Enhancement of Adaptive Immunity to Neisseria Gonorrhoeae

8/12/2019 Enhancement of Adaptive Immunity to Neisseria Gonorrhoeae

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

Enhancement of Adaptive Immunity to

Neisseria gonorrhoeaeby Local IntravaginalAdministration of MicroencapsulatedInterleukin 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, andNeisseria gonorrhoeaeis 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. gonorrhoeaeproactively suppresses host T-helper (Th) 1/

Th2-mediated adaptive immune responses, which can be manipulated to generate protective immunity. Herewe 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 ofspecic 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 ofN. 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 theextensive and rapid antigenic variability of the gono-

coccus and its capacity to resist complement-mediated

bacteriolysis result in immune evasion [1, 68].However,

our recent studies using experimental murine infections

have demonstrated thatN. gonorrhoeae proactively sup-

presses host T-helper (Th) 1and 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

([email protected]; [email protected]).

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:

[email protected].

DOI: 10.1093/infdis/jit354

Immunity toN. gonorrhoeae JID 2013:208 (1 December) 1821
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]


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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.

MATERIALSANDMETHODS

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 pilusexpression, 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 vaginallyon 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

Hanksbuffered 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 ReactionTotal 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 detailedelsewhere [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. gonorrhoeaeinfection 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-tailedttests

to compare the mean values between 2 selected groups. Differ-

ences were considered statistically signicant atP< .05.

RESULTS

Intravaginal Administration of IL-12 Microspheres as

Protection Against Genital TractN. gonorrhoeaeInfection 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 experimentsshowed 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 (Figure1A).

Untreated or blank microsphere-treated mice cleared the

infection in approximately 15 days (Figure1Band1C). 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 infectionby 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 ofN. gonorrhoeae(Figure1Band1C).

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 ofN. gonor-

rhoeae infection but to a lesser extent than IL-12 microspheres

given on the same schedule (Figure1Band1C).

Effects of IL-12 Microsphere Treatment on Th1 and Antibody

Responses to Vaginal Gonococcal InfectionTo 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 byow 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+

T cells were detected (Figure 1D). The IL-12microsphere treatment

markedly enhanced Th1 immune responses toN. 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 microspheresameliorated 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 (Figure1F).

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 gonococcalinfection 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



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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 serumsamples 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 (unpairedttest inDH).



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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 (Figure1G).

IL-12 Microsphere Treatment Induction of Protective

Anamnestic Immunity Against SecondaryN. gonorrhoeae

InfectionWe 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. gonorrhoeaewere 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 withN. 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 (Figure2Aand2B). 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 secondaryNeisseria gonorrhoeaeinfection.

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 previouslytreated 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 (unpairedttest inCF).



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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 (Figure2Aand2B). However, previous IL-

12 microsphere treatment of sham-infected mice did not induce

protection against subsequent infection (Figure2Aand2B). This

result also excluded the possibility that any persisting micro-

spheres still affected the secondaryN. gonorrhoeaeinfection.

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 (Figure2Cand2D). 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 (Figure2Eand2F).

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 (Figure2AF).

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 dayprotected 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 and3B). 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

(Figure3Dand3E). Local antiTGF-or antiIL-10 microsphere

treatment was also associated with enhanced Th1 (IFN-) and

Th2 (IL-4) responses in ILNs (Figure3Cand3F).

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 ofN. gonorrhoeaeon 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 werereinfected after previous IL-12 microsphere treatment were sig-

nicantly higher than those from the same mice during

primaryN. gonorrhoeaeinfection, 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



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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 circulationwas 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-

rhoeaeby 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 againstN. 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 gonorrhoeaeinfection 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 inDplotted 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 (unpairedttest in

CandF).



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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. gonorrhoeaehas 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 ofN.

gonorrhoeaeon 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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Enhancement of Adaptive Immunity to Neisseria Gonorrhoeae