].
In the current study, we have assessed the effect of local in-
travaginal treatment with microencapsulated IL-12 in a murine
gonococcal infection model and found that IL-12 microspheres
dramatically accelerated the clearance of infection. The treat-
ment was associated with signicant enhancement of Th1 cel-
lular and specic antibody responses, and it induced protective
immunity against reinfection. We have further found similar
therapeutic effects when mice were treated with vaginal instilla-
tion of low doses of antiTGF- or antiIL-10 antibodies en-
capsulated in sustained-release microspheres.
MATERIALS ANDMETHODS
Mice
BALB/c mice were purchased from Jackson Laboratories and
were maintained under standard conditions in the Laboratory
Animal Facility at the University at Buffalo. All animal use pro-
tocols were approved by the Institutional Animal Care and Use
Committee of the University at Buffalo.
Bacteria
Neisseria gonorrhoeae FA1090 was kindly provided by Janne
Cannon, PhD, (University of North Carolina at Chapel Hill)
and was cultured on GC agar supplemented with hemoglobin
and Isovitalex (BD Diagnostic Systems). 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 as detailed elsewhere [11]. Opa ex-
pression, as determined previously [11], was Opa A, B/D/G,
and E/K.
Microspheres
Cytokines and antibodies were encapsulated into polylactic
acid (PLA) microspheres using the phase inversion nanoencap-
sulation technology as described elsewhere, except that bovine
serum albumin was replaced by sucrose (0.1%, wt/wt) [27]. Five
formulations were produced: (1) control microspheres containing
no cytokine or antibody, (2) murine IL-12 (0.25-g/mg parti-
cles), (3) murine IL-17 (0.25-g/mg particles), (4) murine anti-
mouse TGF- (10-g/mg particles), and (5) rat anti-mouse
IL-10 (10-g/mg particles).
Mouse Vaginal Infection Model
Female mice between 7 and 9 weeks old were infected vaginally
on day 0 with live N. gonorrhoeae FA1090, as described else-
where [10, 28]. Vaginal mucus was quantitatively cultured daily
on GC agar supplemented with selective antibiotics to deter-
mine the bacterial colonization loads. The limit of detection
was 100 colony-forming units recovered per mouse. Intravagi-
nal treatments with microsphere preparations were given every
second day from day 0 to day 8, by instillation of 40-L suspen-
sions in phosphate-buffered saline of microspheres containing
IL-12, IL-17, antiTGF-, antiIL-10, or control microspheres.
Cell Isolation and Flow Cytometry
Mice were killed, and their iliac lymph nodes (ILNs) and
genital tracts were excised aseptically. The ILNs were teased in
Hanks buffered salt solution to release cells. Vaginal single-cell
suspensions were prepared by enzymatic digestion, as described
elsewhere [10, 29]. Isolated cells were washed with staining
buffer twice, then incubated with the indicated antibodies for
30 minutes on ice, washed twice, and analyzed on a FACS
Calibur cytometer. For determination of intracellular cytokine
expression, cells were restimulated with phorbol myristate
acetateionomycinGolgiStop (eBioscience) for 5 hours, and
then xed with Cytox/Cytoperm (eBioscience). Antibodies to
mouse CD4, CD8, CD19, CD11b, CD11c, NKG2D, Gr-1, inter-
feron (IFN) , interleukin 4 (IL-4), and IL-17A conjugated with
uorescein isothiocyanate, phycoerythrin, or allophycocyanin
were purchased from eBioscience.
Cytokine Enzyme-Linked Immunosorbent Assay
Levels of IL-12p70, IFN-, IL-4, interleukin 5, and IL-17A in
serum or vaginal wash samples were measured in triplicate
using enzyme-linked immunosorbent assay (ELISA) kits pur-
chased from eBioscience.
Real-Time Reverse-Transcription Polymerase Chain Reaction
Total cellular RNA of whole vaginas harvested from the mice
was isolated with RNeasy Mini Kits (Qiagen) and transcribed
to complementary DNA (cDNA) using the iScript cDNA syn-
thesis kit (Bio-Rad). Real-time reverse-transcription polymer-
ase chain reaction (RT-PCR) was performed on an iCycler iQ
detection system (Bio-Rad) using Sybergreen (Bio-Rad) for
real-time monitoring of the PCR. The sequences of IFN-, IL-4,
IL-17A, and -actin primers used were same as described else-
where [12]. Relative quantication of target genes was analyzed
1822 JID 2013:208 (1 December) Liu et al

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based on the threshold cycle determined by Bio-Rad iQ5
optical system software.
Assay of Serum and Mucosal Antibodies
Saliva, vaginal wash, and serum samples were collected from
individual mice on day 15 after inoculation. Gonococcus-
specic IgA, IgG, and immunoglobulin M (IgM) in saliva,
serum, and vaginal wash samples and total IgA, IgG, and IgM
concentrations in secretions were assayed by ELISA, as detailed
elsewhere [10].
Statistical Analysis
Data are expressed as means standard errors of the mean.
Data on the effects of IL-12, IL-17, antiTGF-, and
antiIL-10loaded vs blank microsphere treatments on vaginal
N. gonorrhoeae infection were analyzed using repeated-measures
analysis of variance with Bonferroni corrected post hoc testing of
pairwise comparisons. Kaplan-Meier analysis with log-rank
testing was also used to compare infection clearance. Data from
in vitro experiments were analyzed with unpaired 2-tailed t tests
to compare the mean values between 2 selected groups. Differ-
ences were considered statistically signicant at P < .05.
RESULTS
Intravaginal Administration of IL-12 Microspheres as
Protection Against Genital Tract N. gonorrhoeae Infection in
Mice
To examine the therapeutic effect of IL-12loaded micro-
spheres, 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 contain-
ing 1.0 g of IL-12 every second day was sufcient to accelerate
clearance of the infection relative to treatment with blank mi-
crospheres; no further enhancement of clearance was obtained
with 2.0 g of microencapsulated IL-12, and lower doses were
progressively less effective (Figure 1A).
Untreated or blank microsphere-treated mice cleared the
infection in approximately 15 days (Figure 1B and 1C). Intrava-
ginal instillation of microencapsulated IL-12 at the optimal 1.0-
g dose signicantly reduced the recoverable N. gonorrhoeae
load starting from day 4, and these mice cleared the infection
by day 7, or 8 days earlier than blank microsphere-treated or
untreated mice (Figure 1B and 1C). 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 (Figure 1B and 1C).
Our previous studies in the same murine model had demon-
strated that N. gonorrhoeae mainly elicits Th17 responses,
which are involved in the recruitment of host innate defense
mechanisms, including the inux of neutrophils to the genital
tract [13]. We therefore investigated whether local IL-17 treat-
ment could also promote the clearance of gonococcal infection.
Intravaginal administration of IL-17loaded microspheres at
the optimal dose (1.0 g) also accelerated clearance of N. gonor-
rhoeae infection but to a lesser extent than IL-12 microspheres
given on the same schedule (Figure 1B and 1C).
Effects of IL-12 Microsphere Treatment on Th1 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-12loaded
or blank microspheres. Single-cell suspensions were prepared
from ILNs 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 ow cytometry to detect intracellular IFN-, IL-4,
and IL-17. Starting on day 3 after inoculation, IL-17
+
/CD4
+
T cells were observed in the local draining ILNs, with produc-
tion peaking at day 5 and continuing for the duration of infec-
tion. At day 5, approximately 22% of CD4
+
T cells present in
the ILNs of control-treated infected mice were IL-17
+
, whereas
only approximately 3.5% were IFN-
+
and few IL-4
+
/CD4
+
Tcells were detected(Figure 1D). The IL-12microsphere treatment
markedly enhanced Th1 immune responses to N. gonorrhoeae,
indicated by signicantly increased numbers of IFN-
+
/CD4
+
T cells (Figure 1D). In contrast, IL-12 microspheres did not
change Th2 or Th17 responses, because the numbers of IL-4
+
/
CD4
+
and IL-17
+
/CD4
+
T cells in ILNs were similar between
the treated groups (Figure 1D). The RT-PCR analyses showed
that IFN- but not IL-4 or IL-17 messenger RNA expression
was elevated in the vaginas of infected mice after IL-12 micro-
sphere treatment (Figure 1E). Although IL-17 microspheres
ameliorated gonococcal infection, this treatment was not asso-
ciated with enhanced Th1 or Th2 responses (Figure 1D and
1E), but there was increased inux of Gr-1
+
neutrophils into
the genital tract (Figure 1F).
We also used ELISA to measure IL-12p70, IFN-, IL-4, and
IL-17 concentrations in vaginal wash and serum samples col-
lected 7 days after inoculation. We detected IL-12 (176.5 48.6
pg/mL) in vaginal wash samples from infected mice treated
with IL-12 microspheres. Low levels of IL-12 (41.7 10.7 pg/mL)
were found in the serum samples from these mice, suggesting
that the effects of IL-12 microsphere treatment on gonococcal
infection did not result primarily from the passage of the cyto-
kine into the circulation. Consistent with the ow cytometric
studies, IFN- was present in the vaginal wash (32.6 9.8 pg/
mL) and serum (43.3 11.5 pg/mL) samples from 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.
Interleukin 12 can stimulate humoral immune responses in
an IFN-dependent manner or directly [16]. We therefore
Immunity to N. gonorrhoeae JID 2013:208 (1 December) 1823

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determined whether IL-12 microsphere treatment during N.
gonorrhoeae infection led to the production of anti-gonococcal
antibodies in vaginal wash, saliva, and serum samples collected
15 days after inoculation. The IgM antibodies were at low levels
with little difference between experimental groups (data not
shown). No salivary gonococcus-specic antibody was detected in
any group of mice (data not shown). As reported elsewhere [10],
N. gonorrhoeae infection of control-treated mice did not
Figure 1. Effect of intravaginal IL-12 microsphere (ms) treatment on primary gonococcal infection in BALB/c mice. A, interleukin 12 (IL-12) ms dose opti-
mization experiment. Microspheres containing the stated doses of IL-12 were given on days 0, 2, 4, 6, and 8 (8 mice per group). The Neisseria gonorrhoeae
burden was monitored daily by vaginal swab culture. Signicant differences in infection burdens were found between mice treated with 2.0 g (P < .01),
1.0 g (P < .01), or 0.5 g (P < .05) of microencapsulated IL-12 and controls (analysis of variance). B, Time course of infection in mice treated with IL-12 ms,
soluble IL-12, interleukin 17 (IL-17) ms, or control ms or in untreated mice (cytokine dose, 1.0 g given on days 1, 1, 3, 5, and 7; 8 mice per group). Signi-
cant differences in infection burdens were found between mice treated with IL-12 ms (P < .01) or IL-17 ms (P < .02) and controls (analysis of variance). C,
Data from the experiment shown in B, plotted as percentage of mice remaining infected with the indicated cytokine treatments. Infection was cleared sig-
nicantly faster in mice treated with IL-12 ms (P < .001) or IL-17 ms (P < .001) than in controls (Kaplan-Meier analysis). D, Cytokine expression in isolated
iliac lymph node cells from sham-infected or infected mice with IL-12 ms, IL-17 ms, or control ms treatment (7 mice per group). Expression of interferon
(IFN) , interleukin 4 (IL-4), and IL-17 in CD4
+
T cells isolated at day 5 after infection was analyzed with ow cytometry. E, Reverse-transcription polymerase
chain reaction (RT-PCR) analysis of IFN-, IL-4, and IL-17 messenger RNA (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 (7 mice per group). Cytokine gene expression levels detected with RT-PCR were normalized relative
to expression of -actin and set at 1.0 for the sham-infected group. F, Phenotypic prole of vaginal cells isolated on day 5 from sham-infected or infected
mice treated with IL-17 ms or control ms (7 mice per group). G, H, Vaginal (G) and (H) serum anti-gonococcal immunoglobulin A (IgA) and immunoglobulin
G (IgG) antibody responses in sham-infected or infected mice with IL-12 ms, IL-17 ms, or control ms treatment (7 mice per group). Vaginal wash and serum
samples were collected 15 days after inoculation, and gonococcus-specic and total IgA and IgG were measured with enzyme-linked immunosorbent
assay. Results from 1 of 3 independent experiments are shown. Data shown as means SEM.
#
P < .05; *P < .01 (unpaired t test in DH).
1824 JID 2013:208 (1 December) Liu et al

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signicantly elevate gonococcus-specic IgA or IgG antibodies
in either vaginal wash or serum samples. However, IL-12 mi-
crosphere treatment increased vaginal and serum specic IgG
antibody (Figure 1G and 1H), as well as vaginal specic IgA
antibody production (Figure 1G).
IL-12 Microsphere Treatment Induction of Protective
Anamnestic Immunity Against Secondary N. gonorrhoeae
Infection
We further assessed whether IL-12 microsphere treatment re-
sulted in the generation of immune memory and protective
immunity against reinfection. Groups of mice infected with
N. gonorrhoeae were treated with IL-12loaded or blank micro-
spheres, and after the infection had run its course, the mice
were treated with ceftriaxone (300 g intraperitoneal) on day
15 to ensure complete elimination of the gonococci. An addi-
tional group of sham-infected mice treated with IL-12 micro-
spheres was used to evaluate the possible persistent effect of
IL-12 in the absence of infection; 56 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 sec-
ondary gonococcal infection in previously blank microsphere-
treated mice were the same as for the primary infection of
age-matched naive mice (Figure 2A and 2B). In contrast, intra-
vaginal treatment with IL-12loaded microspheres during pri-
mary infection protected the mice against secondary infection;
Figure 2. Effect of intravaginal interleukin 12 (IL-12) microsphere (ms) treatment during primary infection on secondary Neisseria gonorrhoeae infection.
A, Time course of secondary infection in mice treated with IL-12 ms, soluble IL-12, interleukin 17 (IL-17) ms, or control ms during primary infection or in
previously sham-infected mice with or without IL-12 ms treatment (8 mice per group). Signicant differences in infection burdens were found between
mice previously treated with IL-12 ms (P < .02) and controls (analysis of variance). 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 signicantly faster in mice previously
treated with IL-12 ms (P < .001) than in controls (Kaplan-Meier analysis). C, Flow cytometric analysis of cytokine expression in iliac lymph node 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) (7 mice per group). D, Reverse-transcription polymerase chain reaction (RT-PCR) analysis of in-
terferon (IFN) , interleukin 4 (IL-4), and IL-17 messenger RNA (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 (7 mice per group). Cytokine gene expression levels detected by RT-PCR were normal-
ized relative to expression of -actin and set at 1.0 for sham-reinfected group. E, F, Vaginal (E) and (F) serum anti-gonococcal immunoglobulin A (IgA) and
immunoglobulin G (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 (7 mice per group). Vaginal wash and serum samples were collected 15 days after inoculation, and gonococcus-specic and total
IgA and IgG were measured with enzyme-linked immunosorbent assay. Results from 1 of 3 independent experiments are shown. Data shown as means
SEM.
#
P < .05; *P < .01 (unpaired t test in CF).
Immunity to N. gonorrhoeae JID 2013:208 (1 December) 1825

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reinfected mice that had been treated with IL-12 microspheres
during the primary infection resisted the challenge more effec-
tively than controls (Figure 2A and 2B). However, previous IL-
12 microsphere treatment of sham-infected mice did not induce
protection against subsequent infection (Figure 2A and 2B). This
result also excluded the possibility that any persisting micro-
spheres still affected the secondary N. gonorrhoeae infection.
Flow cytometric and RT-PCR analyses of ILN cells and
vaginas performed 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 signicantly enhanced Th1 (IFN-)
responses (Figure 2C and 2D). There was also a robust specic
secondary antibody response in IL-12 microspheretreated
mice after they were rechallenged with N. gonorrhoeae. Gono-
coccus-specic IgA and IgG antibodies in vaginal wash
samples and IgG antibodies in serum samples from reinfected
mice previously treated with IL-12 microspheres were signi-
cantly higher than those of control groups (Figure 2E and 2F).
In contrast to the effects of IL-12 microsphere treatment, treat-
ment with IL-17 microspheres during primary gonococcal in-
fection did not lead to any protective immunity to secondary
gonococcal infection, or induce any anamnestic T-cell or anti-
body responses (Figure 2AF).
Protective Effects of AntiTGF- and AntiIL-10 Microsphere
Treatments
We next ascertained whether the local application of microspheres
could be used to deliver other therapeutic agents for the immuno-
modulatory treatment of vaginal gonococcal infection. We had
previously found that treatment with 300 g of antiTGF- or
antiIL-10 antibody by systemic injection every second day
protected mice against genital gonococcal infection and induced
specic protective immunity [10, 12]. We therefore treated
N. gonorrhoeae-infected mice with PLA microspheres containing
antiTGF- or antiIL-10 antibody by intravaginal instillation.
Treatment with 10-fold lower amounts of antiTGF- or anti
IL-10 antibody (30 g) encapsulated in microspheres achieved a
similar therapeutic effect as observed previously with parenteral
injection of these antibodies (Figure 3A and 3B). No signicant
weight loss (10%) or signs of distress (hunched posture, inactivi-
ty, rufed fur) were observed. In addition, treatment with anti
TGF-- or antiIL-10loaded microspheres during primary gono-
coccal infection induced resistance to secondary infection
(Figure 3D and 3E). Local antiTGF- or antiIL-10 microsphere
treatment was also associated with enhanced Th1 (IFN-) and
Th2 (IL-4) responses in ILNs (Figure 3C and 3F).
DISCUSSION
Although it has long been recognized that gonorrhea can be con-
tracted repeatedly with no apparent development of protective
immunity or immunological memory, the reasons for this are
poorly understood. Our recent studies have demonstrated that
the lack of protective immunity to N. gonorrhoeae is due not
only to immunoevasion through antigenic variation and other
strategies, but also to the ability of the pathogen to suppress
host-specic immune responses in the rst place [9]. Induction
of immunosuppressive cytokines, TGF- and IL-10, and type 1
regulatory T cells play an important role in this process [10
12]. Importantly, our results demonstrate that the natural in-
hibitory effect of N. gonorrhoeae on Th1/Th2-mediated specic
immunity can be immunologically reversed.
The current study showed that intravaginal administration of
microencapsulated IL-12 elicited the development of adaptive
immunity against N. gonorrhoeae genital infection in mice.
Consistent with its well-known type 1stimulatory properties,
IL-12 treatment signicantly enhanced Th1 immune responses
to N. gonorrhoeae, indicated by increased production of IFN-

+
/CD4
+
T cells in both ILNs and the genital tracts of treated
mice compared with those from control mice. The treatment
also signicantly increased the production of gonococcus-
specic vaginal IgA and IgG and serum IgG antibodies, al-
though it did not up-regulate Th2 (IL-4) responses. It has been
reported that IL-12 promotes the production of IgG2a, IgG3
and mucosal IgA antibodies through both IFN-dependent
and direct actions [reviewed in 16]. It initially stimulates Th1
and natural killer cells to secrete large amount of IFN-, which
induces selective isotype switching in B cells and further stimu-
lates antibody production [30]. The IL-12 receptor is expressed
on murine and human B cells through all stages of maturation
[31], and direct interaction of IL-12 with B cells leads to activa-
tion of the p50 and c-Rel NF-B family members, B-cell prolif-
eration and differentiation, and antibody production [3234].
All these mechanisms may contribute to the induction of
humoral immune responses to N. gonorrhoeae infection by
IL-12 microsphere treatment, in the absence of an elevated Th2
(IL-4) response.
Treatment of primary gonococcal infection with IL-12
loaded microspheres also protected the mice against subse-
quent N. gonorrhoeae infection. There were both strong Th1
and specic antibody responses in IL-12 microsphere-treated
mice after they were reexposed to N. gonorrhoeae without
further IL-12 treatment. The levels of gonococcus-specic anti-
bodies in vaginal wash and serum samples from mice that were
reinfected after previous IL-12 microsphere treatment were sig-
nicantly higher than those from the same mice during
primary N. gonorrhoeae infection, suggesting that the treatment
had resulted in the generation of immune memory.
However, intravaginal treatment of mice with the same dose
and schedule of free, soluble IL-12 did not show any protective
effect against either primary or secondary gonococcal infection,
possibly because the cytokine was susceptible to proteolytic
degradation or had limited vaginal mucosal uptake. The potential
1826 JID 2013:208 (1 December) Liu et al

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advantages of local, targeted microencapsulated drug delivery
include reduction of harmful side effects, decreased dosages
needed, and continuous maintenance of drug levels in a thera-
peutically effective range. In our studies the passage of intrava-
ginally delivered microencapsulated IL-12 into the circulation
was minimal, yet the levels achieved in the genital tract were
sufcient to induce acquired immunity to gonococcal infection.
In addition, we determined whether the enhancement of
natural Th17-governed innate immune responses to N. gonor-
rhoeae by local IL-17 microsphere treatment could protect the
mice against gonococcal infection. Consistent with our previ-
ous ndings that antiIL-17 antibody treatment, or deciency
of IL-17RA, leads to prolonged genital tract infection with
N. gonorrhoeae [13], intravaginal instillation of IL-17 micro-
spheres ameliorated primary gonococcal infection. However, it
was less effective than treatment with IL-12 microspheres; fur-
thermore it did not signicantly protect against subsequent in-
fection or induce Th1/Th2 responses. Whereas antiTGF-
treatment interferes with Th17 responses [10, 11], adminis-
tration of IL-12 microspheres enhanced Th1-driven adaptive
responses without affecting IL-17 production and might there-
fore have the advantage of promoting both adaptive and innate
defense against N. gonorrhoeae [35].
We also explored the use of PLA microspheres for intravagi-
nal delivery of other potential therapeutic agents in the treat-
ment of gonococcal infection. We had shown elsewhere that
systemic administration of TGF-- or IL-10-neutralizing anti-
bodies could elicit the generation of Th1/Th2 adaptive immunity
Figure 3. Effect of intravaginal antitransforming growth factor (TGF) antibody or antiinterleukin 10 (IL-10) antibody microsphere (ms) treatment
during primary infection on primary and secondary genital Neisseria gonorrhoeae infection in BALB/c mice. A, Time course of primary infection in mice
treated with antiTGF- ms, antiIL-10 ms, or control ms (8 mice per group). Signicant differences in infection burdens were found between mice treated
with antiTGF- ms (P < .02) or antiIL-10 ms (P < .05) and controls (analysis of variance). B, Data from experiment shown in A plotted as percentage of
mice remaining infected with the indicated treatments. Infection was cleared signicantly faster in mice treated with antiTGF- ms (P < .01) or antiIL-10
ms (P < .01) than in controls (Kaplan-Meier analysis). C, Flow cytometric analysis of cytokine expression in isolated iliac lymph node CD4
+
T cells from
sham-infected or infected mice with antiTGF- antibody ms, antiIL-10 antibody ms, or control ms treatment (7 mice per group). D, Time course of sec-
ondary infection in mice treated with antiTGF- antibody ms, antiIL-10 antibody ms, or control ms during primary infection (8 mice per group). Signicant
differences in infection burdens were found between mice previously treated with antiTGF- ms (P < .02) or antiIL-10 ms (P < .02) and controls (analysis
of variance). E, Data from experiment shown in D plotted as percentage of mice remaining infected after reinfection under the indicated treatments during
primary infection. Infection was cleared signicantly faster in mice previously treated with antiTGF- ms (P < .01) or antiIL-10 ms (P < .001) than in con-
trols (Kaplan-Meier analysis). F, Flow cytometric analysis of cytokine expression in iliac lymph node CD4
+
T cells isolated at day 5 from sham-reinfected or
reinfected mice treated with antiTGF- antibody ms, antiIL-10 antibody ms, or control ms during primary infection (7 mice per group). Results from 1 of
3 independent experiments are shown. Data shown as means SEM. Abbreviations: IFN, interferon; IL-4, interleukin 4.
#
P < .05; *P < .01 (unpaired t test in
C and F).
Immunity to N. gonorrhoeae JID 2013:208 (1 December) 1827

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to N. gonorrhoeae [10, 12]. However, systemic treatment with
these antibodies is unlikely to be a viable therapeutic option for
gonococcal infection owing to the high dosages needed and po-
tential toxicity. Our current results showed that intravaginally
administered microencapsulated antiTGF-- or antiIL-10 at
much lower doses similarly protected the mice against genital
tract N. gonorrhoeae infection, demonstrating the clinical po-
tential of this technology. Because it is known that IL-12 can
antagonize some of the effects of IL-10 and reverse the regula-
tory effects of TGF- [3638], these results are consistent, and
it is possible that treatment with IL-12 microspheres could be
combined with antiIL-10 or anti TGF- microspheres to
achieve a greater effect.
Despite public health measures, gonorrhea remains one of the
most frequent reportable infectious diseases for which we have
no effective vaccine. N. gonorrhoeae has steadily developed resis-
tance to each class of antibiotics deployed against it, including
uoroquinolones and, most recently, even cephalosporins [5]; as
a result, there are serious concerns that gonorrhea might become
untreatable [3]. The results from the current study indicate that
local manipulation of cytokines that control Th1, Th2, and regu-
latory immune responses can reverse the inhibitory effect of N.
gonorrhoeae on adaptive immunity and may serve as novel, safe
approaches to the treatment and prevention of this very
common sexually transmitted infection.
In addition, our ndings may be useful in formulating novel
approaches to gonococcal vaccine development. Indeed it can
be argued that intravaginal administration of IL-12 micro-
spheres serves as an adjuvant that converts gonococcal infec-
tion into a live vaccine. This has the added advantage of
targeting the vaccine toward a population that is at risk of
repeat infection with gonorrhea.
Notes
Acknowledgments. We are grateful to the assistance of the Confocal
Microscope and Flow Cytometry Facility in the School of Medicine and Bi-
omedical Sciences, University at Buffalo. We thank Ann E. Jerse, PhD, for
helpful advice and valuable discussions during the course of this work,
Terry Mashtare, MS, for statistical analysis and advice, and Thomas Russo,
MD, for critical reading of the manuscript.
Financial support. This work was supported by the National Institute
of Allergy and Infectious Diseases at the National Institutes of Health
(grant AI074791 to M. W. R., and grant AI104067 to Y. L.), and by a grant
from The John R. Oishei Foundation, Buffalo (grant to M. W. R.).
Potential conicts of interest. N. K. E. has ownership interest in Thera-
pyX, which is developing sustained-release particulate formulations of
IL-12 for cancer therapy. All other authors report no potential conicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conicts of Interest. Conicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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